Methods for Treating Central Nervous System Disorders

Abstract

The present invention provides a method for preventing or treating a CNS disease. The method includes administering to a subject in need of treatment 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, an inner shell optionally comprising one or more flavonoid oligomers, and a drug encapsulated within the shell. The method of the present invention allows a therapeutically active substance to cross the blood-brain barrier to treat the CNS disease. The method of the present invention is effective in treating CNS diseases such as brain tumors, stroke, and neurodegenerative diseases.

Description

[Technical field]

[0001] The present invention provides a method for treating a central nervous system (CNS) disease, comprising 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 drug encapsulated within the shell. [Background technology]

[0002] A central nervous system (CNS) disease is a neurological disorder that affects the structure or function of the brain or spinal cord, which collectively form the CNS. The condition may be an inherited metabolic disorder, a result of nerve damage due to infection, a neurodegenerative condition, stroke, brain tumor, or other problem of unknown or multiple contributing factors. CNS disorders include brain tumors, Alzheimer's disease, Parkinson's disease, Huntington's disease, migraine, infection, multiple sclerosis, addiction, arachnoid cysts, attention deficit / hyperactivity disorder (ADHD), autism, catalepsy, encephalitis, epilepsy / seizures, infection, locked-in syndrome, meningitis, migraine, 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 aneurysm, obsessive-compulsive disorder (OCD), and defects within the cerebral cortex include microgyria, polymicrogyria, bilateral frontoparietal polymicrogyria, and pachygyria.

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

[0004] Stroke is a medical condition in which insufficient blood flow to the brain causes cell death. There are two main types of stroke: ischemic, due to lack of blood flow, and hemorrhagic, due to bleeding. The main risk factor for stroke is high blood pressure. Other risk factors include high blood cholesterol, smoking, obesity, diabetes mellitus, previous transient ischemic attack, end-stage renal disease, and atrial fibrillation. There is no treatment to regenerate dead brain cells after a stroke. The only FDA-approved drug for ischemic stroke is tPA, which breaks down 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 mainly affect neurons in the human brain. Examples of neurodegenerative diseases are Alzheimer's disease (AD) and other dementias, Parkinson's disease (PD) and Parkinson syndromes, prion diseases, motor neuron diseases (MND), Huntington's disease (HD), spinocerebellar degeneration (SCA), and spinal muscular atrophy (SMA). The two major neurodegenerative diseases are Alzheimer's disease and Parkinson's disease. Some neurodegenerative disorders are caused by genetic changes. Most neurodegenerative disorders are due to a combination of genetic and environmental factors. This makes it difficult to predict who will develop the disease.

[0006] Alzheimer's disease (AD) is the most common neurodegenerative disease, which usually begins slowly and worsens over time. As the disease progresses, symptoms may include problems with language, disorientation (e.g., getting lost easily), mood swings, loss of motivation, neglect, and behavioral problems. Alzheimer's disease is thought to occur when abnormal amounts of amyloid beta (Aβ) (accumulating extracellularly as amyloid plaques and tau protein, or intracellularly as neurofibrillary tangles) form in the brain, affecting neuronal function and connectivity, leading to a progressive loss of brain function. Currently, there is no treatment to stop or reverse its progression, but 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. PD is sometimes referred to as a type of neurodegenerative disease called synucleinopathy, which is caused by abnormal accumulation of the protein α-synuclein in the brain. The most obvious early symptoms of PD are tremor, rigidity, bradykinesia, and difficulty walking. Cognitive and behavioral problems can occur, with depression, anxiety, and blunted affect occurring in many people with PD. Parkinson's dementia often occurs in the advanced stages of the disease. There is no known cure for PD. Treatment aims to reduce the impact of the symptoms.

[0008] The blood-brain barrier (BBB) ​​is a highly selective, semi-permeable boundary of endothelial cells of the central nervous system (CNS) that prevents solutes in the circulating blood from non-selectively entering the CNS where neurons reside. Thus, the BBB is a barrier that prevents effective drugs from accumulating in the brain.

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

[0010] Flavonoids have a general structure of a 15-carbon skeleton consisting of two phenyl rings (A and B) and one heterocyclic ring (C, containing oxygen within the ring).

[0011] [ka] This carbon structure can be abbreviated as C6-C3-C6. According to the IUPAC nomenclature, flavonoids can be classified as follows: Flavonoids or bioflavonoids Isoflavonoids derived from the 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure · Neoflavonoids derived from the 4-phenylcoumarin (4-phenyl-1,2-benzopyrone) structure. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows one embodiment of a MINC (Multi-pathway Immune-modulating Nanocomplex Combination therapy) agent, which is a micelle with a polymer-flavonoid conjugate, e.g., a PEG-EGCG conjugate, in the shell and with an encapsulated drug.

[0013] [Diagram 2] Another embodiment of a MINC agent is shown, which is a micelle comprising a polymer-flavonoid conjugate, e.g., a PEG-EGCG conjugate, in the outer shell and a flavonoid oligomer, e.g., oligomeric EGCG (OEGCG), in the inner shell, with an encapsulated drug.

[0014] [Diagram 3] 1 shows that MINC-doxorubicin (PEG-EGCG, OEGCG, and doxorubicin) penetrated the surrogate BBB Transwell model more than doxorubicin alone.

[0015] [Figure 4] We show the tumor-inhibitory efficacy of OEGCG in both HER2-positive and -negative gliomas and that OEGCG overcomes temozolomide resistance.

[0016] [Diagram 5] 1 shows 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.

[0017] [Figure 6] 1 shows that OEGCG and MINC-BSA suppress triple-negative breast cancer proliferation.

[0018] [Figure 7] 1 shows the ability of OEGCG in protecting against Aβ-induced cell death.

[0019] [Figure 8] It is shown that different polymer-flavonoid conjugates and different flavonoid oligomers were all successful in generating MINC-anti-HER2 with similar particle sizes of approximately 100 nm.

[0020] [Figure 9] FIG. 1 demonstrates that different polymers can be used in the polymer-flavonoid conjugate to successfully generate MINC-BSA. The different polymers are PEG (A), HA (B) and dextran (C).

[0021] [Figure 10] We show that MINC-anti-Aβ was successfully formulated.

[0022] [Figure 11] We demonstrate the successful formulation of MINC-anti-α-syn. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description of the Invention definition The term "about" is defined as ±10%, preferably ±5% of the stated value.

[0024] The term "cytokine" refers to small proteins (approximately 5-70 kDa) that are important in cell signaling. Cytokines have been shown to be involved in autocrine, paracrine, and endocrine signaling as immunomodulatory agents. Cytokines include interferons, interleukins, lymphokines, tumor necrosis factors, and chemokines.

[0025] The term "epigallocatechin gallate" refers to an ester of epigallocatechin and gallic acid and is used interchangeably with "epigallocatechin-3-gallate" or EGCG.

[0026] The term "oligomeric EGCG" (OEGCG) refers to 3 to 20 covalently linked EGCG monomers. OEGCG preferably contains 4 to 12 EGCG monomers.

[0027] The term "polyethylene glycol-epigallocatechin gallate conjugate" or "PEG-EGCG" refers to polyethylene glycol (PEG) bound to one or two EGCG molecules. The term "PEG-EGCG" refers to both PEG-mEGCG conjugates (monomeric EGCG) and PEG-dEGCG (dimeric EGCG) conjugates.

[0028] The present invention provides a method for treating a CNS disease comprising administering to a subject in need of treatment 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, optionally an inner shell formed by one or more flavonoid oligomers, and a drug encapsulated within the shell.

[0029] Flavonoids Flavonoids suitable for the present invention have the general structure of Formula I: [ka] [In the formula, R1 is H or phenyl; R2 is H, OH, gallate, or phenyl; the phenyl is optionally substituted with one or more (e.g., 2-3) hydroxyls; R3 is H, OH, or =O (oxo); or R1 and R2 together form a closed loop ring structure; or R2 and R3 together form a closed loop ring structure.

[0030] Positions 2, 3, 4, 5, 6, 7, or 8 of formula I can be bonded to a hydrocarbon, halogen, oxygen, nitrogen, sulfur, phosphorus, boron, or metal-containing group.

[0031] Examples of flavonoids of Formula I include: [ka] Preferred flavonoid compounds of formula I include: EGCG (CAS#989-51-5), EC (CAS#490-46-0), EGC (CAS#970-74-1) or ECG (CAS#1257-08-5). [ka]

[0032] Polymer-flavonoid conjugates As used herein throughout this application, a polymer-flavonoid conjugate refers to a conjugate of a hydrophilic polymer and a flavonoid compound of formula I.

[0033] Hydrophilic polymer refers to a polymer that is soluble in a polar solvent and can form hydrogen bonds. Suitable hydrophilic polymers for the polymer-flavonoid conjugates of the present invention include poly(ethylene glycol), aldehyde-derivatized hyaluronic acid, hyaluronic acid, dextran, diethylacetal conjugates (e.g., diethylacetal PEG), D-α-tocopheryl polyethylene glycol succinate, aldehyde-derivatized hyaluronic acid-tyramine, hyaluronic acid-aminoacetylaldehyde diethylacetal conjugate-tyramine, cyclotriphosphazene-core phenoxymethyl (methylhydrazono) dendrimers or thiophosphoryl-core phenoxymethyl (methylhydrazono) dendrimers, acrylamides, oxazolines, imines, acrylic acids, methacrylates, diols, oxiranes, alcohols, amines, anhydrides, esters, lactones, terephthalates, amides and ethers polyacrylamides, poloxamers, poly(N-isopropyl ethers, methyl ester ... Poly(N-(2-hydroxypropyl)methacrylamide), poly(oxazoline), polyethyleneimine, poly(acrylic acid), polymethacrylate, poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol), poly(vinylpyrrolidinone), polyether, poly(allylamine), polyanhydrides, 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 acid-co-glycolic acid), poly(ortho ester), poly(2 oxazoline), poly(sebacic acid), poly(terephthalate-co-phosphate), povidone and copolymers.

[0034] Preferred hydrophilic polymers include poly(ethylene glycol), hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-α-tocopheryl, and polyethylene glycol succinate. The molecular weight of the hydrophilic polymer in the polymer-flavonoid conjugate is generally 1K to 100K daltons, preferably 2K to 40K, 2K to 50K, 2K to 80K, 3K to 80K, or 5K to 40K daltons.

[0035] In one embodiment, the polymer contains an aldehyde group attached to the 5, 6, 7, or 8 position (preferably 6 or 8 position) of the A ring of the flavonoid compound. In another embodiment, the polymer contains a thiol group attached to R1 or R2 of the B ring of the flavonoid (when R1 or R2 is -OH).

[0036] In one embodiment, the polymer-flavonoid conjugate is PEG-EGCG, which is PEG bound to one or two molecules of epigallocatechin gallate (EGCG). For example, PEG-EGCG can be prepared by attaching an aldehyde-terminated PEG to EGCG by attaching the PEG via reaction of a free aldehyde group with the 5, 6, 7, or 8 position (preferably 6 or 8 position) of formula I. See WO 2006 / 124000 and WO 2009 / 054813. PEG-EGCG can also be prepared by attaching a thio-terminated PEG to EGCG by attaching the PEG via reaction of a free thio group with R1 or R2 of formula I (wherein R1 or R2 is a phenyl group). See WO 2015 / 171079.

[0037] Flavonoid Oligomers A flavonoid oligomer is a conjugate of one flavonoid with one or more flavonoids. Flavonoid oligomers can contain the same flavonoid (homo-oligomers) or different flavonoids (hetero-oligomers). Flavonoid oligomers useful in the present invention generally have 2-50 or 2-20, preferably 4-12, flavonoids of one or a mixture of types.

[0038] In some embodiments, the flavonoid oligomer is oligomeric EGC (OEGCG), oligomeric EGC (OEC), oligomeric EGC (OEGC), or oligomeric ECG (OECG). OEGCG refers to 3-20 covalently linked monomers of EGCG. OEGCG can be synthesized at the 5, 6, 7, or 8 position (preferably 6 or 8 position) of the A ring, for example, according to WO 2006 / 124000.

[0039] Since the A ring is present in all of the flavonoids of formula 1, other oligomeric flavonoids can be similarly prepared according to WO 2006 / 124000. For example, OEC, OEGC, and OECG can also be prepared according to WO 2006 / 124000.

[0040] MINC Agents MINC (Multi-pathway 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.

[0041] The MINC platform can encapsulate additional agents to form nanoparticle compositions for treatment. MINC agents are micelles with an outer shell formed by one or more polymer-flavonoid conjugates, an inner shell formed by one or more flavonoid oligomers, and an encapsulated drug within the shell. Agent, as used herein, refers to a molecule (e.g., a drug) that has therapeutic activity. For example, the encapsulated agent can be a variety of molecules, such as small molecules, peptides, proteins, monoclonal antibodies, and vaccines.

[0042] In one embodiment, the MINC agent is a micelle that contains a polymer-flavonoid conjugate, for example a PEG-EGCG conjugate, in the shell in which the drug is encapsulated (see FIG. 1).

[0043] In another embodiment, the MINC agent is a micelle comprising a polymer-flavonoid conjugate, e.g., a PEG-EGCG conjugate, in the outer shell and a flavonoid oligomer, e.g., oligomeric EGCG (OEGCG), in the inner shell, in which the drug is encapsulated (see FIG. 2).

[0044] When the agent is a drug, the MINC composition contains two or more components with therapeutic activity that function in a complementary manner to form a multi-target combination therapy with the scaffold component (flavonoid conjugate or flavonoid oligomer) and the encapsulated drug.

[0045] 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.

[0046] 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.

[0047] In one embodiment, the drug is a small compound, including but not limited to doxorubicin, disulfiram, celecoxib, temsirolimus, everolimus, vorinostat, cabozantinib, marizomib, fimepinostat, acetazolamide, metformin, vinblastine, and cyclophosphamide.

[0048] For example, the MINC agent is an anti-HER2 encapsulated within a micelle formed by the polymer-flavonoid conjugate PEG-EGCG and the flavonoid oligomer OEGCG (see WO 2009 / 054813 for structure and formulation methods).

[0049] For example, a MINC agent is doxorubicin encapsulated within a micelle formed by the polymer-flavonoid conjugate PEG-EGCG (see WO 2011 / 112156 for structure and formulation methods).

[0050] Pharmaceutical Compositions The present invention employs a pharmaceutical composition comprising a polymer-flavonoid conjugate, flavonoid oligomer, or MINC agent described in this application, and optionally one or more pharma- ceutically acceptable excipients. The nanoparticle component in the pharmaceutical composition is generally about 1-100% or 1-90%, preferably 20-90%, or 30-80%, for tablet, powder, or parenteral formulations. The polymer-flavonoid conjugate, flavonoid oligomer, or MINC agent composition in the pharmaceutical composition is generally 1-100%, preferably 20-100%, 50-100%, or 70-100%, for capsule formulations. The nanoparticle composition in the pharmaceutical composition is generally 1-50%, 5-50%, or 10-40%, for liquid suspension formulations.

[0051] In one embodiment, the pharmaceutical composition may be in the form of tablets, capsules, granules, fine granules, powders, suspensions, liquids, patches, parenteral preparations, injections, etc. The pharmaceutical composition may be prepared by a conventional method.

[0052] Pharmaceutically acceptable carriers that are non-active ingredients can be selected by those skilled in the art using conventional criteria. Pharmaceutically acceptable carriers include 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 hydroxides, phosphates, citrates, acetates, borates, and trolamine; antioxidants, such as bisulfites, sulfites, metabisulfites, thiosulfites, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol, and ascorbyl palmitate salts, acids, and / or bases; surfactants, such as lecithin and phospholipids, including, but not limited to, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol; poloxamers and poloxamines; polysorbates. polysorbate 80, polysorbate 60, and polysorbate 20; polyethers, such as polyethylene glycol and polypropylene glycol; polyvinyls, such as polyvinyl alcohol and polyvinylpyrrolidone (PVP, povidone); cellulose derivatives, such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose, and salts thereof; petroleum derivatives, such as mineral oil, and white petrolatum; fats, such as lanolin, peanut oil, palm oil, and soybean oil; monoglycerides, diglycerides, and triglycerides; polysaccharides, such as dextran; and glycosaminoglycans, such as sodium hyaluronate. Such pharma- ceutically acceptable carriers may be preserved against microbial contamination through the use of 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 unpreserved preparations for single or multiple use.

[0053] For example, a tablet, capsule, or parenteral formulation of an active compound may contain other excipients that are not biologically active and do not react with the active compound. Tablet or capsule excipients may include fillers, binders, lubricants and glidants, disintegrants, wetting agents, and release rate modifiers. Examples of tablet or capsule excipients include, but are not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide, poly(acrylic acid, and polyvinylpyrrolidone.

[0054] 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.

[0055] The pharmaceutical composition can be applied by local administration and systemic administration. Local administration includes topical administration. Systemic administration includes oral, parenteral (e.g., intravenous, intramuscular, subcutaneous, or rectal), and other systemic administration routes. In systemic administration, the active compound first reaches the plasma and then distributes to the target tissue. Parenteral administration, such as intravenous bolus injection or intravenous infusion, and oral administration are preferred administration routes.

[0056] Treatment method The present invention relates to methods of preventing or treating a CNS disorder by administering to a subject in need thereof a polymer-flavonoid conjugate, a flavonoid oligomer, or a MINC agent as described above.

[0057] Suitable CNS disorders to be treated by the present invention include, but are not limited to, neurodegenerative disorders, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, spongiform encephalopathies, West Nile virus encephalitis, multiple sclerosis, brain injury, spinal cord injury, primary brain cancer, and metastatic brain cancer, Bell's palsy, headaches (the most common brain disorder), 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), and defects in the cerebral cortex include microgyria, polymicrogyria, bilateral frontoparietal polymicrogyria, and pachygyria.

[0058] One important function of polymer-flavonoid conjugates and flavonoid oligomers is to increase drug delivery 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. The drug molecules are encapsulated and not exposed to the BBB, and therefore do not affect their entry into the CNS. This brain delivery applies to all kinds of brain diseases, including brain tumors (gliomas, choroid plexus tumors, pineal tumors, brain metastases, meningiomas, pituitary tumors, neuronal tumors, central nervous system lymphomas), neuroinflammatory diseases (encephalitis, meningitis), neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, 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 frontoparietal polymicrogyria, and pachygyria).

[0059] Another function of the 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. Ex6

[0060] Another function of polymer-flavonoid conjugates and flavonoid oligomers is to reduce oxidative stress and inflammation in neuronal cells, and to 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 oxidative stress in neuronal cells reduces neuronal cell death, and treats neuroinflammatory diseases (encephalitis, meningitis), motor neuron disease ALS, locked-in syndrome, catalepsy, epilepsy or seizures, CNS diseases with spinal cord injury (acute spinal cord injury, myelopathy, multiple sclerosis), CNS diseases caused by circulatory system disorders (stroke, cerebral aneurysm), cerebral cortical disorders (microgyria, polymicrogyria, bilateral frontoparietal polymicrogyria, and pachygyria), and neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, Huntington's disease).

[0061] Another function of the polymer-flavonoid conjugates and flavonoid oligomers is to reduce the accumulation of abnormal proteins (e.g., β-amyloid, tau, and α-synuclein) and to slow and / or reduce disease progression and / or recurrence. These processes treat neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, and Huntington's disease).

[0062] Yet another function of the polymer-flavonoid conjugates and flavonoid oligomers is to activate the immune system of the patient and reduce the incidence of brain tumors. These processes are useful in the treatment of brain tumors (gliomas, choroid plexus tumors, pineal tumors, brain metastases, meningiomas, pituitary tumors, neurotumors, CNS lymphomas).

[0063] Polymer-Flavonoid In a first aspect of the invention, the method comprises administering to a subject in need of treatment an effective amount of a polymer-flavonoid conjugate to treat a CNS disease, the CNS disease being a brain tumor, a circulatory system disorder, a CNS disease associated with spinal cord injury, a neuroinflammatory disease, a motor neuron disease, or a cerebral cortical disorder.

[0064] An "effective amount" as used in this application is an amount effective to treat a disease by ameliorating a pathological condition or alleviating a symptom of the disease.

[0065] The polymer-flavonoid conjugates of the present invention can cross the blood-brain barrier (BBB) ​​from the circulatory vessels to the brain. The polymer-flavonoid conjugates have immune and disease-modulating functions for treating CNS disorders. Furthermore, the polymer-flavonoid conjugates have neuronal cell repair or regeneration activities for treating CNS disorders.

[0066] In one embodiment, the flavonoid is EGCG, EC, EGC, or ECG.

[0067] 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-α-tocopheryl, and polyethylene glycol succinate.

[0068] A preferred polymer-flavonoid conjugate is PEG-EGCG.

[0069] In one embodiment, the CNS disease is a brain tumor selected from the group consisting of glioma, brain metastasis tumor, neuronal tumor, central nervous system lymphoma, choroid plexus tumor, pineal tumor, meningioma, and pituitary tumor.

[0070] In one embodiment, the CNS disease is a CNS disease caused by a circulatory system disorder selected from the group consisting of stroke and cerebral aneurysm.

[0071] 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. In one embodiment, the CNS disorder is a neuroinflammatory disorder of encephalitis or meningitis.

[0072] 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.

[0073] In one embodiment, the CNS disease is a cerebral cortical disorder selected from the group consisting of microgyria, polymicrogyria, bilateral frontoparietal polymicrogyria, and pachygyria.

[0074] The dosage for injection of polymer-flavonoid, such as PEG-EGGC, is generally 0.1-5000 mg / kg (total weight of polymer-flavonoid / body weight of subject), or 1-1000 mg / kg.

[0075] Flavonoid Oligomers In a second aspect of the invention, the method comprises administering to a subject in need of treatment an effective amount of a flavonoid oligomer to treat a CNS disease, the CNS disease being a brain tumor, a circulatory system disorder, a CNS disease associated with spinal cord injury, a neuroinflammatory disease, a motor neuron disease, or a cerebral cortical disorder.

[0076] The flavonoid oligomer of the present invention can cross the BBB from the circulatory blood vessels to the brain.The flavonoid oligomer has immune and disease-regulating functions for treating CNS disorders.Furthermore, the flavonoid oligomer has neuronal cell repair or regeneration activity for treating CNS disorders.

[0077] In one embodiment, the flavonoid oligomer is an oligomer of EGCG, EC, EGC, or ECG.

[0078] In one embodiment, the flavonoid oligomer comprises 4 to 12 flavonoids of EGCG, EC, EGC, or ECG.

[0079] In one embodiment, the CNS disease is a brain tumor selected from the group consisting of glioma, brain metastasis tumor, neuronal tumor, central nervous system lymphoma, choroid plexus tumor, pineal tumor, meningioma, and pituitary tumor.

[0080] In one embodiment, the CNS disease is a CNS disease caused by a circulatory system disorder selected from the group consisting of stroke and cerebral aneurysm.

[0081] 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. In one embodiment, the CNS disorder is a neuroinflammatory disorder of encephalitis or meningitis.

[0082] 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.

[0083] In one embodiment, the CNS disease is a cerebral cortical disorder selected from the group consisting of microgyria, polymicrogyria, bilateral frontoparietal polymicrogyria, and pachygyria.

[0084] The dosage for injection of a flavonoid oligomer, such as OEGCG, is generally 0.1-10,000 mg / kg (total weight of flavonoid oligomers / body weight of subject), or 1-100 mg / kg.

[0085] MINC Drugs In a third aspect of the present invention, the method comprises administering to a subject in need of treatment an effective amount of a micelle having an outer shell comprising one or more polymer-flavonoid conjugates, an inner shell optionally comprising one or more flavonoid oligomers, and a drug encapsulated within the shell, for treating a CNS disease. 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.

[0086] 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 neuron disease (MND), or cerebral aneurysm.

[0087] The polymer-flavonoid conjugates or flavonoid oligomers, as drug delivery vehicles, can cross the BBB from the circulation to the brain and deliver said drugs to treat or diagnose CNS disorders.

[0088] 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-tocopheryl, and polyethylene glycol succinate.

[0089] In one embodiment, the flavonoid oligomer comprises 2 to 20 flavonoids of EGCG, EC, EGC, or ECG. In one embodiment, the shell is formed by PEG-EGCG.

[0090] In one embodiment, the shell is formed by PEG-EGCG and OEGCG.

[0091] In one embodiment, the CNS disorder is Alzheimer's disease and the drug is anti-β amyloid, anti-tau, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, or BDNF.

[0092] In one embodiment, the CNS disorder is Parkinson's disease and the drug is anti-alpha-synuclein, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, GDNF, NRTN, PDGF-BB, CDNF, or BDNF.

[0093] 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.

[0094] In one embodiment, the CNS disease is a brain tumor and the drug is doxorubicin, disulfiram, celecoxib, temsirolimus, everolimus, vorinostat, cabozantinib, marizomib, fimepinostat, 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.

[0095] In one embodiment, the CNS disease is stroke and the drug is an 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.

[0096] In one embodiment, the CNS disorder is Huntington's disease and the drug is anti-mHtt, anti-alpha-synuclein, anti-SEMA4D, anti-TNFα, tetrabenazine, deutetrabenazine, valbenazine, bevantolol, pridopidine, branapram, nilotinib, mitoconix, or azathioprine. 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-alpha4 integrin, anti-IL-2R, LINGO-1, or anti-NOGO-A.

[0097] 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-ataxin 2, anti-TDP43, anti-hnRNPs, a CK-1 inhibitor, anti-FET or HDAC inhibitor, EPO, or IL-2.

[0098] In one embodiment, the CNS disease is acute spinal cord injury and the drug is the extracellular domain of the Nogo receptor, 5-HT1A receptor, FGF, a GSK-3bβ inhibitor, anti-IN-1, TNF-α, IL-12, SDF-1α, SOD1, NEC-1, anti-P-selectin, or anti-CD11d.

[0099] 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.

[0100] In one embodiment, the CNS disorder 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, an anti-CASPR2, or an anti-LGI-1.

[0101] In one embodiment, the CNS disease is meningitis and the drug is C1 inhibitor, anti-C5, anti-MASP-2, anti-PD-L1, anti-CTLA-4, or anti-PD-1.

[0102] In one embodiment, the CNS disease is a motor neuron disease (MND) and the drug is anti-SOD1, anti-TDP-43, anti-C90RF72, 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.

[0103] In one embodiment, the CNS disease is cerebral aneurysm and the drug is a TNF-α inhibitor, an MMP inhibitor, an MCP-1 inhibitor, a phosphodiesterase-4 inhibitor, a mast cell degranulation inhibitor, or an anti-IL-1β.

[0104] The dose of the MINC agent is based on the known dose of the agent to treat the particular disease and condition of the subject. The dose may be a dose approved by the Food and Drug Administration (FDA) or a dose used in clinical trials.

[0105] In the MINC formulation, the dose of PEG-EGCG in combination with OEGCG is generally 10 μg / kg to 100 mg / kg.

[0106] The concentration of the encapsulated drug agent can be as low as 10 μg / kg (e.g., for cytokine drugs, rhBDNF) and as high as 100 mg / kg (for antibody drugs, e.g., anti-alpha-synuclein antibodies at this level).

[0107] For example, when treating brain metastatic breast cancer in adult humans, anti-HER2 (trastuzumab) is administered at 6-10 mg / kg IV every 3 weeks. The same dose range of effective doses of MINC-anti-HER2 can be used to treat brain metastatic breast cancer.

[0108] Liposomal doxorubicin at 40 mg / m for treating gliomas 2 It is given IV every 4 weeks. Effective doses of MINC-doxorubicin within the same dose range can be used to treat gliomas.

[0109] When treating Alzheimer's disease, anti-beta amyloid (aducanumab) is given at 10 mg / kg IV every 4 weeks. The same dose range of effective doses of MINC-anti-beta amyloid can be used to treat Alzheimer's disease.

[0110] For treating Alzheimer's disease, anti-tau antibody (semolinemab) is given at 3-30 mg / kg IV every 4 weeks. Effective doses of MINC-anti-tau in the same dose range can be used to treat Alzheimer's disease.

[0111] For treating Parkinson's disease, anti-alpha-synuclein antibodies are given at 1500-4500 mg IV every 4 weeks. The same dose range of effective doses of MINC-anti-alpha-synuclein can be used to treat Parkinson's disease.

[0112] To promote neuronal cell regeneration caused by stroke and neurodegenerative diseases, BDNF (rhBDNF) is given daily at 25-100 μg / kg intrathecally or by IV injection. The same effective dose range of MINC-BDNF can be used to treat stroke and neurodegenerative diseases that cause neuronal cell death.

[0113] In addition to reducing misfolded protein aggregation in β-amyloid for Alzheimer's disease and α-synuclein for Parkinson's disease, EGCG also exerts neuroprotective functions in these and other central nervous system diseases.Mechanistically, EGCG has direct neuroprotective functions on neuronal cells.These functions 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 neuronal cells from toxin-induced damage.

[0114] The invention is useful for treating humans and non-human animals. For example, the invention is useful in treating mammalian subjects such as humans, horses, pigs, cats, and dogs.

[0115] The present invention provides use of a composition in preventing or treating a CNS disease or maintaining the health of a subject's CNS, 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 within the shell, and a micelle having an agent encapsulated within the shell, wherein the agent is a drug.

[0116] The following examples further illustrate the present invention. These examples are intended to be merely illustrative of the present invention and should not be construed as limiting. EXAMPLES

[0117] Active ingredients OEGCG: OEGCG is oligomerized EGCG. OEGCG is prepared according to WO 2006 / 124000.

[0118] PEG-EGCG: PEG-EGCG is PEG conjugated with one or two EGCG moieties. PEG-EGCG is prepared according to WO 2006 / 124000, WO 2009 / 054813, or WO 2015 / 171079.

[0119] MINC-doxorubicin: MINC-doxorubicin is doxorubicin encapsulated in PEG-EGCG and is prepared according to WO 2011 / 112156.

[0120] Other MINC agents: MINC agents are made according to WO 2011 / 112156 or WO 2015 / 171079. Alternatively, MINC agents can be prepared by encapsulating the drug within micelles formed by PEG-EGCG and OEGCG according to the methods of WO 2006 / 124000 or WO 2009 / 054813.

[0121] MINC-Drugs Deliver Drugs Across the BBB to Treat CNS Diseases (Examples 1-3) Example 1: MINC-doxorubicin delivers doxorubicin to the zebrafish brain material OEGCG is oligomerized EGCG. OEGCG is prepared according to WO 2006 / 124000.

[0122] PEG-EGCG is PEG conjugated with one or two EGCG moieties. PEG-EGCG is prepared according to WO 2006 / 124000, WO 2009 / 054813, or WO 2015 / 171079.

[0123] MINC-doxorubicin is doxorubicin encapsulated in PEG-EGCG and was prepared according to WO 2011 / 112156.

[0124] Zebrafish (Zebrafish International Resource Center, University of Oregon, USA).

[0125] method Doxorubicin does not efficiently penetrate the BBB into the brain parenchyma. We formulated MINC-doxorubicin and used a zebrafish model to test whether the MINC formulation could deliver encapsulated doxorubicin across the BBB to the brain.

[0126] Briefly, Tg(fli1a:EGFP) transgenic zebrafish obtained from the Zebrafish International Resource Center were maintained at 28.5 °C under a photoperiod of 14 h light and 10 h dark. After fertilization, eggs were collected and cultured in aquaria. Embryos that developed to the 48-h post-fertilization (hpf) stage were used for cardiac sinus 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 MINC-doxorubicin (doxorubicin is a red fluorescent compound) was injected using a NANOLITER2000 microinjector equipped with a Micromanipulators 3301R manipulator, the distribution of doxorubicin (red fluorescence) was observed under a Leica DM 2500 fluorescence microscope using the filter CHROMA 41004 (mcherry).

[0127] result Under a fluorescent microscope, the MINC platform was shown to deliver more doxorubicin to the brain than the doxorubicin control (no MINC).

[0128] This experiment used a transparent zebrafish model to visualize the drug doxorubicin, which is unable to enter the brain, but was able to enter the brain in large amounts (bright red) after MINC formulation.

[0129] Example 2: The MINC platform delivers drugs across the BBB in a surrogate cell model material OEGCG, PEG-EGCG and MINC-doxorubicin were the same as those described in Example 1.

[0130] method To confirm the effectiveness of the MINC platform in carrying drugs across the blood-brain barrier, we selected doxorubicin for an in vitro BBB Transwell study.

[0131] In short, 3 x 10 4 Caco-2 cells were seeded on the luminal side of the inserts of a 24-well Transwell plate (Falcon). The medium was changed every 3 days. The transepithelial electrical resistance (TEER) value, which indicates the BBB barrier integrity of each well, was measured using a Millicell ERS Voltohmmeter (Millipore, MA, USA). The TEER value of each insert was 250 Ω. * cm 2 When the cell culture medium reached 470 nm, either (1) 5 μg / mL of unencapsulated free doxorubicin or (2) MINC-doxorubicin with a fluorescence intensity equivalent to 5 μg / mL of doxorubicin was added to the top of the insert. After 8 h of incubation, the medium in the upper insert and the lower culture well was collected, and the fluorescence signal (relative fluorescence units, RFU) at Ex / Em=470 / 595 nm was detected by Spectramax i3x. The drug permeation rate was calculated according to the following formula: Permeation (%)=(RFU) 下部 ×7)÷(RFU 上部 ×7+RFU 下部 ×7)

[0132] result The results are shown in Figure 3: more fluorescent signal was observed in the MINC-doxorubicin treated group compared to the doxorubicin group. This result demonstrates that more MINC-doxorubicin penetrated the surrogate BBB Transwell model than doxorubicin alone.

[0133] Example 3: MINC platform delivers anti-HER2 to the mouse brain material OEGCG and PEG-EGCG are described in Example 1.

[0134] Cyanine 5.5 NHS ester (Cy5.5) (Aladdin).

[0135] Anti-HER2-Cy5.5 conjugates are prepared by reacting anti-HER2 with Cy5.5-NHS ester according to the manufacturer's instructions (Aladdin).

[0136] MINC-anti-HER2-Cy5.5 is anti-HER2-Cy5.5 encapsulated in PEG-EGCG and OEGCG and is prepared according to WO 2009 / 054813.

[0137] method Antibody drugs cannot efficiently penetrate the BBB and enter the brain parenchyma. Using fluorophore-labeled anti-HER2 (trastuzumab) as an example, we demonstrated that MINC formulations can deliver antibody drugs to the brain in mouse models.

[0138] 6-week-old athymic Nude-Foxn1 nu Female mice were used and divided into two groups. One group (n=3) received 10mg / kg anti-HER2-Cy5.5 iv bolus via tail vein as a control. The other group (n=3) received an equivalent anti-HER2-Cy5.5 dose as MINC-anti-HER2-Cy5.5 iv bolus via tail vein. Live images were observed at 8 hours after drug administration using an IVIS (in vivo imaging system) Lumina III XRMS at 674 / 692nm ex / em.

[0139] result The results showed that fluorescent signals were observed in brain regions of mice administered MINC-anti-HER2-Cy5.5 (STM-001) but not in anti-HER2-Cy5.5 (trastuzumab) treated mice.

[0140] Trastuzumab can treat HER2+ breast cancer, but is not approved for gliomas due to poor delivery into the brain. This example shows that MINC-Trastuzumab can deliver Trastuzumab into the brain of mice.

[0141] Flavonoid oligomers (Example 4) and MINC agents (Examples 5-6) are effective in treating difficult-to-treat CNS disorders Example 4: OEGCG inhibits glioma cell proliferation material OEGCG is prepared according to Example 1.

[0142] CCK-8 kit (Targetmol, Shanghai, China)

[0143] A172 cell line (HER2+ glioma cell line) (CRL-1620, ATCC)

[0144] U87 cell line (HER2-glioma cell line) (HTB-14, ATCC)

[0145] method We used an in vitro tumor suppression assay to study the effect of OEGCG on glioma growth. Both HER2 positive and negative human glioma cell lines A172 (HER2+) and U87 (HER2-) obtained from ATCC were used to test the antitumor ability of OEGCG against glioma. Temozolomide-resistant cells derived from A172 and U87 were used to investigate the possibility of OEGCG overcoming temozolomide resistance. Briefly, cells were seeded at a density of 5000 cells / well in 96-well plates and allowed to attach to the surface for 24 hours. The cells were then treated with 0, 30, 60, and 120 μM OEGCG for 72 hours. After treatment, the treated cells were incubated with CCK-8 reagent for 1 hour at 37° C., and the absorbance value at 450 nm was detected using a SpectraMax® iD3 reader. Results were reported as the mean ± standard deviation of at least two replicates.

[0146] result Figure 4 shows the tumor suppression efficacy of OEGCG in two glioma cell lines, A172 (HER2+) and U87 (HER2-). The results demonstrate that OEGCG has broad efficacy for treating glioma cells regardless of their HER2 expression level (Figures 4A and 4C). Furthermore, OEGCG is also effective in treating temozolomide-resistant glioma cells (Figures 4B and 4D). Temozolomide is the first-line treatment for high-grade gliomas, and the results support that OEGCG can overcome temozolomide resistance and can be used as a combination therapy with additional drugs encapsulated within MINCs.

[0147] Example 5: MINC-anti-HER2 efficacy study in glioma mouse models material OEGCG and PEG-EGCG are prepared according to Example 1.

[0148] MINC-anti-HER2-Cy5.5 is anti-HER2 encapsulated in PEG-EGCG and OEGCG and is prepared according to WO 2009 / 054813.

[0149] A172 glioma cell line (CRL1620, ATCC); D-luciferin (Sigma-Aldrich)

[0150] method We used an orthotopic glioma mouse model to confirm the efficacy of MINC-anti-HER2 in treating glioma. A172 cell line was engineered with luciferase gene (A172-Luc) to image tumor size. For the preparation of the mouse model, a skull hole was made in the right frontal brain region. Then, a superfine needle was inserted to a depth of 3 mm using a stereotactic guide device, and then 1 × 10 6 A172-Luc cells (suspended in 3 μL of DMEM) were slowly injected into the brains of mice. The mice were divided into two groups: a vehicle group (saline as a no-treatment control) and a MINC-anti-HER2 group.

[0151] Two weeks after tumor implantation, saline or MINC-anti-HER2 was injected iv via tail vein at 10 mg / kg twice a week for 43 days. Tumor size was examined every other week using an in vivo imaging system (IVIS). After anesthetizing the mice, they were injected intraperitoneally with luciferin solution and then transferred to the IVIS chamber for image acquisition.

[0152] result Figure 5 shows that mice treated with MINC-anti-HER2 had reduced luciferase signal in A172 gliomas compared to saline-treated controls. The data demonstrate that MINC-anti-HER2 effectively inhibited glioma growth.

[0153] Example 6: OEGCG and MINC-BSA suppress triple-negative cancer cell proliferation material OEGCG is prepared according to Example 1.

[0154] MINC-BSA is BSA encapsulated in PEG-EGCG and OEGCG. Prepared according to WO 2009 / 054813.

[0155] Acid phosphatase (ACP) assay kit (LSBio)

[0156] MDA-MB-231 cell line (CRM-HTB-26D, ATCC)

[0157] MDA-MB-468 cell line (HTB-132, ATCC)

[0158] BT-20 cell line (HTB-19, ATCC)

[0159] method We used an in vitro tumor suppression assay to study 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 at a density of 3000-10,000 cells / well in 96-well plates and allowed to attach to the surface for 24 hours. Cells were then treated with 0, 256, 320, 400 and 500 μM OEGCG for 72 hours or 0, 4.2, 8.3 and 16.7 μM MINC-BSA for 72 hours. After treatment, treated cells were incubated with pNPP reagent for 30 minutes at 37°C and absorbance values ​​at 410 nm were detected using a SpectraMax® iD3 reader. Results were reported as the mean ± standard deviation of at least three replicates. Acid phosphatase (ACP) is an enzyme that catalyzes the cleavage of phosphate groups from other molecules during digestion.

[0160] The enzyme level 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 after addition of a stopping reagent is directly proportional to the enzyme activity and reflects the viability of cancer cells.

[0161] result Figure 6 shows the tumor suppression efficacy of OEGCG and MINC-BSA in three triple-negative breast cancer cell lines. The results demonstrate that OEGCG (Figure 6A) and MINC-BSA (Figure 6B) have significant tumor suppression effects in triple-negative breast cancer cell lines. Triple-negative breast cancer is non-responsive to anti-HER2 therapy. These results support that OEGCG and MINC nanoparticle platform (MINC-BSA, BSA has no anti-cancer effect alone) can overcome anti-HER2 therapy resistance and that MINC can be used with encapsulated additional drugs. Considering that 25-46% of triple-negative breast cancer patients have brain metastasis problems, the results also indicate that the MINC platform can be used for brain metastasis cancer patients.

[0162] Alzheimer's Disease (Examples 7-8) Example 7. OEGCG suppresses Aβ-induced cell death material OEGCG is prepared according to Example 1.

[0163] Recombinant Aβ(1-42) peptide, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) are purchased from Genscript, Thermo Fisher, or any publicly available supplier. HT-22 cells were obtained from Millipore (Bedford, MA, USA).

[0164] method To confirm the efficacy of OEGCG in protecting against Aβ-induced neuronal cell death, an in vitro MTT assay was performed. Briefly, HT-22 cells were plated in 24-well dishes at 2 × 10 per well. 5 Cells were seeded at 100x and maintained in logarithmic growth phase for 3 days. Cells were then treated with 2.5 μM prepared oligo Aβ, Aβ+OEGCG for 24 hours. After incubation, cells were washed once with warm PBS to remove test substances, and tetrazolium salt was added for 30 minutes at room temperature. Formazan product was then measured spectrophotometrically at 550 nm. Viability was calculated as a percentage of control cells treated with vehicle alone (mock).

[0165] result Figure 7 shows the ability of OEGCG in protecting against Aβ-induced cell death. Pathogenic Aβ accumulation can induce neuronal cell death and lead to neurodegeneration. Reduction of Aβ accumulation has therapeutic potential for treating Alzheimer's disease. The results demonstrate that OEGCG reduces Aβ-induced cell death, which is the cause and primary target for treating Alzheimer's disease.

[0166] Example 8. OEGCG and PEG-EGCG suppress Aβ-induced oxidative stress. material OEGCG and PEG-EGCG are prepared according to Example 1.

[0167] Recombinant Aβ(1-42) peptide, DCFH-DA, is purchased from Genscript, Thermo Fisher, or any publicly available supplier.

[0168] HT-22 cells were obtained from Millipore (Bedford, MA, USA).

[0169] method To confirm the effectiveness of OEGCG and PEG-EGCG in reducing Aβ-induced oxidative stress, an in vitro cell reactive oxygen species (ROS) staining test was performed. HT-22 cells were seeded at 2 × 105 cells per well in 24-well dishes 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, Aβ peptides were dissolved at 1 mM in 100% 1,1,1,3,3,3-hexafluoro-2-propanol and dried using a vacuum desiccator. Aβ was then resuspended in dimethyl sulfoxide (DMSO) at a concentration of 5 mM and stored at -20 °C. To obtain oligomers, Aβ peptides were 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 h, and immediately added to cell cultures at a final concentration of 2.5 μM. After cells were treated with Aβ for 1 h, 50.9, 25.4, and 12.7 μg / mL OEGCG and 105.7, 52.4, and 26.4 μg / mL PEG-EGCG were added to the cells and incubated for 6 h. Next, cells were treated with 20 μM DCFH-DA for 0.5 h at 37 °C and 5% CO2. After DCFH-DA staining, cells were washed twice with DMEM and once with phosphate-buffered saline to remove background signals. Fluorescence images were also collected with a fluorescence microscope (DP72 / CKX41, Olympus), and all images were used under the same fluorescence conditions and exposure times.

[0170] result Aβ induces oxidative stress in neuronal cells, therefore more reactive oxygen species (ROS) signals can be observed by DCFH-DA staining (green). Fluorescence image results show 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 showed 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 Alzheimer's disease progression.

[0171] Evidence for Different Flavonoid Oligomers and Polymer-Flavonoid Conjugates in the Formation of MINC Drugs (Examples 9-12) Example 9: Methods for preparing MINC-anti-HER2 using different flavonoids in flavonoid oligomers and polymer-flavonoid conjugates material OEGCG is oligomerized EGCG. OECG is oligomerized ECG. These flavonoid oligomers were prepared according to WO 2006 / 124000.

[0172] PEG-EGCG is PEG conjugated with one or two EGCGs. PEG-EC is PEG conjugated with one or two ECs. PEG-ECG is PEG conjugated with one or two ECGs. These polymer-flavonoids were prepared according to WO 2006 / 124000, WO 2009 / 054813, or WO 2015 / 171079.

[0173] Anti-HER2 is trastuzumab obtained from Eirgenix.

[0174] method MINC anti-HER2 nanoparticles were prepared according to WO 2009 / 054813. Briefly, anti-HER2 was incubated in PBS. Different flavonoid oligomers, including OEGCG or OECG, were then added to anti-HER2, followed by different polymer-flavonoids, including PEG-EGCG, PEG-ECG, or PEG-EC. After the mixture was incubated at room temperature, unreacted oligomeric flavonoids and polymer-flavonoids were removed using a 10K MWCO centrifugal filter. The nanoparticle size was measured using DLS (Anton Paar Litesizer 500). The results are shown in Figure 8.

[0175] result Figure 8 shows that the different polymer-flavonoid conjugates and different flavonoid oligomers all successfully produced MINC-anti-HER2 micelles with one unimodal peak of similar particle size around 100 nm. The results demonstrated the successful formation of homogenous nanoparticles (micelles) with the expected size, with no small peak of unencapsulated anti-HER2 (around 5-10 nm) observed.

[0176] In this example, the different flavonoid oligomers used included OEGCG (Figures 8A, 8C, and 8D) and OECG (Figure 8B); the different polymer-flavonoids used included PEG-EGCG (Figures 8A and 8B), PEG-EC (Figure 8C), and PEG-ECG (Figure 8D).

[0177] These data support that MINC nanoparticles can be formed by different flavonoid oligomers and different polymer-flavonoid conjugates.

[0178] Example 10: Methods for preparing MINC-BSA using different polymers in the polymer-flavonoid conjugate material OEGCG is oligomerized EGCG and is prepared according to WO 2006 / 124000.

[0179] PEG-EGCG is PEG conjugated with one or two EGCGs. HA-EGCG is HA conjugated with one or two EGCGs. Dextran-EGCG is dextran conjugated with one or two EGCGs. These different polymer-flavonoids are prepared according to WO 2006 / 124000, WO 2009 / 054813, or WO 2015 / 171079.

[0180] BSA is purchased from Sigma Aldrich.

[0181] method MINC (Multi-targeted immune nanocarrier combination)-BSA nanoparticles were prepared according to WO 2009 / 054813. Briefly, BSA was incubated in PBS. Subsequently, OEGCG or OEGCG was added to BSA, followed by the addition of different polymer-flavonoids, such as PEG-EGCG, HA-EGCG and dextran-EGCG. After the mixture was incubated at room temperature, unreacted OEGCG and polymer-flavonoids were removed using a 10K MWCO centrifugal filter. DLS (Anton Paar Litesizer 500) was used to measure the nanoparticle size.

[0182] result Figure 9 demonstrates that different polymers can be used in the polymer-flavonoid conjugates to successfully generate MINC-BSA. The results demonstrate the successful formation of homogenous nanoparticles (micelles), and the small peak of unencapsulated BSA (approximately 5-10 nm) was not observed. These different polymers are PEG (Figure 9A), HA (Figure 9B), and dextran (Figure 9C). Taken together, these data support that MINC nanoparticles can be formed by different polymer-flavonoid conjugates.

[0183] Example 11: Method for preparing MINC-anti-Aβ. material OEGCG is oligomerized EGCG and is prepared according to WO 2006 / 124000.

[0184] PEG-EGCG is PEG conjugated to one or two EGCG moieties, prepared according to WO 2006 / 124000, WO 2009 / 054813, or WO 2015 / 171079.

[0185] Anti-Aβ is purchased from Biolegend.

[0186] method 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 (Anton Paar Litesizer 500).

[0187] result FIG. 10 shows the successful formulation of MINC-anti-Aβ.

[0188] Example 12: Method for preparing MINC-anti-α-syn material OEGCG is oligomerized EGCG and is prepared according to WO 2006 / 124000.

[0189] PEG-EGCG is PEG conjugated to one or two EGCG moieties, prepared according to WO 2006 / 124000, WO 2009 / 054813, or WO 2015 / 171079.

[0190] Anti-α-syn is purchased from Biolegend.

[0191] method MINC-anti-α-syn nanoparticles (PEG-EGCG, OEGCG and anti-α-syn) were prepared according to Example 8. DLS (Anton Paar Litesizer 500) was used to measure the size of the MINC-anti-α-syn nanoparticles.

[0192] result FIG. 11 shows successful formulation of MINC-anti-α-syn.

[0193] [Table 1-1] [Table 1-2]

[0194] The invention, and the manner and process of making and using it, have been described in such full, clear, concise and exact terms as to enable any person skilled in the art to which the invention pertains to make and use the same. It is to be understood that the foregoing has described preferred embodiments of the invention, and that changes can be made therein without departing from the scope of the invention as set forth in the appended claims. To particularly point out and distinctly claim the subject matter which is regarded as the invention, the following claims conclude this specification.

Claims

1. A pharmaceutical composition for treating central nervous system (CNS) diseases comprising an effective amount of micelles having an outer shell containing one or more polymer-flavonoid conjugates, an inner shell optionally containing one or more flavonoid oligomers, and a drug encapsulated within the shell, 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-tocopheryl, and polyethylene glycol succinate. The flavonoid is EGCG, EC, EGC, or ECG, as shown in the following structure. 【Chemistry 1】 The flavonoid oligomer contains 2 to 20 flavonoids of EGCG, EC, EGC, or ECG. The aforementioned CNS disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lewy body dementia, brain tumor, stroke, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), acute spinal cord injury, encephalitis, epilepsy, seizure, meningitis, motor neuron disease (MND), and cerebral aneurysm, and is a pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the micelle has an outer shell containing PEG-EGCG and an inner shell containing an EGCG oligomer.

3. The pharmaceutical composition according to 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 pharmaceutical composition according to 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 pharmaceutical composition according to 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 pharmaceutical composition according to claim 1 or 2, wherein the CNS disease is a brain tumor, and the drug is doxorubicin, disulfiram, celecoxib, temsirolimus, everolimus, vorinostat, cabozantinib, marizomib, fimepinostat, 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 pharmaceutical composition according to claim 1 or 2, wherein the CNS disease is stroke, and the drug is an MMP inhibitor, an eNOS inhibitor, an anti-TLR4, an anti-HSP, an anti-IL6, an anti-IL-12, S100β, fibronectin, MCP-1, MMP9, UCH-L1, BDNF, GDNF, NRTN, PDGF-BB, or CDNF.

8. The method according to 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, duetetrabenazine, valbenazine, bevantrol, pridopidine, branapram, nilotinib, mitoconix, or azathioprine.

9. The pharmaceutical composition according to 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 pharmaceutical composition according to claim 1 or 2, wherein the CNS disease is amyotrophic lateral sclerosis (ALS), and the drug is anti-NOGO-A, PKC inhibitor, IGF-1, NOGO-A, GDNF, VEGF, anti-SOD1, S1R, GLT-1, anti-ataxin 2, anti-TDP43, anti-hnRNPs, CK-1 inhibitor, anti-FET or HDAC inhibitor, EPO, or IL-2.

11. The pharmaceutical composition according to claim 1 or 2, wherein the CNS disease is acute spinal cord injury, and the drug is the extracellular domain of the Nogo receptor, 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 pharmaceutical composition according to 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 pharmaceutical composition according to claim 1 or 2, wherein the CNS disease is epilepsy or seizure, and the drug is an mTOR inhibitor, a PI3K inhibitor, a GABA inhibitor, an anti-Glu3B peptide antibody, an anti-NR1 antibody, an anti-CASPR2, or an anti-LGI-1.

14. The pharmaceutical composition according to 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 pharmaceutical composition according to claim 1 or 2, wherein the CNS disease is motor neuron disease (MND), and the drug is anti-SOD1, anti-TDP-43, anti-C90RF72, 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 pharmaceutical composition according to claim 1 or 2, wherein the CNS disease is a cerebral aneurysm, and the drug is a TNF-α inhibitor, an MMP inhibitor, an MCP-1 inhibitor, a phosphodiesterase-4 inhibitor, a mast cell degranulation inhibitor, or an anti-IL-1β.

17. A pharmaceutical composition for treating CNS diseases, comprising an effective amount of polymer-flavonoid conjugate, The flavonoid is EGCG, EC, EGC, or ECG, as shown in the following structure. 【Chemistry 2】 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-tocopheryl, and polyethylene glycol succinate. A pharmaceutical composition in which the CNS disease is selected from the group consisting of brain tumors, cardiovascular disorders, CNS diseases accompanied by spinal cord injury, neuroinflammatory diseases, motor neuron diseases, and cerebral cortical disorders.

18. The pharmaceutical composition according to claim 17, wherein the polymer-flavonoid conjugate is PEG-EGCG.

19. A pharmaceutical composition for treating CNS diseases, comprising an effective amount of flavonoid oligomers, The flavonoid is EGCG, EC, EGC, or ECG, as shown in the following structure. 【Transformation 3】 The flavonoid oligomer contains 4 to 12 flavonoids of EGCG, EC, EGC, or ECG. A pharmaceutical composition in which the CNS disease is selected from the group consisting of brain tumors, cardiovascular disorders, CNS diseases accompanied by spinal cord injury, neuroinflammatory diseases, motor neuron diseases, and cerebral cortical disorders.

20. The pharmaceutical composition according to claim 19, wherein the flavonoid oligomer is an oligomer of EGCG.

21. The pharmaceutical composition 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 metastatic tumor, neurotumor, central nervous system lymphoma, choroid plexus tumor, pineal gland tumor, meningioma, and pituitary tumor.

22. The pharmaceutical composition 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 pharmaceutical composition according to any one of claims 17 to 20, wherein the CNS disease is a CNS disease accompanied by spinal cord injury selected from the group consisting of multiple sclerosis, acute spinal cord injury, and myelopathy.

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

25. The pharmaceutical composition according to 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 pharmaceutical composition according to any one of claims 17 to 20, wherein the CNS disease is a cerebral cortical disorder selected from the group consisting of cerebellar gyri, polycerebellar gyri, bilateral frontoparietal polycerebellar gyri, and pachygyri.

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