Methods for treating autoimmune diseases

Abstract

The present invention provides a method of treating autoimmune diseases, including type 1 diabetes, inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, psoriasis, myasthenia gravis, and hashimoto thyroiditis. The method comprises the step of administering to a subject in need thereof an effective amount of a micelle having an outer shell comprising one or more polymer-flavonoid conjugates, optionally an inner shell comprising one or more flavonoid oligomers, and a drug encapsulated within these shells. The invention also provides a method of treating an autoimmune disease by administering to a subject in need thereof an effective amount of one or more polymer-flavonoid conjugates or one or more flavonoid oligomers.

Description

Technical Field

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

[0002] Autoimmune diseases are conditions caused by an abnormal immune response to a functional body part. Common autoimmune diseases include celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Hashimoto's thyroiditis, vitiligo, Sjögren's syndrome, myositis, chronic inflammatory demyelinating polyneuropathy (CIDP), dermatomyositis, Guillain-Barre syndrome, psoriatic arthritis, ulcerative colitis, and vasculitis.

[0003] Rheumatoid arthritis

[0004] Rheumatoid arthritis (RA) is a long-term autoimmune disease that primarily affects the joints. It typically causes warmth, swelling, and pain in the joints. While the cause of RA is unknown, it is believed to involve a combination of genetic and environmental factors. The underlying mechanism involves the body's immune system attacking the joints. This causes inflammation and thickening of the joint capsule.

[0005] Inflammatory bowel disease

[0006] Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the colon and small intestine, with Crohn's disease and ulcerative colitis being the main types. Crohn's disease affects the small and large intestine, as well as the mouth, esophagus, stomach and anus, while ulcerative colitis primarily affects the colon and rectum. Although Crohn's disease and UC are very different diseases, both may present any of the following symptoms: abdominal pain, diarrhea, rectal bleeding, severe internal cramps / muscle cramps in the pelvic region, and weight loss. Anemia is the most common extraintestinal complication of inflammatory bowel disease. Related conditions or diseases include arthritis, pyoderma gangrenosum, primary sclerosing cholangitis, and non-thyroid disease syndrome (NTIS).

[0007] Multiple sclerosis

[0008] Multiple sclerosis (MS), also known as disseminated encephalomyelitis, is the most common demyelinating disease in which the insulating covering of nerve cells in the brain and spinal cord is damaged. This damage destroys the ability of parts of the nervous system to transmit signals, leading to a series of signs and symptoms, including physical, psychological and sometimes psychiatric problems. Specific symptoms may include double vision, blindness in one eye, muscle weakness and difficulty in feeling or coordination. MS has several forms, with new symptoms appearing in isolated attacks (relapsing forms) or accumulating over time (developing forms). Between attacks, symptoms may disappear completely, yet permanent neurological problems usually remain, especially as the disease progresses. Although the cause is unclear, it is believed that the underlying mechanism is the destruction of the immune system or the exhaustion of myelin-producing cells.

[0009] Type 1 diabetes

[0010] Type 1 diabetes (T1D), formerly known as juvenile diabetes, is an autoimmune disease that originates when the islets of Langerhans (containing beta cells) in the pancreas produce very little or no insulin. Insulin is a hormone that cells need to use blood sugar as energy, and it helps regulate normal glucose levels in the bloodstream. Before treatment, this leads to high blood sugar levels in the body. Common symptoms of this elevated blood sugar are frequent urination, increased thirst, increased hunger, weight loss, and other serious complications. Additional symptoms can include blurred vision, fatigue, and slow wound healing. Symptoms usually develop over a short period of time, usually a few weeks. The cause of type 1 diabetes is unknown, but it is believed to involve a combination of genetic and environmental factors. The underlying mechanism involves autoimmune destruction of the beta cells in the pancreas that produce insulin.

[0011] Systemic lupus erythematosus

[0012] Systemic lupus erythematosus (SLE) is an autoimmune disease in which the body's immune system mistakenly attacks healthy tissue in many parts of the body. Symptoms vary from person to person and can be mild to severe. Common symptoms include joint pain and swelling, fever, chest pain, hair loss, mouth ulcers, swollen lymph nodes, feeling tired, and a rash that is most common on the face. There are usually disease phases called flares and remission phases during which there are almost no symptoms. Systemic lupus erythematosus affects multiple organ systems and is characterized by a widespread loss of immune tolerance.

[0013] psoriasis

[0014] Psoriasis is a long-term, non-infectious autoimmune disease characterized by raised areas of abnormal skin. These areas are red or purple, dry, itchy and scaly in some people with darker skin. The severity of psoriasis varies from small local spots to whole body coverage. Damage to the skin can trigger psoriatic skin changes at this point, which is called Koebner phenomenon. The cause of psoriasis is not fully understood. At present, there is no cure for psoriasis, but there are some treatment options, local agents are usually used for mild diseases, light therapy is used for moderate diseases, and systemic agents are used for severe diseases. Flavonoids have a general structure of a 15-carbon skeleton, which is composed of two benzene rings (A and B) and a heterocyclic ring (C, which contains embedded oxygen).

[0015] Typical treatment

[0016] Treatment depends on the type and severity of the disease. Most autoimmune diseases are chronic and do not have a definite cure, but symptoms can be alleviated and controlled by treatment. In general, the goal of various therapeutic methods is to alleviate the symptoms presented to alleviate and control the autoimmune response of the body while still maintaining the ability of the patient to resist the disease they may encounter. Traditional treatment options may include immunosuppressant drugs that weaken the overall immune response, such as: nonsteroidal anti-inflammatory drugs (NSAIDs) that reduce inflammation, glucocorticoids that reduce inflammation, and disease-modifying antirheumatic drugs (DMARDs) that reduce the damage tissue and organ effects of inflammatory autoimmune responses.

[0017] Flavonoids

[0018]

[0019] This carbon structure can be abbreviated as C6-C3-C6. According to the IUPAC nomenclature, flavonoids can be classified as:

[0020] Flavonoids or bioflavonoids

[0021] Isoflavonoids derived from the structure of 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) New flavonoids derived from the structure of 4-phenylcoumarin (4-phenyl-1,2-benzopyrone) BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 One embodiment of a MINC (Multi-channel Immunomodulatory Nanocomplex Combination Therapy)-agent is described, which is a micelle having a polymer-flavonoid conjugate (eg, PEG-EGCG conjugate) in a shell and having an encapsulated agent.

[0023] Figure 2Another embodiment of a MINC-agent is described, which is a micelle comprising a polymer-flavonoid conjugate (eg, PEG-EGCG conjugate) in an outer shell and a flavonoid oligomer (eg, oligomeric EGCG (OEGCG)) in an inner shell and having an encapsulated agent.

[0024] Figure 3 It was shown that OEGCG and PEG-EGCG inhibited LPS-induced inflammatory cytokine TNF-α production in rheumatoid arthritis synovial fibroblasts.

[0025] Figure 4 OEGCG and PEG-EGCG were shown to reduce the production of inflammatory cytokine genes IL-6 and COX2 in a cellular model of inflammatory bowel disease.

[0026] Figure 5 MINC-anti-CD3 was shown to have reduced toxicity compared to anti-CD3.

[0027] Figure 6 It was shown that different flavonoids in the polymer-flavonoid conjugates as well as different flavonoid oligomers successfully generated MINC-anti-HER2 micelles with a particle size of approximately 100 nm.

[0028] Figure 7 It was shown that different polymers in the polymer-flavonoid conjugates successfully generated MINC-BSA micelles with a particle size of approximately 100 nm. DETAILED DESCRIPTION

[0029] definition

[0030] The term "about" is defined as ±10%, preferably ±5% of the stated value.

[0031] The term "epigallocatechin gallate" refers to the ester of epigallocatechin and gallic acid, and is used interchangeably with "epigallocatechin-3-gallate" or EGCG.

[0032] The term "oligomeric EGCG" (OEGCG) refers to 3 to 30, preferably 2 to 20, covalently linked EGCG monomers. OEGCG preferably contains 4 to 12 EGCG monomers.

[0033] The term "polyethylene glycol-epigallocatechin gallate conjugate" or "PEG-EGCG" refers to polyethylene glycol (PEG) conjugated to one or two EGCG molecules. The term "PEG-EGCG" refers to both PEG-mEGCG conjugate (monomeric EGCG) and PEG-dEGCG (dimeric EGCG) conjugates.

[0034] The present invention provides a method for treating autoimmune diseases. The method comprises the step of administering to a subject in need thereof an effective amount of (i) a polymer-flavonoid conjugate, (ii) a flavonoid oligomer, or (iii) a micelle having an outer shell formed by one or more polymer-flavonoid conjugates and an optional inner shell formed by one or more flavonoid oligomers and a drug encapsulated in these shells.

[0035] Flavonoids

[0036] The flavonoids suitable for use in the present invention have the general structure of Formula I:

[0037]

[0038] in:

[0039] R 1 is H or phenyl;

[0040] R 2 is H, OH, gallate or phenyl; wherein phenyl is optionally substituted with one or more (e.g., 2 to 3) hydroxyl groups;

[0041] R 3 is H, OH or =O (oxo); or

[0042] R 1 and R 2 together to form a closed loop structure; or

[0043] R 2 and R 3 Together they form a closed loop structure.

[0044] The 2-, 3-, 4-, 5-, 6-, 7- or 8-position of Formula I may be bonded to a group containing hydrocarbon, halogen, oxygen, nitrogen, sulfur, phosphorus, boron or a metal.

[0045] Examples of flavonoids of Formula I include:

[0046]

[0047] Preferred flavonoid compounds of formula I include:

[0048] EGCG(CAS#989-51-5), EC(CAS#490-46-0), EGC(CAS#970-74-1) or ECG(CAS#1257-08-5).

[0049]

[0050] Polymer-flavonoid conjugates

[0051] As used herein throughout this application, polymer-flavonoid conjugate refers to a conjugate of a hydrophilic polymer and a flavonoid compound of Formula I.

[0052] Hydrophilic polymers refer to polymers that are soluble in polar solvents and can form hydrogen bonds. Hydrophilic polymers suitable for use in the polymer-flavonoid conjugates of the present invention include, but are not limited to, poly(ethylene glycol), aldehyde-derivatized hyaluronic acid, hyaluronic acid, dextran, diethyl acetal conjugates (e.g., diethyl acetal PEG), D-α-tocopheryl polyethylene glycol succinate, aldehyde-derivatized hyaluronic acid-tyramine, hyaluronic acid-aminoacetaldehyde diethyl acetal conjugate-tyramine, cyclotriphosphazene core phenoxymethyl (methylhydrazono) dendrimers or thiophosphoryl core phenoxymethyl (methylhydrazono) dendrimers, acrylamide, oxazoline, imine, acrylic acid, methacrylate, glycol, ethylene oxide, alcohol, amine, anhydride, ester, lactone, terephthalate, amide and ether, polyacrylamide, pol Sham, poly(N-isopropylacrylamide), poly(oxazoline), polyethyleneimine, poly(acrylic acid), polymethacrylate, poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol), poly(vinyl pyrrolidone), polyether, poly(allylamine), polyanhydride, poly(β-amino ester), poly(butylene succinate), polycaprolactone, polycarbonate, polydioxanone, poly(glycerol), polyglycolic acid, poly(3-hydroxypropionic acid), poly(2-hydroxyethyl methacrylate), poly(N-(2-hydroxypropyl) methacrylamide), polylactic acid, poly(lactic-co-glycolic acid), poly(orthoesters), poly(2-oxazoline), poly(sebacic acid), poly(terephthalate-co-phosphate), povidone and copolymers.

[0053] Preferred hydrophilic polymers include poly(ethylene glycol), hyaluronic acid, dextran, polyethylene imine, poloxamer, povidone, D-α-tocopherol and polyethylene glycol succinate. The molecular weight of the hydrophilic polymer in the polymer-flavonoid conjugate is generally 1K to 100K Daltons, preferably 2K to 40K Daltons, 2K to 50K Daltons, 2K to 80K Daltons, 3K to 80K Daltons or 5K to 40K Daltons.

[0054] In one embodiment, the polymer contains an aldehyde group conjugated to position 5, 6, 7 or 8 (preferably position 6 or 8) of the A ring of the flavonoid compound.

[0055] In another embodiment, the polymer contains an R 1 or R 2 Conjugated thiol (when R 1 or R 2 is -OH).

[0056] In one embodiment, the polymer-flavonoid conjugate is PEG-EGCG, which is a PEG conjugated to one or two molecules of epigallocatechin gallate (EGCG). For example, PEG-EGCG can be prepared by reacting the aldehyde-terminated PEG to EGCG via a free aldehyde group with position 5, 6, 7, or 8 (preferably position 6 or 8) of formula I, by connecting PEG. See WO2006 / 124000 and WO2009 / 054813. PEG-EGCG can also be prepared by reacting the sulfo-terminated PEG to EGCG via a free sulfo group with R1 or R2 (wherein R1 or R2 is phenyl) of formula I, by connecting PEG, by conjugating the sulfo-terminated PEG to EGCG. See WO2015 / 171079.

[0057] Flavonoid oligomers

[0058] Flavonoid oligomers are conjugates of a flavonoid with one or more flavonoids. Flavonoid oligomers can contain the same flavonoids (homo-oligomers) or different flavonoids (hetero-oligomers). Flavonoid oligomers that can be used in the present invention generally have 2 to 50 or 2 to 20, preferably 4 to 12 flavonoids of one type or mixed types.

[0059] In some embodiments, the flavonoid oligomer is oligomeric EGC (OEGCG), oligomeric EC (OEC), oligomeric EGC (OEGC) or oligomeric ECG (OECG). OEGCG refers to 3 to 20 covalently linked EGCG monomers. For example, OEGCG can be synthesized according to WO2006 / 124000 at position 5, 6, 7 or 8 (preferably position 6 or 8) of the A ring.

[0060] Since the A ring is present in all flavonoids according to formula 1, other oligomeric flavonoids can be prepared analogously according to WO 2006 / 124000. For example, OEC, OEGC and OECG can also be prepared according to WO 2006 / 124000.

[0061] MINC-Pharmacy

[0062] MINC (Multichannel Immunomodulatory Nanocomplex Combination Therapy) is a platform technology that exploits the biological activity of polymer-flavonoid conjugates or flavonoid oligomers that form micelles in solution.

[0063] The MINC platform can encapsulate additional agents to form nanoparticle compositions for combination therapy. A MINC-agent is 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 an agent encapsulated within the shell. As used herein, an agent refers to a molecule having therapeutic activity for treating an autoimmune disease, including but not limited to an anti-inflammatory agent, such as an anti-inflammatory antibody, an anti-inflammatory cytokine, an anti-inflammatory compound, or an antibody or compound.

[0064] The MINC platform can reduce the toxicity of the encapsulated agent, which expands the effective dose window of the agent in autoimmune diseases. For example, anti-CD3 is a broad-spectrum immunosuppressant used to treat various autoimmune diseases. However, anti-CD3 has high toxicity, which limits its clinical application. MINC-anti-CD3 formulations reduce the toxicity of anti-CD3. MINC-anti-CD3 is a safe drug for the treatment of autoimmune diseases.

[0065] In one embodiment, the MINC-agent is a micelle ( Figure 1 ).

[0066] In another embodiment, the MINC-agent is a micelle ( Figure 2 ).

[0067] When the agent is a drug, the MINC-agent composition comprises two or more therapeutically active components that are functionally complementary to form a multi-targeted combination therapy through its backbone components (flavonoid conjugates or flavonoid oligomers) and encapsulated drugs. MINC-agents are stable in hydrophilic environments, such as blood circulation.

[0068] In one embodiment, the agent in the MINC-agent is an anti-inflammatory antibody, including but not limited to anti-IL-1, anti-IL-1α, anti-IL-1β, anti-IL-1R, anti-IL-2, anti-IL-6, anti-IL-6R, anti-IL-7, anti-IL-7R, anti-IL-10, anti-IL-11, anti-IL-12, anti-IL-17, anti-IL-18, anti-IL-23, anti-IFN-α, anti-IFN-β, anti-IFN-γ, anti-TNF-α, anti-CD3, anti-CD20, anti-TGF-β, anti-T lymphocyte globulin (ATG). Such MINC-agents are suitable for treating rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, type 1 diabetes, systemic lupus erythematosus and psoriasis.

[0069] In one embodiment, the agent in the MINC-agent is an anti-inflammatory antibody, including but not limited to adalimumab, infliximab, golimumab, rituximab, certolizumab pegol, tocilizumab, salizumab, vedolizumab, edrelizumab, natalizumab, PF-00547659, ustekinumab, alemtuzumab, daclizumab, ocrelizumab, canakinumab, dapirolizumab, secukinumab, ixekizumab. Such MINC-agents are suitable for treating rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, type 1 diabetes, systemic lupus erythematosus and psoriasis.

[0070] In one embodiment, the agent in the MINC-agent is an antibody, including but not limited to adalimumab, infliximab, golimumab, rituximab, certolizumab pegol, tocilizumab, salirumab. Such MINC-agents are suitable for treating rheumatoid arthritis.

[0071] In one embodiment, the agent in the MINC-agent is an antibody, including but not limited to vedolizumab, edrolizumab, natalizumab, PF-00547659, ustekinumab, golimumab, adalimumab, certolizumab pegol, natalizumab, infliximab. Such MINC-agents are suitable for treating inflammatory bowel disease.

[0072] In one embodiment, the agent in the MINC-agent is an antibody, including but not limited to natalizumab, alemtuzumab, daclizumab, ocrelizumab. Such MINC-agents are suitable for treating multiple sclerosis.

[0073] In one embodiment, the agent in the MINC-agent is an antibody, including but not limited to canakinumab, ustekinumab, rituximab, otelizumab, vesilizumab. Such MINC-agents are suitable for treating type 1 diabetes.

[0074] In one embodiment, the agent in the MINC-agent is an antibody, including but not limited to pedalitumumab, belimumab, anirumab, BIIB059. Such MINC-agents are suitable for treating systemic lupus erythematosus.

[0075] In one embodiment, the agent in the MINC-agent is an antibody, including but not limited to infliximab, adalimumab, ustekinumab, secukinumab, ixekizumab. Such MINC-agents are suitable for treating psoriasis.

[0076] In one embodiment, the agent in the MINC-agents is an anti-inflammatory compound, including but not limited to leflunomide, aspirin, naproxen, diclofenac, ibuprofen, methotrexate, sulfasalazine, hydroxychloroquine, sulfasalazine, baricitinib, tofacitinib, azathioprine, cyclosporine, minocycline, ozanimod, mesalazine, olsalazine, balsalazide, prednisone, hydrocortisone, budesonide, 6-mercaptopurine, methotrexate, metronidazole, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, laquinimod, mitoxantrone, dalfampridine, metformin, tolbutamide, chlorpropamide, tolsulfuronamide, glyburide, glipizide, glimepiride, regulatide The invention relates to linaglinide, nateglinide, sitagliptin, saxagliptin, linagliptin, alogliptin, rosiglitazone, pioglitazone, acarbose, miglitol, canagliflozin, dapagliflozin, empagliflozin, BT-063, fenacinib, ipomitamide, cinaimod, ivotinib, bortezomib, ibuprofen, naproxen, chloroquine, quinacrine, betamethasone propionate, prednisolone, methylprednisolone sodium succinate, cyclophosphamide, mycophenolate mofetil, prasterone, dehydroepiandrosterone, apremilast, ruxolitinib, baricitinib, ASP015K, sotrastaurin, fostamatinib, tamatinib. Such MINC-agents are suitable for treating rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, type 1 diabetes, systemic lupus erythematosus and psoriasis.

[0077] In one embodiment, the agent in the MINC-agent is a compound including, but not limited to, leflunomide, aspirin, naproxen, diclofenac, ibuprofen, methotrexate, sulfasalazine, hydroxychloroquine, sulfasalazine, baricitinib, tofacitinib, azathioprine, cyclosporine, minocycline. Such MINC-agents are suitable for treating rheumatoid arthritis.

[0078] In one embodiment, the agent in the MINC-agent is a compound including, but not limited to, tofacitinib, ozanimod, sulfasalazine, mesalamine, olsalazine, balsalazide, prednisone, hydrocortisone, budesonide, azathioprine, 6-mercaptopurine, methotrexate, cyclosporine, metronidazole, methylprednisolone. Such MINC-agents are suitable for treating inflammatory bowel disease.

[0079] In one embodiment, the agent in the MINC-agent is a compound including, but not limited to, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, laquinimod, mitoxantrone, dalfampridine. Such MINC-agents are suitable for treating multiple sclerosis.

[0080] In one embodiment, the agent in the MINC-agent is a compound drug, including but not limited to metformin, tolbutamide, chlorpropamide, tolsulfuronamide, glibenclamide, glipizide, glimepiride, repaglinide, nateglinide, sitagliptin, saxagliptin, linagliptin, alogliptin, rosiglitazone, pioglitazone, acarbose, miglitol, canagliflozin, dapagliflozin, empagliflozin. Such MINC-agents are suitable for treating type 1 diabetes.

[0081] In one embodiment, the agent in the MINC-agent is a compound including, but not limited to, hydroxychloroquine, prednisone, methylprednisolone, cyclophosphamide, azathioprine, mycophenolate mofetil, BT-063, fenacinib, ipomitamide, cinamod, ivostimib, bortezomib, ibuprofen, naproxen, aspirin, chloroquine, quinacrine, betamethasone propionate, prednisone, prednisolone, methylprednisolone sodium succinate, cyclophosphamide, mycophenolate mofetil, cyclosporine, methotrexate, prasterone, dehydroepiandrosterone. Such MINC-agents are suitable for treating systemic lupus erythematosus.

[0082] In one embodiment, the agent in the MINC-agent is a compound including, but not limited to, apremilast, tofacitinib, ruxolitinib, baricitinib, ASP015K, sotrastaurin, BMS-582949, fostamatinib, tamatinib. Such MINC-agents are suitable for treating psoriasis.

[0083] In one embodiment, the agent in the MINC-agent is an anti-inflammatory protein, including but not limited to etanercept, abatacept, mongosan, exenatide, liraglutide, albiglutide, dulaglutide, abatacept, alecept, IL-2, GCSF, RSLV-132, RC18, AMG 592. Such MINC-agents are suitable for treating rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, type 1 diabetes, systemic lupus erythematosus and psoriasis.

[0084] For example, MINC-agent is an anti-IL-1 encapsulated in micelles formed by PEG-EGCG and flavonoid oligomer OEGCG. For structure and formulation methods, see WO2009 / 054813.

[0085] For example, the MINC-agent is etanercept encapsulated in micelles formed by PEG-EGCG and the flavonoid oligomer OEGCG. For structure and formulation methods, see WO2009 / 054813.

[0086] For example, the MINC-agent is leflunomide encapsulated in micelles formed by PEG-EGCG. For structure and formulation methods, see WO2011 / 112156.

[0087] Pharmaceutical composition

[0088] The present invention uses a pharmaceutical composition comprising a hydrophilic polymer-flavonoid conjugate, flavonoid oligomer or MINC-agent composition as described in the present application, and optionally one or more pharmaceutically acceptable carriers. For tablets, powders or parenteral preparations, the nanoparticle composition in the pharmaceutical composition is generally about 1% to 90%, preferably 20% to 90% or 30% to 80%. For capsule preparations, the PEG-flavonoid conjugate, flavonoid oligomer or MINC-agent composition in the pharmaceutical composition is generally 1% to 100%, preferably 20% to 100%, 50% to 100% or 70% to 100%. For liquid suspension preparations, the nanoparticle composition in the pharmaceutical composition is generally 1% to 50%, 5% to 50% or 10% to 40%.

[0089] In one embodiment, the pharmaceutical composition may be in the form of tablets, capsules, granules, fine granules, powders, suspensions, patches, parenterals, injectables, etc. The pharmaceutical composition may be prepared by conventional methods.

[0090] Pharmaceutically acceptable carriers are inactive ingredients that can be selected by those skilled in the art using conventional criteria. Pharmaceutically acceptable carriers can contain a variety of ingredients, including but not limited to saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents such as sodium chloride, potassium chloride, glycerol and glucose; pH adjusters and buffers such as salts of hydroxides, phosphates, citrates, acetates, borates and triethanolamine; antioxidants such as salts, acids and / or bases of bisulfites, sulfites, metabisulfites, thiosulfites, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol and ascorbyl palmitate; surfactants such as lecithin and phospholipids, including but not limited to phosphatidylcholine, phosphatidylethanolamine and phosphatidylcholine. alcohols; poloxamers and poloxamines; polysorbates such as polysorbate 80, polysorbate 60 and polysorbate 20; polyethers such as polyethylene glycol and polypropylene glycol; polyethylenes such as polyvinyl alcohol and polyvinyl pyrrolidone (PVP, povidone); cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose and hydroxypropylmethylcellulose and their salts; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, soybean oil; mono-, di- and triglycerides; polysaccharides such as dextran; and glycosaminoglycans such as sodium hyaluronate. Such pharmaceutically acceptable carriers may be preserved to prevent bacterial contamination with well-known preservatives including, but not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or may be formulated as non-preserved preparations for single or multiple use.

[0091] For example, the tablet, capsule or parenteral preparation of the active compound may contain other excipients that are not biologically active and do not react with the active compound. The excipients of tablets or capsules may include fillers, binders, lubricants and glidants, disintegrants, wetting agents and release rate regulators. Examples of excipients of tablets or capsules include but are not limited to carboxymethyl cellulose, cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, karaya gum, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide, poly (acrylic acid) and polyvinyl pyrrolidone.

[0092] 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.

[0093] The pharmaceutical composition can be applied by partial administration and systemic administration. Partial administration includes topical administration. Systemic administration includes oral administration, parenteral administration (such as intravenous administration, intramuscular administration, subcutaneous administration or rectal administration) and other systemic administration routes. In systemic administration, the active compound first reaches the plasma and is then distributed to the target tissue. Parenteral administration (such as intravenous bolus injection or intravenous infusion) and oral administration are preferred routes of administration.

[0094] Treatment

[0095] The present invention relates to a method for preventing or treating autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, type 1 diabetes, systemic lupus erythematosus and psoriasis.

[0096] Polymer-Flavonoid

[0097] In a first aspect of the invention, the method comprises the step of administering to a subject in need thereof an effective amount of a polymer-flavonoid conjugate to treat an autoimmune disease. As used herein, "effective amount" is an amount effective to treat a disease by improving pathological conditions or reducing disease symptoms.

[0098] The polymer-flavonoid conjugates of the present invention have immunomodulatory and anti-inflammatory activities for the treatment of autoimmune diseases.

[0099] In one embodiment, the flavonoid is EGCG, EC, EGC or ECG.

[0100] In one embodiment, the polymer is a hydrophilic polymer having a molecular weight of 1,000 to 100,000 Daltons and is selected from the group consisting of PEG, hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-alpha-tocopherol, and polyethylene glycol succinate.

[0101] A preferred polymer-flavonoid conjugate is PEG-EGCG.

[0102] In one embodiment, the autoimmune disease is associated with the joints and muscles and is rheumatoid arthritis, psoriatic arthritis, Sjögren's syndrome, or systemic lupus erythematosus.

[0103] In one embodiment, the autoimmune disease is associated with the digestive tract and is inflammatory bowel disease, celiac disease, or ulcerative colitis.

[0104] In one embodiment, the autoimmune disease is associated with the endocrine system and is type 1 diabetes, Graves' disease, Hashimoto's thyroiditis, or Addison's disease.

[0105] In one embodiment, the autoimmune disease is associated with the skin and is psoriasis, dermatomyositis, or vitiligo.

[0106] In one embodiment, the autoimmune disease is associated with the nervous system and is multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, or Guillain-Barre syndrome.

[0107] In one embodiment, the autoimmune disease is myasthenia gravis, autoimmune vasculitis, pernicious anemia, vasculitis, or myositis.

[0108] The polymer-flavonoid (eg, PEG-EGGC) for injection is typically administered at 0.1 mg / kg to 10000 mg / kg, 0.6 mg / kg to 1200 mg / kg (total weight of PEG-flavonoid / body weight of subject), or 1 mg / kg to 1000 mg / kg.

[0109] Flavonoid oligomers

[0110] In a second aspect of the present invention, the method comprises the step of administering an effective amount of flavonoid oligomers to a subject in need thereof. The flavonoid oligomers of the present invention have immunomodulatory and anti-inflammatory activities useful for treating autoimmune diseases.

[0111] In one embodiment, the flavonoid oligomer is an oligomer of EGCG, EC, EGC or ECG.

[0112] In one embodiment, the flavonoid oligomer comprises 4 to 12 flavonoids of EGCG, EC, EGC or ECG.

[0113] In one embodiment, the autoimmune disease is associated with the joints and muscles and is rheumatoid arthritis, psoriatic arthritis, Sjögren's syndrome, or systemic lupus erythematosus.

[0114] In one embodiment, the autoimmune disease is associated with the digestive tract and is inflammatory bowel disease, celiac disease, or ulcerative colitis.

[0115] In one embodiment, the autoimmune disease is associated with the endocrine system and is type 1 diabetes, Graves' disease, Hashimoto's thyroiditis, or Addison's disease.

[0116] In one embodiment, the autoimmune disease is associated with the skin and is psoriasis, dermatomyositis, or vitiligo.

[0117] In one embodiment, the autoimmune disease is associated with the nervous system and is multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, or Guillain-Barre syndrome.

[0118] In one embodiment, the autoimmune disease is myasthenia gravis, autoimmune vasculitis, pernicious anemia, vasculitis, or myositis.

[0119] The flavonoid oligomer for injection (eg, OEGCG) is generally administered at 0.1 mg / kg to 1000 mg / kg, 0.1 mg / kg to 100 mg / kg (total weight of flavonoid oligomer / body weight of subject), or 1 mg / kg to 100 mg / kg.

[0120] MINC-Drugs

[0121] In a third aspect of the invention, the method includes administering to a subject in need thereof an effective amount of micelles to treat an autoimmune disease, the micelles having an outer shell comprising one or more polymer-flavonoid conjugates and an optional inner shell comprising one or more flavonoid oligomers and a drug encapsulated in these shells. In one embodiment, the micelles comprise both an outer shell and an inner shell. In another embodiment, the micelles do not have an inner shell and have only one shell comprising one or more polymer-flavonoids.

[0122] The polymer-flavonoid conjugate or flavonoid oligomer provides its own therapeutic effect and further delivers the agent for treating autoimmune diseases.

[0123] In one embodiment, the polymer is a hydrophilic polymer having a molecular weight of 1,000 to 100,000 Daltons and is selected from the group consisting of poly(ethylene glycol) (PEG), hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-α-tocopherol, and polyethylene glycol succinate;

[0124] In one embodiment, the flavonoid oligomer comprises 2 to 50 or 2 to 20 flavonoids of EGCG, EC, EGC or ECG;

[0125] In one embodiment, the shell is formed from PEG-EGCG.

[0126] In one embodiment, the shell is formed from PEG-EGCG and OEGCG.

[0127] In one embodiment, the autoimmune disease is type 1 diabetes, and the drug is anti-CD3, anti-IL-1β, anti-IL-1R, GAD, HSP60, B9-23 peptide, MIP, IL-2, anti-CTLA4, anti-CD40, anti-CD20, anti-PD1, TKI, GLP-1, anti-STAT1, anti-S1P, anti-12 / 15-LOX, IL-35, HDAC inhibitor, anti-DYRK1A, NFAT, substance P, LRH-1, IL-10, IGF, IGFBP1, ARX, GCSF, VMAT-2, or RAE-1.

[0128] In one embodiment, the autoimmune disease is inflammatory bowel disease, and the drug is anti-CD3, anti-ITGA4, anti-IL-12B, anti-TNF, anti-JAK2, anti-PTGS1 / 2, PPAR-γ, anti-ITGB7, NR3C1, anti-JAK3, anti-ALOX5, anti-TYK2, anti-PPAT, VDR, anti-MMP1, anti-MMP7, anti-DHFR, anti-MMP13, anti-ATP4A, S1PR1, anti-IL-36, anti-IL-6, anti-IL-6R, anti-TLR9, IL-10, anti-TGF-β, anti-IL-9, anti-IL-12, anti-smad7, anti-integrin, anti-MDR1, anti-PPAR-γ, anti-IL-35, anti-IL-27, or anti-IL17.

[0129] In one embodiment, the autoimmune disease is rheumatoid arthritis and the drug is anti-TNF-α, anti-IL-1R, anti-IL-1, anti-IL-6R, anti-IL-6, anti-IL-2, IL-10, IL-15, IL-18, anti-IL-17, anti-IL-17R, anti-IFN-γ, anti-CCL2, anti-CCR9, anti-CX3CL1, anti-CCR1, anti-CCR2, anti-CCR5, anti-TLR4, anti-GRK2, anti-MEK, anti-MMP9, anti-CD3, anti-CD80, anti-BTK, anti-IL-23, anti-GM-CSF, anti-CXCL10, anti-CXCL12, anti-CXCL13, anti-CXCL16, anti-CXCR1 / 2, anti-CXCL3, anti-CXCR4, anti-CXCR7, anti-CCR7, anti-JAK, anti-p38 MAPK, anti-IRAK-4, anti-CD20, anti-CD11 a, or anti-CD19.

[0130] In one embodiment, the autoimmune disease is multiple sclerosis and the drug is a ROS scavenger, anti-NF-κB, anti-NOX2, anti-iNOS, MDA, HNE, 4-HHR inhibitor, IFN-β, NRF2 activator, PGC-1α, anti-IL-1β, anti-TNF-α, anti-IL-6, anti-IL-6R, anti-MMP-9, anti-IL-12, anti-IFN-γ, CNTF, anti-caspase 3, anti-MIP-1α, anti-TLR2, anti-CD3, anti-ICAM-1, anti-CCR6, anti-IL2, anti-IL-9, anti-17, TGF-β, IL-4 or FoxP3 activator.

[0131] In one embodiment, the autoimmune disease is systemic lupus erythematosus and the drug is anti-CD20, anti-CD19, anti-CD22, a proteasome inhibitor, anti-BAFF, anti-BTK, anti-CD28, anti-CD40L, anti-CD40, anti-IL12, anti-IL23, anti-IL17, IL2, an mTOR inhibitor, a calcineurin inhibitor, anti-JAK, anti-IFN-α, anti-IFNAR, anti-IFN-γ, anti-TLR7, anti-TLR9, anti-TLR8, anti-CTLA4, anti-IL6, or anti-CD3.

[0132] In one embodiment, the autoimmune disease is psoriasis and the drug is anti-CD3, anti-TNF, anti-IL-17A, anti-IL-12, anti-IL-23, anti-36, anti-PDE4, anti-JAK, anti-RORgT, anti-IRAK-4, anti-CCL20, anti-CXCL8, anti-IL-1β, anti-iNOS, anti-IL-8, anti-IFNγ, anti-S1PR1, anti-Tyk2, anti-A3 adenosine receptor, or an mTOR inhibitor.

[0133] In one embodiment, the autoimmune disease is myasthenia gravis and the drug is anti-CD3, anti-FcRN, anti-CD20, anti-CD19, anti-CD154, a proteasome inhibitor, anti-CD38, anti-CD40, anti-IL6, anti-TNF, anti-BARF, anti-BTK, anti-IL-6R, anti-IL-17A, anti-C5, or anti-AChR.

[0134] In one embodiment, the autoimmune disease is Hashimoto's thyroiditis and the drug is anti-CD3, anti-TSHR, anti-TPO, anti-Tg, anti-NIS, anti-IL-12, anti-IFN-γ, anti-IL-4, anti-IL-5, anti-IL-10, anti-IL-17, anti-IL-6, anti-FLT3L, anti-LypR620W, anti-ICAM1, anti-ILv7, anti-IL-23, anti-RHOH, anti-RNASET2, anti-SLAMF6, anti-TNF-α, or anti-FcRN.

[0135] The dosing of the MINC-agent is based on the known doses of the agent used to treat the specific disease and the condition of the subject. The dose can be a dose approved by the Food and Drug Administration (FDA) or a dose used in a clinical trial.

[0136] In the MINC-medicament, typically the dose of PEG-EGCG combined with OEGCG is between 10 μg / kg and 100 mg / kg.

[0137] The concentration of encapsulated drug agent can be as low as 1 μg / kg (eg, for cytokine drugs, G-CSF) and as high as 10 mg / kg (for antibody drugs, eg, certolizumab is at this level).

[0138] For example, when the MINC-agent comprises an antibody for treating rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus and psoriasis (including but not limited to anti-IL-1, anti-IL-1α, anti-IL-1β, anti-IL-1R, anti-IL-2, anti-IL-6, anti-IL-6R, anti-IL-7, anti-IL-7R, anti-IL-10, anti-IL-11, anti-IL-12, anti-IL-17, anti-IL-18, anti-IL-23, anti-IFN-α, anti-IFN-β, anti-IFN-γ, anti-TNF-α, anti-CD20, anti-TGF-β, anti-T lymphocyte globulin (ATG)), the antibody can be administered IV once every one to four weeks in the range of 0.1 mg / kg to 10 mg / kg.

[0139] For example, to treat rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, psoriasis or other autoimmune diseases in adults, 0.1 mg / kg to 10 mg / kg of anti-TNF-α is administered IV once every one to four weeks. MINC-anti-TNF-α containing the same dosage range of anti-TNF-α can be used to treat these diseases.

[0140] For example, to treat rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, psoriasis or other autoimmune diseases in adults, 0.1 mg / kg to 10 mg / kg of etanercept is administered IV once every one to four weeks. MINC-Etanercept contains etanercept in the same dosage range that can be used to treat these diseases.

[0141] For example, to treat rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, and psoriasis in adults, 0.5 mg / kg to 50 mg / kg of methylprednisolone is administered IV once every one to four weeks. MINC-Methylprednisolone, including methylprednisolone in the same dosage range, can be used to treat these diseases.

[0142] For example, when the MINC-agent comprises an anti-inflammatory compound used to treat rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, and psoriasis (including but not limited to leflunomide, aspirin, naproxen, diclofenac, ibuprofen, methotrexate, sulfasalazine, hydroxychloroquine, sulfasalazine, baricitinib, tofacitinib, azathioprine, cyclosporine, minocycline, ozanimod, mesalazine, olsalazine, balsalazide, prednisone, hydrocortisone, budesonide, 6-mercaptopurine, metronidazole, methylprednisolone, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, laquinimod, mitoxantrone, dalfampridine, metformin, tolbutamide, chlorpropamide, When the anti-inflammatory compound is administered IV in the range of 0.1 mg / kg to 10 mg / kg once every one to four weeks, the anti-inflammatory compound may be administered IV in the range of 0.1 mg / kg to 10 mg / kg when the anti-inflammatory compound is administered IV in the range of 0.1 mg / kg to 10 mg / kg once every one to four weeks.

[0143] For example, when the MINC-agent comprises a nonsteroidal anti-inflammatory drug (NSAID) used to treat rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, and psoriasis (including but not limited to ibuprofen, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin, or aspirin), the anti-inflammatory drug can be administered IV once to twice daily in the range of 0.1 mg / kg to 10 mg / kg.

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

[0145] The following examples further illustrate the present invention. These examples are intended only to illustrate the present invention and should not be construed as limiting.

[0146] Example

[0147] Active ingredients

[0148] OEGCG :

[0149] OEGCG is oligomerized EGCG. OEGCG was prepared according to WO2006 / 124000.

[0150] PEG-EGCG :

[0151] PEG-EGCG is PEG conjugated to one or two EGCGs. PEG-EGCG is prepared according to WO2006 / 124000, WO2009 / 054813 or WO2015 / 171079.

[0152] MINC-Pharmacy :

[0153] According to the methods in WO2006 / 124000 or WO2009 / 054813, the MINC-agents were prepared by encapsulating the agent in micelles formed by PEG-EGCG and OEGCG.

[0154] Example 1: OEGCG and PEG-EGCG inhibit LPS-induced inflammatory response in synovial fibroblasts of rheumatoid arthritis Cytokine TNF-α production

[0155] Material

[0156] TNF-α ELISA Kit, Human (Sino biologicalal, catalog: KIT10602)

[0157] Rheumatoid arthritis synovial fibroblasts (RASF) (from RIKEN)

[0158] method

[0159] Rheumatoid arthritis synovial fibroblasts (RASF) were cultured at 1×10 4 Cells / well were seeded in 96-well plates and incubated overnight. On the second day, cells were pretreated with different concentrations of OEGCG or PEG-EGCG for 20 minutes and then incubated with 1 μg / mL LPS for 48 hours. At the end of the treatment, cell supernatants were collected and human TNF-α ELISA kits were used according to the manufacturer's instructions.

[0160] result

[0161] RASF cells were stimulated with LPS and the concentration of TNF-α was increased. Treatment with OEGCG or PEG-EGCG significantly inhibited TNF-α production in a concentration-dependent manner ( Figure 3 RASF cells can secrete inflammatory cytokines, including TNF-α, which promotes immune cell activation and joint and bone destruction. Reduction of TNF-α by OEGCG and PEG-EGCG treatment has therapeutic benefits in the treatment of rheumatoid arthritis.

[0162] Example 2: OEGCG and PEG-EGCG reduce inflammation in a cellular model of inflammatory bowel disease

[0163] Material

[0164] TRIzol-A+ reagent (Tiagen)

[0165] Caco-2 from ATCC (HTB-37)

[0166] method

[0167] Caco-2 cells were cultured in growth medium at 37°C in a humidified chamber with 5% CO2. Caco-2 cells were plated at 2×10 6 Cells / well were seeded in 6-well plates overnight and pretreated with OEGCG (3.3 μM) or PEG-EGCG (10 μM) for 24 h, and then cultured for another 8 h in the presence or absence of LPS (20 μg / mL). After treatment, total RNA was extracted from Caco-2 cells using TRIzol-A+ reagent according to the manufacturer's instructions. The extracted RNA was reverse transcribed into cDNA according to the manufacturer's protocol (Tiangen, Beijing, China). The cDNA was analyzed by QuantStudio TM qRT-PCR was performed using the 6Fle Real-Time PCR System (Life Technologies, USA), with initial denaturation conditions set at 95°C for 3 minutes, followed by 35 to 50 cycles of denaturation at 95°C for 10 seconds, annealing at 56°C for 10 seconds, and extension at 72°C for 60 seconds. Cycle threshold (Ct) values ​​were calculated and normalized to the level of the housekeeping gene GAPDH. Forward and reverse primers were used as follows: Wu et al. (Inflammation. 2019 Dec; 42 (6) -2215, 2225).

[0168] result

[0169] The mRNA levels of IL-6 and COX2 in Caco-2 cells were analyzed by qRT-PCR assay. Compared with the untreated group, the treatment of OEGCG and PEG-EGCG decreased the mRNA expression of IL-6 and COX2 ( Figure 4 These results suggest that OEGCG and PEG-EGCG treatment can slow down disease progression by inhibiting the production of inflammatory cytokines.

[0170] Example 3: MINC-anti-CD3 reduces the toxicity of anti-CD3

[0171] Material

[0172] Anti-CD3 antibody was purchased from Biolegend (Catalog #317325).

[0173] MINC-anti-CD3 was prepared according to WO2009 / 054813.

[0174] method

[0175] Six to eight week old Balb / c mice were maintained in a specific pathogen-free environment. After 3 days of acclimation, treatment was initiated on the day after baseline calculation, designated as day 0. Anti-CD3 or MINC-anti-CD3 was administered to mice at doses of 5 μg / mouse, 25 μg / mouse, and 125 μg / mouse. Daily iv for 2 days. Mice were randomly assigned to one of three treatment groups: 1) saline; 2) anti-CD3; 3) MINC-anti-CD3, n=5. Body weight, survival, and clinical signs were measured every other day for eight consecutive days. Statistical analysis was performed using GraphPad Prizm. Two-way ANOVA was used to assess differences between groups, with p<0.05 considered significant.

[0176] result

[0177] Free anti-CD3 antibody significantly reduced the weight of mice from day 2 to day 5 after two injections of treatment ( Figure 5 A). Four of the five high-dose group and three of the five medium-dose group died between days 5 and 7 ( Figure 5 B). 125 μg / mouse of MINC-anti-CD3 antibody showed moderate weight loss on day 5 and recovered on day 7 ( Figure 5 A). No animals died in the MINC-anti-CD3 group ( Figure 5 B). The results showed that MINC encapsulation significantly reduced toxicity and tolerated doses up to 25 times the clinical dose (equivalent to 5 μg / mouse).

[0178] Anti-CD3 is a broad-spectrum immunosuppressant used to treat various autoimmune diseases. However, its high toxicity limits its clinical application. This example shows that the MINC formulation successfully reduces the toxicity of anti-CD3. MINC-anti-CD3 is a safe drug for the treatment of autoimmune diseases.

[0179] Different flavonoid oligomers and polymer-flavonoid conjugates in the formation of MINC-drugs (Examples 4 to 5)

[0180] Example 4: Preparation of different flavonoids for use in flavonoid oligomers and polymer-flavonoid conjugates MINC-Anti-HER2 Approach .

[0181] Material

[0182] Anti-HER2 was trastuzumab from Eirgenix.

[0183] method

[0184] MINC anti-HER2 nanoparticles were prepared according to WO2009 / 054813. Briefly, anti-HER2 was incubated in PBS. Subsequently, different flavonoid oligomers including OEGCG or OECG were added to anti-HER2, followed by different polymer-flavonoids including PEG-EGCG, PEG-ECG or PEG-EC. After incubation of the mixture at room temperature, unreacted oligomeric flavonoids and polymer-flavonoids were removed using a 10K MWCO centrifugal filter. Nanoparticle size was measured using DLS (Anton Paar Litesizer 500), and the results are shown in Table 1. Figure 6 shown.

[0185] result

[0186] Figure 6 It was shown that different polymer-flavonoid conjugates and different flavonoid oligomers successfully generated MINC-anti-HER2 micelles with a particle size of about 100 nm. The results showed that homogeneous nanoparticles (micelles) with expected sizes (about 5 nm to 10 nm) different from those of unencapsulated anti-HER antibodies were successfully formed.

[0187] In this example, different flavonoid oligomers used included OEGCG ( Figure 6 A, 6C and 6D) and OECG ( Figure 6 B); and different polymer-flavonoids used including PEG-EGCG ( Figure 6 A and 6B), PEG-EC ( Figure 6 C) and PEG-ECG ( Figure 6 D).

[0188] These data support that MINC nanoparticles can be formed from different flavonoid oligomers and different polymer-flavonoid conjugates.

[0189] Example 5: Method for preparing MINC-BSA using different polymers in polymer-flavonoid conjugates Law .

[0190] Material

[0191] BSA was purchased from Sigma-Aldrich.

[0192] method

[0193] MINC (multi-target immune nanocarrier combination)-BSA nanoparticles were prepared according to WO2009 / 054813. In brief, BSA was incubated in PBS. Subsequently, OEGCG was added to BSA, followed by different polymer-flavonoids, including 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. Nanoparticle size was measured using DLS (Anton Paar Litesizer 500).

[0194] result

[0195] Figure 7 Different polymers in the polymer-flavonoid conjugates were used to successfully generate MINC-BSA. The results showed that homogeneous nanoparticles (micelles) were successfully formed. These different polymers were PEG ( Figure 7 A) HA Figure 7 B) and dextran ( Figure 7 C). In summary, the data support that MINC nanoparticles can be formed from different polymer-flavonoid conjugates.

[0196] Example 6: MINC-anti-CD3 study in type 1 diabetes (T1D) (hypothetical example)

[0197] Purpose

[0198] This experiment aims to show that MINC-anti-CD3 treatment can treat T1D by improving blood glucose, insulin sensitivity, HbA1c levels, and C-peptide levels compared with the saline group. This experiment aims to show that MINC-anti-CD3 is effective in the treatment of type 1 diabetes.

[0199] Material

[0200] Anti-CD3 antibody was purchased from Biolegend (Catalog #317325).

[0201] MINC-anti-CD3 was prepared according to WO2009 / 054813.

[0202] method

[0203] Diabetic female NOD mice (NOD / ShiLtJ strain) aged 8 to 15 weeks were monitored for diabetic status twice weekly. Diabetic status was defined as blood glucose ≥ 200 mg / dL by tail bleeding on 2 consecutive days. Mice were assigned to groups including a control group and treatment groups consisting of different doses of MINC-anti-CD3 or free anti-CD3. Treatment groups were treated with MINC-anti-CD3 or anti-CD3 via iv injection at 1 μg, 5 μg and 25 μg of anti-CD3 equivalents for 5 consecutive days. Weight loss, survival, fasting blood glucose and C-peptide were measured once a week. HbA1c was measured on days 14, 28 and 42 after the first injection. The values ​​of the MINC-anti-CD3 treated group and the anti-CD3 treated group were compared by one-way ANOVA (α=0.05).

[0204] Example 7. MINC-anti-CD3 in the treatment of rheumatoid arthritis (RA) (hypothetical example)

[0205] Material

[0206] Rat anti-CD3 antibody was purchased from Biolegend.

[0207] The MINC-anti-CD3 antibody was prepared according to WO2009 / 054813.

[0208] Purpose

[0209] This experiment aims to demonstrate that MINC-anti-CD3 treatment reduces joint swelling and maintains bone integrity compared to the saline group. Histopathological examination aims to show that MINC-anti-CD3 is better at reducing immune cell infiltration, inflammatory cytokine production, and better outcomes compared to saline alone. This experiment aims to show that MINC-anti-CD3 is effective in treating rheumatoid arthritis.

[0210] method

[0211] Eighty 10-week-old male Wistar rats were purchased from BioLasco. The rats were maintained in a facility free of specific pathogens. In brief, on day 0, 0.2 mL of emulsified bovine type II collagen was subcutaneously injected with Freund's adjuvant into the base of the tail, followed by a booster injection on day 7. On the same day, rats received saline or 0.25 mg / kg to 6.25 mg / kg of MINC-anti-CD3 via intravenous injection once a week. After administration, the bone performance of the rats was examined by micro-CT, and joint swelling was examined by measuring the diameter at the ankle and hind paw. At the end point, the rats were killed on day 28 for histopathological examination. Rat ankle joint samples were fixed with 10% formalin. After decalcification in hydrochloric acid, the samples were embedded and fixed in paraffin. The sections were stained with hematoxylin and eosin (H&E) to examine joint morphology. TNF-α expression levels and inflammatory cell infiltration levels were detected by anti-TNF-α and anti-CD38 immunohistochemical staining.

[0212] Example 8. MINC-anti-TNF-α in the treatment of rheumatoid arthritis (RA) (hypothetical example)

[0213] Purpose

[0214] This experiment aims to demonstrate that MINC-anti-TNF-α treatment reduces joint swelling and maintains bone integrity compared to the saline group. Histopathological examination aims to show that MINC-anti-TNF-α treatment can reduce immune cell infiltration, reduce TNF-α levels and have better results than saline controls. This experiment aims to show that MINC-anti-TNF-α is effective in the treatment of rheumatoid arthritis.

[0215] Material

[0216] Rat anti-TNF-α antibody was purchased from Biolegend.

[0217] The MINC-anti-TNF-α antibody was prepared according to WO2009 / 054813.

[0218] method

[0219] Eighty 10-week-old male Wistar rats were purchased from BioLasco. The rats were maintained in a specific pathogen-free facility. In brief, on day 0, 0.2 mL of emulsified bovine type II collagen was subcutaneously injected with Freund's adjuvant into the base of the tail, followed by a booster injection on day 7. On the same day, rats received two injections of 2 mg / kg to 20 mg / kg of MINC-anti-TNF-α via intravenous injection once a week. After administration, the bone performance of the rats was examined by micro-CT, and joint swelling was examined by measuring the diameter at the ankle and hind paw. At the end point, the rats were killed on day 28 for histopathological examination. Rat ankle joint samples were fixed with 10% formalin. After decalcification in hydrochloric acid, the samples were embedded in paraffin. The sections were stained with hematoxylin and eosin (H&E) to examine joint morphology. TNF-α expression levels and inflammatory cell infiltration levels were detected by anti-TNF-α and anti-CD38 immunohistochemical staining.

[0220] Example 9. MINC-anti-CD3 in the treatment of inflammatory bowel disease (IBD) (hypothetical example)

[0221] Purpose

[0222] This experiment was designed to show that the DAI scores of rats in the MINC-anti-CD3 treated group were significantly lower than those of rats in the saline group. Histopathological examination was designed to show that ulcers, edema, and inflammatory cell infiltration with mucosal congestion were reduced in the MINC-anti-CD3 treated group. Overall, this experiment was designed to show that MINC-anti-CD3 has therapeutic efficacy in inflammatory bowel disease.

[0223] Material

[0224] Rat anti-CD3 antibody was purchased from Biolegend.

[0225] The MINC-anti-CD3 antibody was prepared according to WO2009 / 054813.

[0226] method

[0227] Rats were fasted for 48 hours and anesthetized with 3% sodium pentobarbital. In brief, on the first day, an lavage needle was inserted into the anus and the needle was advanced to 8 cm from the anal verge. Then, 1.5 ml to 2.0 ml of 8% acetic acid was injected into the colon, and an additional 3 ml to 5 ml of saline was added to diffuse the acetic acid in the colon. After 24 hours, the rats were grouped and treated with saline or 0.25 mg / kg to 6.25 mg / kg of MINC-anti-CD3 via iv injection on the 2nd and 5th days. On the 8th day, the rats were killed and the tissues of the colon and adjacent ulcers or congested areas were collected. The fixed and paraffin-embedded tissues were stained with hematoxylin and eosin for subsequent histological examination. The disease activity index (DAI) was scored according to the following criteria. No weight loss is scored as 0 points, 1% to 5% weight loss is scored as 1 point, 5% to 10% weight loss is scored as 2 points, 10% to 15% weight loss is scored as 3 points, and more than 15% weight loss is scored as 4 points.

[0228] Example 10 MINC-anti-TNF-α in the treatment of inflammatory bowel disease (IBD) (hypothetical example)

[0229] Purpose

[0230] This experiment was designed to show that the DAI scores of rats in the MINC-anti-TNF-α treated group were significantly lower than those of rats in the saline group. Histopathological examination was designed to show that ulcers, edema, and inflammatory cell infiltration with mucosal congestion were reduced in the MINC-anti-TNF-α treated group. Overall, this experiment was designed to show that MINC-anti-TNF-α has therapeutic efficacy in inflammatory bowel disease.

[0231] Material

[0232] Rat anti-TNF-α antibody was purchased from Biolegend.

[0233] The MINC-anti-TNF-α antibody was prepared according to WO2009 / 054813.

[0234] method

[0235] Rats were fasted for 48 hours and anesthetized with 3% sodium pentobarbital. Briefly, an lavage needle was inserted into the anus and the needle was advanced to 8 cm from the anal verge. Next, 1.5 ml to 2.0 ml of 8% acetic acid was injected into the colon, and an additional 3 ml to 5 ml of saline was added to diffuse the acetic acid in the colon. After 24 hours, the rats were grouped and treated with saline or 0.5 mg / kg to 20 mg / kg of MINC-anti-TNF-α via iv injection on the 2nd and 5th days. On the 8th day, the rats were sacrificed and the tissues of the colon and adjacent ulcers or congested areas were collected. The fixed and paraffin-embedded tissues were stained with hematoxylin and eosin for subsequent histological examination. The disease activity index (DAI) was based on the following criteria. No weight loss was scored as 0 points, 1% to 5% weight loss was scored as 1 point, 5% to 10% weight loss was scored as 2 points, 10% to 15% weight loss was scored as 3 points, and more than 15% weight loss was scored as 4 points.

[0236] List of abbreviations

[0237] 4-HHR 4-Hydroxyhexenal reductase

[0238] AChR Acetylcholine Receptor

[0239] ALOX5 Arachidonic acid 5-lipoxygenase

[0240] ARX ​​Aristaless-related homeobox

[0241] ATP4A ATPase H+ / K+ transporter α subunit

[0242] BAFF B cell activating factor

[0243] BTK Bruton's tyrosine kinase

[0244] C5 complement component 5

[0245] CCL2 CC motif chemokine ligand 2

[0246] CCR CC chemokine receptor type

[0247] CNTF Ciliary neurotrophic factor

[0248] CTLA4 Cytotoxic T lymphocyte-associated protein 4

[0249] CX3CL1 CX3C chemokine ligand 1

[0250] CXCL10 CXC motif chemokine ligand 10

[0251] CXCR1 / 2 CXC chemokine receptor type 1 / 2

[0252] DHFR dihydrofolate reductase

[0253] DYRK1A Dual specificity tyrosine phosphorylation regulated kinase 1A

[0254] FcRN neonatal Fc receptor

[0255] FLT3L FMS-like tyrosine kinase 3 ligand

[0256] FoxP3 Forkhead box protein P3

[0257] GAD Glutamate decarboxylase

[0258] GCSF Granulocyte colony stimulating factor

[0259] GLP-1 Glucagon-like peptide 1

[0260] GM-CSF Granulocyte-macrophage colony-stimulating factor

[0261] GRK2 G protein-coupled receptor kinase 2

[0262] HDAC Histone deacetylase

[0263] HNE 4-Hydroxynonenal

[0264] HSP60 Heat shock protein 60

[0265] ICAM-1 intercellular adhesion molecule 1

[0266] IFNAR interferon alpha / beta receptor

[0267] IGF Insulin-like growth factor

[0268] IGFBP1 Insulin-like growth factor binding protein 1

[0269] IL- interleukin-

[0270] iNOS Inducible nitric oxide synthase

[0271] IRAK-4 Interleukin-1 receptor-associated kinase 4

[0272] ITGA4 Integrin α4

[0273] ITGB7 Integrin β7

[0274] JAK2 Janus kinase 2

[0275] JAK3 Janus kinase 3

[0276] LRH-1 Liver receptor homolog-1

[0277] LypR620W Lymphatic protein tyrosine phosphatase R620W

[0278] MDA Malondialdehyde

[0279] MDR1 Multidrug resistance protein 1

[0280] MEK Mitogen-activated protein kinase kinase

[0281] MIP macrophage inflammatory protein

[0282] MMP1 matrix metalloproteinase 1

[0283] NFAT Nuclear factor of activated T cells

[0284] NF-κB Nuclear factor κB

[0285] NIS Sodium / iodide symporter

[0286] NOX2 NADPH oxidase 2

[0287] NR3C1 Nuclear receptor subfamily 3C member 1 (also known as glucocorticoid receptor)

[0288] NRF2 Nuclear factor erythroid 2-related factor 2

[0289] p38 MAPK p38 mitogen-activated protein kinase

[0290] PD1 Programmed cell death protein 1

[0291] PDE4 Phosphodiesterase 4

[0292] PGC-1α Peroxisome proliferator-activated receptor gamma coactivator 1-alpha

[0293] PPAR-γ Peroxisome proliferator-activated receptor gamma

[0294] PPAT phosphoribosylpyrophosphate amidotransferase

[0295] PTGS1 / 2 Prostaglandin-endoperoxide synthase 1 / 2 (also known as COX-1 / 2)

[0296] RAE-1 retinoic acid early transcript 1

[0297] RHOH Ras homolog family member H

[0298] RNASET2 Ribonuclease T2

[0299] RORgT Retinoic acid receptor-related orphan receptor gamma T

[0300] S1P Sphingosine-1-phosphate

[0301] S1PR1 Sphingosine-1-phosphate receptor 1

[0302] SLAMF6 signaling lymphocyte activation molecule family member 6

[0303] STAT1 signal transducer and activator of transcription 1

[0304] TGF-β Transforming Growth Factor Beta

[0305] TKI Tyrosine kinase inhibitor

[0306] TLR9 Toll-like receptor 9

[0307] TNF Tumor necrosis factor

[0308] TPO Thrombopoietin

[0309] TSHR thyroid stimulating hormone receptor

[0310] TYK2 Tyrosine kinase 2

[0311] VDR Vitamin D receptor

[0312] VMAT-2 Vesicular monoamine transporter 2

[0313] The invention and the manner and process of making and using the same are now described in full, clear, concise and precise terms to enable any person skilled in the art to make and use the invention. It should be understood that the foregoing describes preferred embodiments of the invention and that modifications may be made therein without departing from the scope of the invention as set forth in the claims. In order to particularly point out and distinctly claim the subject matter regarded as inventive, the specification ends with the following claims.

Claims

1. A method for treating an autoimmune disease, the method comprising the step of administering to a subject in need thereof an effective amount of a micelle having an outer shell comprising one or more polymer-flavonoid conjugates, an optional inner shell comprising one or more flavonoid oligomers, and a drug encapsulated within the shell; wherein the polymer is a hydrophilic polymer having a molecular weight of 1,000 to 100,000 Daltons and is selected from the group consisting of poly(ethylene glycol) (PEG), hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-α-tocopherol and polyethylene glycol succinate; The flavonoid is EGCG, EC, EGC or ECG, as shown in the following structure: The flavonoid oligomers contain 2 to 20 flavonoids of EGCG, EC, EGC or ECG; The autoimmune disease is selected from the group consisting of type 1 diabetes, inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, psoriasis, myasthenia gravis and Hashimoto's thyroiditis.

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

3. The method of claim 1 or 2, wherein the autoimmune disease is type 1 diabetes, and the drug is anti-CD3, anti-IL-1β, anti-IL-1R, GAD, HSP60, B9-23 peptide, MIP, IL-2, anti-CTLA4, anti-CD40, anti-CD20, anti-PD1, TKI, GLP-1, anti-STAT1, anti-S1P, anti-12 / 15-LOX, IL-35, HDAC inhibitor, anti-DYRK1A, NFAT, substance P, LRH-1, IL-10, IGF, IGFBP1, ARX, GCSF, VMAT-2 or RAE-1.

4. The method of claim 1 or 2, wherein the autoimmune disease is inflammatory bowel disease, and the drug is anti-CD3, anti-ITGA4, anti-IL-12B, anti-TNF, anti-JAK2, anti-PTGS1 / 2, PPAR-γ, anti-ITGB7, NR3C1, anti-JAK3, anti-ALOX5, anti-TYK2, anti-PPAT, VDR, anti-MMP1, anti-MMP7, anti-DHFR, anti-MMP13, anti-ATP4A, S1PR1, anti-IL-36, anti-IL-6, anti-IL-6R, anti-TLR9, IL-10, anti-TGF-β, anti-IL-9, anti-IL-12, anti-smad7, anti-integrin, anti-MDR1, anti-PPAR-γ, anti-IL-35, anti-IL-27 or anti-IL17.

5. The method of claim 1 or 2, wherein the autoimmune disease is rheumatoid arthritis, and the drug is anti-TNF-α, anti-IL-1R, anti-IL-1, anti-IL-6R, anti-IL-6, anti-IL-2, IL-10, IL-15, IL-18, anti-IL-17, anti-IL-17R, anti-IFN-γ, anti-CCL2, anti-CCR9, anti-CX3CL1, anti-CCR1, anti-CCR2, anti-CCR5, anti- TLR4, anti-GRK2, anti-MEK, anti-MMP9, anti-CD3, anti-CD80, anti-BTK, anti-IL-23, anti-GM-CSF, anti-CXCL10, anti-CXCLl2, anti-CXCL13, anti-CXCL16, anti-CXCR1 / 2, anti-CXCL3, anti-CXCR4, anti-CXCR7, anti-CCR7, anti-JAK, anti-p38MAPK, anti-IRAK-4, anti-CD20, anti-CD11a or anti-CD19.

6. The method of claim 1 or 2, wherein the autoimmune disease is multiple sclerosis, and the drug is a ROS scavenger, anti-NF-κB, anti-NOX2, anti-iNOS, MDA, HNE, 4-HHR inhibitor, IFN-β, NRF2 activator, PGC-1α, anti-IL-1β, anti-TNF-α, anti-IL-6, anti-IL-6R, anti-MMP-9, anti-IL-12, anti-IFN-γ, CNTF, anti-caspase 3, anti-MIP-1α, anti-TLR2, anti-CD3, anti-ICAM-1, anti-CCR6, anti-IL2, anti-IL-9, anti-17, TGF-β, IL-4 or FoxP3 activator.

7. The method of claim 1 or 2, wherein the autoimmune disease is systemic lupus erythematosus, and the drug is anti-CD20, anti-CD19, anti-CD22, a proteasome inhibitor, anti-BAFF, anti-BTK, anti-CD28, anti-CD40L, anti-CD40, anti-IL12, anti-IL23, anti-IL17, IL2, an mTOR inhibitor, a calcineurin inhibitor, anti-JAK, anti-IFN-α, anti-IFNAR, anti-IFN-γ, anti-TLR7, anti-TLR9, anti-TLR8, anti-CTLA4, anti-IL6, or anti-CD3.

8. The method of claim 1 or 2, wherein the autoimmune disease is psoriasis, and the drug is anti-CD3, anti-TNF, anti-IL-17A, anti-IL-12, anti-IL-23, anti-IL-36, anti-PDE4, anti-JAK, anti-RORgT, anti-IRAK-4, anti-CCL20, anti-CXCL8, anti-IL-1β, anti-iNOS, anti-IL-8, anti-IFNγ, anti-S1PR1, anti-Tyk2, anti-A3 adenosine receptor or mTOR inhibitor.

9. The method of claim 1 or 2, wherein the autoimmune disease is myasthenia gravis and the drug is anti-CD3, anti-FcRN, anti-CD20, anti-CD19, anti-CD154, a proteasome inhibitor, anti-CD38, anti-CD40, anti-IL6, anti-TNF, anti-BARF, anti-BTK, anti-IL-6R, anti-IL-17A, anti-C5 or anti-AChR.

10. The method of claim 1 or 2, wherein the autoimmune disease is Hashimoto's thyroiditis, and the drug is anti-CD3, anti-TSHR, anti-TPO, anti-Tg, anti-NIS, anti-IL-12, anti-IFN-γ, anti-IL-4, anti-IL-5, anti-IL-10, anti-IL-17, anti-IL-6, anti-FLT3L, anti-LypR620W, anti-ICAM1, anti-IL17, anti-IL-23, anti-RHOH, anti-RNASET2, anti-SLAMF6, anti-TNF-α, or anti-FcRN.

11. A method of treating an autoimmune disease, the method comprising the step of administering to a subject in need thereof an effective amount of one or more polymer-flavonoid conjugates, wherein the polymer is a hydrophilic polymer having a molecular weight of 1,000 to 100,000 Daltons and is selected from the group consisting of PEG, hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-α-tocopherol and polyethylene glycol succinate; and the flavonoid is EGCG, EC, EGC or ECG, as shown in the following structure: and The autoimmune disease is selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, Sjögren's syndrome, systemic lupus erythematosus, inflammatory bowel disease, celiac disease, ulcerative colitis, type 1 diabetes, Graves' disease, Hashimoto's thyroiditis, Addison's disease, psoriasis, dermatomyositis, vitiligo, multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, myasthenia gravis, autoimmune vasculitis, pernicious anemia, vasculitis and myositis.

12. The method of claim 11, wherein the polymer-flavonoid conjugate is PEG-EGCG.

13. A method for treating an autoimmune disease, the method comprising the step of administering an effective amount of one or more flavonoid oligomers to a subject in need thereof, Wherein the flavonoid is EGCG, EC, EGC or ECG, as shown in the following structure: The flavonoid oligomers contain 4 to 12 flavonoids of EGCG, EC, EGC or ECG; and The autoimmune disease is selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, Sjögren's syndrome, systemic lupus erythematosus, inflammatory bowel disease, celiac disease, ulcerative colitis, type 1 diabetes, Graves' disease, Hashimoto's thyroiditis, Addison's disease, psoriasis, dermatomyositis, vitiligo, multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, myasthenia gravis, autoimmune vasculitis, pernicious anemia, vasculitis and myositis. The method according to claim 13 , wherein the flavonoid oligomer is an oligomer of EGCG.

15. The method according to any one of claims 11 to 14, wherein the autoimmune disease is associated with joints and muscles and is selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, Sjögren's syndrome and systemic lupus erythematosus.

16. The method according to any one of claims 11 to 14, wherein the autoimmune disease is associated with the digestive tract and is selected from the group consisting of: inflammatory bowel disease, celiac disease and ulcerative colitis.

17. The method according to any one of claims 11 to 14, wherein the autoimmune disease is associated with the endocrine system and is selected from the group consisting of: type 1 diabetes, Graves' disease, Hashimoto's thyroiditis and Addison's disease.

18. The method according to any one of claims 11 to 14, wherein the autoimmune disease is associated with the skin and is selected from the group consisting of: psoriasis, dermatomyositis and vitiligo.

19. The method according to any one of claims 11 to 14, wherein the autoimmune disease is associated with the nervous system and is selected from the group consisting of: multiple sclerosis, chronic inflammatory demyelinating polyneuropathy and Guillain-Barre syndrome.

20. The method of any one of claims 11 to 14, wherein the autoimmune disease is myasthenia gravis, autoimmune vasculitis, pernicious anemia, vasculitis, or myositis.

Images