Treatment methods for neurodegenerative diseases

Abstract

The present invention provides a method for treating neurodegenerative diseases. The method comprises administering to a subject in need a nanocomplex having an effective amount of a polymer-flavonoid complex, or an outer shell containing one or more polymer-flavonoid complexes, and optionally an inner shell containing one or more flavonoid oligomers, and a drug such as an anti-CD3 antibody or anti-CD33 antibody encapsulated within the shell. The method delivers the therapeutic agent across the blood-brain barrier to treat neurodegenerative diseases. The method is effective in treating neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Lewy body dementia, and Huntington's disease.

Description

Field of Invention

[0001] The present invention provides a method for treating or preventing the recurrence of symptoms of neurodegenerative diseases. The method includes administering to a subject in need a nanocomplex having a shell containing an effective amount of a polymer-flavonoid complex, or one or more polymer-flavonoid complexes, or one or more flavonoid oligomers, or a combination thereof, with an antibody drug encapsulated within the shell. Background of the Invention

[0002] Neurodegenerative diseases are caused by the progressive loss of structure or function of nerve cells in a process known as neurodegeneration. Neurodegenerative diseases are a group of diseases that primarily affect nerve cells in the human brain and spinal cord. Examples of neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Lewy body dementia, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Creutzfeldt-Jakob disease, Friedreich's ataxia, motor neuron disease, Batten's disease, tauopathy, prion diseases, spinal muscular atrophy, and spinocerebellar ataxia. The two major neurodegenerative diseases are Alzheimer's disease and Parkinson's disease. Some neurodegenerative diseases are caused by hereditary genetic changes. Most neurodegenerative diseases result from a combination of genetic and environmental factors. Therefore, it is difficult to predict who will develop dementia. In 2019, 55 million people worldwide suffered from dementia, and this number is estimated to increase to 139 million by 2050.

[0003] There is no good way to reverse the degeneration of nerve cells. Oxidative stress and inflammation are the two major causes of neurodegeneration. Recent research has revealed that these diseases share many similarities and are caused by the accumulation of atypical protein assemblies that trigger cell death.

[0004] Alzheimer's disease (AD) is the most common neurodegenerative disease, usually developing slowly and gradually worsening. As the disease progresses, symptoms such as speech difficulties, disorientation (e.g., getting lost easily), mood swings, decreased motivation, self-deprecation, and behavioral problems may appear. Alzheimer's disease is thought to be caused by the accumulation of abnormal amounts of amyloid-beta (Aβ) in the brain as amyloid plaques outside cells, and the accumulation of tau protein inside cells as neurofibrillary tangles. These affect the function and connectivity of nerve cells, leading to a progressive decline in brain function. Currently, there is no treatment that can stop or reverse the progression of symptoms, but there are several treatments that can temporarily improve symptoms.

[0005] Parkinson's disease (PD) is a long-term degenerative disease of the central nervous system that primarily affects the motor system. It is sometimes classified as a type of neurodegenerative disease called a synucleinopathy, due to the abnormal accumulation of the protein alpha-synuclein in the brain. The most prominent early symptoms of PD are tremors, muscle rigidity, slowness of movement, and difficulty walking. Many PD patients also experience cognitive and behavioral impairments such as depression, anxiety, and apathy. Dementia is common in the advanced stages of PD. There is no cure for PD; treatment aims to mitigate the effects of these symptoms.

[0006] Lewy body dementia (LBD) is the second most common type of dementia after Alzheimer's disease. While the exact cause is unknown, it is believed to involve the formation of abnormal protein deposits in the brain, damaging nerve cells. These abnormal protein aggregates are called Lewy bodies, and their main component is alpha-synuclein. Lewy bodies can also be found in other types of dementia, including Alzheimer's disease, multiple system atrophy, and Parkinson's disease. LBD is a progressive form of dementia, causing a decline in thinking and reasoning abilities, as well as problems with motor skills, behavior, and mood. These symptoms typically begin after the age of 50. The progression of symptoms varies depending on the individual's health. The average life expectancy for LBD patients is 5-8 years, and currently there is no cure for LBD.

[0007] Huntington's disease (HD) is a genetic disorder in which a protein called huntingtin damages nerve cells in the brain. HD causes a decline in physical, mental, and emotional abilities. Symptoms typically appear in middle age, and there is currently no cure. People with HD exhibit uncontrollable, dance-like movements (chorea), abnormal posture, behavior, emotions, thoughts, and personality problems. Cognitive changes worsen as the disease progresses, eventually making it impossible to work, drive, or care for oneself.

[0008] Anti-CD3 antibodies target surface receptors on T cells, suppressing the inflammatory response during disease onset. Anti-CD3 antibodies have been shown to regulate T cells and promote the differentiation of microglia from the M1 to the M2 state. M1 microglia secrete inflammatory mediators, causing inflammation and neurotoxicity. Conversely, M2 microglia secrete anti-inflammatory mediators, promoting anti-inflammatory responses and neuroprotection.

[0009] Clinically, treatment with anti-CD3 antibodies is accompanied by widespread toxic side effects that occur almost immediately after the first dose. This reaction, known as cytokine release syndrome (CRS), occurs in many patients treated with anti-CD3 antibodies. CRS is a serious side effect that causes a life-threatening systemic inflammatory response in patients. Long-term administration of anti-CD3 antibodies can cause leukopenia (a decrease in white blood cell count), leading to an increased risk of infection. For these reasons, the treatment of neurodegenerative diseases and other autoimmune diseases with anti-CD3 antibodies currently has clinical limitations.

[0010] The blood-brain barrier (BBB) ​​is a highly selective, semipermeable boundary of the endothelial cells of the central nervous system (CNS) that prevents solutes in the circulating blood vessels from non-selectively entering the central nervous system where nerve cells reside. Therefore, the BBB acts as a barrier that prevents many drugs effective in treating brain diseases from reaching the brain.

[0011] There are four pathways for blood-brain barrier (BBB) ​​permeability: passive diffusion, carrier-mediated transport, receptor-mediated transcytosis, and adsorption-mediated transcytosis (AMT). The AMT pathway utilizes caveolae as a transport medium. Endocytosis via caveolae is an important transport mechanism for the uptake of macromolecules from the bloodstream into the central nervous system (CNS).

[0012] Flavonoids have a general structure of 15 carbon skeletons, which consist of two phenyl rings (A and B) and one heterocycle (C, an embedded oxygen-containing ring). JPEG2026519767000002.jpg88156 This carbon structure is abbreviated as C6-C3-C6. According to 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 explanation of the drawing]

[0013] Figure 1 illustrates one embodiment of a MINC (Multi-pathway Immunomodulatory Nanocomplex Combination Therapy) agent. This is a nanocomplex having a polymer-flavonoid complex (e.g., PEG-EGCG complex) as a shell, with the drug encapsulated within it.

[0014] Figure 2 illustrates another embodiment of the MINC agent. This is, This nanocomposite contains a polymer-flavonoid complex (e.g., PEG-EGCG complex) as its outer shell and a flavonoid oligomer (e.g., oligomeric EGCG (OEGCG)) as its inner shell, with the drug encapsulated within it.

[0015] Figure 3 shows the nanoparticle size distribution of the MINC-anti-CD3 antibody nanocomposite composition.

[0016] Figure 4 shows that MINC-Dox penetrated the BBB better than Dox.

[0017] Figure 5 shows that MINC-anti-CD3 antibody protected neurons and inhibited Aβ-induced cell death.

[0018] Figure 6 shows that OEGCG, PEG-GCGC, and MINC-anti-CD3 antibody promoted the proliferation of neurons.

[0019] Figure 7 shows that MINC-anti-CD3 antibody decreased the secretion of IL-6 from microglia by PLS stimulation.

[0020] Figure 8 shows that MINC-anti-CD3 antibody reduced the toxicity of anti-CD3 in the body weight test and survival test.

[0021] Figure 9 shows that the treatment with MINC-anti-CD3 antibody was safe in animals and did not change the composition of blood cells compared to the control group treated with saline. DETAILED DESCRIPTION OF THE INVENTION

[0022] Definitions The term "about" is defined as ±10%, preferably ±5% of the stated value.

[0023] The term "cytokine" refers to a small protein (<80 kDa) important in cell signaling. Cytokines have been shown to be involved in autocrine, paracrine, and endocrine signaling as immunomodulatory factors. Cytokines include interferons, interleukins, lymphokines, tumor necrosis factors, and chemokines.

[0024] The term "epigallocatechin gallate" refers to an ester of epigallocatechin and gallic acid and is used interchangeably with "epigallocatechin-3-gallate" or EGCG.

[0025] The term "nanocomposite" refers to a nanometer-sized (1–999 nm) complex containing multiple different components linked together by ionic interactions, hydrophobic interactions, and non-covalent bonds such as hydrogen bonds.

[0026] The term "oligomeric EGCG" (OEGCG) refers to a monomer consisting of 3 to 20 covalently bonded EGCG molecules. OEGCG preferably contains 4 to 12 EGCG molecules.

[0027] The term "polyethylene glycol-epigallocatechin gallate complex" or "PEG-EGCG" refers to polyethylene glycol (PEG) complexed with one or two molecules of EGCG. The term "PEG-EGCG" refers to both PEG-mEGCG complexes (monomerized EGCG) and PEG-dEGCG complexes (dimerized EGCG).

[0028] This disclosure provides a method for treating neurodegenerative diseases. The method comprises administering to a subject in need of treatment an effective amount of (i) a polymer-flavonoid complex, (ii) a flavonoid oligomer, or (iii) a nanocomplex having (a) an outer shell containing one or more polymer-flavonoid complexes, (b) an inner shell optionally containing one or more flavonoid oligomers, and (c) an antibody encapsulated within the shell. Anti-CD3 antibodies are preferred antibody drugs in this method. Preferred antibodies in this method include anti-CD3 antibodies and anti-CD33 antibodies.

[0029] Free anti-CD3 antibodies are known to cause life-threatening side effects in patients, such as cytokine storms, which are abnormal releases of lethal cytokines. This nanocomplex reduces the toxicity of anti-CD3 antibodies and is effective in treating neurodegenerative diseases such as AD, PD, HD, and LBD.

[0030] Flavonoids A flavonoid suitable for the present invention has the general structure of formula I. JPEG2026519767000003.jpg89107In formula, R1 is either H or a phenyl group; R2 is H, OH, a gallic acid ester group, or a phenyl group; where the phenyl group is optionally substituted with one or more (e.g., two to three) hydroxyl groups; R3 is H, OH, or =O (oxo group); or R1 and R2 together form a closed loop structure; or R2 and R3 together form a closed-loop ring structure. The 2nd, 3rd, 4th, 5th, 6th, 7th, or 8th positions of formula I can be bonded to groups containing hydrocarbons, halogens, oxygen, nitrogen, sulfur, phosphorus, boron, or metals.

[0031] Examples of flavonoids of formula I include the following: JPEG2026519767000004.jpg95134

[0032] Preferred flavonoid compounds of formula I include EGCG (CAS number: 989-51-5), EC (CAS number: 490-46-0), EGC (CAS number: 970-74-1), or ECG (CAS number: 1257-08-5). JPEG2026519767000005.jpg109138

[0033] Polymer-flavonoid complex As used throughout this specification, the polymer-flavonoid complex refers to a complex of a hydrophilic polymer with a flavonoid compound of formula I.

[0034] Hydrophilic polymers are polymers that are soluble in polar solvents and can form hydrogen bonds. Suitable hydrophilic polymers for this polymer-flavonoid complex include poly(ethylene glycol), aldehyde-derivative hyaluronic acid, hyaluronic acid, dextran, diethyl acetal complex (e.g., diethyl acetal PEG), D-α-tocopheryl polyethylene glycol succinate, aldehyde-derivative hyaluronic acid-tyramine, hyaluronic acid-aminoacetylaldehyde diethyl acetal complex-tyramine, cyclotriphosphazen core phenoxymethyl (methylhydrazono) dendrimer or thiophosphoryl core phenoxymethyl (methylhydrazono) dendrimer, acrylamide, oxazoline, imine, acrylic acid, methacrylate, diol, oxirane, alcohol, amine, anhydride, ester, lactone, terephthalate, amide and ether polyacrylamide, poloxamer, poly(N-isopropyl) Examples of poly(acrylamide), poly(oxazoline), polyethyleneimine, poly(acrylic acid), polymethacrylate, poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol), poly(vinylpyrrolidone), polyether, poly(allylamine), polyanhydride, poly(β-aminoester), 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-glycolic acid copolymer), poly(orthoester), poly(2-oxazoline), poly(sebacic acid), poly(terephthalate-phosphate copolymer), povidone, and copolymers are, but are not limited thereto.

[0035] 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 complex is generally 1K to 100K daltons, preferably 2K to 40K, 2K to 50K, 2K to 80K, 3K to 80K, or 5K to 40K daltons.

[0036] In one embodiment, the polymer contains an aldehyde group complexed at the 5th, 6th, 7th, or 8th position (preferably the 6th or 8th position) of the A ring of the flavonoid compound.

[0037] In another embodiment, the polymer contains a thiol group complexed with R1 or R2 of the B ring of the flavonoid (if R1 or R2 is -OH).

[0038] In one embodiment, the polymer-flavonoid complex is PEG-EGCG, which is a complex of PEG with one or two molecules of epigallocatechin gallate (EGCG). For example, PEG-EGCG can be prepared by complexing aldehyde-terminated PEG with EGCG by linking PEG via a reaction with a free aldehyde group at position 5, 6, 7, or 8 (preferably position 6 or 8) of formula I. See WO2006 / 124000 and WO2009 / 054813. PEG-EGCG can also be prepared by complexing thio-terminated PEG with EGCG by linking PEG via a reaction with a free thio group at R1 or R2 (where R1 or R2 is a phenyl group) of formula I. See WO2015 / 171079.

[0039] Flavonoid oligomers A flavonoid oligomer is a complex of one flavonoid and one or more flavonoids. A flavonoid oligomer may contain the same flavonoid (homo-oligomer) or different flavonoids (hetero-oligomer). Flavonoid oligomers useful in the present invention typically contain 2 to 50 or 2 to 20, preferably 4 to 12, single or mixed types of flavonoids.

[0040] 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 bonded EGCG monomers. For example, OEGCG can be synthesized at position 5, 6, 7, or 8 (preferably 6 or 8) of the A ring according to WO2006 / 124000.

[0041] According to formula I, since all flavonoids contain an A ring, other oligomeric flavonoids can be similarly produced according to WO2006 / 124000. For example, OEC, OEGC, and OECG can also be produced according to WO2006 / 124000.

[0042] MINC agent MINC (Multi-pathway Immunomodulatory Nanocomplex Combination Therapy) is a platform technology that utilizes the physiological activity of polymer-flavonoid complexes or flavonoid oligomers that form nanocomplexes in solution.

[0043] The MINC platform can encapsulate drugs and form therapeutic nanoparticle compositions. A MINC agent is a nanocomplex comprising an outer shell containing one or more polymer-flavonoid complexes, an inner shell optionally containing one or more flavonoid oligomers, and a drug within these shells. The drug, as used herein, refers to a molecule (e.g., a drug) having therapeutic activity.

[0044] In one embodiment, the MINC agent is in the form of micelles.

[0045] In one embodiment, the MINC agent is a nanocomposite containing a polymer-flavonoid complex (e.g., a PEG-EGCG complex) and a drug encapsulated within the shell (see Figure 1).

[0046] In another embodiment, the MINC agent is a nanocomposite containing a polymer-flavonoid complex (e.g., PEG-EGCG complex) in the outer shell and a flavonoid oligomer (e.g., oligomeric EGCG (OEGCG)) in the inner shell, with the drug encapsulated within these shells (see Figure 2).

[0047] If the agent is a drug, the composition of the MINC agent comprises two or more components having therapeutic activity, these components having complementary functions, and together with their skeletal components (flavonoid complexes or flavonoid oligomers) and encapsulated drugs, it forms a multi-target combination therapy.

[0048] In one embodiment, the agent in the MINC agent is an antibody that includes, but is not limited to, an anti-CD3 antibody, an anti-CD39 antibody, an anti-CD73 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-GZM A antibody, an anti-GZM B antibody, an anti-CD33 antibody, an anti-TAM antibody, an anti-FcγRI antibody, an anti-CD36 antibody, an anti-RAGE antibody, an anti-APOE antibody, or an anti-CR1 antibody.

[0049] For example, the MINC agent is an anti-CD3 antibody, the polymer-flavonoid complex is PEG-EGCG, and the flavonoid oligomer is OEGCG. This is one preferred composition for treating neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Lewy body dementia, and Huntington's disease.

[0050] Pharmaceutical composition The present invention provides a pharmaceutical composition comprising a polymer-flavonoid complex, a flavonoid oligomer, or a MINC agent described in this application, and optionally one or more pharmaceutically acceptable excipients. The nanoparticle component in the pharmaceutical composition is generally about 1-100% or 1-90%, preferably 20-90%, or 30-80%, in the case of tablets, powders, or parenteral formulations. The composition of polymer-flavonoid complex, flavonoid oligomer, or MINC agent in the pharmaceutical composition is generally 1-100%, preferably 20-100%, 50-100%, or 70-100%, in the case of capsule formulations. The nanoparticle composition in the pharmaceutical composition is generally 1-50%, 5-50%, or 10-40%, in the case of liquid suspension formulations.

[0051] In one embodiment, the pharmaceutical composition can take the form of tablets, capsules, granules, fine granules, powders, suspensions, solutions, patches, parenteral preparations, injections, etc. The above-mentioned pharmaceutical composition can be prepared by conventional methods.

[0052] A pharmaceutically acceptable carrier, which is an inactive component, can be selected by those skilled in the art using conventional criteria. Pharmacochemically acceptable carriers include physiological saline and electrolyte solutions; ionic and nonionic osmotic agents (sodium chloride, potassium chloride, glycerol, and glucose); pH adjusters and buffers (hydroxides, phosphates, citrates, acetates, borates, trolamine); antioxidants (bisulfites, sulfites, metabisulfites, thiosulfites, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol, ascorbyl palmitate salts, acids, and / or bases); surfactants (lecithin and phospholipids, including but not limited to phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol); poloxamers and poloxamines; polysorbates (polysorbate 80, polysorbate 60, and polysorbate 20); polyethers (polyethylene glycol and polypropylene glycol); polyvinyl (polyvinyl alcohol and poly The pharmaceutically acceptable carrier may contain, but is not limited to, vinylpyrrolidone (PVP, povidone); cellulose derivatives (methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose and their salts); petroleum derivatives (mineral oil and white petrolatum); fats (lanolin, peanut oil, palm oil, and soybean oil); monoglycerides, diglycerides, and triglycerides; polysaccharides (dextran); and glycosaminoglycans (sodium hyaluronate). Such pharmaceutically acceptable carriers can be protected from bacterial contamination using 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 they can be formulated as preservative-free formulations for single or multiple use.

[0053] For example, tablets, capsules, or parenteral formulations of the active compound may contain other excipients that are not physiologically active and do not react with the active compound. Excipients for tablets or capsules may include fillers, binders, lubricants, flow enhancers, disintegrants, wetting agents, and release rate modifiers. Examples of excipients for tablets or capsules include, but are not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide, polyacrylic acid, and polyvinylpyrrolidone. For example, tablet formulations may contain inert components such as colloidal silicon dioxide, crospovidone, hypromellose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, sodium starch glycolate, and titanium dioxide. Capsule formulations may contain inert components such as gelatin, magnesium stearate, and titanium dioxide. The powdered oral formulation may contain inactive ingredients such as silica gel, sodium benzoate, sodium citrate, sucrose, and xanthan gum.

[0054] The pharmaceutical composition may be administered by local or systemic administration. Local administration includes topical administration. Systemic administration includes oral administration, parenteral administration (intravenous, intramuscular, subcutaneous, or rectal administration), and other routes of systemic administration. 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 routes of administration.

[0055] Treatment method The present invention relates to a method for treating or preventing the recurrence of neurodegenerative diseases by administering a polymer-flavonoid complex, flavonoid oligomer, or MINC agent as described above to a subject in need.

[0056] Appropriate neurodegenerative diseases treatable by the present invention include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), Lewy body dementia (LBD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Creutzfeldt-Jakob disease (CJD), Friedreich's ataxia (FA), motor neuron disease (MND), Batten's disease, spinal muscular atrophy (SMA), and spinocerebellar ataxia (SCA). This method is particularly useful for the treatment of AD, PD, LBD, and HD.

[0057] Polymer-flavonoid complexes, flavonoid oligomers The present invention relates to a method for treating neurological disorders by administering one or more polymer-flavonoid complexes and / or one or more flavonoid oligomers.

[0058] The method comprises administering an effective amount of one or more polymer-flavonoid complexes and / or one or more flavonoid oligomers to a subject in need of treatment for neurodegenerative diseases. The polymer-flavonoid complexes and one or more flavonoid oligomers have already been described in this application.

[0059] EGCG can reduce misfolded β-amyloid and tau protein aggregation in Alzheimer's disease, α-synuclein in Parkinson's disease and Lewy body dementia, and huntingtin in Huntington's disease. EGCG also exhibits neuroprotective functions in these neurodegenerative diseases. Mechanismally, EGCG exerts neuroprotective effects directly on nerve cells. EGCG exerts antioxidant effects by acting as a free radical scavenger and also exhibits anti-apoptotic effects by suppressing the expression of apoptosis-inducing genes. EGCG contained in MINC agents protects nerve cells from toxic damage. Furthermore, EGCG is known to promote nerve cell proliferation and regeneration.

[0060] In one embodiment, the polymer is a hydrophilic polymer with 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.

[0061] In one embodiment, the flavonoid oligomer contains 2 to 50 flavonoids of EGCG, EC, EGC, or ECG.

[0062] In one embodiment, the shell is formed by PEG-EGCG.

[0063] In one embodiment, the shell is formed by PEG-EGCG and OEGCG.

[0064] One of the key functions of polymer-flavonoid complexes and flavonoid oligomers is to facilitate drug delivery to the brain (by crossing the blood-brain barrier) and enhance therapeutic effects. This function is due to the ability of polymer-flavonoid complexes to cross the BBB. Because the drug molecules are encapsulated, they are not exposed to the BBB and do not affect their transition to the central nervous system (CNS). This brain delivery is applicable to neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Lewy body dementia, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Creutzfeldt-Jakob disease, Friedreich's ataxia, motor neuron disease, Batten disease, spinal muscular atrophy, and spinocerebellar ataxia).

[0065] Another function of polymer-flavonoid complexes and flavonoid oligomers is to restore cognitive behavior by suppressing neuronal cell death and promoting cell regeneration. This function is used in the treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Lewy body dementia, and Huntington's disease.

[0066] Another function of polymer-flavonoid complexes and flavonoid oligomers is to reduce oxidative stress and inflammation in nerve cells caused by abnormal protein aggregates. Oxidative stress causes neurotoxicity and is involved in the development of neurodegenerative diseases. By reducing oxidative stress in nerve cells, neuronal cell death is reduced, and neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Lewy body dementia, and Huntington's disease) can be treated.

[0067] Another function of polymer-flavonoid complexes and flavonoid oligomers is to reduce the accumulation of abnormal proteins (β-amyloid, tau, and α-synuclein), slow disease progression, and / or mitigate relapses. These processes treat neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Lewy body dementia, and Huntington's disease).

[0068] MINC agent The present invention relates to a method for treating neurological diseases by administering a MINC agent. The method comprises administering to a subject in need of treatment for a neurodegenerative disease an effective amount of a nanocomplex comprising (a) an outer shell containing one or more polymer-flavonoid complexes, (b) optionally an inner shell containing one or more flavonoid oligomers, and (c) an anti-CD3 antibody or anti-CD33 antibody encapsulated within the shell.

[0069] In one embodiment, the outer shell is formed by one or more polymer-flavonoid complexes. In one embodiment, the inner shell is formed by one or more flavonoid oligomers. In one embodiment, the drug is an antibody.

[0070] Neurodegenerative diseases suitable for treatment with this method include Alzheimer's disease, Parkinson's disease, Lewy body dementia, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Creutzfeldt-Jakob disease, Friedreich's ataxia, motor neuron disease, Batten disease, spinal muscular atrophy, and spinocerebellar ataxia.

[0071] As already explained above, polymer-flavonoid complexes or flavonoid oligomers can cross the blood-brain barrier from the circulatory system to the brain, serving as drug delivery vehicles for the treatment of neurodegenerative diseases.

[0072] Other functions of polymer-flavonoid complexes or flavonoid oligomers have already been described above.

[0073] In one embodiment, the agents (or drugs) in the MINC agent can modulate the activity of immune T cells and microglia. These agents include, but are not limited to, the following antibodies. Preferred antibodies include anti-CD3 antibodies.

[0074] Anti-CD3 antibodies can reduce neuroinflammation by regulating T cells and microglia, making them effective in treating Alzheimer's disease (AD). Anti-CD3 antibodies improve AD by protecting nerve cells from damage caused by Aβ accumulation and differentiating the phenotype of brain microglia from M1 (inflammatory, damaging type) to M2 (protective type that removes plaque).

[0075] Anti-CD3 antibodies are found in the blood of CD3 + Reducing the total number of T cells is useful in treating Parkinson's disease (PD). The severity of PD is determined by the observed CD3 count. + It is significantly correlated with a decrease in T cells. Improvement in Parkinson's disease symptoms includes a reduction in tremors, muscle rigidity, neuralgia, and / or dystonia.

[0076] Huntington's disease (HD) is a hereditary neurodegenerative disorder characterized by the abnormal accumulation of huntingtin protein in the brain. Early drug intervention is expected. In HD, several preclinical and clinical trials of potential immunomodulatory agents, including anti-CD3 antibodies, are being investigated. The abnormal inflammatory state and neuronal damage in the central nervous system in HD are similar to those in other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In HD, several preclinical and clinical trials of potential immunomodulatory agents, including anti-CD3 antibodies, are being investigated. Suppression of serum immune activation markers and improvement in cortical motor activity have been observed.

[0077] However, free anti-CD3 antibodies are known to cause life-threatening side effects, such as cytokine storms, which are lethal abnormal cytokine releases in patients. This MINC-anti-CD3 antibody reduces the toxicity of anti-CD3 antibodies and is effective in the treatment of AD, PD, HD, and LBD.

[0078] Anti-CD33 antibodies reduce the phagocytic ability of microglia and inhibit the excessive surface expression of CD33, which contributes to the risk of Alzheimer's disease.

[0079] Anti-CD39 antibodies regulate ectonucleotidase, thereby breaking down ATP and suppressing the production of inflammatory cytokines.

[0080] Anti-CD73 antibodies regulate ectonucleotidase, thereby breaking down ATP and suppressing the production of inflammatory cytokines.

[0081] Anti-PD-1 antibodies function as systemic PD-1 inhibitors, promoting the removal of brain amyloid-beta plaques and improving memory in Alzheimer's disease (AD).

[0082] Anti-PD-L1 antibodies increase the levels of effector memory T cells in the periphery, leading to an increase in the number of monocyte-derived macrophages in the brain parenchyma and the secretion of the anti-inflammatory cytokine IL-10.

[0083] Anti-PD-L2 antibodies inhibit T cell proliferation by disrupting cell cycle progression, thereby reducing macrophage and B cell infiltration into the brain during the progression of multiple sclerosis and Alzheimer's disease.

[0084] Anti-CTLA-4 antibodies activate immune tolerance in T cells. This stimulation is beneficial in autoimmune diseases such as AD and MS.

[0085] Anti-GZM A / B antibodies are released from activated T cells and inhibit granzyme B, a cytotoxic protease associated with acute and subacute inflammatory brain diseases in neurodegenerative diseases.

[0086] Interleukin-2 antibodies improve amyloid pathology, synaptic dysfunction, and memory in Alzheimer's disease.

[0087] Anti-TREM2 antibodies induce microglial activation and improve cognitive function in Alzheimer's disease.

[0088] Anti-TAM antibodies function as agonists to maintain brain homeostasis, promoting the phagocytosis of apoptotic cells by microglia.

[0089] Anti-scavenger receptors regulate microglia and induce phagocytosis of Aβ.

[0090] Anti-FcγR antibodies inhibit FcγR-induced pro-inflammatory responses, including the release of cytokines and other mediators in microglia, during the progression of Alzheimer's disease (AD).

[0091] Anti-CD36 antibodies act as inhibitors of the binding of CD36 to amyloid-beta, thereby preventing microglial activation in the progression of Alzheimer's disease (AD).

[0092] Anti-RAGE antibodies inhibit the downstream RAGE-Aβ interaction and ROS production in microglia, astrocytes, and endothelial cells during the progression of Alzheimer's disease (AD).

[0093] Anti-APOE antibodies can inhibit plaque-induced microglial reactivity and lipid metabolism, thereby promoting inflammation.

[0094] Anti-CR1 antibodies regulate microglia in the immune clearance of Aβ in the brain of Alzheimer's disease.

[0095] Anti-CD38 antibodies regulate microglial multifunction, NAD metabolism, and inflammatory cytokine production, thereby reducing neuroinflammation associated with the progression of Alzheimer's disease (AD).

[0096] In one embodiment, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, or Lewis body dementia, and the drug is an anti-CD3 antibody, anti-CD39 antibody, anti-CD73 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-GZM A antibody, anti-GZM-B antibody, anti-CD33 antibody, anti-TAM antibody, anti-FcγRI antibody, anti-CD36 antibody, anti-RAGE antibody, anti-APOE antibody, anti-CR1 antibody, and anti-CD38 antibody.

[0097] The dosage of the MINC agent is determined based on known dosages of drugs used to treat specific diseases and the subject's condition. The dosage may be a dosage approved by the Food and Drug Administration (FDA) or a dosage used in clinical trials.

[0098] For MINC preparations, the typical combined dosage of PEG-EGCG and OEGCG is 10 μg / kg to 10 mg / kg.

[0099] The concentration of the drug in MINC is a minimum of 10 μg / kg and a maximum of 10 mg / kg.

[0100] To treat Alzheimer's disease, MINC-anti-CD3 antibody can be administered intravenously once every 1 to 4 weeks at a dose ranging from 0.01 to 10 mg of anti-CD3 antibody / kg. Following administration of MINC-anti-CD3 antibody, oxidative stress in the brain is reduced, and neuronal regeneration is promoted. This helps alleviate symptoms such as difficulties with communication, judgment, understanding, memory, and motor skills. To prevent recurrence of these symptoms, repeated administration of 0.005 to 5 mg every 3 to 6 months may be appropriate. In conjunction with brain imaging, the concentrations of biomarkers such as Aβ42, Aβ40, t-tau, p-tau, and inflammatory cytokines in the peripheral circulation should be monitored.

[0101] To treat Parkinson's disease, MINC-anti-CD3 antibody can be administered intravenously at a dose of 0.01–10 mg of anti-CD3 antibody / kg every 1–4 weeks. Treatment with MINC-anti-CD3 antibody inhibits the accumulation of abnormal proteins in the areas of the brain that control motor function. After MINC-anti-CD3 antibody treatment, improvement in symptoms such as rhythmic tremors, bradykinesia (slowness of movement), and muscle rigidity is observed. To prevent recurrence of these symptoms, repeated administration of 0.005–5 mg every 3–6 months may be appropriate. In conjunction with brain imaging, the concentrations of biomarkers (α-synuclein and inflammatory cytokines) in the peripheral circulation should be monitored.

[0102] To treat Huntington's disease, MINC-anti-CD3 antibody can be administered intravenously once every 1 to 4 weeks at a dose ranging from 0.01 to 10 mg of anti-CD3 antibody / kg. MINC-anti-CD3 antibody inhibits the localization of Huntington's plaque formation and reduces oxidative stress in the patient's brain. MINC-anti-CD3 antibody slows the progression of the disease by alleviating major symptoms, including depression, mood swings and personality changes, stumbling and clumsiness, involuntary twitching of the limbs, or restless movements. To prevent recurrence of these symptoms, repeated administration of 0.005 to 5 mg every 3 to 6 months may be appropriate. Monitoring of biomarkers (mHtt and inflammatory cytokines) in peripheral circulation should be performed in conjunction with brain imaging.

[0103] To treat Lewy body dementia, MINC-anti-CD3 antibody can be administered intravenously once every 1 to 4 weeks at a dose ranging from 0.01 to 10 mg of anti-CD3 antibody / kg. MINC-anti-CD3 antibody reduces the aggregation of Lewy bodies in the patient's brain. Patients recover from major disease symptoms such as hallucinations, fluctuations in cognitive function, sleep behavior disturbances, and spontaneous changes in attention. To prevent recurrence of these symptoms, repeated administration of 0.005 to 5 mg every 3 to 6 months may be appropriate. Monitoring of biomarkers (such as inflammatory cytokines) in peripheral circulation should be performed in conjunction with brain imaging.

[0104] Anti-CD3 antibodies bind to CD3 receptors on T cells, suppressing inflammation, reducing the secretion of IL-1β and TNFα from microglia, and differentiating microglia from (damaging) M1 to (protective) M2.

[0105] Anti-CD3 monoclonal antibodies have been used alone to treat type 1 diabetes and Alzheimer's disease. However, severe clinical toxicity has hindered further clinical development. By encapsulating anti-CD3 antibodies with OE / PE, the immunomodulatory function of these antibodies is maintained, and the overall MINC-anti-CD3 antibody complex becomes non-toxic.

[0106] Clinically, anti-CD3 monoclonal antibodies are approved for the treatment of TID, but due to drug toxicity, current treatment courses are limited to a single lifetime administration of 1-20 μg / kg intravenous injection for 14 days without repeated doses. In the case of TID and neurodegenerative diseases (AD, PD, HD, and LBD), there are no toxicity issues, so repeated administration of MINC-anti-CD3 antibodies in the same dose range can be used to promote long-term cognitive rejuvenation.

[0107] The present invention is useful for the treatment of humans and non-human animals. For example, the present invention is useful for the treatment of subjects such as humans, horses, pigs, cats, dogs, or rodents.

[0108] The following embodiments further illustrate the present invention. These embodiments are intended to illustrate the present invention only and should not be construed as limiting it. [Examples]

[0109] Active ingredients (for all examples) OEGCG: OEGCG is oligomerized EGCG, prepared according to WO2009 / 054813.

[0110] PEG-EGCG: PEG-EGCG is PEG complexed with one or two EGCG molecules and prepared according to WO2009 / 054813.

[0111] MINC agent: The MINC agent was prepared according to WO2009 / 054813. Alternatively, the MINC agent may also be prepared by encapsulating the agent within a nanocomposite formed from PEG-EGCG and OEGCG, according to the method of WO2009 / 054813.

[0112] Example 1: Method for preparing MINC-anti-CD3 antibody

[0113] material The anti-CD3 antibody was purchased from Biolegend. The MINC-anti-CD3 antibody was prepared according to WO2009 / 054813. MINC-anti-CD3 antibody nanoparticles (PEG-EGCG, OEGCG, and anti-CD3 antibody) were prepared according to WO2011 / 112156.

[0114] method The size of the MINC-anti-CD3 antibody nanoparticles was measured using DLS (Anton Paar Litesizer 500).

[0115] result The size of the nanoparticles was measured using DLS (Anton Paar Litesizer 500). Figure 3 shows the final particle size, with a median nanoparticle size of 87.08 nm. The standard deviation was 0.945. More than 95% of the nanoparticles were distributed in the range of 50–300 nm.

[0116] Example 2: The MINC drug platform delivers drugs and permeates the blood-brain barrier in an alternative cell model.

[0117] material OEGCG, PEG-EGCG, and MINC-doxorubicin are the same as those described in Example 1.

[0118] method To confirm the effectiveness of the MINC platform in delivering drugs and crossing the blood-brain barrier, doxorubicin (a chemotherapy drug that cannot penetrate one type of blood-brain barrier) was selected for the in vitro BBB Transwell trial.

[0119] Simply put, 3x10 4 Caco-2 cells were seeded in the luminal side of an insert in a 24-well Transwell plate (Falcon) to simulate a blood-brain barrier (BBB). The culture medium was changed every 3 days. The transepithelial electrical resistance (TEER), which indicates the integrity of the BBB in each well, was measured using a Millicell ERS Voltohmmeter (Millipore, MA, USA). The TEER value for each insert was 250 Ω*cm. 2 Once the target temperature was reached, (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 hours of incubation, the medium from the upper insert and lower culture wells was collected, and the fluorescence signal (relative fluorescence units, RFU) at ex / em = 470 / 595 nm was detected using Spectramax i3x. The drug transmission rate was calculated using the following formula: Transmission Rate (%) = (RFU 下部 × 7) ÷ (RFU 上部 × 7 + RFU下部 The calculation was performed according to × 7).

[0120] result The results are shown in Figure 4. Significantly more fluorescence signals were observed in the MINC-doxorubicin treatment group compared to the doxorubicin-alone control group (n = 3, p<0.001). This result suggests that our MINC drug platform enhanced the BBB permeability of doxorubicin in an alternative BBB transwell model.

[0121] Example 3: The MINC drug platform delivers the antibody drug, an anti-HER2 antibody, to the brain in mice.

[0122] material OEGCG and PEG-EGCG were prepared according to Example 1. Cyanine 5.5 NHS ester (Cy 5.5) (manufactured by Aladdin) was used. The anti-HER2 antibody-Cy5.5 conjugate was prepared by reacting the anti-HER2 antibody with the Cy5.5-NHS ester according to the manufacturer's (Aladdin) instructions. MINC-anti-HER2 antibody-Cy5.5 is anti-HER2 antibody-Cy5.5 encapsulated in PEG-EGCG and OEGCG, and was prepared according to WO2009 / 054813.

[0123] method Antibody drugs cannot efficiently penetrate the brain parenchyma. Antibody drugs exhibit similar structures and molecular sizes (approximately 150 kDa) consisting of Fab and Fc regions. In this example, a fluorescently labeled anti-HER2 antibody (trastuzumab) was selected as an example to demonstrate that MINC formulations can deliver antibody drugs to the brain in a mouse model.

[0124] 6-week-old athymoid nude Foxn1 nuFemale mice were used. These mice were divided into two groups. One group (n = 3) received a tail vein bolus injection of 10 mg / kg of anti-HER2 antibody-Cy5.5 as a control. The other group (n = 3) received a tail vein bolus injection of the same amount of anti-HER2 antibody-Cy5.5 in MINC-anti-HER2 antibody-Cy5.5. Eight hours after drug administration, live images were observed at ex / em = 674 / 692 nm using an IVIS (in vivo imaging system) Lumina III XRMS.

[0125] Results The results showed that a significantly stronger fluorescence signal was observed in the brain regions of mice administered MINC-anti-HER2 antibody-Cy5.5 (STM-001), but not in mice administered anti-HER2 antibody-Cy5.5.

[0126] Trastuzumab is approved by the FDA as a therapeutic agent for breast cancer, but is not approved as a therapeutic agent for glioma due to its low penetration into the brain. This example shows that the MINC drug platform can deliver antibody drugs into the brain in mice. + The mechanism by which the MINC drug platform delivers anti-HER2 antibodies into the brain is also applicable to other antibodies including anti-CD3 antibodies.

[0127] Example 4: MINC-anti-CD3 antibody inhibited Aβ-induced neuronal cell death.

[0128] Materials

[0129] Recombinant Aβ(1-42) peptide and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Genscript and Thermo Fisher. HT-22 cells were obtained from Millipore (Bedford, MA, USA). MINC-anti-CD3 antibody was prepared according to Example 1. ​

[0130] method To confirm the effect of MINC-anti-CD3 antibody in suppressing Aβ-induced neuronal cell death, an in vitro MTT assay was performed. Briefly, HT-22 cells were placed in a 24-well dish at a rate of 2 × 10⁶ per well. 5 Cells were seeded individually and cultured for 3 days during the logarithmic growth phase. These cells were then treated with a prepared 2.5 μM oligo-Aβ and Aβ+MINC-anti-CD3 antibody for 24 hours. After incubation, the cells were washed once with warmed PBS to remove the test material, and tetrazolium salt was added over 30 minutes at room temperature. The formazan product was then measured at 550 nm using a spectrophotometer. Viability was calculated as a percentage of untreated control cells.

[0131] result Figure 5 shows that the MINC-anti-CD3 antibody exhibits a protective effect on nerve cells treated with Aβ. When nerve cell lines were treated with Aβ peptide, cell viability (percentage of cells) decreased to less than 40% compared to the untreated group. After adding the MINC-anti-CD3 antibody to the Aβ-treated cells, the MINC-anti-CD3 antibody showed a protective effect, and cell viability significantly increased to over 50%.

[0132] The accumulation of abnormal proteins (Aβ and α-synuclein) in the brain is common in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and Lowie's body dementia. This data demonstrates that the MINC-anti-CD3 antibody protects nerve cells from damage caused by the accumulation of abnormal proteins.

[0133] Example 5: OEGCG and PEG-EGCG suppress Aβ-induced oxidative stress.

[0134] material Recombinant Aβ(1-42) peptide, DCFH-DA, was purchased from Genscript and Thermo Fisher. HT-22 cells were obtained from Millipore (Bedford, MA, USA).

[0135] method This experiment aimed to test the effects of OEGCG, PEG-EGCG, and MINC-anti-CD3 antibody on reducing Aβ-induced oxidative stress. In vitro reactive oxygen species (ROS) staining tests were performed. HT-22 cells were placed in a 24-well dish at a rate of 2 × 10⁶ per well. 5 Cells were seeded individually and cultured for 3 days. Afterward, cells were treated with Aβ, Aβ+OEGCG, Aβ+PEG-EGCG, and Aβ+MINC-anti-CD3 antibody. Oligo-Aβ was prepared. Briefly, the Aβ peptide was dissolved in 100% 1,1,1,3,3,3-hexafluoro-2-propanol to 1 mM and dried using a vacuum desiccator. Next, the Aβ was resuspended in dimethyl sulfoxide (DMSO) at a concentration of 5 mM and stored at -20°C. To obtain the oligomer, the Aβ peptide was diluted to a final concentration of 100 μM in Dulbecco's Modified Eagle Medium (DMEM, Gibco), gently shaken at 4°C for 24 hours, and then immediately added to the cell culture to a final concentration of 2.5 μM. After treating cells with Aβ for 1 hour, 10 μM OEGCG or PEG-EGCG was added to the cells and incubated for 6 hours. Next, the cells were treated with 20 μM DCFH-DA for 0.5 hours 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 acquired using a fluorescence microscope (DP72 / CKX41, Olympus), with the same fluorescence conditions and exposure time used for all images.

[0136] result The level of oxidative stress induced by Aβ stimulation was measured using the fluorescent dye DCFH-DA. Cells with high oxidative stress showed high fluorescence intensity. These results indicate that the control group treated with Aβ had the highest number of fluorescently positive cells (94.4% of all cells) and the highest fluorescence intensity of the positive cells, while the Aβ+OEGCG (20 μM) group had the lowest number of fluorescently positive cells (12.5% ​​of all cells) and the lowest fluorescence intensity of the positive cells, and the Aβ+PEG-ECGC (20 μM) group had the lowest number of fluorescently positive cells (11.3% of all cells) and the lowest fluorescence intensity of the positive cells.

[0137] Oxidative stress is known to cause neuronal cell death in various neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and Lewy body dementia. The aforementioned data demonstrate that the MINC components OEGCG, PEG-EGCG, and MINC agents protect neurons from oxidative stress-mediated nerve damage in these neurodegenerative diseases.

[0138] Example 6: OEGCG, PEG-ECGC, and MINC-anti-CD3 antibody promote the proliferation / regeneration of nerve cells.

[0139] material OEGCG and PEG-EGCG were prepared according to Example 1. MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Abcam. HT-22 cells were obtained from Millipore (Bedford, MA, USA). The MINC-anti-CD3 antibody was prepared according to Example 1.

[0140] method We confirmed that OE, PE, and MINC-anti-CD3 antibodies can promote the proliferation / regeneration of nerve cells using an in vitro cell proliferation assay (MTT assay). Nerve cells (HT-22) were treated with OE, PE, and MINC-anti-CD3 antibodies, and the in vitro MTT assay was performed.

[0141] Simply put, HT-22 cells are placed in DMEM medium supplemented with 5% FBS in a 96-well plate at a rate of 2.5 × 10⁶ 3 Cells were seeded at a rate of one cell per well and cultured for 24 hours during the logarithmic growth phase. Subsequently, cells were treated with OE (2.2 μg / mL), PE (4 μg / mL), and MINC-anti-CD3 antibody (0.74 μg / mL) for 48 hours. After culturing, cells were washed once with warmed PBS to remove test substances, and MTT reagent (tetrazolium salt) was added over 4 hours at 37°C. The formazan product was then measured at 550 nm using a spectrophotometer. A higher OD value indicates a higher cell count and higher cell viability. Cell proliferation rate was calculated as a percentage of untreated control cells.

[0142] result Figure 6 shows that, compared to the group treated with physiological saline (blank, 100%), the cell count increased by 150% in the OEGCG-treated group, by 170% in the PEG-ECGC-treated group, and by 140% in the MINC-anti-CD3 antibody-treated group. These results support the ability of OEGCG, PEG-ECGC, and MINC-anti-CD3 antibody to promote neuronal cell proliferation / regeneration.

[0143] Nerve cell damage directly leads to a decline in cognitive abilities in patients. This is one of the common symptoms of Alzheimer's disease, Parkinson's disease, Huntington's disease, and Lewy body dementia. Currently, there are no drugs that increase the number of nerve cells in the event of nerve cell damage. The data from this example show that the MINC-anti-CD3 antibody stimulates nerve cell proliferation / regeneration, which could be applied to various neurodegenerative diseases.

[0144] Example 7: MINC-anti-CD3 antibody differentiates microglia from M1 to M2.

[0145] material OEGCG and PEG-EGCG were prepared according to Example 1. Recombinant Aβ(1-42) peptide, MTT(3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Abcam. HT-22 cells were obtained from Millipore (Bedford, MA, USA). The MINC-anti-CD3 antibody was prepared according to Example 1.

[0146] method LPS was used to stimulate microglia into an inflammatory M1 phenotype (IL-6 secretion is characteristic of the M1 phenotype). MINC-anti-CD3 antibody was used to reduce the inflammatory M1 phenotype in microglia.

[0147] In short, BV-2 cells were seeded at a rate of 10,000 cells per well in a 96-well plate and incubated at 37°C for 16 hours. The cells were then treated with LPS (100 ng / mL) or LPS + MINC-anti-CD3 antibody (10 μg / mL) for 24 hours. After treatment, the supernatant was collected for an IL-6 ELISA assay. Results were normalized to an untreated control group. Cell viability in each group was also measured using an MTT assay.

[0148] result LPS differentiated microglial cells into the inflammatory M1 phenotype, as indicated by IL-6 secretion. Figure 7 shows a decrease in IL-6 levels after treatment with MINC-anti-CD3 antibody. This suggests a reduction in the M1 phenotype of microglia and a shift to the M2 phenotype, which is an anti-inflammatory and neuroprotective microglial phenotype (representative tests were used to calculate the fold change). MTT assays were also performed on all cell groups, but the data showed no difference in cell number in the MINC-anti-CD3 antibody-treated group. This result indicates that the MINC-anti-CD3 antibody reduced IL-6 secretion rather than cell viability (cell number).

[0149] Clinical data indicate that the phenotype of microglia in the brain is critically important for the progression of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and Lowie's body dementia. M1 microglia release inflammatory cytokines (IL-6, IL-1β, and TNF-α) and are neurotoxic. On the other hand, M2 microglia release anti-inflammatory mediators (IL-4, IL-10, and TGF-β) and have neuroprotective activity. In addition, M2 microglia take up and remove abnormal protein deposits, protecting nerve cells from damage. In the brains of neurodegenerative diseases, microglia are predominantly differentiated into the M1 type. This example demonstrates that the MINC-anti-CD3 antibody can treat neurodegenerative diseases by reducing M1-stage microglia. Recovery of inflammation in the brain after MINC-anti-CD3 antibody treatment is suggested.

[0150] Example 8: MINC-anti-CD3 antibody reduces the toxicity of anti-CD3 antibodies and restores the animal's health and survival rate.

[0151] material The anti-CD3 antibody was purchased from Biolegend. The MINC-anti-CD3 antibody was prepared according to WO2011 / 112156. I purchased the BalbB / c mouse from BioLesco.

[0152] method Thirty-five male Balb / c mice, 6–8 weeks old, were housed in a pathogen-free environment. After a 3-day acclimatization period, treatment was initiated the day after baseline calculation (day 0). Free anti-CD3 antibody or MINC-anti-CD3 antibody was administered intravenously daily for 2 days at doses of 5, 25, and 125 μg per mouse. Mice were randomly assigned to one of three treatment groups: 1) saline, 2) anti-CD3 antibody, or 3) MINC-anti-CD3 antibody (n = 5). Body weight, survival rate, and clinical symptoms were measured every other day for 8 consecutive days. Statistical analysis was performed using GraphPad Prizm. Two-way ANOVA was used to evaluate group differences, with p < 0.05 considered statistical significance.

[0153] result As shown in Figure 8, injection of free anti-CD3 antibody was toxic at all three doses, causing not only a significant decrease in animal body weight but also failure to recover to the normal range. Even in the group receiving the lowest dose of 5 μg of free anti-CD3 antibody, animal body weight decreased dramatically on days 0 and 1 after injection. This weight loss was irreversible and was similar in the 125 μg / mouse dose group. In contrast, there was no significant change in body weight after administration of 5 μg and 25 μg of MINC-anti-CD3 antibody. Even at the highest dose of 125 μg, animal body weight recovered to a level comparable to the control group treated with saline.

[0154] The survival rate of the animal group treated with free anti-CD3 antibody (125 μg) decreased on day 4 (animal deaths occurred). On the other hand, the survival rate of the MINC-anti-CD3 antibody remained at 100% until the end of the experiment.

[0155] These results demonstrate that MINC encapsulation significantly reduces the toxicity of free anti-CD3 antibodies, and that they are well-tolerated even at doses up to 25 times the clinically appropriate dose (5 μg / mouse). Due to the low toxicity of MINC-anti-CD3 antibodies, repeated administration to patients may be helpful in restoring cognitive abilities after long-term treatment.

[0156] Example 9: Animal blood cell analysis has shown that the MINC-anti-CD3 antibody is safe.

[0157] material The anti-CD3 antibody was purchased from Biolegend. The MINC-anti-CD3 antibody was prepared according to Example 1. The C57BL / 6 mice were purchased from the National Center for Experimental Animals (NLAC).

[0158] method Fourteen-week-old C57BL / 6 mice were administered either a control (physiological saline), 5 μg, or 25 μg of STM-003 (in 50 μL of physiological saline) once daily via tail vein injection for two days. Complete blood counts were analyzed on day 4 after injection. Red blood cells (RBCs), platelets (PLTs), white blood cells (WBCs), neutrophils (NEUTs), and lymphocytes (LYMPHs) were compared among the three groups.

[0159] result Figure 9 shows that treatment with MINC-anti-CD3 antibody was safe in animals and did not alter blood cell composition compared to the control group treated with physiological saline.

[0160] Previous clinical trials have shown that cytokine release syndrome (CRS) frequently occurs in patients treated with free anti-CD3 antibodies. CRS is primarily due to an increase in the white blood cell count in the patient's blood. No significant changes were observed in the cell counts of white blood cells, red blood cells, and platelets. This further demonstrates the safety of treatment with MINC-anti-CD3 antibodies. Due to its high safety profile, MINC-anti-CD3 antibodies can be applied to the long-term treatment (repeated administration) of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and Lewy body dementia, and can prevent relapses of these diseases.

[0161] Example 10: In a surrogate Transwell cell model, the MINC platform delivers anti-CD3 and anti-CD33 antibodies across the blood-brain barrier (BBB) ​​(predicted example).

[0162] the purpose This example demonstrates that the MINC platform delivers antibody drugs to the brain by crossing the blood-brain barrier (BBB). An in vitro surrogate Transwell cell model simulates tight junctions between cells in the BBB. MINC-anti-CD3 antibodies or MINC-drugs are expected to exhibit stronger fluorescence intensity compared to non-MINC formulations. This implies an increased amount of drug crossing the BBB structure.

[0163] Anti-CD3 and anti-CD33 antibodies can be replaced with other antibodies that target T cells and microglia in the brain (including anti-CD39, anti-CD73, anti-PD-L2, anti-CTLA-4, anti-GZM A, anti-GZM B, anti-CD33, anti-TAM, anti-FcγRI, anti-CD36, anti-RAGE, anti-APOE, anti-CR1, and anti-CD38). These antibodies are labeled with Cy5.5 according to the manufacturer's instructions and encapsulated in the MINC platform using the method described in the active ingredient section.

[0164] material Anti-CD3-Cy5.5 and anti-CD33-Cy5.5 complexes were prepared by reacting anti-CD3 antibody and anti-CD33 antibody with Cy5.5-NHS ester according to the manufacturer's instructions. MINC-anti-CD3 antibody, MINC-anti-CD33 antibody, and other MINC nanoparticles were prepared according to WO2011 / 112156.

[0165] method To confirm the effectiveness of the MINC platform in enabling drugs to cross the blood-brain barrier, MINC-anti-CD3 antibody and MINC-anti-CD33 antibody were selected as examples using the in vitro BBB Transwell assay. Details are described below.

[0166] In short, bEnd.3 cells or Caco-2 cells were seeded in the luminal side of an insert in a 12-well transwell plate (Falcon). The TEER value of each well was measured using a Millicell ERS Voltohmmeter (Millipore, MA, USA). The TEER value of each insert was 200 Ω*cm. 2 If the threshold was exceeded, either unencapsulated free drugs or drugs with MINC encapsulated (n=3) were added to the top of the insert, and basal extravasation medium was collected after 3, 6, 12, and 24 hours. The medium from the upper well (before permeation) and lower well (after permeation) of each treatment group was used for quantification of the drug that permeated the blood-brain barrier by fluorescence intensity, ELISA, spectrophotometric method, or mass spectrum.

[0167] Example 11: The MINC platform delivers anti-CD3 to the mouse brain (predicted example).

[0168] the purpose This example demonstrates that the MINC-anti-CD3 antibody is delivered to the mouse brain. The amount of fluorescently labeled anti-CD3 antibody in brain regions is measured using an in vivo mouse model. Compared to non-MINC anti-CD3 antibodies, the MINC-anti-CD3 antibody is expected to increase blood-brain barrier (BBB) ​​permeability compared to the unencapsulated drug alone.

[0169] material MINC-anti-CD3 antibody-Cy5.5 is anti-CD3 antibody-Cy5.5 encapsulated in PEG-EGCG and OEGCG, and was prepared according to Example 4.

[0170] method In a mouse model, a fluorescently labeled anti-CD3 antibody was used as an example to demonstrate that MINC formulations can deliver antibody drugs to the brain.

[0171] 6-week-old athymoid nude Foxn1 nuFemale mice were used. The mice were divided into two groups. One group (n=3) received a tail vein bolus of 10 mg / kg of anti-CD3 antibody-Cy5.5 as a control. The other group (n=3) received the same amount of MINC-anti-CD3 antibody-Cy5.5 as a tail vein bolus. Eight hours after administration, live images were observed using an IVIS (in vivo imaging system) Lumina III XRMS at ex / em = 674 / 692 nm.

[0172] Example 12: Method for preparing MINC-anti-CD3 antibodies using different flavonoids in flavonoid oligomers and polymer-flavonoid complexes (predicted example)

[0173] the purpose This example demonstrates that the MINC platform can encapsulate anti-CD3 antibodies using different flavonoids and formulate nanoparticles. Flavonoid oligomers are formed using EC, ECG, and EGCG, or they are complexed with PEG to form PEG-EC, PEG-ECG, and PEG-EGCG. DLS is used to demonstrate that different flavonoids can form nanoparticles.

[0174] method MINC-anti-CD3 antibody nanoparticles were prepared according to WO2009 / 054813. Briefly, various flavonoid oligomers, including OEGCG or OECG, were added to a PBS solution of anti-CD3 antibody, followed by the addition of various polymer-flavonoids, including PEG-EGCG, PEG-ECG, and PEG-EC. After incubation of the mixture at room temperature, unreacted oligomer-flavonoids and polymer-flavonoids were removed using a 10K MWCO centrifuge filter. The size of the nanoparticles was measured using a DLS (Anton Paar Litesizer 500).

[0175] Example 13: Method for preparing MINC-anti-CD3 antibodies using different hydrophilic polymers in polymer-flavonoid complexes (predicted example)

[0176] the purpose This example demonstrates that the MINC platform can encapsulate anti-CD3 antibodies using different hydrophilic polymers and formulate nanoparticles. PEG, HA, and dextran are used to conjugate with EGCG, forming PEG-EGCG, HA-EGCG, and dextran-EGCG. DLS is used to demonstrate that different polymers can form nanoparticles.

[0177] material OEGCG is oligomerized EGCG, prepared according to WO2006 / 124000. PEG-EGCG is PEG complexed with one or two EGCG molecules. HA-EGCG is HA complexed with one or two EGCG molecules. Dextran-EGCG is dextran complexed with one or two EGCG molecules. These different polymer-flavonoids were prepared according to WO2006 / 124000, WO2009 / 054813, or WO2015 / 171079. The anti-CD3 antibody was purchased from Biolegend.

[0178] method Nanoparticles of MINC (multi-target immune nanocarrier combination)-anti-CD3 antibody were prepared according to WO2009 / 054813. Briefly, the anti-CD3 antibody was incubated in PBS. Subsequently, OEGCG or OEGCG was added to the anti-CD3 antibody, followed by the addition of various polymer-flavonoids, including PEG-EGCG, HA-EGCG, and dextran-EGCG. After incubation of the mixture at room temperature, unreacted OEGCG and polymer-flavonoids were removed using a 10K MWCO centrifuge filter. The size of the nanoparticles was measured using DLS (Anton Paar Litesizer 500).

[0179] Example 14: MINC-anti-CD3 antibody test in an animal model of Alzheimer's disease (predicted example)

[0180] the purpose This example demonstrates the efficacy of MINC-anti-CD3 antibody in the treatment of Alzheimer's disease. Brain Aβ content and behavioral tests in mice or rats treated with the solvent, anti-CD3 antibody, or MINC-anti-CD3 antibody showed improvements in spatial working memory and exploratory behavior.

[0181] material OEGCG and PEG-EGCG were prepared according to Example 1. The anti-CD3 antibody was purchased from Biolegend. The MINC-anti-CD3 antibody was prepared according to Example 1.

[0182] method In short, we used Tg APPsw mice, APP / PS1 mice, or Wistar rats. These mice or rats were divided into several groups. Each group received intravenous injections twice a week for 2-3 months of either a solvent (PBS or saline, as the untreated group), 0.1-100 mg / kg of anti-CD3 antibody, or MINC-anti-CD3 antibody at a concentration equivalent to anti-CD3 antibody. The concentration of anti-β-amyloid antibody ranged from 0.01 to 10 μg / mL. To analyze brain Aβ levels and Aβ loading, these mice or rats were sacrificed at 6-24 months of age. Quantitative Aβ imaging analysis was performed using anti-β-amyloid antibody (clone 4G8).

[0183] The Morris Water Maze (MWM) test was performed to measure spatial working memory and exploratory activity. The open-field water maze procedure, in which mice learn to escape from opaque water to a hidden platform, is an established model for testing the cognitive function of mice. The formation and retention of spatial memory were assessed using the MWM assay. A 10 cm escape platform was submerged 1 cm below the water surface in a circular plastic pool filled with opaque water. Three visual cues were placed on the walls surrounding the pool. A digital camera was placed above the center of the maze. Images were acquired and transmitted to a PC running tracking software. For the first three days (pre-training), mice were trained using the visible platform (the platform was placed above the water surface). To assess the formation of spatial memory, mice were trained for eight consecutive days to find the hidden platform. Escape latency for each trial (four times a day, at 3-5 minute intervals) was recorded and analyzed using tracking software. On the third and ninth days of training, the platform was removed, and memory retention in the probe trials was evaluated by analyzing the search patterns used by each mouse over a fixed 45-second period. Escape latency, which represents the time it took for the mouse to find the hidden platform, was measured and analyzed, and the average value of four trials per day was used.

[0184] Example 15: MINC-anti-CD33 antibody test in an animal model of Alzheimer's disease (predicted example)

[0185] the purpose This example demonstrates the efficacy of MINC-anti-CD33 antibody in the treatment of Alzheimer's disease. Brain Aβ content and behavioral tests of mice or rats treated with the solvent, anti-CD33 antibody, or MINC-anti-CD33 antibody can show improvements in spatial working memory and exploratory behavior. This example is expected to demonstrate the therapeutic effect of MINC-anti-CD33 antibody on Alzheimer's disease.

[0186] material OEGCG and PEG-EGCG were prepared according to Example 1. The anti-CD33 antibody was purchased from Biolegend. The MINC-anti-CD33 antibody was prepared according to Example 1.

[0187] method To confirm the effect of MINC-anti-CD33 antibody on suppressing Aβ accumulation or on restoring β-amyloid-induced behavioral disorders, an in vivo Alzheimer's disease model was used.

[0188] In short, we used Tg APPsw mice, APP / PS1 mice, or Wistar rats. These mice or rats were divided into several groups. Each group received intravenous injections of either 0.1–100 mg / kg of anti-CD33 antibody, or MINC-anti-CD33 antibody at a concentration equivalent to anti-CD33 antibody, twice a week for 2–3 months, in a solvent (PBS or saline, as an untreated control). The concentration of anti-β-amyloid antibody was 0.01–10 μg / mL. These mice or rats were sacrificed at 6–24 months of age to analyze brain Aβ levels and Aβ loadings. Quantitative Aβ imaging analysis was performed using anti-β-amyloid antibody (clone 4G8).

[0189] The Morris Water Maze (MWM) test was performed to measure spatial working memory and exploratory activity. The open-field water maze procedure, in which mice learn to escape from opaque water to a hidden platform, is an established model for testing the cognitive function of mice. The formation and retention of spatial memory were assessed using the MWM assay. A 10 cm escape platform was submerged 1 cm below the water surface in a circular plastic pool filled with opaque water. Three visual cues were placed on the walls surrounding the pool. A digital camera was placed above the center of the maze. Images were acquired and transmitted to a PC running tracking software. For the first three days (pre-training), mice were trained using the visible platform (the platform was placed above the water surface). To assess the formation of spatial memory, mice were trained for eight consecutive days to find the hidden platform. Escape latency for each trial (four times a day, at 3-5 minute intervals) was recorded and analyzed using tracking software. On the third and ninth days of training, the platform was removed, and memory retention in the probe trials was evaluated by analyzing the search patterns used by each mouse over a fixed 45-second period. Escape latency, which represents the time it took for the mouse to find the hidden platform, was measured and analyzed, and the average value of four trials per day was used.

[0190] Example 16: MINC-anti-CD3 antibody test in an animal model of Parkinson's disease (predicted example)

[0191] the purpose This example demonstrates the efficacy of MINC-anti-CD3 antibody in the treatment of Parkinson's disease. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine synthesis. A decrease in TH activity or expression is crucial in the development of Parkinson's disease. In an animal model of MPTP neurotoxic PD, mice treated with MINC-anti-CD3 antibody are expected to maintain higher levels of TH expression or enzyme activity compared to an untreated control group. Therefore, this example aims to demonstrate the protection of dopaminergic neuronal function by MINC-anti-CD3 antibody. MINC-anti-CD3 antibody has therapeutic effects on Parkinson's disease.

[0192] material The MINC-anti-CD3 antibody was prepared according to WO2009 / 054813. The anti-CD3 antibody and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were purchased from Genscript, Abcam, Thermo Fisher, or Sigma.

[0193] method To confirm the efficacy of MINC-anti-CD3 antibody in the treatment of Parkinson's disease (PD), an in vivo PD model was used. Briefly, C57 / BL mice with MPTP-induced PD were used. The mice were divided into several groups. Each group received intravenous injection of either 0.1–100 mg / kg of anti-CD3 antibody, or MINC-anti-CD3 antibody at a concentration equivalent to anti-CD3 antibody, in a solvent (PBS or saline, as an untreated control), for 5–10 days. Subsequently, the mice received intraperitoneal injection of 1–100 mg / kg of MPTP for 3–5 days. The mice or rats were sacrificed 3 days after the final injection. The striatum from the posterior part of the mouse brain was used to prepare homogenates for measuring tyrosine hydroxylase activity. Tyrosine hydroxylase protein levels were analyzed using Western blotting.

[0194] Example 17: MINC-anti-CD33 antibody test in an animal model of Parkinson's disease (predicted example)

[0195] the purpose This example demonstrates the efficacy of MINC-anti-CD33 antibody in the treatment of Parkinson's disease. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine synthesis. A decrease in TH activity or expression is crucial in the development of Parkinson's disease. In an animal model of MPTP neurotoxic PD, mice treated with MINC-anti-CD33 antibody are expected to maintain higher levels of TH expression or enzyme activity compared to an untreated control group. Therefore, this example aims to demonstrate the protection of dopaminergic neuronal function by MINC-anti-CD33 antibody. MINC-anti-CD33 antibody has therapeutic effects on Parkinson's disease.

[0196] material The MINC-anti-CD33 antibody was prepared according to WO2009 / 054813. The anti-CD33 antibody and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were purchased from Genscript, Abcam, Thermo Fisher, Sigma, or any other supplier.

[0197] method To confirm the efficacy of MINC-anti-CD33 antibody in the treatment of Parkinson's disease (PD), an in vivo PD model was used. Briefly, C57 / BL mice with MPTP-induced PD were used. The mice were divided into several groups. Each group was intravenously injected with either 0.1–100 mg / kg of anti-CD33 antibody or MINC-anti-CD33 antibody at a concentration equivalent to anti-CD33 antibody, in a solvent (PBS or saline, as an untreated control), for 5–10 days. Subsequently, the mice were intraperitoneally injected with 1–100 mg / kg of MPTP for 3–5 days. The mice or rats were sacrificed 3 days after the final injection. The striatum from the posterior part of the mouse brain was used to prepare homogenates for measuring tyrosine hydroxylase activity. Tyrosine hydroxylase protein levels were analyzed using Western blotting.

Claims

1. A method for treating neurodegenerative diseases, The process includes administering an effective amount of a nanocomplex having (a) an outer shell containing one or more polymer-flavonoid complexes, (b) an optional inner shell containing one or more flavonoid oligomers, and (c) an anti-CD3 antibody or anti-CD33 antibody encapsulated within the shell, to a subject in need. The flavonoid is EGCG, EC, EGC, or ECG, as shown in the following structure: The polymer is a hydrophilic polymer with a molecular weight of 1,000 to 100,000 daltons, selected from the group consisting of polyethylene glycol (PEG), hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-α-tocopheryl, and polyethylene glycol succinate. The flavonoid oligomer contains 2 to 20 flavonoids of EGCG, EC, EGC, or ECG. The neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lewy body dementia, and Huntington's disease. method.

2. The method according to claim 1, wherein the method reduces the toxicity of anti-CD3 antibodies, promotes the proliferation of nerve cells, prevents nerve cell death, reduces oxidative stress on nerve cells, differentiates microglia from the M1 state to the M2 state, and / or reduces abnormal protein aggregation.

3. The method according to claim 1, wherein the neurodegenerative disease is Alzheimer's disease.

4. The method according to claim 1, wherein the neurodegenerative disease is Parkinson's disease.

5. The method according to claim 1, wherein the neurodegenerative disease is Lewy body dementia.

6. The method according to claim 1, wherein the neurodegenerative disease is Huntington's disease.

7. The method according to any one of claims 1 to 6, wherein the anti-CD3 antibody is encapsulated within the shell.

8. The method according to any one of claims 1 to 7, wherein the flavonoid is EGCG and the hydrophilic polymer is PEG.

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