Methods for treating central nervous system diseases

Abstract

The invention provides a method for preventing or treating CNS diseases. The method comprises the step of administering to a subject in need thereof an effective amount of (i) a polymer-flavonoid conjugate, (ii) a flavonoid oligomer, or (iii) a micelle having an outer shell comprising one or more polymer-flavonoid conjugates and optionally an inner shell comprising one or more flavonoid oligomers, and a drug encapsulated within these shells. The method allows a therapeutically effective material to pass through the blood-brain barrier to treat CNS disease. The methods are effective in treating CNS diseases, such as brain tumors, stroke, neurodegenerative diseases.

Description

Technical Field

[0001] The present invention provides a method for treating a central nervous system (CNS) disease. The method comprises the step of administering to a subject in need thereof an effective amount of (i) a polymer-flavonoid conjugate, (ii) a flavonoid oligomer, or (iii) a micelle having a shell formed by one or more polymer-flavonoid conjugates or one or more flavonoid oligomers or a combination thereof and having a pharmaceutical agent encapsulated in the shell. Background Art

[0002] Central nervous system (CNS) diseases are neurological disorders that affect the structure or function of the brain or spinal cord (which together form the CNS). The condition can be a genetic metabolic disorder, the result of nerve damage from infection, a neurodegenerative disorder, a stroke, a brain tumor, or other problems from unknown or multiple factors. CNS diseases include brain tumors, Alzheimer's disease, Parkinson's disease, Huntington's disease, migraines, infections, multiple sclerosis, addiction, arachnoid cysts, attention-deficit / hyperactivity disorder (ADHD), autism, catalepsy, encephalitis, epilepsy / seizures, infections, locked-in syndrome, meningitis, migraines, myelopathy, Tourette's syndrome, Bell's palsy, headaches (largest brain disease), autoimmune disorders, cerebral palsy, motor neuron disease (MND), neurofibromatosis, epilepsy and seizures, acute spinal cord injury, amyotrophic lateral sclerosis (ALS), ataxia, Bell's palsy, cerebral aneurysms, obsessive-compulsive disorder (OCD), defects in the cerebral cortex including microgyria, polymicrogyria, bilateral frontal polymicrogyria, and pachygyria.

[0003] Brain tumors occur when abnormal cells form in the brain. Brain tumors can be primary brain cancer, metastatic brain cancer, or benign brain tumors. Generally, brain tumors develop when there is a problem with cell division. Problems with the body's immune system can lead to brain tumors.

[0004] A stroke is a medical condition in which poor blood flow to the brain causes cell death. There are two main types of stroke: ischemic stroke, which is caused by lack of blood flow, and hemorrhagic stroke, which is caused by bleeding. The main risk factor for stroke is high blood pressure. Other risk factors include high blood cholesterol, smoking, obesity, diabetes, a previous transient ischemic attack, end-stage renal disease, and atrial fibrillation. There is no treatment that regenerates dead brain cells after a stroke. The only FDA-approved drug for ischemic stroke is tPA, which breaks up blood clots in the brain. Prevention is an important clinical strategy. Oral anticoagulants such as warfarin are the mainstay of stroke prevention.

[0005] Neurodegenerative diseases are a group of diseases that primarily affect neurons in the human brain. Examples of neurodegenerative diseases are Alzheimer's disease (AD) and other dementias, Parkinson's disease (PD) and Parkinson's syndrome, prion diseases, motor neuron disease (MND), Huntington's disease (HD), spinocerebellar ataxia (SCA) and spinal muscular atrophy (SMA). Two major neurodegenerative diseases are Alzheimer's disease and Parkinson's disease. Some neurodegenerative disorders are caused by inherited genetic changes. Most neurodegenerative disorders are due to a combination of genetic and environmental factors. This makes it difficult to predict who will become ill.

[0006] Alzheimer's disease (AD) is the most common neurodegenerative disease, which is usually slow in onset and gradually worsens. Along with the development of the disease, symptoms may include language problems, disorientation (including being easily lost), mood swings, loss of motivation, self-neglect and behavioral problems. It is believed that when an abnormal amount of amyloid beta (A β) is formed in the brain, Alzheimer's disease occurs, and the amyloid beta accumulates as amyloid plaques and tau protein in the extracellular space, or accumulates as neurofibrillary tangles in the cell, thereby affecting neuronal function and connection, causing the progressive loss of brain function. At present, there is no treatment to stop or reverse its progress, although some treatments may temporarily improve symptoms.

[0007] Parkinson's disease (PD) is a long-term degenerative disorder of the central nervous system that primarily affects the motor system. Parkinson's disease is sometimes referred to as a class of neurodegenerative diseases called synucleinopathies due to abnormal accumulation of the protein alpha-synuclein in the brain. The most obvious early symptoms of PD are tremors, rigidity, slowed movements, and difficulty walking. In many people with PD, cognitive and behavioral problems may also occur along with depression, anxiety, and apathy. Parkinson's disease dementia becomes common in the late stages of the disease. There is currently no cure for PD; treatment aims to reduce the impact of the symptoms.

[0008] The blood-brain barrier (BBB) ​​is a highly selective semipermeable boundary of central nervous system (CNS) endothelial cells that prevents solutes in the circulating blood vessels from non-selectively crossing into the CNS where neurons reside. Therefore, the BBB is a barrier that impedes the accumulation of effective drugs in the brain.

[0009] There are 4 pathways for BBB permeation (passive diffusion, carrier-mediated transport, receptor-mediated transcytosis, and adsorption-mediated transcytosis). Adsorption-mediated transcytosis (AMT) is the major pathway. The AMT pathway uses caveolae as transport vehicles. Caveolae are a subset of lipid rafts present in BBB endothelial cells. Caveolae-mediated endocytosis is a key transport mechanism for the uptake of macromolecules from the bloodstream into the CNS.

[0010] Flavonoids have a general structure of a 15-carbon backbone consisting of two benzene rings (A and B) and a heterocyclic ring (C, which contains an embedded oxygen).

[0011]

[0012] This carbon structure can be abbreviated as C6-C3-C6. According to the IUPAC nomenclature, flavonoids can be classified as:

[0013] Flavonoids or bioflavonoids

[0014] Isoflavonoids, which are derived from the structure of 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone)

[0015] New flavonoids derived from the structure of 4-phenylcoumarin (4-phenyl-1,2-benzopyrone) BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 One embodiment of a MINC (Multi-channel Immunomodulatory Nanocomplex Combination Therapy)-agent is described, which is a micelle having a polymer-flavonoid conjugate (eg, PEG-EGCG conjugate) in a shell and having an encapsulated agent.

[0017] Figure 2 Another embodiment of a MINC-agent is described, which is a micelle comprising a polymer-flavonoid conjugate (eg, PEG-EGCG conjugate) in an outer shell and a flavonoid oligomer (eg, oligomeric EGCG (OEGCG)) in an inner shell and having an encapsulated agent.

[0018] Figure 3 It was shown that more MINC-doxorubicin (PEG-EGCG, OEGCG and doxorubicin) penetrated the surrogate BBB transwell model compared to doxorubicin alone.

[0019] Figure 4 The tumor suppressive efficacy of OEGCG in HER2-positive and -negative gliomas was shown, and OEGCG overcame temozolomide resistance.

[0020] Figure 5 It was shown that mice treated with MINC-anti-HER2 (PEG-EGCG, OEGCG and anti-HER2) had reduced luciferase signal in A172 glioma compared to saline-treated controls.

[0021] Figure 6 showed that OEGCG and MINC-BSA inhibited the growth of triple-negative breast cancer.

[0022] Figure 7 The ability of OEGCG in protecting against Aβ-induced cell death is shown.

[0023] Figure 8 It was shown that different polymer-flavonoid conjugates and different flavonoid oligomers all successfully generated MINC-anti-HER2 with similar particle size of approximately 100 nm.

[0024] Fig. 9 It was shown that different polymers in the polymer-flavonoid conjugates can be used to successfully generate MINC-BSA. These different polymers are PEG (A), HA (B) and dextran (C).

[0025] Fig.10 Successful formulation of MINC-anti-Aβ is shown.

[0026] Fig.11 Successful formulation of MINC-anti-α-syn is shown. DETAILED DESCRIPTION

[0027] definition

[0028] The term "about" is defined as ±10%, preferably ±5% of the stated value.

[0029] The term "cytokine" refers to small proteins (about 5 kDa to 70 kDa) that are important in cell signaling. Cytokines have been shown to participate in autocrine, paracrine, and endocrine signaling as immunomodulators. Cytokines include interferons, interleukins, lymphokines, tumor necrosis factors, and chemokines.

[0030] The term "epigallocatechin gallate" refers to the ester of epigallocatechin and gallic acid, and is used interchangeably with "epigallocatechin-3-gallate" or EGCG.

[0031] The term "oligomeric EGCG" (OEGCG) refers to 3 to 20 covalently linked EGCG monomers. OEGCG preferably contains 4 to 12 EGCG monomers.

[0032] The term "polyethylene glycol-epigallocatechin gallate conjugate" or "PEG-EGCG" refers to polyethylene glycol (PEG) conjugated to one or two EGCG molecules. The term "PEG-EGCG" refers to both PEG-mEGCG conjugate (monomeric EGCG) and PEG-dEGCG (dimeric EGCG) conjugates.

[0033] The present invention provides a method for treating CNS diseases. The method comprises the step of administering to a subject in need thereof an effective amount of (i) a polymer-flavonoid conjugate, (ii) a flavonoid oligomer, or (iii) a micelle having an outer shell formed by one or more polymer-flavonoid conjugates and an optional inner shell formed by one or more flavonoid oligomers and a drug encapsulated in these shells.

[0034] Flavonoids

[0035] The flavonoids suitable for use in the present invention have the general structure of Formula I:

[0036]

[0037] in:

[0038] R1 is H or phenyl;

[0039] R2 is H, OH, gallate or phenyl; wherein the phenyl is optionally substituted with one or more (e.g., 2 to 3) hydroxyl groups;

[0040] R3 is H, OH or =O (oxo); or

[0041] R1 and R2 together form a closed loop structure; or

[0042] R2 and R3 together form a closed loop structure.

[0043] The 2-, 3-, 4-, 5-, 6-, 7- or 8-position of Formula I may be bonded to a group containing hydrocarbon, halogen, oxygen, nitrogen, sulfur, phosphorus, boron or a metal.

[0044] Examples of flavonoids of Formula I include:

[0045]

[0046] Preferred flavonoid compounds of formula I include:

[0047] EGCG(CAS#989-51-5), EC(CAS#490-46-0), EGC(CAS#970-74-1) or ECG(CAS#1257-08-5)

[0048]

[0049] Polymer-flavonoid conjugates

[0050] As used herein throughout this application, polymer-flavonoid conjugate refers to a conjugate of a hydrophilic polymer and a flavonoid compound of Formula I.

[0051] Hydrophilic polymers refer to polymers that are soluble in polar solvents and can form hydrogen bonds. Hydrophilic polymers suitable for use in the polymer-flavonoid conjugates of the present invention include, but are not limited to, poly(ethylene glycol), aldehyde-derivatized hyaluronic acid, hyaluronic acid, dextran, diethyl acetal conjugates (e.g., diethyl acetal PEG), D-α-tocopheryl polyethylene glycol succinate, aldehyde-derivatized hyaluronic acid-tyramine, hyaluronic acid-aminoacetaldehyde diethyl acetal conjugate-tyramine, cyclotriphosphazene core phenoxymethyl (methylhydrazono) dendrimers or thiophosphoryl core phenoxymethyl (methylhydrazono) dendrimers, acrylamide, oxazoline, imine, acrylic acid, methacrylate, glycol, ethylene oxide, alcohol, amine, anhydride, ester, lactone, terephthalate, amide and ether, polyacrylamide, pol Sham, poly(N-isopropylacrylamide), poly(oxazoline), polyethyleneimine, poly(acrylic acid), polymethacrylate, poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol), poly(vinyl pyrrolidone), polyether, poly(allylamine), polyanhydride, poly(β-amino ester), poly(butylene succinate), polycaprolactone, polycarbonate, polydioxanone, poly(glycerol), polyglycolic acid, poly(3-hydroxypropionic acid), poly(2-hydroxyethyl methacrylate), poly(N-(2-hydroxypropyl) methacrylamide), polylactic acid, poly(lactic-co-glycolic acid), poly(orthoesters), poly(2-oxazoline), poly(sebacic acid), poly(terephthalate-co-phosphate), povidone and copolymers.

[0052] Preferred hydrophilic polymers include poly(ethylene glycol), hyaluronic acid, dextran, polyethylene imine, poloxamer, povidone, D-α-tocopherol and polyethylene glycol succinate. The molecular weight of the hydrophilic polymer in the polymer-flavonoid conjugate is generally 1K-100K Dalton, preferably 2K-40K Dalton, 2K-50K Dalton, 2K-80K Dalton, 3K-80K Dalton or 5K-40K Dalton.

[0053] In one embodiment, the polymer contains an aldehyde group conjugated to position 5, 6, 7 or 8 (preferably position 6 or 8) of the A ring of the flavonoid compound.

[0054] In another embodiment, the polymer contains a thiol group conjugated to R1 or R2 of the B ring of the flavonoid (when R1 or R2 is -OH).

[0055] In one embodiment, the polymer-flavonoid conjugate is PEG-EGCG, which is a PEG conjugated to one or two molecules of epigallocatechin gallate (EGCG). For example, PEG-EGCG can be prepared by reacting the aldehyde-terminated PEG to EGCG via a free aldehyde group with position 5, 6, 7, or 8 (preferably position 6 or 8) of formula I, by connecting PEG. See WO2006 / 124000 and WO2009 / 054813. PEG-EGCG can also be prepared by reacting the sulfo-terminated PEG to EGCG via a free sulfo group with R1 or R2 (wherein R1 or R2 is phenyl) of formula I, by connecting PEG, by conjugating the sulfo-terminated PEG to EGCG. See WO2015 / 171079.

[0056] Flavonoid oligomers

[0057] Flavonoid oligomers are conjugates of a flavonoid with one or more flavonoids. Flavonoid oligomers can contain the same flavonoids (homo-oligomers) or different flavonoids (hetero-oligomers). Flavonoid oligomers that can be used in the present invention generally have 2 to 50 or 2 to 20, preferably 4 to 12 flavonoids of one type or mixed types.

[0058] In some embodiments, the flavonoid oligomer is oligomeric EGC (OEGCG), oligomeric EC (OEC), oligomeric EGC (OEGC) or oligomeric ECG (OECG). OEGCG refers to 3 to 20 covalently linked EGCG monomers. For example, OEGCG can be synthesized according to WO2006 / 124000 at position 5, 6, 7 or 8 (preferably position 6 or 8) of the A ring.

[0059] Since the A ring is present in all flavonoids according to formula 1, other oligomeric flavonoids can be prepared analogously according to WO 2006 / 124000. For example, OEC, OEGC and OECG can also be prepared according to WO 2006 / 124000.

[0060] MINC-Pharmacy

[0061] MINC (Multichannel Immunomodulatory Nanocomplex Combination Therapy) is a platform technology that exploits the biological activity of polymer-flavonoid conjugates or flavonoid oligomers that form micelles in solution.

[0062] The MINC platform can encapsulate additional agents to form nanoparticle compositions for therapy. MINC-agents are micelles having an outer shell formed by one or more polymer-flavonoid conjugates and an optional inner shell formed by one or more flavonoid oligomers and drugs encapsulated in these shells. As used herein, an agent refers to a molecule (e.g., a drug) with therapeutic activity. For example, the encapsulated agent can be a variety of molecules, including small molecules, peptides, proteins, monoclonal antibodies, and vaccines.

[0063] In one embodiment, the MINC-agent is a micelle comprising a polymer-flavonoid conjugate (e.g., PEG-EGCG conjugate) in a shell and having an encapsulated agent (see Figure 1 ).

[0064] In another embodiment, the MINC-agent is a micelle comprising a polymer-flavonoid conjugate (e.g., PEG-EGCG conjugate) in an outer shell and a flavonoid oligomer (e.g., oligomeric EGCG (OEGCG)) in an inner shell and having an encapsulated agent (see Figure 2 ).

[0065] When the agent is a drug, the MINC-agent composition comprises two or more therapeutically active components that are functionally complementary to form a multi-targeted combination therapy through its backbone components (flavonoid conjugates or flavonoid oligomers) and the encapsulated agents.

[0066] In one embodiment, the agent in the MINC-agent is an antibody, including but not limited to anti-HER2, anti-EGFR, anti-PD-L1, anti-PDGFRA, anti-VEGFR2, anti-beta amyloid, anti-tau, or anti-alpha-synuclein.

[0067] In one embodiment, the agent in the MINC-agent is a cytokine, including but not limited to IL-2, IL-4, IL-12, IFN-α, IFN-β, IFN-γ, TNF-α, GM-CSF, GDNF, NRTN, PDGF-BB, CDNF.

[0068] In one embodiment, the pharmaceutical agent is a small compound, including but not limited to doxorubicin, disulfiram, celecoxib, temsirolimus, everolimus, vorinostat, cabozantinib, marizomib, fimepinostat, acetazolamide, metformin, vinblastine, and cyclophosphamide.

[0069] For example, MINC-agent is an anti-HER2 encapsulated in micelles formed by the polymer-flavonoid conjugate PEG-EGCG and the flavonoid oligomer OEGCG. For structure and formulation methods, see WO2009 / 054813.

[0070] For example, the MINC-agent is doxorubicin encapsulated in micelles formed by the polymer-flavonoid conjugate PEG-EGCG. For structure and formulation methods, see WO2011 / 112156.

[0071] Pharmaceutical composition

[0072] The present invention uses a pharmaceutical composition comprising a polymer-flavonoid conjugate, flavonoid oligomer or MINC-agent as described in the present application, and optionally one or more pharmaceutically acceptable excipients. For tablets, powders or parenteral formulations, the nanoparticle component in the pharmaceutical composition is generally about 1% to 100% or about 1% to 90%, preferably 20% to 90% or 30% to 80%. For capsule formulations, the polymer-flavonoid conjugate, flavonoid oligomer or MINC-agent composition in the pharmaceutical composition is generally 1% to 100%, preferably 20% to 100%, 50% to 100% or 70% to 100%. For liquid suspension formulations, the nanoparticle composition in the pharmaceutical composition is generally 1% to 50%, 5% to 50% or 10% to 40%.

[0073] In one embodiment, the pharmaceutical composition may be in the form of tablets, capsules, granules, fine granules, powders, suspensions, solutions, patches, parenterals, injectables, etc. The pharmaceutical composition may be prepared by conventional methods.

[0074] Pharmaceutically acceptable carriers are inactive ingredients that can be selected by those skilled in the art using conventional criteria. Pharmaceutically acceptable carriers can contain a variety of ingredients, including but not limited to saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents such as sodium chloride, potassium chloride, glycerol and glucose; pH adjusters and buffers such as salts of hydroxides, phosphates, citrates, acetates, borates and triethanolamine; antioxidants such as salts, acids and / or bases of bisulfites, sulfites, metabisulfites, thiosulfites, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol and ascorbyl palmitate; surfactants such as lecithin and phospholipids, including but not limited to phosphatidylcholine, phosphatidylethanolamine and phosphatidylcholine. alcohols; poloxamers and poloxamines; polysorbates such as polysorbate 80, polysorbate 60 and polysorbate 20; polyethers such as polyethylene glycol and polypropylene glycol; polyethylenes such as polyvinyl alcohol and polyvinyl pyrrolidone (PVP, povidone); cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose and hydroxypropylmethylcellulose and their salts; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, soybean oil; mono-, di- and triglycerides; polysaccharides such as dextran; and glycosaminoglycans such as sodium hyaluronate. Such pharmaceutically acceptable carriers may be preserved to prevent bacterial contamination with well-known preservatives including, but not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or may be formulated as non-preserved preparations for single or multiple use.

[0075] For example, the tablet, capsule or parenteral preparation of the active compound may contain other excipients that are not biologically active and do not react with the active compound. The excipients of tablets or capsules may include fillers, binders, lubricants and glidants, disintegrants, wetting agents and release rate regulators. Examples of excipients of tablets or capsules include but are not limited to carboxymethyl cellulose, cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, karaya gum, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide, poly (acrylic acid) and polyvinyl pyrrolidone.

[0076] For example, tablet formulations may contain inactive ingredients such as colloidal silicon dioxide, crospovidone, hypromellose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, sodium starch glycolate, and titanium dioxide. Capsule formulations may contain inactive ingredients such as gelatin, magnesium stearate, and titanium dioxide. Powder oral formulations may contain inactive ingredients such as silica gel, sodium benzoate, sodium citrate, sucrose, and xanthan gum.

[0077] The pharmaceutical composition can be applied by partial administration and systemic administration. Partial administration includes topical administration. Systemic administration includes oral administration, parenteral administration (such as intravenous administration, intramuscular administration, subcutaneous administration or rectal administration) and other systemic administration routes. In systemic administration, the active compound first reaches the plasma and is then distributed to the target tissue. Parenteral administration (such as intravenous bolus injection or intravenous infusion) and oral administration are preferred routes of administration.

[0078] Treatment

[0079] The present invention relates to a method for preventing or treating a CNS disease by administering the polymer-flavonoid conjugate, flavonoid oligomer or MINC-agent as described above to a subject in need thereof.

[0080] CNS diseases suitable for treatment by the present invention include, but are not limited to, neuronal degenerative diseases, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, spongiform encephalopathies, West Nile virus encephalitis, multiple sclerosis, brain injury, spinal cord injury, primary and metastatic brain cancer, Bell's palsy, headaches (the largest brain disease), autoimmune disorders, cerebral palsy, motor neuron disease (MND), neurofibromatosis, epilepsy and seizures, acute spinal cord injury, amyotrophic lateral sclerosis (ALS), ataxia, Bell's palsy, cerebral aneurysm, obsessive-compulsive disorder (OCD), defects in the cerebral cortex including microgyria, polymicrogyria, bilateral frontal polymicrogyria and pachygyria.

[0081] An important function of polymer-flavonoid conjugates and flavonoid oligomers is to increase the delivery of drugs to the brain (through the BBB) to enhance therapeutic efficacy. This function is due to the ability of polymer-flavonoid conjugates to penetrate the BBB. Drug molecules are encapsulated, and they are not exposed to the BBB, so they have no effect on entering the CNS. This brain delivery is applicable to all kinds of brain diseases, including brain tumors (gliomas, choroid plexus tumors, pineal tumors, brain metastases, meningiomas, pituitary tumors, neurological tumors, central nervous system lymphomas), neuroinflammatory diseases (encephalitis, meningitis), neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Lewy body dementia, Huntington's disease), motor neuron diseases (ataxia, neurofibromatosis, amyotrophic lateral sclerosis (ALS), catalepsy, epilepsy / seizures, locked-in syndrome), CNS diseases with spinal cord injury (acute spinal cord injury, myelopathy, multiple sclerosis), CNS diseases caused by circulatory system disorders (stroke, cerebral aneurysm), and cerebral cortical disorders (microgyria, polymicrogyria, bilateral frontal polymicrogyria, and pachygyria).

[0082] Another function of polymer-flavonoid conjugates and flavonoid oligomers is to reduce neuronal cell death and enhance cell regeneration to restore cognitive behavior. This function treats neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Lewy body dementia and Huntington's disease. Example 6

[0083] Another function of polymer-flavonoid conjugates and flavonoid oligomers is to reduce neuronal oxidative stress and inflammation, and reduce disease progression and recurrence. Oxidative stress leads to neurotoxicity and is involved in the development of neurodegenerative diseases and CNS inflammatory diseases (including ischemic and hemorrhagic stroke). Reducing neuronal oxidative stress will reduce neuronal cell death and treat neuroinflammatory diseases (encephalitis, meningitis), motor neuron disease ALS, locked-in syndrome, catalepsy or seizures, CNS diseases with spinal cord injury (acute spinal cord injury, myelopathy, multiple sclerosis), CNS diseases caused by circulatory disorders (stroke, cerebral aneurysm), cerebral cortical disorders (microgyria, polymicrogyria, bilateral frontal polymicrogyria and pachygyria) and neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Lewy body dementia, Huntington's disease).

[0084] Another function of polymer-flavonoid conjugates and flavonoid oligomers is to reduce the accumulation of abnormal proteins (e.g., beta-amyloid, Tau and alpha-synuclein) and delay and / or reduce the progression and / or recurrence of the disease. These methods treat neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Lewy body dementia and Huntington's disease).

[0085] Another function of polymer-flavonoid conjugates and flavonoid oligomers is to activate the patient's immune system and reduce the development of brain tumors. These methods can be used to treat brain tumors (gliomas, choroid plexus tumors, pineal tumors, brain metastases, meningiomas, pituitary tumors, neural tumors, CNS lymphomas).

[0086] Polymer-Flavonoid

[0087] In a first aspect of the invention, the method comprises the step of administering an effective amount of a polymer-flavonoid conjugate to a subject in need thereof to treat a CNS disease. The CNS disease is a brain tumor, a circulatory disorder, a CNS disease with spinal cord injury, a neuroinflammatory disease, a motor neuron disease or a cerebral cortex disorder.

[0088] As used in this application, an "effective amount" is an amount effective for treating a disease by ameliorating the pathological condition or reducing the symptoms of the disease.

[0089] The polymer-flavonoid conjugate of the present invention can pass through the blood-brain barrier (BBB) ​​from the circulatory blood vessels to reach the brain. The polymer-flavonoid conjugate has immune function and disease regulation function for treating CNS disorders. In addition, it has neuronal cell repair or regeneration activity for treating CNS disorders.

[0090] In one embodiment, the flavonoid is EGCG, EC, EGC or ECG.

[0091] In one embodiment, the polymer is a hydrophilic polymer having a molecular weight of 1,000 to 100,000 Daltons and is selected from the group consisting of PEG, hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-alpha-tocopherol, and polyethylene glycol succinate.

[0092] A preferred polymer-flavonoid conjugate is PEG-EGCG.

[0093] In one embodiment, the CNS disease is a brain tumor selected from the group consisting of glioma, brain metastasis, neurological tumor, central nervous system lymphoma, choroid plexus tumor, pineal tumor, meningioma and pituitary tumor.

[0094] In one embodiment, the CNS disease is a CNS disease caused by a circulatory disorder selected from the group consisting of: stroke and cerebral aneurysm.

[0095] In one embodiment, the CNS disease is caused by a spinal cord injury selected from the group consisting of multiple sclerosis, acute spinal cord injury and myelopathy,

[0096] In one embodiment, the CNS disease is a neuroinflammatory disease of encephalitis or meningitis.

[0097] In one embodiment, the CNS disease is a motor neuron disease selected from the group consisting of amyotrophic lateral sclerosis (ALS), epilepsy, seizures, catalepsy, ataxia, and locked-in syndrome.

[0098] In one embodiment, the CNS disease is a cerebral cortical disorder selected from the group consisting of microgyria, polymicrogyria, bilateral frontal polymicrogyria, and pachygyria.

[0099] The polymer-flavonoid (eg, PEG-EGGC) for injection is typically administered at 0.1 mg / kg to 5000 mg / kg (total weight of polymer-flavonoid / body weight of subject) or 1 mg / kg to 1000 mg / kg.

[0100] Flavonoid oligomers

[0101] In a second aspect of the invention, the method comprises the step of administering an effective amount of flavonoid oligomers to a subject in need thereof to treat a CNS disease. The CNS disease is a brain tumor, a circulatory disorder, a CNS disease with spinal cord injury, a neuroinflammatory disease, a motor neuron disease or a cerebral cortex disorder.

[0102] The flavonoid oligomers of the present invention can reach the brain through the BBB from the circulatory blood vessels. The flavonoid oligomers have immune functions and disease regulation functions for treating CNS disorders. In addition, they have neuronal cell repair or regeneration activity for treating CNS disorders.

[0103] In one embodiment, the flavonoid oligomer is an oligomer of EGCG, EC, EGC or ECG.

[0104] In one embodiment, the flavonoid oligomer comprises 4 to 12 flavonoids of EGCG, EC, EGC or ECG.

[0105] In one embodiment, the CNS disease is a brain tumor selected from the group consisting of glioma, brain metastasis, neurological tumor, central nervous system lymphoma, choroid plexus tumor, pineal tumor, meningioma and pituitary tumor.

[0106] In one embodiment, the CNS disease is a CNS disease caused by a circulatory disorder selected from the group consisting of: stroke and cerebral aneurysm.

[0107] In one embodiment, the CNS disease is caused by a spinal cord injury selected from the group consisting of multiple sclerosis, acute spinal cord injury and myelopathy,

[0108] In one embodiment, the CNS disease is a neuroinflammatory disease of encephalitis or meningitis.

[0109] In one embodiment, the CNS disease is a motor neuron disease selected from the group consisting of amyotrophic lateral sclerosis (ALS), epilepsy, seizures, catalepsy, ataxia, and locked-in syndrome.

[0110] In one embodiment, the CNS disease is a cerebral cortical disorder selected from the group consisting of microgyria, polymicrogyria, bilateral frontal polymicrogyria, and pachygyria.

[0111] The flavonoid oligomer for injection (eg, OEGCG) is usually administered at 0.1 mg / kg to 1000 mg / kg (total weight of flavonoid oligomer / body weight of subject) or 1 mg / kg to 100 mg / kg.

[0112] MINC- medicine thing

[0113] In a third aspect of the invention, the method comprises administering to a subject in need thereof an effective amount of micelles to treat a CNS disease, the micelles having an outer shell comprising one or more polymer-flavonoid conjugates and an optional inner shell comprising one or more flavonoid oligomers and a drug encapsulated in the shells. In one embodiment, the outer shell is formed by one or more polymer-flavonoid conjugates. In one embodiment, the inner shell is formed by one or more flavonoid oligomers.

[0114] The CNS disease is Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, brain tumor, stroke, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS) or acute spinal cord injury, encephalitis, epilepsy, seizures, meningitis, motor neurone disease (MND) or cerebral aneurysm.

[0115] The polymer-flavonoid conjugate or flavonoid oligomer can be used as a delivery vehicle for drugs from the circulating blood vessels across the BBB to the brain to deliver the drug for the treatment or diagnosis of CNS disorders.

[0116] In one embodiment, the polymer is a hydrophilic polymer having a molecular weight of 1,000 to 100,000 Daltons and is selected from the group consisting of poly(ethylene glycol) (PEG), hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-α-tocopherol, and polyethylene glycol succinate;

[0117] In one embodiment, the flavonoid oligomer comprises 2 to 20 flavonoids of EGCG, EC, EGC or ECG;

[0118] In one embodiment, the shell is formed from PEG-EGCG.

[0119] In one embodiment, the shell is formed from PEG-EGCG and OEGCG.

[0120] In one embodiment, the CNS disease is Alzheimer's disease and the drug is anti-β-amyloid, anti-tau, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, or BDNF.

[0121] In one embodiment, the CNS disease is Parkinson's disease and the drug is anti-α-synuclein, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, GDNF, NRTN, PDGF-BB, CDNF, or BDNF.

[0122] In one embodiment, the CNS disease is dementia with Lewy bodies and the drug is anti-β-amyloid or anti-α-synuclein, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, or BDNF.

[0123] In one embodiment, the CNS disease is a brain tumor and the drug is doxorubicin, disulfiram, celecoxib, temsirolimus, everolimus, vorinostat, cabozantinib, marizomib, filmenostat, acetazolamide, metformin, vinblastine, cyclophosphamide, anti-HER2, anti-EGFR, anti-PD-1, anti-PD-L1, anti-PDGFRA, anti-VEGFR2, IL-2, IL-4, IL-12, IFN-α, IFN-β, IFN-γ, or TNF-α.

[0124] In one embodiment, the CNS disease is stroke and the drug is a MMP inhibitor, an eNOS inhibitor, anti-TLR4, anti-HSP, anti-IL6, anti-IL-12, S100β, fibronectin, MCP-1, MMP9, UCH-L1, BDNF, GDNF, NRTN, PDGF-BB, or CDNF.

[0125] In one embodiment, the CNS disease is Huntington's disease and the drug is anti-mHtt, anti-α-synuclein, anti-SEMA4D, anti-TNFα, Tetrabenazine, deutetrabenazine, valbenazine, bevantolol, pridopidine, branaplam, nilotinib, mitoconix, or azathioprine.

[0126] In one embodiment, the CNS disease is multiple sclerosis and the drug is anti-CD4, anti-IL-17, anti-CD19, anti-CD20, anti-CD25, anti-CD52, anti-RGMA, anti-IL-12, anti-IL-23, anti-α4 integrin, anti-IL-2R, LINGO-1, or anti-NOGO-A.

[0127] In one embodiment, the CNS disease is amyotrophic lateral sclerosis (ALS) and the drug is anti-NOGO-A, a PKC inhibitor, IGF-1, NOGO-A, GDNF, VEGF, anti-SOD1, S1R, GLT-1, anti-Ataxin2, anti-TDP43, anti-hnRNP, CK-1 inhibitor, anti-FET or HDAC inhibitor, EPO or IL-2.

[0128] In one embodiment, the CNS disease is acute spinal cord injury and the drug is Nogo receptor extracellular domain, 5-HT1A receptor, FGF, GSK-3bβ inhibitor, anti-IN-1, TNF-α, IL-12, SDF-1α, SOD1, NEC-1, anti-P-selectin or anti-CD11d.

[0129] In one embodiment, the CNS disease is encephalitis and the drug is anti-FcRn, anti-IL-6, anti-CD20, anti-CD19, anti-CD38, anti-C5, or IL-2.

[0130] In one embodiment, the CNS disease is epilepsy or seizures and the drug is an mTOR inhibitor, a PI3K inhibitor, a GABA inhibitor, an anti-Glu3B peptide antibody, an anti-NR1 antibody, anti-CASPR2, or anti-LGI-1.

[0131] In one embodiment, the CNS disease is meningitis and the drug is a C1 inhibitor, anti-C5, anti-MASP-2, anti-PD-L1, anti-CTLA-4, or anti-PD-1.

[0132] In one embodiment, the CNS disease is motor neuron disease (MND) and the drug is anti-SOD1, anti-TDP-43, anti-C9ORF72, anti-Nogo-A, anti-MuSK, anti-IL-6R, anti-NRP-1, anti-myostatin, anti-CD40L, anti-DR-6, anti-IFN-g, anti-GD1a, anti-CTGF, or anti-HMGB1.

[0133] In one embodiment, the CNS disease is cerebral aneurysm and the drug is a TNF-α inhibitor, a MMP inhibitor, a MCP-1 inhibitor, a phosphodiesterase-4 inhibitor, a mast cell degranulation inhibitor, an anti-IL-1β.

[0134] The dosing of the MINC-agent is based on the known doses of the agent used to treat the specific disease and the condition of the subject. The dose can be a dose approved by the Food and Drug Administration (FDA) or a dose used in a clinical trial.

[0135] In the MINC-medicament, typically the dose of PEG-EGCG combined with OEGCG is between 10 ug / kg and 100 mg / kg.

[0136] The concentration of encapsulated drug agent can be as low as 10 μg / kg (eg, for a cytokine drug, rhBDNF) and as high as 100 mg / kg (for an antibody drug, eg, anti-α-synuclein antibody is at this level).

[0137] For example, to treat breast cancer metastases to the brain in adults, 6 mg / kg to 10 mg / kg of anti-HER2 (trastuzumab) is administered IV once every 3 weeks. Effective doses of MINC-anti-HER2 within the same dose range can be used to treat breast cancer metastases to the brain.

[0138] For the treatment of glioma, 40 mg / m2 once every 4 weeks 2 Liposomal-doxorubicin is administered IV. Effective doses of MINC-doxorubicin within the same dose range can be used to treat gliomas.

[0139] For the treatment of Alzheimer's disease, anti-beta amyloid (Aducanumab) is given IV at 10 mg / kg every four weeks. Effective doses of MINC-anti-beta amyloid within the same dose range can be used to treat Alzheimer's disease.

[0140] For the treatment of Alzheimer's disease, an anti-tau antibody (Semorinemab) is given IV at 3 mg / kg to 30 mg / kg once every four weeks. Effective doses of MINC-anti-tau within the same dose range can be used to treat Alzheimer's disease.

[0141] For the treatment of Parkinson's disease, the anti-alpha-synuclein antibody is administered IV at 1500 mg to 4500 mg once every four weeks. An effective dose of MINC-anti-alpha-synuclein within the same dose range can be used to treat Parkinson's disease.

[0142] To promote neuronal cell regeneration caused by stroke and neurodegenerative diseases, BDNF (rhBDNF) is administered daily at 25 μg / kg to 100 μg / kg intrathecally or by IV injection. Effective doses of MINC-BDNF within the same dose range can be used to treat stroke and neurodegenerative diseases that cause neuronal cell death.

[0143] In addition to reducing misfolded protein aggregation in beta-amyloid and tau proteins in Alzheimer's disease and alpha-synuclein in Parkinson's disease, EGCG also plays a neuroprotective function in these and other central nervous system diseases. Mechanistically, EGCG has neuroprotective functions directly on neuronal cells. These include antioxidant activity by acting as a free radical scavenger and anti-apoptotic activity by reducing the expression of pro-apoptotic genes. EGCG in polymer-flavonoid conjugates and MINC-agents provides the additional benefit of protecting nerve cells from toxin damage.

[0144] The present invention can be used to treat humans and non-human animals. For example, the present invention can be used to treat mammalian subjects, such as humans, horses, pigs, cats and dogs.

[0145] The present invention provides use of a composition for preventing or treating a CNS disease or maintaining the health of a CNS subject, wherein the composition comprises (i) a polymer-flavonoid conjugate, (ii) one or more flavonoid oligomers, or (iii) a micelle having an outer shell formed by one or more polymer-flavonoid conjugates and optionally an inner shell formed by one or more flavonoid oligomers, and a drug encapsulated in these shells, wherein the agent is a drug.

[0146] The following examples further illustrate the present invention. These examples are intended only to illustrate the present invention and should not be construed as limiting.

[0147] Example

[0148] Active ingredients

[0149] OEGCG :

[0150] OEGCG is oligomerized EGCG. OEGCG was prepared according to WO2006 / 124000.

[0151] PEG-EGCG :

[0152] PEG-EGCG is PEG conjugated to one or two EGCGs. PEG-EGCG is prepared according to WO2006 / 124000, WO2009 / 054813 or WO2015 / 171079.

[0153] MINC-doxorubicin :

[0154] MINC-doxorubicin is doxorubicin encapsulated within PEG-EGCG and was prepared according to WO2011 / 112156.

[0155] Other MINC-Pharmacy :

[0156] MINC-agents are prepared according to WO2011 / 112156 or WO2015 / 171079. Alternatively, MINC-agents may be prepared by encapsulating the agent within micelles formed by PEG-EGCG and OEGCG according to the methods in WO2006 / 124000 or WO2009 / 054813.

[0157] MINC-Drugs allow drugs to cross the BBB and treat CNS diseases (Examples 1-3)

[0158] Example 1: MINC-doxorubicin delivery of doxorubicin to the zebrafish brain .

[0159] Material

[0160] OEGCG is oligomerized EGCG. OEGCG was prepared according to WO2006 / 124000.

[0161] PEG-EGCG is PEG conjugated to one or two EGCGs. PEG-EGCG is prepared according to WO2006 / 124000, WO2009 / 054813 or WO2015 / 171079.

[0162] MINC-doxorubicin is doxorubicin encapsulated within PEG-EGCG and was prepared according to WO2011 / 112156.

[0163] Zebrafish (Zebrafish International Resource Center, University of Oregon, USA).

[0164] method

[0165] Doxorubicin cannot effectively penetrate the BBB to enter the brain parenchyma. We formulated MINC-doxorubicin and used a zebrafish model to test whether the MINC formulation could enable encapsulated doxorubicin to cross the BBB and reach the brain.

[0166] In brief, Tg (flila: EGFP) transgenic zebrafish obtained from the Zebrafish International Resource Center were kept at 28.5°C under a photoperiod of 14 hours of light and 10 hours of darkness. After fertilization, eggs were collected and cultured in an aquarium. Embryos that developed to the 48 hours post-fertilization (hpf) stage were used for cardiac sinus venosus microinjection. For microinjection, MINC-doxorubicin was diluted to 386.37 μM in ddH2O containing phenol red (1%) as an injection tracer. The same amount of doxorubicin was used as a control. After the injection of MINC-doxorubicin (doxorubicin is a red fluorescent compound) using a NANOLITER 2000 microinjector with a Micromanipulators 3301R operator, the distribution of doxorubicin (red fluorescence) was observed under a Leica DM 2500 fluorescence microscope using a filter CHROMA 41004 (mcherry).

[0167] result

[0168] Under fluorescence microscopy, the MINC platform delivered more doxorubicin to the brain compared to a control doxorubicin (without MINC).

[0169] This experiment used a transparent zebrafish model to visualize the red drug doxorubicin, which is unable to enter the brain. Doxorubicin is able to enter the brain in large quantities after MINC formulation (bright red).

[0170] Example 2: MINC platform delivers drugs to penetrate the BBB in a surrogate cell model .

[0171] Material

[0172] OEGCG, PEG-EGCG and MINC-doxorubicin were the same as described in Example 1.

[0173] method

[0174] To confirm the efficacy of the MINC platform in enabling drugs to cross the BBB, we selected doxorubicin for in vitro BBB transwell studies.

[0175] In short, 3×10 4 Caco-2 cells were seeded into the lumen side of the insert of a 24-well transwell plate (Falcon). The culture medium was changed every 3 days. The transepithelial electrical resistance (TEER) value, which represents the integrity of the BBB barrier in each well, was measured by a Millicell ERS volt-ohmmeter (Millipore, Massachusetts, USA). When the TEER value of each insert reached 250Ω*cm 2 At the same time, (1) 5 μg / mL unencapsulated free doxorubicin or (2) MINC-doxorubicin with a fluorescence intensity equivalent to 5 μg / mL doxorubicin was added to the top of the insert. After incubation for 8 hours, the culture medium in the upper insert and the lower culture well was collected and the fluorescence signal (relative fluorescence unit, RFU) at Ex / Em=470 / 595nm was detected by Spectramax i3x. The drug penetration percentage was calculated according to the following formula: Permeation (%) = (RFU 下 ×7)÷(RFU 上 ×7+RFU 下 ×7)

[0176] result

[0177] The results are as follows Figure 3 Shown: More fluorescent signals were shown in the MINC-doxorubicin treated group compared to the doxorubicin group. The results demonstrated that more MINC-doxorubicin penetrated the surrogate BBB transwell model compared to doxorubicin alone.

[0178] Example 3: MINC platform delivers anti-HER2 to the brain of mice .

[0179] Material

[0180] OEGCG and PEG-EGCG were as described in Example 1.

[0181] Cyanine 5.5 NHS ester (Cy 5.5) (Aladdin)

[0182] Anti-HER2-Cy5.5 conjugate was prepared by reacting anti-HER2 antibody with Cy5.5-NHS ester according to the manufacturer's instructions (Aladdin).

[0183] MINC-anti-HER2-Cy5.5 is anti-HER2-Cy5.5 encapsulated in PEG-EGCG and OEGCG, and it was prepared according to WO2009 / 054813.

[0184] method

[0185] Antibody drugs cannot effectively penetrate the BBB to enter the brain parenchyma. We used fluorophore-labeled anti-HER2 (trastuzumab) as an example to demonstrate that the MINC formulation can deliver antibody drugs into the brain in a mouse model.

[0186] Use 6-week-old athymic nude mice-Foxnl nu Female mice were divided into two groups. One group (n=3) received 10 mg / kg anti-HER2-Cy5.5 as a control by tail vein iv bolus. The other group (n=3) received an equivalent anti-HER2-Cy5.5 dose in MINC-anti-HER2-Cy5.5 by tail vein iv bolus. After drug administration, live images were observed at 8 hours using IVIS (in vivo imaging system) Lumina III XRMS with ex / em at 674 / 692 nm.

[0187] result

[0188] The results showed that fluorescent signals were observed in the brain regions of mice receiving MINC-anti-HER2-Cy5.5 (STM-001), but not in mice treated with anti-HER2-Cy5.5 (trastuzumab).

[0189] Trastuzumab is able to treat HER2+ breast cancer, but is not approved for glioma due to poor delivery to the brain.This example shows that MINC-Trastuzumab can deliver trastuzumab to the brain of mice.

[0190] Flavonoid-oligomers (Example 4) and MINC-agents (Examples 5-6) in the treatment of refractory CNS diseases Effective

[0191] Example 4: OEGCG inhibits glioma cell growth .

[0192] Material

[0193] OEGCG was prepared according to Example 1.

[0194] CCK-8 kit (Targetmol, Shanghai, China)

[0195] A172 cell line (HER2+ glioma cell line) (CRL-1620, ATCC)

[0196] U87 cell line (HER2-glioma cell line) (HTB-14, ATCC)

[0197] method

[0198] We used an in vitro tumor inhibition assay to study the role of OEGCG in glioma growth. Both HER2-positive and -negative human glioma cell lines A172 (HER2+) and U87 (HER2-) obtained from ATCC were used to test the anti-tumor ability of OEGCG against glioma. Temozolomide-resistant cells derived from A172 and U87 were used to study the potential of OEGCG to overcome temozolomide resistance. Briefly, cells were seeded in 96-well plates at a density of 5000 cells / well and allowed to adhere to the surface for 24 hours. The cells were then treated with 0μM, 30μM, 60μM, 120μM of OEGCG for 72 hours. After treatment, the treated cells were incubated with CCK-8 reagent at 37°C for 1 hour and used The absorbance was detected at 450 nm using an iD3 reader. Results are reported as the mean ± standard deviation of at least two replicates.

[0199] result

[0200] Figure 4 The tumor suppressive efficacy of OEGCG in two glioma cell lines, A172 (HER2+) and U87 (HER2-), was shown. The results demonstrated that OEGCG has broad efficacy in treating glioma cells regardless of their HER2 expression levels ( Figure 4 A and 4C). In addition, OEGCG is also effective in treating temozolomide-resistant glioma cells ( Figure 4 B and 4D). Temozolomide is a first-line treatment for high-grade gliomas, and these results support that OEGCG can overcome temozolomide resistance and be used as a combination therapy with additional drugs encapsulated in MINC.

[0201] Example 5: MINC-anti-HER2 efficacy study in a glioma mouse model .

[0202] Material

[0203] OEGCG and PEG-EGCG were prepared according to Example 1.

[0204] MINC-anti-HER2-Cy5.5 is anti-HER2 encapsulated in PEG-EGCG and OEGCG, and it was prepared according to WO2009 / 054813.

[0205] A172 glioma cell line (CRL1620, ATCC); D-luciferin (Sigma-Aldrich)

[0206] method

[0207] We used an orthotopic glioma mouse model to confirm the efficacy of MINC-anti-HER2 in the treatment of glioma. To image tumor size, the A172 cell line was engineered with the luciferase gene (A172-Luc). To generate the mouse model, a cranial burr hole was created in the right frontal brain region. An ultrafine needle was then inserted to a depth of 3 mm using a stereotactic guidance device, and 1 × 10 6 A172-Luc cells (suspended in 3 μl of DMEM) were slowly injected into the mouse brain. The mice were divided into two groups, a vehicle group (saline as a no-treatment control) and a MINC-anti-HER2 group.

[0208] Two weeks after tumor implantation, saline or MINC-anti-HER2 was injected iv at 10 mg / kg via tail vein twice a week for 43 days. Tumor size was examined every two weeks using an in vivo imaging system (IVIS). After anesthetizing the mice, luciferin solution was injected intraperitoneally into the mice and then transferred to an IVIS chamber for image acquisition.

[0209] result

[0210] Figure 5 The results showed that mice treated with MINC-anti-HER2 had reduced luciferase signal in A172 glioma compared to saline-treated controls. The data demonstrated that MINC-anti-HER2 effectively inhibited glioma growth.

[0211] Example 6: OEGCG and MINC-BSA inhibit the growth of triple-negative cancer cells .

[0212] Material

[0213] OEGCG was prepared according to Example 1.

[0214] MINC-BSA is BSA encapsulated in PEG-EGCG and OEGCG.

[0215] Prepared according to WO2009 / 054813.

[0216] Acid phosphatase (ACP) assay kit (LSBio).

[0217] MDA-MB-231 cell line (CRM-HTB-26D, ATCC).

[0218] MDA-MB-468 cell line (HTB-132, ATCC).

[0219] BT-20 cell line (HTB-19, ATCC).

[0220] method

[0221] We used an in vitro tumor inhibition assay to investigate the effects of OEGCG and MINC-BSA in triple-negative breast cancer. Human MDA-MB-231, MDA-MB-468, and BT-20 triple-negative breast cell lines obtained from ATCC were used. Briefly, cells were seeded in 96-well plates at a density of 3,000 cells / well to 10,000 cells / well and allowed to adhere to the surface for 24 hours. The cells were then treated with 0 μM, 256 μM, 320 μM, 400 μM, and 500 μM of OEGCG for 72 hours, or with 0 μM, 4.2 μM, 8.3 μM, and 16.7 μM of MINC-BSA for 72 hours. After treatment, the treated cells were incubated with the pNPP reagent at 37°C for 30 minutes and cultured using The absorbance was measured at 410 nm using an iD3 reader. Results are reported as mean ± SD of at least three replicates. Acid phosphatase (ACP) is an enzyme that catalyzes the cleavage of phosphate groups from other molecules during digestion.

[0222] Enzyme levels can be used as a biomarker for cancer. This non-radioactive, colorimetric ACP assay is based on the cleavage of p-nitrophenol from a synthetic substrate. The increase in absorbance at 405 nm following the addition of a stop reagent is proportional to the enzyme activity and thus reflects the viability of the cancer cells.

[0223] result

[0224] Figure 6 The tumor suppressive efficacy of OEGCG and MINC-BSA in three triple-negative breast cancer cell lines was shown. Figure 6 A) and MINC-BSA( Figure 6B) has a significant tumor inhibitory effect in triple-negative breast cancer cell lines. Triple-negative breast cancer is unresponsive to anti-HER2 therapy. These results support that OEGCG and MINC nanoparticle platforms (MINC-BSA, BSA itself does not have an anti-cancer effect) can overcome anti-HER2 therapy resistance, and MINC can be used with encapsulated additional drugs. Considering that 25% to 46% of triple-negative breast cancer patients have brain metastasis problems, the results also show that the MINC platform can be used for brain metastasis cancer patients.

[0225] Alzheimer's disease (Examples 7-8)

[0226] Example 7. OEGCG inhibits Aβ-induced cell death

[0227] Material

[0228] OEGCG was prepared according to Example 1.

[0229] Recombinant Aβ(1-42) peptide MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Genscript, Thermo Fisher Scientific, or any publicly available supplier. HT-22 cells were obtained from Millipore (Bedford, MA, USA).

[0230] method

[0231] To confirm the efficacy of OEGCG in protecting Aβ-induced neuronal cell death, an in vitro MTT assay was performed. Briefly, HT-22 cells were cultured at 2 × 10 5 / well was inoculated in a 24-well plate and maintained in the logarithmic growth phase for 3 days. The cells were then treated with 2.5 μM oligo-Aβ, Aβ+OEGCG prepared for 24 hours. After incubation, the cells were washed once with warm PBS to remove the test material, and tetrazolium salts were added at room temperature for 30 minutes. The formazan product was then measured with a spectrophotometer at 550 nm. Viability was calculated as the percentage of control cells treated with a single vehicle (simulation).

[0232] result

[0233] Figure 7 The ability of OEGCG in protecting against Aβ-induced cell death is shown. Pathogenic Aβ accumulation can induce neuronal cell death and lead to neurodegeneration. The reduction of Aβ accumulation has therapeutic potential for treating Alzheimer's disease. The results prove that OEGCG reduces Aβ-induced cell death, which is the cause and main target for treating Alzheimer's disease.

[0234] Example 8. OEGCG and PEG-EGCG inhibit Aβ-induced oxidative stress.

[0235] Material

[0236] OEGCG and PEG-EGCG were prepared according to Example 1.

[0237] Recombinant Aβ(1-42) peptide DCFH-DA was purchased from GenScript Biotech, Thermo Fisher Scientific, or any publicly available supplier.

[0238] HT-22 cells were obtained from Millipore Corporation (Bedford, Massachusetts, USA).

[0239] method

[0240] To confirm the efficacy of OEGCG and PEG-EGCG in reducing Aβ-induced oxidative stress. In vitro cellular reactive oxygen species (ROS) staining tests were performed. HT-22 cells were seeded in 24-well plates at 2×105 cells / well and maintained for 3 days. The cells were then treated with Aβ with or without OEGCG (OE) and PEG-EGCG (PE). Oligo-Aβ was prepared according to previous experience. Briefly, the Aβ peptide was dissolved in 1 mM of 100% 1,1,1,3,3,3-hexafluoro-2-propanol and dried using a vacuum desiccator. Next, Aβ was resuspended in dimethyl sulfoxide (DMSO) to a concentration of 5 mM and stored at -20 μ. To obtain oligomers, the Aβ peptide was diluted to a final concentration of 100 μM using Dulbecco's modified Eagle medium (DMEM; Gibco), incubated at 4 °C after gentle shaking for 24 hours, and immediately added to the cell culture at a final concentration of 2.5 μM. After the cells were treated with Aβ for 1 hour, 50.9μg / mL, 25.4μg / mL, 12.7μg / mL of OEGCG and 105.7μg / mL, 52.4μg / mL and 26.4μg / mL of PEG-EGCG were added to the cells and incubated for 6 hours. The cells were then treated with 20μM DCFH-DA for 0.5 hour at 37°C and 5% CO2. After DCFH-DA staining, the cells were washed twice with DMEM and once with phosphate-buffered saline to remove background signals. Fluorescence images were also collected by fluorescence microscopy (DP72 / CKX41, Olympus Corporation (Olympus)), and all images used the same fluorescence conditions and exposure time.

[0241] result

[0242] Aβ induces oxidative stress in neuronal cells, so more reactive oxygen species (ROS) signals can be observed by DCFH-DA staining (green). The results of fluorescence images showed that both OEGCG and PEG-EGCG significantly reduced ROS production. ROS production is a risk factor for inducing brain inflammation and cell damage. The results show that OEGCG and PEG-EGCG have therapeutic potential to protect neuronal cells from Aβ-induced oxidative stress, which is the cause of neuronal death during the progression of Alzheimer's disease.

[0243] Evidence for different flavonoid oligomers and polymer-flavonoid conjugates in the formation of MINC-drugs (Examples 9-12)

[0244] Example 9: Preparation of different flavonoids for use in flavonoid oligomers and polymer-flavonoid conjugates MINC-Anti-HER2 Approach .

[0245] Material

[0246] OEGCG is oligomerized EGCG. OECG is oligomerized ECG. These flavonoid oligomers were prepared according to WO2006 / 124000.

[0247] PEG-EGCG is PEG conjugated to one or two EGCGs. PEG-EC is PEG conjugated to one or two ECs. PEG-ECG is PEG conjugated to one or two ECGs. These polymer-flavonoids are prepared according to WO2006 / 124000, WO2009 / 054813 or WO2015 / 171079.

[0248] Anti-HER2 was trastuzumab from Eirgenix.

[0249] method

[0250] MINC anti-HER2 nanoparticles were prepared according to WO2009 / 054813. Briefly, anti-HER2 was incubated in PBS. Subsequently, different flavonoid oligomers including OEGCG or OECG were added to anti-HER2, followed by different polymer-flavonoids including PEG-EGCG, PEG-ECG or PEG-EC. After incubation of the mixture at room temperature, unreacted oligomeric flavonoids and polymer-flavonoids were removed using a 10K MWCO centrifugal filter. Nanoparticle size was measured using DLS (Anton Paar Litesizer 500), and the results are shown in Table 1. Figure 8 shown.

[0251] result

[0252] Figure 8It was shown that different polymer-flavonoid conjugates and different flavonoid oligomers successfully generated MINC-anti-HER2 micelles with a single peak of similar particle size of about 100 nm. The results demonstrated that homogeneous nanoparticles (micelles) of the expected size were successfully formed, and no small peak of unencapsulated anti-HER2 (about 5 nm to 10 nm) was observed.

[0253] In this example, different flavonoid oligomers used included OEGCG ( Figure 8 A, 8C and 8D) and OECG ( Figure 8 B); and different polymer-flavonoids used including PEG-EGCG ( Figure 8 A and 8B), PEG-EC ( Figure 8 C) and PEG-ECG ( Figure 8 D).

[0254] These data support that MINC nanoparticles can be formed from different flavonoid oligomers and different polymer-flavonoid conjugates.

[0255] Example 10: Method for preparing MINC-BSA using different polymers in polymer-flavonoid conjugates Law .

[0256] Material

[0257] OEGCG is oligomeric EGCG, which was prepared according to WO2006 / 124000.

[0258] PEG-EGCG is PEG conjugated to one or two EGCGs. HA-EGCG is HA conjugated to one or two EGCGs. Dextran-EGCG is dextran conjugated to one or two EGCGs. These different polymer-flavonoids are prepared according to WO2006 / 124000, WO2009 / 054813 or WO2015 / 171079.

[0259] BSA was purchased from Sigma-Aldrich.

[0260] method

[0261] MINC (multi-target immune nanocarrier combination)-BSA nanoparticles were prepared according to WO2009 / 054813. In brief, BSA was incubated in PBS. Subsequently, OEGCG or OEGCG was added to BSA, and then different polymer-flavonoids, including PEG-EGCG, HA-EGCG and dextran-EGCG, were added. After the mixture was incubated at room temperature, unreacted OEGCG and polymer-flavonoids were removed using a 10K MWCO centrifugal filter. Nanoparticle size was measured using DLS (Anton PaarLitesizer 500).

[0262] result

[0263] Fig. 9 The results showed that different polymers in the polymer-flavonoid conjugates can be used to successfully generate MINC-BSA. The results demonstrated that homogeneous nanoparticles (micelles) were successfully formed and no small peak (about 5nm to 10nm) of unencapsulated BSA was observed. These different polymers were PEG ( Fig. 9 A) HA Fig. 9 B) and dextran ( Fig. 9 C) Taken together, these data support that MINC nanoparticles can be formed from different polymer-flavonoid conjugates.

[0264] Example 11: Method for preparing MINC-anti-Aβ .

[0265] Material

[0266] OEGCG is oligomeric EGCG, which was prepared according to WO2006 / 124000.

[0267] PEG-EGCG is PEG conjugated to one or two EGCGs and is prepared according to WO2006 / 124000, WO2009 / 054813 or WO2015 / 171079.

[0268] Anti-Aβ was purchased from Biolegend.

[0269] method

[0270] MINC-anti-Aβ nanoparticles (PEG-EGCG, OEGCG and anti-Aβ) were prepared according to Example 9. The size of the MINC-anti-Aβ nanoparticles was measured using DLS (AntonPaar Litesizer 500).

[0271] result

[0272] Fig.10Successful formulation of MINC-anti-Aβ is shown.

[0273] Example 12: Method for preparing MINC-anti-α-syn .

[0274] Material

[0275] OEGCG is oligomeric EGCG, which was prepared according to WO2006 / 124000.

[0276] PEG-EGCG is PEG conjugated to one or two EGCGs and is prepared according to WO2006 / 124000, WO2009 / 054813 or WO2015 / 171079.

[0277] Anti-α-syn was purchased from Biogen Biotech.

[0278] method

[0279] MINC-anti-α-syn nanoparticles (PEG-EGCG, OEGCG and anti-α-syn) were prepared according to Example 8. The size of the MINC-anti-α-syn nanoparticles was measured using DLS (Anton Paar Litesizer 500).

[0280] result

[0281] Fig.11 Successful formulation of MINC-anti-α-syn is shown.

[0282] List of abbreviations

[0283] ALS amyotrophic lateral sclerosis

[0284] Aβ β-amyloid protein

[0285] α-syn α-synuclein

[0286] BBB blood-brain barrier

[0287] BDNF Brain-derived neurotrophic factor

[0288] C1 complement component 1

[0289] C5 complement component 5

[0290] CASPR2 Contactin-associated protein-like 2

[0291] CDNF Brain dopamine neurotrophic factor

[0292] CNS Central Nervous System

[0293] CTGF Connective tissue growth factor

[0294] DR-6 Death receptor 6

[0295] EGFR Epidermal Growth Factor Receptor

[0296] eNOS Endothelial nitric oxide synthase

[0297] EPO erythropoietin

[0298] FcRn neonatal Fc receptor

[0299] FET Field Effect Transistor

[0300] FGF Fibroblast Growth Factor

[0301] GABA Gamma-aminobutyric acid

[0302] GDNF Glial cell line-derived neurotrophic factor

[0303] GLT1 Glutamate transporter-1

[0304] Glu3B Glutamate [NMDA] receptor subunit 3B

[0305] GSK-3β inhibitors Glycogen synthase kinase-3β inhibitors

[0306] HA Hyaluronic Acid

[0307] HDAC inhibitors Histone deacetylase inhibitors

[0308] HER2 Human epidermal growth factor receptor 2

[0309] HMGB1 High mobility group box 1

[0310] hnRNPs heterogeneous nuclear ribonucleoproteins

[0311] HSP Heat Shock Protein Antibody

[0312] HT1A receptor Serotonin receptor 1a

[0313] IFN-α Interferon-α

[0314] IL-1β Interleukin-1B

[0315] IL6R Interleukin-6 receptor

[0316] LGI-1 Leucine-rich glioma inactivated protein 1

[0317] LINGO-1 Leucine-rich repeat and Ig domain-containing 1

[0318] MASP-2 Mannan-binding lectin serine protease 2

[0319] MCP-1 Monocyte chemoattractant protein-1

[0320] MINC multi-channel immunomodulatory nanocomplex combination therapy

[0321] mHtt mutant huntingtin protein

[0322] MMP matrix metalloproteinase

[0323] MMP9 Matrix metalloproteinase 9

[0324] MND Motor Neurone Disease

[0325] mTOR mammalian target of rapamycin

[0326] NOGO-A Neurite Outgrowth Inhibitor A

[0327] NR1 N-methyl-D-aspartate receptor subunit 1

[0328] NRTN neurotrophic factor

[0329] PD-1 Programmed death-1

[0330] PDGF-BB Platelet-derived growth factor-Bb

[0331] PDGFRA Platelet-derived growth factor receptor A

[0332] PD-L1 Programmed death ligand 1

[0333] PEG Polyethylene glycol

[0334] PI3K Phosphatidylinositol 3-kinase

[0335] RGMA repulsive guidance molecule Bmp co-receptor A

[0336] S1R Sigma-1 receptor

[0337] SEMA4D axon guidance protein 4D

[0338] SOD1 Superoxide dismutase 1

[0339] TLR4 Toll-like receptor 4 antibody

[0340] TNFα Tumor necrosis factor alpha

[0341] TREM2 Triggering receptor expressed on myeloid cells 2

[0342] UCH-L1 Ubiquitin C-terminal hydrolase L1

[0343] VEGFR2 Vascular endothelial growth factor receptor 2

[0344] The invention and the manner and process of making and using the same are now described in full, clear, concise and precise terms to enable any person skilled in the art to make and use the invention. It should be understood that the foregoing describes preferred embodiments of the invention and that modifications may be made therein without departing from the scope of the invention as set forth in the claims. In order to particularly point out and distinctly claim the subject matter regarded as inventive, the specification ends with the following claims.

Claims

1. A method for treating a central nervous system (CNS) disease, the method comprising the step of administering to a subject in need thereof an effective amount of a micelle having an outer shell comprising one or more polymer-flavonoid conjugates, an optional inner shell comprising one or more flavonoid oligomers, and a drug encapsulated within the shell; wherein the polymer is a hydrophilic polymer having a molecular weight of 1,000 to 100,000 Daltons and is selected from the group consisting of poly(ethylene glycol) (PEG), hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-α-tocopherol and polyethylene glycol succinate; The flavonoid is EGCG, EC, EGC or ECG, as shown in the following structure: The flavonoid oligomers contain 2 to 20 flavonoids of EGCG, EC, EGC or ECG; The CNS disease is selected from the group consisting of: Alzheimer's disease, Parkinson's disease, Lewy body dementia, brain tumors, stroke, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), acute spinal cord injury, encephalitis, Epilepsy, seizures, meningitis, motor neurone disease (MND) and brain aneurysms.

2. The method of claim 1, wherein the micelle has an outer shell comprising PEG-EGCG and an inner shell comprising an EGCG oligomer.

3. The method of claim 1 or 2, wherein the CNS disease is Alzheimer's disease and the drug is anti-β-amyloid, anti-tau, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2 or BDNF.

4. The method of claim 1 or 2, wherein the CNS disease is Parkinson's disease and the drug is anti-α-synuclein, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, GDNF, NRTN, PDGF-BB, CDNF or BDNF.

5. The method of claim 1 or 2, wherein the CNS disease is Lewy body dementia and the drug is anti-β-amyloid or anti-α-synuclein, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2 or BDNF.

6. The method of claim 1 or 2, wherein the CNS disease is a brain tumor and the drug is doxorubicin, disulfiram, celecoxib, temsirolimus, everolimus, vorinostat, cabozantinib, marizomib, feminostat, acetazolamide, metformin, vinblastine, cyclophosphamide, anti-HER2, anti-EGFR, anti-PD-1, anti-PD-L1, anti-PDGFRA, anti-VEGFR2, IL-2, IL-4, IL-12, IFN-α, IFN-β, IFN-γ or TNF-α.

7. The method of claim 1 or 2, wherein the CNS disease is stroke, and the drug is a MMP inhibitor, an eNOS inhibitor, anti-TLR4, anti-HSP, anti-IL6, anti-IL-12, S100β, fibronectin, MCP-1, MMP9, UCH-L1, BDNF, GDNF, NRTN, PDGF-BB, or CDNF.

8. The method of claim 1 or 2, wherein the CNS disease is Huntington's disease and the drug is anti-mHtt, anti-α-synuclein, anti-SEMA4D, anti-TNFα, tetrabenazine, deutetrabenazine, valbenazine, bevantolol, pridopidine, brampton, nilotinib, mitocinib, or azathioprine.

9. The method of claim 1 or 2, wherein the CNS disease is multiple sclerosis and the drug is anti-CD4, anti-IL-17, anti-CD19, anti-CD20, anti-CD25, anti-CD52, anti-RGMA, anti-IL-12, anti-IL-23, anti-α4 integrin, anti-IL-2R, LINGO-1, or anti-NOGO-A.

10. The method of claim 1 or 2, wherein the CNS disease is amyotrophic lateral sclerosis (ALS), and the drug is anti-NOGO-A, a PKC inhibitor, IGF-1, NOGO-A, GDNF, VEGF, anti-SOD1, S1R, GLT-1, anti-Ataxin2, anti-TDP43, anti-hnRNP, CK-1 inhibitor, anti-FET or HDAC inhibitor, EPO or IL-2.

11. The method of claim 1 or 2, wherein the CNS disease is acute spinal cord injury, and the drug is Nogo receptor extracellular domain, 5-HT1A receptor, FGF, GSK-3bβ inhibitor, anti-IN-1, TNF-α, IL-12, SDF-1α, SOD1, NEC-1, anti-P-selectin or anti-CD11d.

12. The method of claim 1 or 2, wherein the CNS disease is encephalitis and the drug is anti-FcRn, anti-IL-6, anti-CD20, anti-CD19, anti-CD38, anti-C5 or IL-2.

13. The method of claim 1 or 2, wherein the CNS disease is epilepsy or seizures, and the drug is an mTOR inhibitor, a PI3K inhibitor, a GABA inhibitor, an anti-Glu3B peptide antibody, an anti-NR1 antibody, anti-CASPR2, or anti-LGI-1.

14. The method of claim 1 or 2, wherein the CNS disease is meningitis and the drug is a C1 inhibitor, anti-C5, anti-MASP-2, anti-PD-L1, anti-CTLA-4, or anti-PD-1.

15. The method of claim 1 or 2, wherein the CNS disease is motor neuron disease (MND), and the drug is anti-SOD1, anti-TDP-43, anti-C9ORF72, anti-Nogo-A, anti-MuSK, anti-IL-6R, anti-NRP-1, anti-myostatin, anti-CD40L, anti-DR-6, anti-IFN-g, anti-GD1a, anti-CTGF, or anti-HMGB1.

16. The method of claim 1 or 2, wherein the CNS disease is cerebral aneurysm, and the drug is a TNF-α inhibitor, a MMP inhibitor, a MCP-1 inhibitor, a phosphodiesterase-4 inhibitor, a mast cell degranulation inhibitor, or an anti-IL-1β.

17. A method for treating a CNS disease, the method comprising the step of administering to a subject in need thereof an effective amount of a polymer-flavonoid conjugate, Wherein the flavonoid is EGCG, EC, EGC or ECG, as shown in the following structure: The polymer is a hydrophilic polymer having a molecular weight of 1,000 to 100,000 Daltons and is selected from the group consisting of PEG, hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-α-tocopherol and polyethylene glycol succinate; and The CNS disease is selected from the group consisting of brain tumors, circulatory disorders, CNS diseases associated with spinal cord injury, neuroinflammatory diseases, motor neuron diseases and cerebral cortical disorders.

18. The method of claim 17, wherein the polymer-flavonoid conjugate is PEG-EGCG.

19. A method for treating a CNS disease, the method comprising the step of administering an effective amount of a flavonoid oligomer to a subject in need thereof, Wherein the flavonoid is EGCG, EC, EGC or ECG, as shown in the following structure: The flavonoid oligomers contain 4 to 12 flavonoids of EGCG, EC, EGC or ECG; and The CNS disease is selected from the group consisting of brain tumors, circulatory disorders, CNS diseases associated with spinal cord injury, neuroinflammatory diseases, motor neuron diseases and cerebral cortical disorders.

20. The method of claim 19, wherein the flavonoid oligomer is an oligomer of EGCG.

21. The method according to any one of claims 17 to 20, wherein the CNS disease is a brain tumor selected from the group consisting of glioma, brain metastasis, neurological tumor, central nervous system lymphoma, choroid plexus tumor, pineal tumor, meningioma and pituitary tumor.

22. The method according to any one of claims 17 to 20, wherein the CNS disease is a CNS disease caused by a circulatory disorder selected from the group consisting of: stroke and cerebral aneurysm.

23. The method of any one of claims 17 to 20, wherein the CNS disease is a CNS disease with spinal cord injury selected from the group consisting of multiple sclerosis, acute spinal cord injury, and myelopathy.

24. The method according to any one of claims 17 to 20, wherein the CNS disease is a neuroinflammatory disease of encephalitis or meningitis.

25. The method of any one of claims 17 to 20, wherein the CNS disease is a motor neuron disease selected from the group consisting of amyotrophic lateral sclerosis (ALS), epilepsy, seizures, catalepsy, ataxia, and locked-in syndrome.

26. The method according to any one of claims 17 to 20, wherein the CNS disease is a cerebral cortical disorder selected from the group consisting of microgyria, polymicrogyria, bilateral frontal polymicrogyria and pachygyria.

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