Methods for Treating Autoimmune Diseases

Abstract

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

[0002] Autoimmune diseases are conditions that result from an abnormal immune response against a functioning 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, Sjogren's syndrome, myositis, chronic inflammatory demyelinating polyneuropathy (CIDP), dermatomyositis, Guillain-Barre syndrome, psoriatic arthritis, ulcerative colitis, and vasculitis.

[0003] Rheumatoid arthritis Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects the joints. Typically, RA causes joints to feel hot, swollen, and painful. The cause of RA is unclear, but it is thought to involve a combination of genetic and environmental factors. The underlying mechanism is that the body's immune system attacks the joints. This causes inflammation and thickening of the joint capsule.

[0004] inflammatory bowel disease 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 major types. Crohn's disease affects the small and large intestine as well as the mouth, esophagus, stomach, and anus, while ulcerative colitis affects primarily the colon and rectum. Although Crohn's disease and ulcerative colitis (UC) are entirely different diseases, both may present with any of the following symptoms: abdominal pain, diarrhea, rectal bleeding, severe internal cramps / cramps in the pelvic region, and weight loss. Anemia is the most common extraintestinal complication of inflammatory bowel disease. Associated complaints and diseases include arthritis, pyoderma gangrenosum, primary sclerosing cholangitis, and non-thyroid disease syndrome (NTIS).

[0005] Multiple sclerosis 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 impairs the ability of parts of the nervous system to transmit signals, causing a series of signs and symptoms that include physical, mental, and sometimes psychiatric problems. Specific symptoms can include double vision, blindness in one eye, muscle weakness, and impaired sensation and coordination. There are several forms of MS, some of which occur as isolated attacks of new symptoms (relapsing) and others that accumulate over time (progressive). Between attacks, symptoms may disappear completely, but many are left with permanent neurological problems, especially as the disease progresses. The cause is unknown, but the underlying mechanism is thought to be either destruction by the immune system or a failure of myelin-producing cells.

[0006] type 1 diabetes Type 1 diabetes (T1D), previously known as juvenile onset diabetes, is an autoimmune disease that occurs when the islets of Langerhans (containing beta cells) in the pancreas produce too little or no insulin. Insulin is a hormone necessary for cells to use blood glucose for energy and helps maintain normal glucose levels in the blood. Left untreated, type 1 diabetes causes high blood glucose levels in the body. Common symptoms of this elevated blood glucose level include frequent urination, increased thirst, increased hunger, weight loss, and other serious complications. Additional symptoms may include blurred vision, fatigue, and slow wound healing. Symptoms are typically short-lived, often onset within a matter of weeks. The cause of type 1 diabetes is unknown, but a combination of genetic and environmental factors is thought to be involved. The underlying mechanism involves the autoimmune destruction of insulin-producing beta cells in the pancreas.

[0007] Systemic lupus erythematosus Systemic lupus erythematosus (SLE) is an autoimmune disease in which the body's immune system mistakenly attacks healthy tissues in many parts of the body. Symptoms vary from person to person and can range from mild to severe. Common symptoms include joint pain and swelling, fever, chest pain, hair loss, mouth ulcers, swollen lymph nodes, fatigue, and a red rash, most commonly on the face. There are often periods of disease called flares and periods of remission with few symptoms in between. SLE affects multiple organ systems and is characterized by a widespread loss of immune tolerance.

[0008] psoriasis Psoriasis is a persistent, non-contagious autoimmune disease characterized by areas of abnormal skin projections. These areas are red or, in darker-skinned people, purple, dry, itchy, and scaly. Psoriasis can range in severity from small, localized patches to covering the entire body. Injury to the skin can trigger psoriatic skin changes in those spots, known as the Koebner phenomenon. The causes of psoriasis are not fully understood. Currently, there is no cure available for psoriasis, but several treatment options exist, typically using topical agents in mild cases, phototherapy in moderate cases, and systemic medications in severe cases. Flavonoids have a general formula of a 15-carbon backbone consisting of two phenyl rings (A and B rings) and one heterocyclic ring (C ring, which contains an embedded oxygen).

[0009] Typical treatment Treatment depends on the type and severity of the condition. The majority of autoimmune diseases are chronic and there is no definitive cure, but treatment can reduce and control symptoms. Overall, the goal of various treatments is to manipulate the body's autoimmune response to alleviate symptoms and mitigate them while preserving the patient's ability to fight any illness they may encounter. Traditional treatment options may include immunosuppressants to dampen the overall immune response, such as nonsteroidal anti-inflammatory drugs (NSAIDs) to reduce inflammation, glucocorticoids to reduce inflammation, and disease-modifying antirheumatic drugs (DMARDs) to reduce the effect of the inflammatory autoimmune response in damaging tissues and organs.

[0010] Flavonoids [ka] This carbon structure can be abbreviated as C6-C3-C6. According to the IUPAC nomenclature, flavonoids can be classified as: Flavonoids or bioflavonoids Isoflavonoids derived from the 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure Neoflavonoids derived from the 4-phenylcoumarin (4-phenyl-1,2-benzopyrone) structure [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows one embodiment of a MINC (Multi-pathway Immune-modulating Nanocomplex Combination therapy)-agent, which is a micelle with a polymer-flavonoid conjugate, e.g., PEG-EGCG conjugate, in the shell and an encapsulated agent. [Diagram 2] FIG. 2 shows another embodiment of a MINC-drug, which is a micelle comprising a polymer-flavonoid conjugate, e.g., a PEG-EGCG conjugate, in the outer shell and a flavonoid oligomer, e.g., oligomeric EGCG (OEGCG), in the inner shell, with an encapsulated drug. [Figure 3A] FIG. 3 shows that OEGCG and PEG-EGCG inhibit the production of TNF-α, an inflammatory cytokine, induced by LPS in rheumatoid arthritis synovial fibroblasts. [Figure 3B] FIG. 3 shows that OEGCG and PEG-EGCG inhibit the production of TNF-α, an inflammatory cytokine, induced by LPS in rheumatoid arthritis synovial fibroblasts. [Figure 4] FIG. 4 shows that OEGCG and PEG-EGCG reduce the production of the proinflammatory cytokine genes IL-6 and COX2 in a cell model of inflammatory bowel disease. [Figure 5A] FIG. 5 shows that MINC-anti-CD3 has reduced toxicity compared to anti-CD3. [Figure 5B] FIG. 5 shows that MINC-anti-CD3 has reduced toxicity compared to anti-CD3. [Figure 6A]FIG. 6 shows that MINC-anti-HER2 micelles with particle sizes of around 100 nm can be produced when the polymer-flavonoid conjugates contain different flavonoids and different flavonoid oligomers. [Figure 6B] FIG. 6 shows that MINC-anti-HER2 micelles with particle sizes of around 100 nm can be produced when the polymer-flavonoid conjugates contain different flavonoids and different flavonoid oligomers. [Figure 6C] FIG. 6 shows that MINC-anti-HER2 micelles with particle sizes of around 100 nm can be produced when the polymer-flavonoid conjugates contain different flavonoids and different flavonoid oligomers. [Figure 6D] FIG. 6 shows that MINC-anti-HER2 micelles with particle sizes of around 100 nm can be produced when the polymer-flavonoid conjugates contain different flavonoids and different flavonoid oligomers. [Figure 7A] FIG. 7 shows that when different polymers in the polymer-flavonoid conjugates were used, MINC-BSA micelles with particle sizes of around 100 nm could be produced. [Figure 7B] FIG. 7 shows that when different polymers in the polymer-flavonoid conjugates were used, MINC-BSA micelles with particle sizes of around 100 nm could be produced. [Figure 7C] FIG. 7 shows that when different polymers in the polymer-flavonoid conjugates were used, MINC-BSA micelles with particle sizes of around 100 nm could be produced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] definition The term "about" is defined as ±10%, preferably ±5% of the stated value.

[0013] The term "epigallocatechin gallate" refers to an ester of epigallocatechin and gallic acid and is used synonymously with "epigallocatechin-3-gallate" and EGCG.

[0014] The term "oligomeric EGCG" (OEGCG) refers to 3 to 30 covalently linked EGCG monomers, preferably 2 to 20 EGCG monomers. OEGCG preferably contains 4 to 12 EGCG monomers.

[0015] The term "polyethylene glycol-epigallocatechin gallate conjugate" or "PEG-EGCG" refers to polyethylene glycol (PEG) conjugated to one or two molecules of EGCG. The term "PEG-EGCG" refers to both PEG-mEGCG conjugates (monomeric EGCG) and PEG-dEGCG (dimeric EGCG) conjugates.

[0016] The present invention provides a method for treating an autoimmune disease, comprising administering to a subject in need of treatment an effective amount of (i) a polymer-flavonoid conjugate, (ii) a flavonoid oligomer, or (iii) a micelle having an outer shell formed by one or more polymer-flavonoid conjugates, optionally an inner shell formed by one or more flavonoid oligomers, and a drug encapsulated within the shells.

[0017] Flavonoids Flavonoids suitable for the present invention have the general formula I: [ka] Formula I During the ceremony, R 1 is H or phenyl; R 2 is H, OH, gallic acid, or phenyl; optionally, the phenyl is substituted with one or more (e.g., 2-3) hydroxyls; R3 is H, OH, or =O (oxo); or R 1 and R 2 are taken together to form a closed ring structure; or R 2 and R 3 together form a closed ring structure.

[0018] Positions 2, 3, 4, 5, 6, 7, or 8 of formula I may be linked to a hydrocarbon, halogen, oxygen, nitrogen, sulfur, phosphorus, boron, or metal-containing group.

[0019] Examples of flavonoids of formula I include: [ka]

[0020] Preferred flavonoid compounds of formula I include: EGCG (CAS Registry Number 989-51-5), EC (CAS Registry Number 490-46-0), EGC (CAS Registry Number 970-74-1), or ECG (CAS Registry Number 1257-08-5). [ka]

[0021] Polymer-flavonoid conjugates As used herein, a polymer-flavonoid conjugate refers to a conjugate of a hydrophilic polymer and a flavonoid compound of Formula I.

[0022] Hydrophilic polymer refers to a polymer that is soluble in polar solvents and can form hydrogen bonds. Suitable hydrophilic polymers for the polymer-flavonoid conjugate include, but are not limited to, polyethylene glycol, aldehyde-derivatized hyaluronic acid, hyaluronic acid, dextran, diethylacetal conjugates (e.g., diethylacetal PEG), D-α-tocopheryl polyethylene glycol succinate, aldehyde-derivatized hyaluronic acid-tyramine, hyaluronic acid-aminoacetylaldehyde diethylacetal conjugate-tyramine, cyclotriphosphazene core phenoxymethyl (methylhydrazono) dendrimer or thiophosphoryl core phenoxymethyl (methylhydrazono) dendrimer, acrylamide, oxazoline, imine, acrylic acid, methacrylate, diol, oxirane, alcohol, amine, anhydride, ester, lactone, terephthalate, amide, and ether. Polyacrylamides, poloxamers, poly(N-isopropylacrylamide), poly(oxazolines), polyethyleneimines, poly(acrylic acid), polymethacrylates, polyethylene glycols, poly(ethylene oxide), poly(vinyl alcohol), poly(vinylpyrrolidinone), polyethers, poly(allylamine), polyanhydrides, poly(β-amino esters), polybutylene succinate, polycaprolactone, polycarbonates, polydioxanone, poly(glycerol), polyglycolic acid, poly(3-hydroxypropionic acid), poly(2-hydroxyethyl methacrylate), poly(N-(2-hydroxypropyl)methacrylamide), polylactic acid, lactic acid-glycolic acid copolymers, poly(ortho esters), poly(2 oxazolines), poly(sebacic acid), terephthalic acid-phosphoric acid copolymers, povidone, and copolymers.

[0023] Preferred hydrophilic polymers include polyethylene glycol, hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-α-tocopheryl, and polyethylene glycol succinate. The molecular weight of the hydrophilic polymer of the polymer-flavonoid conjugate is usually 1K to 100K daltons, preferably 2K to 40K, 2K to 50K, 2K to 80K, 3K to 80K, or 5K to 40K daltons.

[0024] In one embodiment, the polymer contains an aldehyde group conjugated to the 5-, 6-, 7-, or 8-position (preferably the 6- or 8-position) of the A ring of the flavonoid compound.

[0025] In another embodiment, the polymer is R 1 or R 2 (R 1 or R 2 is -OH).

[0026] In one embodiment, the polymer-flavonoid conjugate is PEG-EGCG, in which PEG is conjugated to one or two molecules of epigallocatechin gallate (EGCG). PEG-EGCG can be prepared, for example, by conjugating aldehyde-terminated PEG to EGCG by binding of PEG via reaction of a free aldehyde group with the 5-, 6-, 7-, or 8-position (preferably 6- or 8-position) of formula I. See WO 2006 / 124000 and WO 2009 / 054813. PEG-EGCG can also be prepared by conjugating thio-terminated PEG to EGCG by binding of PEG via reaction of a free thio group with R1 or R2 of formula I, where R1 or R2 is a phenyl group. See WO 2015 / 171079.

[0027] Flavonoid Oligomers A flavonoid oligomer is a flavonoid conjugated to one or more flavonoids. Flavonoid oligomers may contain the same flavonoid (homo-oligomer) or different flavonoids (hetero-oligomer). Flavonoid oligomers useful in the present invention usually have 2-50 or 2-20, preferably 4-12, flavonoids of one or mixed types.

[0028] 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 EGCG monomers covalently linked together. OEGCG can be synthesized, for example, at the 5-, 6-, 7-, or 8-position (preferably 6- or 8-position) of the A ring according to WO 2006 / 124000.

[0029] Since the A ring is present in all of the flavonoids of formula 1, other oligomeric flavonoids can be similarly prepared according to WO 2006 / 124000. For example, OEC, OEGC, and OECG can also be prepared according to WO 2006 / 124000.

[0030] MINC-Drugs MINC (Multi-pathway Immune-modulating Nanocomplex Combination therapy) is a platform technology that utilizes the bioactivity of polymer-flavonoid conjugates or flavonoid oligomers that form micelles in solution.

[0031] The MINC platform can encapsulate additional drugs to form nanoparticle compositions for combination therapy. MINC-drugs are micelles with a shell formed by one or more polymer-flavonoid conjugates or one or more flavonoid oligomers, or a combination thereof, with a drug encapsulated within the shell. Drug, as used herein, refers to a molecule with therapeutic activity to treat autoimmune disease, including, but not limited to, anti-inflammatory agents such as anti-inflammatory antibodies, anti-inflammatory cytokines, anti-inflammatory compounds, or antibodies, or compounds.

[0032] The MINC platform can reduce the toxicity of the encapsulated drug, thereby expanding the effective dosage window of the drug in autoimmune diseases. For example, anti-CD3 is a broad-spectrum immunosuppressant for treating various autoimmune diseases. However, anti-CD3 has high toxicity that limits its clinical use. The MINC-anti-CD3 formulation reduces the toxicity of anti-CD3. MINC-anti-CD3 is a safe drug for treating autoimmune diseases.

[0033] In one embodiment, the MINC-drug is a micelle that contains a polymer-flavonoid conjugate, such as a PEG-EGCG conjugate, in its shell and encapsulates the drug (Figure 1).

[0034] In another embodiment, the MINC-agent is a micelle comprising a polymer-flavonoid conjugate, e.g., a PEG-EGCG conjugate, in the outer shell, a flavonoid oligomer, e.g., oligomeric EGCG (OEGCG), in the inner shell, and an agent is encapsulated (Figure 2).

[0035] When the encapsulated agent is a drug, the MINC-drug composition contains two or more components with therapeutic activity, due to its framework component (flavonoid conjugate or flavonoid oligomer) and the encapsulated agent, which are complementary in function to form a multi-targeted combination therapy. The MINC-drug is stable in a hydrophilic environment such as the blood circulation.

[0036] In one embodiment, the agent in the MINC-drug 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-drugs are suitable for treating rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, type 1 diabetes, systemic lupus erythematosus, and psoriasis.

[0037] In one embodiment, the drug in the MINC-drug is an anti-inflammatory antibody, including but not limited to adalimumab, infliximab, golimumab, rituximab, certolizumab, tocilizumab, sarilumab, vedolizumab, etrolizumab, natalizumab, PF-00547659, ustekinumab, alemtuzumab, daclizumab, ocrelizumab, canakinumab, dapirolizumab, secukinumab, ixekizumab. Such MINC-drugs are suitable for treating rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, type 1 diabetes, systemic lupus erythematosus, and psoriasis.

[0038] In one embodiment, the drug in the MINC-drug is an antibody, including but not limited to adalimumab, infliximab, golimumab, rituximab, certolizumab, tocilizumab, sarilumab, etc. Such MINC-drugs are suitable for treating rheumatoid arthritis.

[0039] In one embodiment, the agent in the MINC-agent is an antibody, including but not limited to, vedolizumab, etrolizumab, natalizumab, PF-00547659, ustekinumab, golimumab, adalimumab, certolizumab, natalizumab, infliximab, etc. Such MINC-agents are suitable for treating inflammatory bowel disease.

[0040] In one embodiment, the agent in the MINC-agent is an antibody, including but not limited to natalizumab, alemtuzumab, daclizumab, ocrelizumab, etc. Such MINC-agents are suitable for treating multiple sclerosis.

[0041] In one embodiment, the drug in the MINC-drug is an antibody, including but not limited to canakinumab, ustekinumab, rituximab, otelixizumab, teplizumab, visilizumab, etc. Such MINC-drugs are suitable for treating type 1 diabetes.

[0042] In one embodiment, the agent in the MINC-agent is an antibody, including but not limited to dapirolizumab, belimumab, anifrolumab, BIIB059. Such MINC-agents are suitable for treating systemic lupus erythematosus.

[0043] In one embodiment, the drug in the MINC-drug is an antibody, including but not limited to infliximab, adalimumab, ustekinumab, secukinumab, ixekizumab, etc. Such MINC-drugs are suitable for treating psoriasis.

[0044] In one embodiment, the drug in the MINC-drug is leflunomide, aspirin, naproxen, diclofenac, ibuprofen, methotrexate, sulphasalazine, hydroxychloroquine, sulfasalazine, baricitinib, tofacitinib, azathioprine, cyclosporine, minocycline, ozanimod, mesalamine, olsalazine, balsalazide, prednisone, hydrocortisone, budesonide, 6-mercaptopurine, methotrexate, metronidazole, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, laquinimod, mitoxantrone, dalfampridine, metformin, tolbutamide, chlorpropamide, tolazamide, glyburide, glipizide, glimepiride, repaglinide, nateglinide, Anti-inflammatory compounds include, but are not limited to, rivaroxaban, sitagliptin, saxagliptin, linagliptin, alogliptin, rosiglitazone, pioglitazone, acarbose, miglitol, canagliflozin, dapagliflozin, empagliflozin, BT-063, fenebrutinib, iveldomid, cenerimod, evobrutinib, bortezomib, ibuprofen, naproxen, chloroquine, quinacrine, betamethasone dipropionate, prednisolone, methylprednisolone sodium succinate, cyclophosphamide, mycophenolate mofetil, prasterone, dehydroepiandrosterone, apremilast, ruxolitinib, baricitinib, ASP015K, sotrastaurin, fostamatinib, and tamatinib. Such MINC-drugs are suitable for treating rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, type 1 diabetes, systemic lupus erythematosus, and psoriasis.

[0045] In one embodiment, the drug in the MINC-drug is a compound including, but not limited to, leflunomide, aspirin, naproxen, diclofenac, ibuprofen, methotrexate, sulphasalazine, hydroxychloroquine, sulfasalazine, baricitinib, tofacitinib, azathioprine, cyclosporine, and minocycline. Such MINC-drugs are suitable for treating rheumatoid arthritis.

[0046] In one embodiment, the drug in the MINC-drug 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-drugs are suitable for treating inflammatory bowel disease.

[0047] In one embodiment, the drug in the MINC-medicine is a compound drug, including but not limited to glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, laquinimod, mitoxantrone, and dalfampridine. Such MINC-medicines are suitable for treating multiple sclerosis. In one embodiment, the drug in the MINC-medicine is a compound drug, including but not limited to metformin, tolbutamide, chlorpropamide, tolazamide, glyburide, glipizide, glimepiride, repaglinide, nateglinide, sitagliptin, saxagliptin, linagliptin, alogliptin, rosiglitazone, pioglitazone, acarbose, miglitol, canagliflozin, dapagliflozin, and empagliflozin. Such MINC-medicines are suitable for treating type 1 diabetes.

[0048] In one embodiment, the drug in the MINC-drug is a compound including, but not limited to, hydroxychloroquine, prednisone, methylprednisolone, cyclophosphamide, azathioprine, mycophenolic acid, BT-063, fenebrutinib, iveldimide, cenerimod, evobrutinib, bortezomib, ibuprofen, naproxen, aspirin, chloroquine, quinacrine, betamethasone dipropionate, prednisone prednisolone, methylprednisolone sodium succinate, cyclophosphamide, mycophenolate mofetil, cyclosporine, methotrexate, prasterone, dehydroepiandrosterone. Such MINC-drug is suitable for treating systemic lupus erythematosus.

[0049] In one embodiment, the drug in the MINC-drug is a compound including, but not limited to, apremilast, tofacitinib, ruxolitinib, baricitinib, ASP015K, sotrastaurin, BMS-582949, fostamatinib, tamatinib. Such MINC-drug is suitable for treating psoriasis.

[0050] In one embodiment, the drug in the MINC-drug is an anti-inflammatory protein, including but not limited to etanercept, abatacept, mongersen, exenatide, liraglutide, albiglutide, dulaglutide, abatacept, alefacept, IL-2, GCSF, RSLV-132, RC18, AMG 592. Such MINC-drugs are suitable for treating rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, type 1 diabetes, systemic lupus erythematosus, and psoriasis.

[0051] For example, MINC-drug is an anti-IL-1 encapsulated in micelles formed by PEG-EGCG and flavonoid oligomer OEGCG (see WO 2009 / 054813 for structure and formulation).

[0052] For example, a MINC-drug is etanercept encapsulated in micelles formed by PEG-EGCG and the flavonoid oligomer OEGCG. See WO 2009 / 054813 for structure and formulation.

[0053] For example, a MINC-drug is leflunomide encapsulated in micelles formed by PEG-EGCG. See WO 2011 / 112156 for structure and formulation.

[0054] Pharmaceutical Compositions The present invention uses a pharmaceutical composition comprising a hydrophilic polymer-flavonoid conjugate, flavonoid oligomer, or MINC-drug composition as described herein, and optionally one or more pharma- ceutically acceptable carriers. In the case of a tablet, powder, or parenteral formulation, the nanoparticle composition in the pharmaceutical composition is usually about 1-90%, preferably 20-90%, or 30-80%. In the case of a capsule formulation, the PEG-flavonoid conjugate, flavonoid oligomer, or MINC-drug composition in the pharmaceutical composition is usually 1-100%, preferably 20-100%, 50-100%, or 70-100%. In the case of a suspension formulation, the nanoparticle composition in the pharmaceutical composition is usually 1-50%, 5-50%, or 10-40%.

[0055] In one embodiment, the pharmaceutical composition may be in the form of tablets, capsules, granules, microgranules, powders, suspensions, patches, parenterals, injections, or the like. The pharmaceutical compositions described above may be prepared by conventional methods.

[0056] Pharmaceutically acceptable carriers are inert ingredients and can be selected by those skilled in the art using conventional criteria. Pharmaceutically acceptable carriers include saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents, such as sodium chloride, potassium chloride, glycerol, and glucose; pH adjusters and buffers, such as hydroxide salts, phosphate salts, citrate salts, acetate salts, borates, and salts of trolamine; antioxidants, such as bisulfite, sulfite, metabisulfite, thiosulfite, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol, and salts of ascorbyl palmitate, acids, and / or bases; surfactants, such as lecithin and phospholipids, including, but not limited to, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol; poloxamers and poloxamines; polysorbate 80, ... It may contain ingredients including, but not limited to, polysorbates such as resorbate 60 and polysorbate 20; polyethers such as polyethylene glycol and polypropylene glycol; polyvinyls such as polyvinyl alcohol and polyvinylpyrrolidone (PVP, povidone); cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose, and their salts; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, and soybean oil; monoglycerides, diglycerides, and triglycerides; polysaccharides such as dextran; and glycosaminoglycans such as sodium hyaluronate. Such pharma- ceutically acceptable carriers may be preserved against microbial contamination through the use of 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 unpreserved preparations for single or multiple use.

[0057] For example, tablets, capsules, or parenteral formulations of an active compound may contain other excipients that are not biologically active and do not react with the active compound. Tablet or capsule excipients may include fillers, binders, lubricants and glidants, disintegrants, wetting agents, and release rate modifiers. Examples of tablet or capsule excipients include, but are not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide, poly(acrylic acid), and polyvinylpyrrolidone. 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. Powdered oral formulations may contain inactive ingredients such as silica gel, sodium benzoate, sodium citrate, sucrose, and xanthan gum.

[0058] The pharmaceutical composition can be applied by local and systemic administration. Local administration includes topical administration. Systemic administration includes oral, parenteral (such as intravenous, intramuscular, subcutaneous, or rectal), and other systemic routes of 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 drip, and oral administration are preferred routes of administration.

[0059] Treatment methods The present invention relates to methods for preventing or treating autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, type 1 diabetes, systemic lupus erythematosus, and psoriasis.

[0060] Polymer-Flavonoid In a first aspect of the present invention, the method comprises administering to a subject in need of treatment for an autoimmune disease an effective amount of a polymer-flavonoid conjugate to treat the autoimmune disease. An "effective amount," as used herein, is an amount effective to treat the disease by ameliorating a pathological condition or reducing a symptom of the disease.

[0061] The polymer-flavonoid conjugates of the present invention have immunomodulatory and anti-inflammatory activities for treating autoimmune diseases.

[0062] In one embodiment, the flavonoid is EGCG, EC, EGC, or ECG.

[0063] In one embodiment, the polymer is a hydrophilic polymer having a molecular weight of 1000 to 100,000 daltons and is selected from the group consisting of PEG, hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-α-tocopheryl, and polyethylene glycol succinate.

[0064] A preferred polymer-flavonoid conjugate is PEG-EGCG.

[0065] In one embodiment, the autoimmune disease is associated with the joints and muscles and is rheumatoid arthritis, psoriatic arthritis, Sjogren's syndrome, or systemic lupus erythematosus.

[0066] In one embodiment, the autoimmune disease is associated with the digestive tract and is inflammatory bowel disease, celiac disease, or ulcerative colitis.

[0067] 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.

[0068] In one embodiment, the autoimmune disease is associated with the skin and is psoriasis, dermatomyositis, or vitiligo.

[0069] In one embodiment, the autoimmune disease is associated with the nervous system and is multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, or Guillain-Barre syndrome.

[0070] In one embodiment, the autoimmune disease is myasthenia gravis, autoimmune vasculitis, pernicious anemia, vasculitis, or myositis.

[0071] The dosage of the polymer-flavonoid for injection, such as PEG-EGGC, is typically 0.1-10000 mg / kg, 0.6-1200 mg / kg (total weight of PEG-flavonoid / body weight of subject), or 1-1000 mg / kg.

[0072] Flavonoid Oligomers In a second aspect of the present invention, the method comprises administering to a subject in need thereof an effective amount of a flavonoid oligomer, the flavonoid oligomer of the present invention having immunomodulatory and anti-inflammatory activity for treating autoimmune diseases.

[0073] In one embodiment, the flavonoid oligomer is an oligomer of EGCG, EC, EGC, or ECG.

[0074] In one embodiment, the flavonoid oligomer comprises 4 to 12 EGCG, EC, EGC, or ECG flavonoids.

[0075] In one embodiment, the autoimmune disease is associated with the joints and muscles and is rheumatoid arthritis, psoriatic arthritis, Sjogren's syndrome, or systemic lupus erythematosus.

[0076] In one embodiment, the autoimmune disease is associated with the digestive tract and is inflammatory bowel disease, celiac disease, or ulcerative colitis.

[0077] 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.

[0078] In one embodiment, the autoimmune disease is associated with the skin and is psoriasis, dermatomyositis, or vitiligo.

[0079] In one embodiment, the autoimmune disease is associated with the nervous system and is multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, or Guillain-Barre syndrome.

[0080] In one embodiment, the autoimmune disease is myasthenia gravis, autoimmune vasculitis, pernicious anemia, vasculitis, or myositis.

[0081] The dosage of a flavonoid oligomer, such as OEGCG, for injection is usually 0.1 to 1000 mg / kg, 0.1 to 100 mg / kg (total weight of flavonoid oligomers / body weight of subject), or 1 to 100 mg / kg.

[0082] MINC drugs In a third aspect of the present invention, the method comprises administering to a subject in need of treatment for an autoimmune disease 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 the shells, to treat the autoimmune disease. In one embodiment, the micelle comprises both an outer shell and an inner shell. In another embodiment, the micelle does not have an inner shell and has only one shell comprising one or more polymer-flavonoids.

[0083] The polymer-flavonoid conjugates or flavonoid oligomers exert their own therapeutic effect and further deliver the drug to treat autoimmune diseases.

[0084] In one embodiment, the polymer is a hydrophilic polymer having a molecular weight of 1000 to 100,000 daltons and is selected from the group consisting of polyethylene glycol (PEG), hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-α-tocopheryl, and polyethylene glycol succinate.

[0085] In one embodiment, the flavonoid oligomer comprises 2 to 50 or 2 to 20 EGCG, EC, EGC, or ECG flavonoids.

[0086] In one embodiment, the shell is formed by PEG-EGCG.

[0087] In one embodiment, the shell is formed by PEG-EGCG and OEGCG.

[0088] In one embodiment, the autoimmune disease is type 1 diabetes and the agent 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 inhibitors, anti-DYRK1A, NFAT, substance P, LRH-1, IL-10, IGF, IGFBP1, ARX, GCSF, VMAT-2, or RAE-1.

[0089] In one embodiment, the autoimmune disease is inflammatory bowel disease and the agent 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.

[0090] In one embodiment, the autoimmune disease is rheumatoid arthritis and the agent 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-CC R2, 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-CD11a, or anti-CD19.

[0091] In one embodiment, the autoimmune disease is multiple sclerosis and the agent 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 a FoxP3 activator.

[0092] In one embodiment, the autoimmune disease is systemic lupus erythematosus and the agent is anti-CD20, anti-CD19, anti-CD22, a proteosome 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.

[0093] In one embodiment, the autoimmune disease is psoriasis and the agent 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.

[0094] In one embodiment, the autoimmune disease is myasthenia gravis and the agent 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.

[0095] In one embodiment, the autoimmune disease is Hashimoto's thyroiditis and the agent 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.

[0096] Dosing of the MINC-drug is based on known doses of the drug to treat a particular disease and the condition of the subject. The doses may be Food and Drug Administration (FDA) approved doses or doses used in clinical trials.

[0097] In the MINC-medicine, the dose of PEG-EGCG combined with OEGCG is usually 10 μg / kg to 100 mg / kg.

[0098] The concentration of the encapsulated drug can be as low as 1 μg / kg (e.g., for the cytokine drug G-CSF) or as high as 10 mg / kg (e.g., for antibody drugs such as certolizumab).

[0099] For example, where the MINC-agent comprises an 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-CD20, anti-TGF-β, anti-T lymphocyte globulin (ATG) for treating rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, and psoriasis, the antibody may be administered IV in the range of 0.1-10 mg / kg once every 1-4 weeks.

[0100] For example, to treat rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, psoriasis, or other autoimmune diseases in adults, anti-TNF-α is administered IV at 0.1-10 mg / kg once every 1-4 weeks. MINC-anti-TNF-α contains the same dose range of anti-TNF-α that can be used to treat these diseases.

[0101] For example, to treat rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, psoriasis, or other autoimmune diseases in adults, etanercept is administered IV at 0.1-10 mg / kg once every 1-4 weeks. MINC-etanercept contains the same dose range of etanercept that can be used to treat these diseases.

[0102] For example, methylprednisolone is administered IV at 0.5-50 mg / kg once every 1-4 weeks to treat rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, and psoriasis in adults. MINC-methylprednisolone contains the same dose range of methylprednisolone that can be used to treat these diseases.

[0103] For example, MINC-drugs include leflunomide, aspirin, naproxen, diclofenac, ibuprofen, methotrexate, sulfasalazine, hydroxychloroquine, sulfasalazine, baricitinib, tofacitinib, azathioprine, cyclosporine, minocycline, ozanimod, mesalamine, olsalazine, balsalazide, prednisone, hydrocortisone, budesonide, 6-mercaptopurine, metronidazole, methylprednisolone, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, laquinimod, mitoxantrone, dalfampyridine, metformin, tolbutamide, chlorpropamide, tolazamide, glyburide, glipizide for treating rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, and psoriasis. , glimepiride, repaglinide, nateglinide, sitagliptin, saxagliptin, linagliptin, alogliptin, rosiglitazone, pioglitazone, acarbose, miglitol, canagliflozin, dapagliflozin, empagliflozin, cyclophosphamide, mycophenolic acid, BT-063, fenebrutinib, iveldmide, cenerimod, evobrutinib, bortezomib, clo When including an anti-inflammatory compound, including but not limited to roquine, quinacrine, betamethasone dipropionate, prasterone, dehydroepiandrosterone, apremilast, ruxolitinib, ASP015K, sotrastaurin, BMS-582949, fostamatinib, or tamatinib, the anti-inflammatory compound may be administered IV in the range of 0.1-10 mg / kg once every 1-4 weeks.

[0104] For example, where the MINC-medication comprises a nonsteroidal anti-inflammatory drug (NSAID) including, but not limited to, ibuprofen, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin, or aspirin for treating rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, and psoriasis, the anti-inflammatory agent may be administered IV in the range of 0.1-10 mg / kg, once to twice daily.

[0105] The invention is useful in treating humans and non-human animals. For example, the invention is useful in treating mammalian subjects such as humans, horses, pigs, cats, and dogs.

[0106] The following examples further illustrate the present invention. These examples are intended merely to illustrate the invention and should not be construed as limiting. EXAMPLES

[0107] Active ingredient OEGCG: OEGCG is oligomerized EGCG. OEGCG is prepared according to WO 2006 / 124000.

[0108] PEG-EGCG: PEG-EGCG is PEG conjugated with one or two EGCGs. PEG-EGCG is prepared according to WO 2006 / 124000, WO 2009 / 054813, or WO 2015 / 171079.

[0109] MINC-Drugs: MINC-drugs are prepared by encapsulating the drug within micelles formed by PEG-EGCG and OEGCG according to the methods in WO 2006 / 124000 or WO 2009 / 054813.

[0110] Example 1: OEGCG and PEG-EGCG inhibit the production of inflammatory cytokine TNF-α in LPS-induced rheumatoid arthritis synovial fibroblasts material TNF-alpha ELISA Kit, Human (Sino Biologicals, Catalog Number: KIT10602) Rheumatoid arthritis synovial fibroblasts (RASF) (RIKEN)

[0111] method Rheumatoid arthritis synovial fibroblasts (RASFs) were cultured at 1 × 10 4 Cells / well were seeded into 96-well plates and incubated overnight. On the second day, cells were pretreated with different concentrations of OEGCG or PEG-EGCG for 20 min, and then co-incubated with 1 μg / mL LPS for 48 h. After treatment, cell supernatants were collected and subjected to human TNF-α ELISA kit according to the manufacturer's instructions.

[0112] result Stimulation of RASF cells with LPS increased the concentration of TNF-α. Treatment with OEGCG or PEG-EGCG significantly inhibited the production of TNF-α in a concentration-dependent manner (Figure 3). RASF cells can secrete proinflammatory cytokines, including TNF-α, which promotes immune cell activation and joint and bone destruction. The reduction of TNF-α by OEGCG and PEG-EGCG treatment has therapeutic effects in the treatment of rheumatoid arthritis.

[0113] Example 2: OEGCG and PEG-EGCG reduce inflammation in a cellular model of inflammatory bowel disease material TRIzol-A+ Reagent (Tiagen) Caco-2 was from ATCC (HTB-37).

[0114] method Caco-2 cells were cultured in growth medium at 37°C in a humidified chamber with 5% CO2. Caco-2 cells were cultured at 2 × 10 6Cells / well were seeded overnight in 6-well dishes and pretreated with OEGCG (3.3 μM) or PEG-EGCG (10 μM) for 24 h, followed by further incubation in the presence or absence of LPS (20 μg / mL) for 8 h. 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 Co., Ltd., Beijing, China). qRT-PCR was performed with a QuantStudio™ 6 Flex Real-Time PCR System (Life Technologies, USA) and the conditions were set as follows: initial denaturation at 95°C for 3 min, followed by denaturation at 95°C for 10 s, annealing at 56°C for 10 s, and extension at 72°C for 60 s for 35–50 cycles. Ct (cycle threshold) values ​​were calculated and normalized to the levels of the housekeeping gene GAPDH. The forward and reverse primers were used according to the publication by Wu et al. (Inflammation. 2019 Dec;42(6):2215-2225.).

[0115] result The mRNA levels of IL-6 and COX2 in Caco-2 cells were analyzed by qRT-PCR assay. Treatment with OEGCG and PEG-EGCG reduced the mRNA expression of IL-6 and COX2 compared with the untreated group (Figure 4). These results suggest that treatment with OEGCG and PEG-EGCG can reduce disease progression through inhibiting inflammatory cytokine production.

[0116] Example 3: MINC-anti-CD3 reduces the toxicity of anti-CD3 material Anti-CD3 antibody was purchased from BioLegend (catalog no. 317325). MINC-anti-CD3 was prepared according to WO 2009 / 054813.

[0117] method Balb / c mice aged 6-8 weeks were housed in a specific pathogen-free environment. After 3 days of acclimation, treatment was initiated the day after baseline calculation (represented as day 0). Anti-CD3 or MINC-anti-CD3 was administered intravenously to mice at doses of 5, 25, and 125 μg / mouse daily 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 once daily and every other day for 8 consecutive days. Statistical analysis was performed using GraphPad Prizm. Two-way ANOVA was used to assess differences between groups, and p<0.05 was considered significant.

[0118] result After two doses of treatment, free anti-CD3 antibody significantly reduced mouse body weight on days 2-5 (Figure 5A). Four of five mice in the high dose group and three of five mice in the medium dose group died between days 5-7 (Figure 5B). In contrast, MINC-anti-CD3 antibody at 125 μg / mouse showed a mild weight loss on day 5 and recovery on day 7 (Figure 5A). No animals in the MINC-anti-CD3 group died (Figure 5B). These results indicate that MINC encapsulation significantly reduced toxicity and tolerated doses up to 25 times the clinical dose (equivalent to 5 μg / mouse).

[0119] Anti-CD3 is a broad-spectrum immunosuppressant for treating various autoimmune diseases. However, the high toxicity of anti-CD3 limits its clinical use. This example shows that MINC formulation can reduce the toxicity of anti-CD3. MINC-anti-CD3 is a safe drug for treating autoimmune diseases.

[0120] Different Flavonoid Oligomers and Polymer-Flavonoid Conjugates in Forming MINC-Drugs (Examples 4-5) Example 4: Methods for preparing MINC-anti-HER2 using different flavonoids in flavonoid oligomers and polymer-flavonoid conjugates material Anti-HER2 was trastuzumab obtained from Eirgenix.

[0121] method MINC-anti-HER2 nanoparticles were prepared according to WO 2009 / 054813. Briefly, anti-HER2 was incubated in PBS. Different flavonoid oligomers, including OEGCG or OECG, were then added to anti-HER2, followed by different polymer-flavonoids, including PEG-EGCG, PEG-ECG, or PEG-EC. The mixture was incubated at room temperature, after which unreacted oligomeric and polymer-flavonoids were removed using a 10K MWCO centrifugal filter. Nanoparticle size was measured using DLS (Anton Paar Litesizer 500). The results are shown in Figure 6.

[0122] result Figure 6 shows that various polymer-flavonoid conjugates and various flavonoid oligomers were all able to generate MINC-anti-HER2 micelles with particle sizes around 100 nm. These results indicate that homogenous nanoparticles (micelles) were able to form with predicted sizes different from those of unencapsulated anti-HER antibodies (approximately 5-10 nm).

[0123] In this example, the various flavonoid oligomers used included OEGCG (Figures 6A, 6C, and 6D) and OECG (Figure 6B); the various polymer-flavonoids used included PEG-EGCG (Figures 6A and 6B), PEG-EC (Figure 6C), and PEG-ECG (Figure 6D).

[0124] These data confirm that MINC nanoparticles can be formed using a variety of flavonoid oligomers and a variety of polymer-flavonoid conjugates.

[0125] Example 5: Methods for preparing MINC-BSA using different polymers for the polymer-flavonoid conjugate material BSA was purchased from Sigma-Aldrich.

[0126] method Multi-target immune nanocarrier combination (MINC)-BSA nanoparticles were prepared according to WO 2009 / 054813. Briefly, BSA was incubated in PBS. OEGCG was then added to the BSA, followed by different polymer-flavonoids, including PEG-EGCG, HA-EGCG, and dextran-EGCG. The mixture was incubated at room temperature, after which unreacted OEGCG and polymer-flavonoids were removed using a 10K MWCO centrifugal filter. Nanoparticle size was measured using DLS (Anton Paar Litesizer 500).

[0127] result Figure 7 shows that MINC-BSA could be produced using various polymers in the polymer-flavonoid conjugates. These results indicate that homogenous nanoparticles (micelles) could be formed. These different polymers were PEG (Figure 7A), HA (Figure 7B), and dextran (Figure 7C). Collectively, these data support the ability of different polymer-flavonoid conjugates to form MINC nanoparticles.

[0128] Example 6: MINC-Anti-CD3 Trial in Type 1 Diabetes (T1D) (Hypothetical Example) the purpose This example is intended to show that MINC-anti-CD3 treatment can treat T1D by improving blood glucose, insulin sensitivity, HbA1c levels, and C-peptide levels compared to the saline group. This example is intended to show that MINC-anti-CD3 is effective in treating type 1 diabetes.

[0129] material Anti-CD3 antibody is purchased from BioLegend (catalog number 317325). MINC-anti-CD3 is prepared according to WO 2009 / 054813.

[0130] method Diabetic female NOD mice (strain NOD / ShiLtJ) aged 8-15 weeks are monitored twice weekly for the presence of diabetes. Diabetic status is defined as blood glucose ≥ 200 mg / dL by tail bleed on two consecutive days. Mice are assigned to groups including one control group and treatment groups consisting of different doses of MINC-anti-CD3 or free anti-CD3. Treatment groups are treated with MINC-anti-CD3 or anti-CD3 by intravenous injection for 5 consecutive days to the equivalent of 1 μg, 5 μg, and 25 μg of anti-CD3. Weight loss, survival, fasting glucose, and C-peptide are measured weekly. HbA1c is measured 14, 28, and 42 days after the first injection. Values ​​for MINC-anti-CD3 and anti-CD3 treatment groups are compared by one-way ANOVA (α = 0.05).

[0131] Example 7. MINC-anti-CD3 in the treatment of rheumatoid arthritis (RA) (hypothetical example) material Rat anti-CD3 antibody is purchased from BioLegend. MINC-anti-CD3 antibodies are prepared according to WO 2009 / 054813.

[0132] the purpose This example is intended to show that MINC-anti-CD3 treatment can reduce joint swelling and maintain bone integrity compared to the saline group. Histopathological examination is intended to show that MINC-anti-CD3 is better at reducing immune cell infiltration, inflammatory cytokine production, and has better outcomes than saline alone. This experiment is intended to show that MINC-anti-CD3 is effective in treating rheumatoid arthritis.

[0133] method Eighty 10-week-old male Wistar rats are purchased from Biolasco. Rats are maintained in a specific pathogen-free facility. Briefly, on day 0, 0.2 mL of emulsified bovine type II collagen plus Freund's adjuvant is injected subcutaneously at the base of the tail, followed by a booster injection on day 7. On the same day, rats receive weekly intravenous injections of saline or 0.25-6.25 mg / kg MINC-anti-CD3. After treatment, rats are examined for bone performance by micro-CT and for joint swelling by measuring ankle and hind paw diameters. At endpoint, rats are sacrificed on day 28 for histopathological examination. Rat ankle joint samples are fixed in 10% formalin. After decalcification in hydrochloric acid, samples are embedded and fixed in paraffin. Sections are stained with hematoxylin and eosin (H&E) to examine joint morphology. The levels of TNF-α expression and inflammatory cell infiltration will be examined by immunohistochemical staining with anti-TNF-α and anti-CD38.

[0134] Example 8. MINC-Anti-TNF-α in the Treatment of Rheumatoid Arthritis (RA) (Hypothetical Example) the purpose This example is intended to show that MINC-anti-TNF-α treatment can reduce joint swelling and maintain bone integrity compared to the saline group. Histopathological examination is intended to show that MINC-anti-TNF-α reduces immune cell infiltration, reduces TNF-α, and leads to better outcomes than saline alone. This experiment is intended to show that MINC-anti-TNF-α is effective in treating rheumatoid arthritis.

[0135] material Rat anti-TNF-α antibody is purchased from BioLegend. MINC-anti-TNF-α antibodies are prepared according to WO 2009 / 054813.

[0136] method Eighty 10-week-old male Wistar rats are purchased from Biolasco. Rats are maintained in a specific pathogen-free facility. Briefly, on day 0, 0.2 mL of emulsified bovine collagen type II plus Freund's adjuvant is injected subcutaneously at the base of the tail, followed by a booster injection on day 7. On the same day, rats receive 2-20 mg / kg MINC-anti-TNF-α intravenously once a week for a total of two doses. After dosing, rats are examined for bone performance by micro-CT and for joint swelling by measuring ankle and hind paw diameters. At endpoint, rats are sacrificed on day 28 for histopathological examination. Rat ankle joint samples are fixed in 10% formalin. After decalcification in hydrochloric acid, samples are embedded in paraffin. Sections are stained with hematoxylin and eosin (H&E) to examine joint morphology. The levels of TNF-α expression and inflammatory cell infiltration will be examined by immunohistochemical staining with anti-TNF-α and anti-CD38.

[0137] Example 9. MINC-anti-CD3 in the treatment of inflammatory bowel disease (IBD) (hypothetical example) the purpose This experiment was intended to show that the DAI scores of rats in the MINC-anti-CD3 treated group were significantly lower than those in the saline group. Histopathological examination was intended to show that ulceration, edema, and inflammatory cell infiltration with mucosal hyperemia were reduced in the MINC-anti-CD3 treated group. Overall, this experiment was intended to show that MINC-anti-CD3 has a therapeutic effect in inflammatory bowel disease.

[0138] material Rat anti-CD3 antibody is purchased from BioLegend. MINC-anti-CD3 antibodies are prepared according to WO 2009 / 054813.

[0139] method Rats are fasted for 48 hours and anesthetized with 3% sodium pentobarbital. Briefly, on day 1, a lavage needle is inserted into the anus and advanced 8 cm from the anus verge. Next, 1.5-2.0 ml of 8% acetic acid is injected into the colon, and an additional 3-5 ml of saline is added to allow the acetic acid to diffuse into the colon. After 24 hours, rats are divided into groups treated with saline or 0.25-6.25 mg / kg MINC-anti-CD3 by intravenous injection on days 2 and 5. On day 8, rats are sacrificed and colons and tissues adjacent to ulcerated or hyperemic areas are harvested. Fixed and paraffin-embedded tissues are stained with hematoxylin and eosin for later histological examination. The disease activity index (DAI) is scored according to the following criteria: No weight loss is scored as 0, 1-5% weight loss is scored as 1, 5-10% weight loss is scored as 2, 10-15% weight loss is scored as 3, and >15% weight loss is scored as 4.

[0140] Example 10: MINC-Anti-TNF-α in the Treatment of Inflammatory Bowel Disease (IBD) (Hypothetical Example) the purpose This experiment was intended to show that the DAI scores of rats in the MINC-anti-TNF-α treated group were significantly lower than those in the saline group. Histopathological examination was intended to show that ulceration, edema, and inflammatory cell infiltration with mucosal hyperemia were reduced in the MINC-anti-TNF-α treated group. Overall, this experiment was intended to show that MINC-anti-TNF-α has a therapeutic effect in inflammatory bowel disease.

[0141] material Rat anti-TNF-α antibody is purchased from BioLegend. MINC-anti-TNF-α antibodies are prepared according to WO 2009 / 054813.

[0142] method Rats are fasted for 48 hours and anesthetized with 3% sodium pentobarbital. Briefly, an irrigation needle is inserted into the anus and advanced 8 cm from the anal verge. Next, 1.5-2.0 ml of 8% acetic acid is injected into the colon, and an additional 3-5 ml of saline is added to allow the acetic acid to diffuse into the colon. After 24 hours, rats are divided into groups treated with saline or 0.5-20 mg / kg MINC-anti-TNF-α by intravenous injection on days 2 and 5. On day 8, rats are sacrificed and colons and tissues adjacent to ulcerated or hyperemic areas are harvested. Fixed and paraffin-embedded tissues are stained with hematoxylin and eosin for later histological examination. Disease Activity Index (DAI) according to the following criteria: No weight loss is scored as 0, 1-5% weight loss is scored as 1, 5-10% weight loss is scored as 2, 10-15% weight loss is scored as 3, and >15% weight loss is scored as 4.

[0143] [Table 1-1] [Table 1-2]

[0144] The invention, and the methods and steps of making and using the same, are described in such full, clear, concise and exact terms as to enable any person skilled in the art to which the invention pertains to make and use the same. The foregoing describes preferred embodiments of the invention, it being understood that changes can be made therein without departing from the scope of the invention as set forth in the appended claims. To particularly point out and distinctly claim the subject matter which is regarded as the invention, the following claims conclude this specification.

Claims

1. 1. A method of treating an autoimmune disease, comprising: administering to a subject in need of treatment for an autoimmune disease an effective amount of a micelle having an outer shell comprising one or more polymer-flavonoid conjugates, and optionally an inner shell comprising one or more flavonoid oligomers, and a drug encapsulated within said shells; the polymer is a hydrophilic polymer having a molecular weight of 1000 to 100,000 Daltons and is selected from the group consisting of polyethylene glycol (PEG), hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-α-tocopheryl, and polyethylene glycol succinate; The flavonoid is EGCG, EC, EGC, or ECG, which has the following structure: 【Chemistry 1】 The flavonoid oligomer comprises 2 to 20 EGCG, EC, EGC, or ECG flavonoids; 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; method.

2. The method of claim 1 , wherein the micelles have an outer shell comprising PEG-EGCG and an inner shell comprising EGCG oligomers.

3. 3. The method of claim 1 or 2, wherein the autoimmune disease is type 1 diabetes, and the agent 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 according to claim 1 or 2, wherein the autoimmune disease is an 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 according to 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-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-CD11a, or anti-CD19.

6. The method according to claim 1 or 2, wherein the autoimmune disease is multiple sclerosis, and the drug is 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. 3. The method of claim 1 or 2, wherein the autoimmune disease is systemic lupus erythematosus and the agent is anti-CD20, anti-CD19, anti-CD22, a proteosome 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. 3. The method of claim 1 or 2, wherein the autoimmune disease is psoriasis, and the agent 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 an mTOR inhibitor.

9. 3. The method of claim 1 or 2, wherein the autoimmune disease is myasthenia gravis and the agent 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 agent 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. 1. A method of treating an autoimmune disease, comprising: administering to a subject in need of treatment for an autoimmune disease an effective amount of one or more polymer-flavonoid conjugates; the polymer is a hydrophilic polymer having a molecular weight of 1000 to 100,000 Daltons and is selected from the group consisting of PEG, hyaluronic acid, dextran, polyethyleneimine, poloxamer, povidone, D-α-tocopheryl, and polyethylene glycol succinate; The flavonoid is EGCG, EC, EGC, or ECG, which has the following structure: 【Chemistry 2】 The autoimmune disease is selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, Sjogren'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; method.

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

13. 1. A method of treating an autoimmune disease, comprising: administering to a subject in need of treatment for an autoimmune disease an effective amount of one or more flavonoid oligomers; The flavonoid is EGCG, EC, EGC, or ECG, which has the following structure: 【Chemistry 3】 The flavonoid oligomer comprises 4 to 12 EGCG, EC, EGC, or ECG flavonoids; The autoimmune disease is selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, Sjogren'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; method.

14. The method of claim 13, wherein the flavonoid oligomer is an oligomer of EGCG.

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

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

17. 15. The method of 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. 15. The method of 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. 15. The method of any one of claims 11 to 14, wherein the autoimmune disease is myasthenia gravis, autoimmune vasculitis, pernicious anemia, vasculitis, or myositis.

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