Preparation and application of a rapamycin prodrug and its nanoformulation

Abstract

The present invention belongs to the field of medical technology, and relates to a rapamycin prodrug and its derivative nanoformulation and its application in preventing or treating transplant rejection, autoimmune diseases, allergic reactions (such as food allergies), and immune responses to therapeutic agents. The structural formula of the rapamycin prodrug is shown in general formula 1. The rapamycin prodrug and its derivative nanoformulation of the present invention are used to treat or prevent one or more of transplant rejection, autoimmune diseases, food allergies, and immune responses to therapeutic agents. In some embodiments, the rapamycin prodrug nanoformulation can be used to deliver antigens or other immunomodulators that are desired to be tolerated. In other embodiments, the rapamycin prodrug nanoformulation can be used to clear circulating proteins or peptides or antibodies associated with one or more of the diseases mentioned above. The present invention also discloses methods and uses of rapamycin prodrug nanoformulations for inducing immune tolerance.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and relates to a rapamycin prodrug and a nano preparation derived therefrom, and applications thereof in preventing or treating transplant rejection, autoimmune diseases, allergic reactions (such as food allergic reactions), and immune responses to therapeutic agents. Background Art

[0002] Rejection of transplanted tissue, autoimmune diseases, allergic reactions (such as food allergies), and immune responses to therapeutic agents are all pathological conditions that cause immune rejection due to the antigenicity of certain biomolecules.

[0003] The rejection reaction of transplanted tissues is that after the recipient undergoes allogeneic tissue or organ transplantation, the foreign tissue or organ transplant is recognized by the recipient's immune system as a "foreign component", which initiates an immunological response to attack, destroy and eliminate the transplant. The mechanism of rejection reaction mainly includes two aspects: cellular immunity and humoral immunity. The most common acute rejection reaction in clinical practice is mainly mediated by cellular immunity, while hyperacute rejection and chronic rejection reactions are mainly mediated by humoral immunity. Graft-versus-host reaction is a reaction caused by the recognition of host antigens by specific lymphocytes in the graft. This reaction not only leads to transplant failure, but can also cause serious consequences for the recipient.

[0004] Autoimmune diseases are disease states caused by the body's immune system's immune response to its own components. The result of the body's immune response to foreign antigens is usually the elimination of the antigens, but when the body's immune response occurs to its own cell or tissue antigens, its own cells or tissues are not easily completely eliminated by the effector cells of the immune system but are constantly attacked, resulting in the body entering a disease state.

[0005] Hypersensitivity: also known as allergy, refers to an abnormal specific immune response characterized by physiological dysfunction or tissue cell damage when the body is stimulated by certain antigens.

[0006] Therapeutic treatments, such as protein or enzyme replacement therapies, often result in an unwanted immune response to the specific therapy. In this case, immune cells will recognize the therapeutic agent as foreign and attempt to neutralize or destroy it, just as they attempt to destroy infectious agents such as bacteria and viruses. This unwanted immune response can neutralize the effectiveness of the therapy or result in an allergic reaction to the therapy.

[0007] At present, although these adverse immune responses can be reduced by using immunosuppressants or regulators, traditional immunosuppressants are broadly effective and the use of broadly effective immunosuppressants may produce serious side effects such as tumors, infections, nephrotoxicity and metabolic disorders. Therefore, the development of new therapies will be beneficial.

[0008] Rapamycin (RAPA) is a macrolide drug with immunosuppressive, anti-proliferative, anti-angiogenic, anti-fungal and anti-inflammatory properties. It is often used clinically to prevent transplant rejection. RAPA can block the calcium-dependent and calcium-independent signal transduction pathways of T lymphocytes and B lymphocytes, thereby achieving immunosuppression. In addition, rapamycin also has the effect of inducing antigen presenting cells (APCs) to transform into a tolerant type and selectively increasing the number and function of Tregs.

[0009] Nanomedicines have the natural ability to be taken up by APCs or other immune cells, and can be used to deliver immunosuppressive drugs and / or antigens, adjuvants or diagnostic agents, or (and) to carry out targeted modification, reduce or eliminate the antigenicity of related biomolecules, thereby achieving the prevention and treatment of transplant rejection, autoimmune diseases, allergic reactions (such as food allergies), and reducing the immune response to therapeutic agents and improving the efficacy of related therapeutic agents. The application of rapamycin prodrugs and their derivative nanoformulations in these treatments has not been reported in the prior art. Summary of the invention

[0010] The purpose of the present invention is to provide a rapamycin prodrug and a nanoformulation derived therefrom and applications thereof in preventing or treating transplant rejection, autoimmune diseases, allergic reactions (such as food allergies), and immune responses to therapeutic agents.

[0011] To achieve the above purpose, the present invention adopts the following technical solution:

[0012] A rapamycin prodrug, the rapamycin prodrug has a structural formula as shown in Formula 1,

[0013]

[0014] Where: A is R1, R2, and R3 can be the same or different and can be selected from hydrocarbon chains, alkanoic acids, and lipid compounds; L is a connecting arm.

[0015] Preferably, in the rapamycin prodrug as shown in Formula 1:

[0016] A is

[0017] L is -C(O)-, -C(O)CH2-, -C(O)CH2CH2-, -C(O)CH2CH2CH2-, -C(O)CH2CH2CH2CH2-

[0018] R1, R2, and R3 can be the same or different and can be selected from saturated C 1-35 Hydrocarbon chain, unsaturated C 1-35 Hydrocarbon chain, C 2-35 Alkanoic acid, cholesterol and their derivatives.

[0019] Further preferably, in the rapamycin prodrug as shown in the general formula 1:

[0020] A is

[0021] L is -C(O)CH2CH2-, -C(O)CH2CH2CH2CH2-

[0022] R1 is a saturated or unsaturated C 10-22 hydrocarbon chains, cholesterol and their derivatives;

[0023] R2 is a saturated C 1-22 hydrocarbon chain;

[0024] R3 is a saturated C 2-22 Alkane acid.

[0025] Furthermore, in the rapamycin prodrug as shown in Formula 1:

[0026] A is

[0027] L is -C(O)CH2CH2-,

[0028] R1 is C 10 H 21 , C 12 H 25 , C 14 H 29 , C 15 H 31 , C 16 H 33 , C 17 H 35 , C 18 H 37 , C 18 H 35 Straight or branched hydrocarbon

[0029] R2 is C 10 H 21 , C 12 H 25 , C14 H 29 , C 15 H 31 , C 16 H 33 , C 17 H 35 , C 18 H 37 , C 22 H 45 Straight or branched chain hydrocarbons;

[0030] R3 is a saturated C 10-22 Alkane acid.

[0031] A method for preparing a rapamycin prodrug comprises reacting rapamycin with an acid anhydride in the presence of a weak base under lipase catalysis to obtain a 4,2-hydroxydicarboxylate, and then subjecting the rapamycin to an amidation reaction (connecting R1) to obtain the prodrug; or directly esterifying rapamycin with an enol ester under lipase catalysis to obtain (connecting R2); or reacting rapamycin under the catalysis of DIPEA to generate an alkynyl-modified rapamycin, and further catalyzing the alkynyl and azide-modified alkanoic acid to a click reaction (connecting R3), and then purifying to obtain the final product.

[0032] Specifically, rapamycin and anhydride are reacted in the presence of a weak base by Novozym 435 enzyme catalysis to obtain 42-hydroxydicarboxylate, which is then subjected to an amidation reaction to obtain the product.

[0033] Specifically, rapamycin and enol ester are directly esterified in the presence of 5-99.9% (by weight) Novozym 435 enzyme content within 4-96 hours.

[0034] Specifically, the propargyl alcohol ethoxy compound is first trifluoromethanesulfonated to obtain 2-propynyloxyethyl trifluoromethanesulfonate, which is then reacted with Rapa under the catalysis of DIPEA to generate Alkynyl-Rapa, which is then further subjected to a click reaction between Alkynyl-Rapa and azide-modified alkanoic acid under the catalysis of CuSO4 and sodium ascorbate, and then purified to obtain the final product.

[0035] A pharmaceutical preparation, comprising the rapamycin prodrug of the general formula; the preparation is an injection, liposome, nanoparticle, emulsion or microemulsion.

[0036] The preparation can be administered as an ophthalmic preparation, a nasal preparation, oral administration, pulmonary administration, ear administration, rectal administration, vaginal administration, intravenous or intramuscular injection or subcutaneous administration.

[0037] The pharmaceutical preparation is a self-assembled nanoparticle prepared from the rapamycin prodrug described in the general formula.

[0038] Furthermore, the prodrug is prepared with an auxiliary agent in the presence or absence of a stabilizer to form nanoparticles with a particle size ranging from 10 to 700 nm.

[0039] The stabilizer is a compound with an amphiphilic structure and surface activity, and the added amount thereof is 0.1% to 50% of the mass of the rapamycin prodrug compound.

[0040] The amphiphilic structure compound with surface activity includes but is not limited to DSPE functionalized phospholipids: distearoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine-polyethylene glycol, distearoyl phosphatidylethanolamine, methoxy polyethylene glycol and other DSPE functionalized phospholipids.

[0041] The above-mentioned nanoparticles can further continue to encapsulate drugs or antigens. The drugs can be drugs that inhibit or reduce the activation of T / B lymphocytes, such as vitamin D3, etc., or immunosuppressants. Immunosuppressants are drugs that have an inhibitory effect on the body's immune response and can inhibit the proliferation and function of cells related to the immune response (T cells and B cells, dendritic cells or macrophages, etc.), such as azathioprine, chlorambucil, cyclophosphamide, cyclosporine, mycophenolate mofetil and methotrexate, etc., or drugs that have the effect of blocking tumor necrosis factor, such as etanercept, infliximab, abatacept, rituximab and adalimumab, or lipid prodrug forms of macrolide analogs such as rapamycin, or some drugs or substances with positive immune regulation, such as diphenylcyclopropenone, etc.; nanoparticles can also encapsulate or modify related antigens and adjuvants. The antigens can be autoimmune disease-related antigens, and the adjuvants are substances that enhance or reduce the immune response.

[0042] A prodrug and its pharmaceutical preparation, the prodrug represented by the general formula or the pharmaceutical preparation is used to prevent or treat transplant rejection, autoimmune diseases, allergic reactions (such as food allergies), immune responses to therapeutic agents

[0043] Furthermore, rapamycin lipid prodrug nanoparticles can be further loaded with antigens, adjuvants, therapeutic or diagnostic agents, or (and) subjected to targeted modification, or (and) subjected to nanoparticle surface antigen modification, and can also be further loaded into nanoformulations as active ingredients for use in the prevention or treatment of transplant rejection, autoimmune diseases, allergic reactions (such as food allergies), and immune responses to therapeutic agents.

[0044] As discussed in more detail below, there are a variety of antigens that can be expected to be tolerated. These can include, but are not limited to, substances that cause an adverse immune response when a subject is exposed to an antigen or certain therapeutic drugs. In some embodiments, the adverse immune response can be the result of antigen ingestion (oral or nasal, or via some other mucosal route), as it can be the case with food antigens. In some embodiments, a therapeutic can be purposefully administered to a subject to treat a disease or condition that the subject suffers from. In additional embodiments, the antigen can be generated by the subject, such as an autoimmune antigen.

[0045] In one aspect, a method is provided, comprising providing a dose of a rapamycin prodrug or a pharmaceutical preparation thereof as described herein, wherein in certain embodiments, the rapamycin prodrug is attached to a synthetic nanocarrier to obtain a nanoformulation, and the nanoformulation is used as a drug to prevent or reduce transplant rejection in a subject, wherein the rapamycin prodrug is any compound described herein, wherein the transplant is selected from heart, lung, heart-lung or liver transplantation, pancreas transplantation, islet cell transplantation, small intestine transplantation, stem cell transplantation, other organ transplantation, etc. In one embodiment, the regimen can reduce infection, transplant rejection and other serious complications and even mortality when the immunosuppressant described herein is administered.

[0046] In one aspect, a method is provided, comprising providing a dose of a rapamycin prodrug or a pharmaceutical preparation thereof as described herein, wherein in certain embodiments, the rapamycin prodrug is attached to a synthetic nanocarrier to obtain a nanoformulation, and these nanocarriers can be further loaded with autoantigens by passive loading or surface modification methods, and can also be subjected to relevant targeted modifications, and used as a drug for the prevention and treatment of autoimmune diseases, wherein the rapamycin prodrug is any compound described herein, and the autoantigen is an antigen derived from the body against which an undesirable immune response can be formed.

[0047] The specific autoimmune disease states described above include, but are not limited to: acute necrotizing and hemorrhagic leukoencephalitis; Addison's disease; psoriatic arthritis; Alzheimer's disease; rheumatoid arthritis; autoimmune aplastic anemia; autoimmune hypophysitis; autoimmune gastritis; autoimmune polyendocrine syndrome type 1; bullous pemphigoid; celiac disease; coxsackie myocarditis; Duhring dermatitis herpetiformis; diabetes mellitus (type 1); epidermolysis bullosa acquisita; giant cell myocarditis; Goodpasture's syndrome; Graves' disease; Hashimoto's thyroiditis; mixed connective tissue disease; multiple sclerosis; myasthenia gravis; neuromyelitis optica; Pernicious angemis; pemphigus vulgaris and variants; pituitary autoimmune diseases; premature ovarian failure; rheumatic heart disease; systemic sclerosis; Sjögren's syndrome; primary biliary cirrhosis; polymyositis; primary sclerosing cholangitis; systemic lupus erythematosus; systemic sclerosis; ulcerative colitis; undifferentiated connective tissue disease; uveitis; vitiligo; Wegener's granulomatosis; psoriasis; rheumatic heart disease; alopecia areata; alopecia universalis; ankylosing spondylitis; juvenile arthritis; atopic dermatitis; autoimmune aplastic anemia.

[0048] Antigens associated with TID may be insulin, proinsulin, preproinsulin, gluten, gliadin, glutamic acid decarboxylase-65 (GAD-65 or glutamic acid decarboxylase 2), GAD-67, glucose-6-phosphatase 2 (IGRP or islet-specific glucose-6-phosphatase catalytic subunit-associated protein), insulinoma-associated protein 2 (IA-2), and insulinoma-associated protein 2β (IA-2B); other antigens include ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen 38, GLIMA 38, chromogranin-A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocarcinoma-entero-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, islet-specific glucose-6-phosphatase catalytic subunit-associated protein, and SST G protein-coupled receptors 1-5.

[0049] In autoimmune thyroid disease, the major antigens in autoimmune thyroid disease (including Hashimoto's thyroiditis and Graves' disease) include thyroglobulin (TG), thyroid peroxidase (TPO), and thyroid stimulating hormone receptor (TSHR); other antigens include sodium iodide transporter (symporter) (NIS) and megalin. In thyroid-associated eye and skin diseases, in addition to thyroid autoantigens (including TSHR), the antigen is insulin-like growth factor 1 receptor. In hypoparathyroidism, the major antigen is calcium-sensing receptor.

[0050] The major antigens in multiple sclerosis include myelin basic protein, myelin oligodendrocyte glycoprotein, and myelin proteolipoprotein.

[0051] In autoimmune hypophysitis, or pituitary autoimmune disease, the major antigens include pituitary-specific protein factor (PGSF) 1a and 2; another antigen is iodinated thyronine deiodinase type 2.

[0052] In rheumatoid arthritis, major antigens include type II collagen, immunoglobulin binding protein, the fragment crystallizable region of immunoglobulin G, double-stranded DNA, and native and citrullinated forms of proteins implicated in the pathology of rheumatoid arthritis, including fibrin / fibrinogen, vimentin, collagens I and II, and α-enolase.

[0053] In autoimmune gastritis, the major antigen is H+, K+-ATPase; in pernicious anemia, the major antigen is intrinsic factor.

[0054] In celiac disease, the major antigens are tissue transglutaminase and native and deamidated forms of gluten or gluten-like proteins, such as α-, γ-, and β-gliadins, glutenins, hordeins, secalin, and avenin. Although the major antigen in celiac disease is α-gliadin, α-gliadin is converted to be more immunogenic in vivo by deamidation by tissue glutaminase, which converts the glutamine of α-gliadin to glutamate. Thus, although α-gliadin is originally a foreign food antigen, it can be characterized as a self-antigen once it is modified in vivo to become more immunogenic.

[0055] In vitiligo, the main antigens are tyrosinase, tyrosinase-related proteins 1 and 2, and Pmel17.

[0056] In myasthenia gravis, the major antigen is the acetylcholine receptor.

[0057] In pemphigus vulgaris and variants, the major antigens are desmogleins 3, 1, and 4; other antigens include pemphigus annexin, desmoglein, plakoglobin, periplatin, desmoplakin, and acetylcholine receptor; in bullous pemphigoid, the major antigens include BP180 and BP230; other antigens include plectin and laminin 5; in Duling dermatitis herpetiformis, the major antigens include endomysial and tissue transglutaminase. In epidermolysis bullosa acquisita, the major antigen is collagen VII.

[0058] In systemic sclerosis, the major antigens include matrix metalloproteinases 1 and 3, the collagen-specific chaperone heat shock protein 47, fibrillin-1, and the PDGF receptor; others include Scl-70, U1RNP, Th / To, Ku, Jol, NAG-2, centromere proteins, topoisomerase I, nucleolar proteins, RNA polymerases I, II, and III, PM-Slc, nucleolar fibrillin, and B23.

[0059] In mixed connective tissue disease, the major antigen is UlsnRNP.

[0060] In Sjögren's syndrome, the major antigens are the nuclear antigens SS-A and SS-B; others include fdrin, poly(ADP-ribose) polymerase and topoisomerase, muscarinic receptors, and Fc-gamma receptor IIIb.

[0061] In systemic lupus erythematosus, the major antigens include nuclear proteins, including the "Smith antigen", SS-A, high mobility group box 1 (HMGB1), nucleosomes, histones, and double-stranded DNA (against which autoantibodies are generated during the disease process).

[0062] In Goodpasture's syndrome, the major antigens include glomerular basement membrane proteins, including collagen IV.

[0063] In type I autoimmune polyendocrine syndrome, antigens include aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine ​​sulfenic acid decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, hepatic P450 cytochromes P450 1A2 and 2A6, SOX-9, SOX-10, calcium sensing receptor protein, and type 1 interferon alpha, beta, and q.

[0064] In neuromyelitis optica, the major antigen is AQP4.

[0065] In uveitis, the major antigens include retinal S-antigen or S-arrestin and interphotoreceptor retinoid binding protein (IRBP) or retinol binding protein 3.

[0066] In one aspect, a method is provided, comprising providing a rapamycin prodrug or a prodrug and a dosage of a pharmaceutical preparation thereof as described in the present invention, wherein in certain embodiments, the rapamycin prodrug is attached to a synthetic nanocarrier to obtain a nanoformulation, and the nanoformulation is used as a drug for desensitization therapy, wherein the rapamycin prodrug is any compound described herein, and the desensitization antigen is a foreign antigen against which an undesirable immune response can be formed, such as a food antigen: conarachidin, allergen II, peanut agglutinin, blue bean protein from peanuts; major allergen / disease resistance protein homolog (Mald2), lipid transport protein precursor (Mald3), major allergen Mald 1.03D (Mald 1) from apples; α-lactalbumin (ALA), lactotransferrin from dairy products; actinidin (Act c1, Actd1), phytocystatin, thaumatin-like protein (Act d 2), kiwellin (Act d 1) from kiwi fruit; 5); ovomucin, ovalbumin, ovotransferrin and lysozyme from egg white; vitellin, apovitillin and vosvetin from egg yolk; 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transport protein (Sin a 3), inhibitory protein (Sin a 4) from mustard; inhibitory protein (Apig 4), high molecular weight glycoprotein (Api g 5) from celery; Pen a 1 allergen (Pen a 1), allergen Pen m 2 (Pen m 2), tropomyosin fast isoform (fast isoform) from shrimp; high molecular weight glutenin, low molecular weight glutenin, α-, γ- and Q-gliadins, hordein, secalin, and / or avenin from wheat and / or other cereals; major strawberry allergen Fra a 1-E (Fra a 1) from strawberry, inhibitory protein (Mus xp 1) from banana. In embodiments utilizing antigens against which an undesirable immune response is developed, such as food antigens, treatments may be provided for reactions to, for example: peanuts, apples, milk, egg whites, egg yolks, mustard greens, celery, shrimp, wheat (and other grains), strawberries, and bananas.Antigens may also be foreign antigens against which an undesirable immune response is developed (e.g., against animal, plant, and environmental antigens), and specific antigens may be, for example, cats, mice, dogs, horses, bees, dust, trees, and goldenrod, including the following proteins or peptides derived from weeds (including ragweed allergens amb a, 1, 2, 3, 5, and 6, and Amb t 5; Chenopodium album Che a 2 and 5; and other weed allergens Par j 1, 2, 3, and Paro 1), grasses (including major allergens Cynd 1, 7, and 12; Dacg 1, 2, and 5; Hol 1.01203; Lol p 1, 2, 3, 5, and 11; Mer a 1; Pha a 1; Poa p 1 and 5); pollen from ragweed and other weeds (including curly dock, lambs allergens, quarters, pigweed, plantain, sheep sorrel, and sagebrush), grasses (including Bermuda, Johnson, Kentucky, Orchard, Sweet vernal, and Timothy grass), and trees (including catalpa, elm, hickory, olive, pecan, sycamore, and walnut); dusts (including major allergens such as Der p 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 18, 20, 21, and 23 from the species Dermatophagoides pteronyssinus; and Der p 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 18, 20, 21, and 23 from the species Dermatophagoides farinae. farina), such as Derf1, 2, 3, 6, 7, 10, 11, 13, 14, 15, 16, 18, 22 and 24; from the species Blomia tropicalis, such as Blot1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 19, and 21; and the allergen Eur m2 from Euroglyphus maynei, Tyr p 13 from Tyrophagus putrescentiae, and the allergens Blag1, 2, and 4; from cockroach Per a1, 3, and 7.Pets (including cats, dogs, rodents, and farm animals, major cat allergens include Fel d 1 to 8, feline IgA, Bla g 2, and feline albumin; major dog allergens include Can f 1 to 6, and dog albumin; bee stings, including major allergens Api m 1 to 12; fungi, including allergens derived from the following species: Aspergillus and Penicillium species, and species Alternaria alternata, Davidiella tassiana, and Trichophyton rubrum.

[0067] The prodrugs in the above-mentioned pharmaceutical preparations can also be mixed with other drugs to prepare corresponding preparations, that is, in one embodiment, one or more of the above antigens and the rapamycin prodrug are co-loaded to obtain a nanoformulation; in another embodiment, one or more of the above antigens and the rapamycin prodrug described herein are respectively loaded into different nanoformulations; in another embodiment, one or more of the above antigens are co-administered with a loaded rapamycin prodrug nanoformulation; according to the embodiment, such methods involve administration before or after exposure to the antigen. In some embodiments, administration before exposure exerts a preventive effect, which in some embodiments substantially avoids or significantly reduces the immune response.

[0068] In one aspect, a method is provided, comprising providing a rapamycin prodrug or a prodrug and a pharmaceutical formulation thereof as described herein, wherein in certain embodiments, the rapamycin prodrug is attached to a nanocarrier to obtain a nanoformulation, and administering the therapeutic macromolecule and the synthetic nanoformulation to a subject simultaneously. In one embodiment, the simultaneous administration is performed according to a regimen that has been shown to reduce anti-therapeutic biopharmaceutical antibody responses and increase the efficacy of the therapeutic drug when administered simultaneously with an immunosuppressant, compared to the therapeutic drug administered without the immunosuppressant.

[0069] The therapeutic biopharmaceuticals described herein include, but are not limited to, therapeutic proteins and polynucleotides. In any one of the provided method or composition embodiments, the therapeutic protein is used for protein replacement or protein supplementation therapy. In another embodiment of any of the provided methods or compositions, the therapeutic macromolecule comprises an infusible or injectable therapeutic protein, enzyme, enzyme cofactor, hormone, blood or blood coagulation factor, cytokine, interferon, growth factor, monoclonal antibody, polyclonal antibody, or protein associated with Pompe disease.

[0070] In one embodiment, the present invention provides a method for treating autoimmune diseases, providing an effective amount of rapamycin prodrug self-assembled nanoparticles to obtain a nanoformulation for treating multiple sclerosis. In the example, an effective amount of rapamycin derivatives and myelin oligodendrocyte glycoprotein are also provided for co-assembly for treating multiple sclerosis.

[0071] In one embodiment, the present invention provides a method for treating alopecia areata (AA), which is a common autoimmune, scarless skin disease that usually causes hair loss on the scalp and other parts of the body (such as eyebrows, eyelashes, etc.), and sometimes even causes nail dents or roughness, which not only affects the patient's aesthetics, but also has a negative impact on the patient's mental health and quality of life. Alopecia areata can occur at any age and has no obvious gender differences, and its etiology is complex, related to genetic, immune, psychological and other factors. The prevalence in my country is about 0.27%, and the lifetime prevalence is about 2%, and it shows a trend of increasing year by year. This disease manifests as varying degrees of hair loss, from focal patchy shedding to the entire scalp and body hair shedding, such as alopecia totalis (AT) and alopecia universalis (AU). Although this disease has the nature of self-healing, it is still difficult to treat and has a high recurrence rate for some patients. In the treatment guidelines that have been published, local corticosteroid administration and contact immunotherapy are mainly recommended as first-line treatments for alopecia areata, depending on age, disease severity, etc. Although topical corticosteroid therapy is effective in the advanced stage, it has a large adverse reaction and is prone to recurrence when used for a long time, so it is difficult to treat severe alopecia areata. In the embodiment, a rapamycin prodrug self-assembled nanoparticle loaded with diphenylcyclopropenone is provided for treating alopecia areata.

[0072] In one embodiment, a rapamycin prodrug and an antigen co-loaded nanoparticle are provided for the treatment of celiac disease. Celiac disease (CD) is an autoimmune disease caused by abnormal sensitivity of the immune system to gluten. In patients with celiac disease, exposure to gluten leads to activation of gluten-specific T cells, ultimately leading to immune-mediated intestinal damage; CD often causes some gastrointestinal symptoms, which may be mild, not obvious, or even completely asymptomatic. People with gastrointestinal symptoms often have a certain degree of the following conditions: diarrhea, foul-smelling stools, abdominal discomfort, flatulence, bloating, mucus or fat droplets in the stool. At the same time, CD often has some non-gastrointestinal symptoms, including some subtle symptoms, such as fatigue, unintentional weight loss, joint pain, neurological symptoms: headache, peripheral neuropathy (tingling / numbness in the hands or feet), psychiatric symptoms: depression, anxiety, itchy rash and other complications. Because gluten-specific T cells are abnormally activated, ultimately leading to immune-mediated intestinal damage, treatment methods that make T cells tolerant to gluten have the potential to cure celiac disease.

[0073] In one embodiment, a method for manufacturing a drug intended to reduce the response of anti-therapeutic macromolecule antibodies is provided. In one embodiment, the drug includes an immunosuppressant and / or a therapeutic macromolecule, wherein the pharmacodynamically effective dose of the therapeutic macromolecule can be reduced. In another embodiment of any one of the manufacturing methods provided herein, the immunosuppressant is attached to a synthetic nanocarrier to obtain a nanoformulation.

[0074] A method is provided, including providing a therapeutic biopharmaceutical that causes or is expected to cause anti-therapeutic macromolecule antibodies after repeated administration in one or more subjects; and providing an immunosuppressant dose, wherein the immunosuppressant dose is attached to a nanocarrier to obtain a nanoformulation. In some embodiments, the method includes repeatedly administering a therapeutic macromolecule to a subject at the same dose or a lower dose while using an immunosuppressant. In some embodiments, the simultaneous administration regimen has been shown to maintain the pharmacodynamic effect of the therapeutic macromolecule at the pharmacodynamic effect of two or more doses of the therapeutic macromolecule on the subject.

[0075] In one embodiment of any of the methods provided above, the method further includes determining a regimen. In another embodiment of any of the methods provided, the method further includes determining a pharmacologically effective dose, such as a reduced or enhanced pharmacologically effective dose. In another embodiment of any of the methods provided, the method further includes evaluating the pharmacodynamic effect of the subject before and / or after administration. In another embodiment of any of the methods provided, simultaneous administration is repeated one or more times. In another embodiment of any of the methods described above, the administration is intravenous, intraperitoneal or subcutaneous. In another embodiment of any of the methods provided, the subject is at risk of an anti-therapeutic macromolecule antibody reaction. In another embodiment of any of the methods provided, the subject is a human or model animal in which an anti-therapeutic macromolecule antibody reaction is expected.

[0076] The advantages of the present invention are:

[0077] The rapamycin lipid prodrug and the nanomedicine derived therefrom of the present invention are used to treat or prevent transplant rejection, autoimmune diseases, food allergies and immune responses to therapeutic agents. Specifically, some embodiments disclosed herein relate to rapamycin prodrugs and nanomedicines derived therefrom, which deliver antigens or / and immunomodulatory drugs to be tolerated to one or more targeted cell types, thereby inducing the processing of antigens and inducing immune tolerance to antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 This is the ESI-MS (m / z) high-resolution mass spectrum of the rapamycin prodrug derivative of Example 1 of the present invention.

[0079] Figure 2 This is the ESI-MS (m / z) high-resolution mass spectrum of the rapamycin prodrug derivative of Example 2 of the present invention.

[0080] Figure 3 This is the ESI-MS (m / z) high-resolution mass spectrum of the rapamycin prodrug derivative of Example 3 of the present invention.

[0081] Figure 4 This is the ESI-MS (m / z) high-resolution mass spectrum of the rapamycin prodrug derivative of Example 4 of the present invention.

[0082] Figure 5 This is the ESI-MS (m / z) high-resolution mass spectrum of the rapamycin prodrug derivative of Example 5 of the present invention.

[0083] Figure 6 This is the ESI-MS (m / z) high-resolution mass spectrum of the rapamycin prodrug derivative of Example 6 of the present invention.

[0084] Figure 7 This is the ESI-MS (m / z) high-resolution mass spectrum of the rapamycin prodrug derivative of Example 7 of the present invention.

[0085] Figure 8 This is the ESI-MS (m / z) high-resolution mass spectrum of the rapamycin prodrug derivative of Example 8 of the present invention.

[0086] Fig. 9 This is the ESI-MS (m / z) high-resolution mass spectrum of the rapamycin prodrug derivative of Example 9 of the present invention.

[0087] Fig.10 This is the ESI-MS (m / z) high-resolution mass spectrum of the rapamycin prodrug derivative of Example 10 of the present invention.

[0088] Fig.11 This is the ESI-MS (m / z) high-resolution mass spectrum of the rapamycin prodrug derivative of Example 11 of the present invention.

[0089] Fig.12 This is the ESI-MS (m / z) high-resolution mass spectrum of the rapamycin prodrug derivative of Example 12 of the present invention.

[0090] Fig.13 This is the long-term stability of the self-assembled nanoparticles of the rapamycin prodrug derivative of Example 1 of the present invention at 4°C.

[0091] Fig.14 This is the long-term stability of the self-assembled nanoparticles of the rapamycin prodrug derivative of Example 2 of the present invention at 4°C.

[0092] Fig.15 This is the long-term stability of the self-assembled nanoparticles of the rapamycin prodrug derivative of Example 4 of the present invention at 4°C.

[0093] Fig.16 This is the long-term stability of the self-assembled nanoparticles of the rapamycin prodrug derivative of Example 7 of the present invention at 4°C.

[0094] Fig.17 This is a diagram for screening composite nanoparticle formulations using the rapamycin prodrug derivative prepared in Example 15.

[0095] Fig.18 This is the long-term stability of the composite nanoparticles of the rapamycin prodrug derivative prepared in Example 15 at 4°C.

[0096] Fig.19 This is a graph showing the titer of anti-KLH IgG antibodies using the self-assembled nanoparticles of Example 1, Example 2, Example 5, Example 7, Example 8, and Example 10 in combination with KLH.

[0097] Fig. 20 To explore the dose-effect relationship of Lipid-Rapa NPs in reducing the immunogenicity of KLH.

[0098] Figure 21-1 The figure shows the efficacy of using the rapamycin prodrug derivatives of Example 1 and Example 2 and loading diphenylcyclopropenone into self-assembled nanoparticles to treat hair growth in alopecia areata.

[0099] Figure 21-2 The figure shows the efficacy of using the rapamycin prodrug derivatives of Example 1 and Example 2 and loading diphenylcyclopropenone into self-assembled nanoparticles for the treatment of alopecia areata on hair growth length.

[0100] Figure 21-3 This is a figure showing the efficacy of using the rapamycin prodrug derivatives of Example 1 and Example 2 and loading diphenylcyclopropenone into self-assembled nanoparticles to treat dermal thickness changes in alopecia areata.

[0101] Figure 22-1 The dose-effect relationship of using the rapamycin prodrug derivative of Example 2 and loading diphenylcyclopropenone into self-assembled nanoparticles for the treatment of alopecia areata and hair growth.

[0102] Figure 22-2 The dose-effect relationship of hair growth length in treating alopecia areata was investigated by using the rapamycin prodrug derivative of Example 2 and loading diphenylcyclopropenone into self-assembled nanoparticles.

[0103] Figure 22-3The dose-effect relationship of dermal thickness change in the treatment of alopecia areata using the rapamycin prodrug derivative of Example 2 and loading diphenylcyclopropenone into self-assembled nanoparticles.

[0104] Figure 23-1 Figure 1 is a clinical scoring diagram of two nanoparticles for treating multiple sclerosis using the rapalogrug derivatives of Example 1 and Example 2 co-formulated with myelin oligodendrocyte glycoprotein and co-formulated with cysteine-modified myelin oligodendrocyte glycoprotein.

[0105] Figure 23-2 Figure 1 shows two nanoparticles prepared by co-combining the rapalogrug derivatives of Example 1 and Example 2 with myelin oligodendrocyte glycoprotein and co-combining the rapalogrug derivatives with cysteine-modified myelin oligodendrocyte glycoprotein for treating multiple sclerosis.

[0106] Fig.24 This is a figure showing the efficacy of the rapamycin prodrug derivative self-assembled nanoparticles of Example 12 for delayed hypersensitivity reactions. DETAILED DESCRIPTION

[0107] The present invention is further illustrated by the following examples, but the present invention is not limited to these examples. Any changes that can be conceived by those skilled in the art should fall within the scope of the present invention.

[0108] Embodiment 1:

[0109] (1R,2R,4S)-4-((2R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5 Synthesis of 2-(6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-Tetrahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azepan-3-yl)propyl)-2-methoxycyclohexyl 4-(decylamino)-4-oxobutanoate

[0110] Rapamycin (compound 1, 250 mg, 0.273 mmol), dihydrofuran-2,5-dione (125 mg, 1.24 mmol), Novozym 435 enzyme (10000 u / g, 1 mg) were added to the reaction bottle, and toluene (15 mL) was added. The reaction solution was heated to 45 ° C and reacted overnight. After the reaction was complete, it was filtered and the filter cake was washed with toluene three times (20 mL × 3). The filtrate was concentrated under reduced pressure and directly used for subsequent reactions without purification. The reaction obtained

[0111] 4-(((1R,2R,4S)-4-((2R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentahydro 1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetradecahydro-3H-23,27-epoxypyridinyl[2,1-c][1]oxacyclohexane-3-yl)propyl)-2-methoxycyclohexyl)oxy)-4-oxybutanoic acid (Compound 2)

[0112] Compound 2 (250 mg, 0.246 mmol), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU, 88.7 mg, 0.294 mmol) and N,N-diisopropylethylamine (30 ul, 0.196 mmol) were added to a reaction bottle, acetonitrile (15 mL) was added and stirred at 25°C for 2 h, acetonitrile was removed by vacuum concentration, 1-decylamine (61.2 mg, 0.39 mmol) and dichloromethane (15 mL) were added, and stirred at 25°C for 2 h. After the reaction was complete, the reaction was stopped and separated by reverse phase preparative chromatography (acetonitrile, 100%) to obtain the compound shown in the following structure of Example 1 (32.2 mg, yield: 51.2%). HRMS: C 65 H 104 N2O 15 Na for [M+Na] + ,calculated,1175.7334,found 1175.7360

[0113]

[0114] Embodiment 2:

[0115] (1R,2R,4S)-4-((2R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5, Synthesis of 6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-Tetrahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azepan-3-yl)propyl)-2-methoxycyclohexyl 4-(dodecylamino)-4-oxobutanoate

[0116] Referring to the synthesis method of Example 1, compound 2 and 1-dodecylamine were used as starting materials and obtained by the same synthesis method with a yield of (55.2%). HRMS: C 67 H 108 N2O 15 Na for [M+Na] + ,calculated,1203.7647,found1203.7662

[0117]

[0118] Embodiment 3:

[0119] (1R,2R,4S)-4-((2R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5, Synthesis of 6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-Tetrahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azepan-3-yl)propyl)-2-methoxycyclohexyl 4-(tetradecylamino)-4-oxobutanoate

[0120] Referring to the synthesis method of Example 1, compound 2 and 1-tetradecylamine were used as starting materials and obtained by the same synthesis method. Yield (51.3%). HRMS: C 69 H 112 N2O 15 Na for [M+Na] +,calculated,1231.7960,found1231.7953

[0121]

[0122] Embodiment 4:

[0123] (1R,2R,4S)-4-((2R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5, Synthesis of 6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-Tetrahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azepan-3-yl)propyl)-2-methoxycyclohexyl 4-(pentadecanylamino)-4-oxobutanoate

[0124] Referring to the synthesis method of Example 1, compound 2 and 1-pentadecanamine were used as starting materials and obtained by the same synthesis method. Yield (55.3%). HRMS: C 70 H 115 N2O 15 for[M+H] + ,calculated,1223.8297,found1223.8333

[0125]

[0126] Embodiment 5:

[0127] (1R,2R,4S)-4-((2R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5, Synthesis of 6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-Tetrahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azepan-3-yl)propyl)-2-methoxycyclohexyl 4-(hexadecylamino)-4-oxobutanoate

[0128] Referring to the synthesis method of Example 1, compound 2 and 1-hexadecylamine were used as starting materials and obtained by the same synthesis method. Yield (53.3%). HRMS: C 71 H 115 N2O 15 for[MH] - ,calculated,1235.8297,found1235.8275

[0129]

[0130] Example 6

[0131] (1R,2R,4S)-4-((2R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,1 Synthesis of 0,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-Tetrahydro-3H-23,27-epoxypyrido[2,1-c][1,4]oxaazahexacyclohexanedione-3-yl)propyl)-2-methoxycyclohexyl 4-((Z)-octadec-9-ene-1-amino)-4-oxobutanoate

[0132] Referring to the synthesis method of Example 1, compound 2 and 9-aminoheptadecane were used as starting materials and obtained by the same synthesis method. Yield (63.5%). HRMS: C 72 H 118 N2O 15 Na for [M+Na] + ,calculated,1273.843,found1273.84385

[0133]

[0134] Example 7

[0135] (1R,2R,4S)-4-((2R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5, Synthesis of 6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-Tetrahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azepan-3-yl)propyl)-2-methoxycyclohexyl 4-(octadecylamino)-4-oxobutanoate

[0136] Referring to the synthesis method of Example 1, compound 2 and 1-octadecylamine were used as starting materials and obtained by the same synthesis method. Yield (54.3%). HRMS: C 73 H 120 N2O 15 Na for [M+Na] + ,calculated,1287.8586,found1287.8590

[0137]

[0138] Example 8

[0139] (1R,2R,4S)-4-((2R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,1 Synthesis of 0,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-Tetrahydro-3H-23,27-epoxypyridinium[2,1-c][4][1]oxazacyclophenanthracenetrioxan-3-yl)propyl)-2-methoxycyclohexyl 4-((E)-octadec-9-en-1-ylamino)-4-oxobutanoate

[0140] Referring to the synthesis method of Example 1, compound 2 and cis-9-octadeceneamine were used as starting materials and obtained by the same synthesis method. Yield (54.3%). HRMS: C 73 H 119 N2O 15 for[M+H] +,calculated,1263.75,found1263.8554

[0141]

[0142] Example 9

[0143] 12-(4-((2-((1R,2R,4S)-4-((2R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6, Synthesis of 9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-Tetrahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azacyclotrialkyl-3-yl)propyl)-2-methoxycyclohexyl)oxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)dodecanoic acid

[0144] Weigh propargyl alcohol ethoxylate (1.50 g, 15 mmol) and dissolve it in dichloromethane (20 ml). Add 2,6-lutidine (2.44 ml, 21 mmol) and trifluoromethanesulfonic anhydride (3.15 ml, 18.7 mmol) at -50 °C under nitrogen protection, and heat to -10 °C and stir for 2 h. Wash the reaction solution three times with saturated NaCl solution, dry the upper layer with anhydrous sodium sulfate, filter and remove the solvent on a rotary evaporator, separate by silica gel column chromatography, and purify with n-hexane and ethyl acetate (9:1 (v / v)) as eluent to obtain product 1: 2-propynyloxyethyl trifluoromethanesulfonate;

[0145] Rapamycin (652 mg, 0.714 mmol) was weighed and dissolved in chloroform (1.5 ml), and trifluoromethanesulfonic acid-2-propynyloxyethyl ester (2.5 g, 5.35 mmol) and DIPEA (6.2 ml, 35.7 mmol) were added thereto under nitrogen protection at -10 °C, and the mixture was stirred at 60 °C for 1-2 h. After the reaction, ethyl acetate and H2O were used for extraction, and the organic layer was collected and washed with saturated NaCl solution 3 times, and then dried with anhydrous Na2SO4 overnight, and separated by silica gel column chromatography, and purified with n-hexane and ethyl acetate (2:1 (v / v)) as eluent to obtain product 2: 2-propynyloxyethyl-modified Rapa (Alkynyl-Rapa)

[0146] Alkynyl-Rapa (9 mg, 9.0 μmol) and azidomyristic acid (12-azidododecanoic acid) (2.18 mg, 9.0 μmol) were weighed and dissolved in methanol (1.0 ml), and 1M copper sulfate pentahydrate (60 μl, 0.060 mM) and sodium ascorbate (75 μl, 0.075 mM) were added and ultrasonically reacted at room temperature for 1-2 hours. After the reaction, the insoluble matter was removed by filtration with a 0.45 μm filter membrane, and finally purified to obtain the final product. The yield was 45.2%. HRMS: C 68 H 108 N4O 16 Na for [M+Na] + ,calculated,1259.7658,found 1259.7679

[0147]

[0148] Example 10

[0149] 15-(4-((2-((1R,2R,4S)-4-((2R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6 Synthesis of 1H-1,2,3-triazol-1-yl)pentadecanoic acid (9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetracarbon dihydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azepano[3-yl)propyl)-2-methoxycyclohexyl)oxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)pentadecanoic acid

[0150] Referring to the synthesis method of Example 9, 2:2-propynyloxyethylated Rapa (Alkynyl-Rapa) and palmitic acid azide (15-azidopentadecanoic acid) were used as starting materials and obtained by the same synthesis method. The yield was 50.3%. HRMS: C 71 H 114 N4O 16 Nafor[M+Na] + ,calculated,1301.8128,found 1301.8120

[0151]

[0152] Embodiment 11

[0153] (1R,2R,4S)-4-((2R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo- Synthesis of 1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-Tetrahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azepan-3-yl)propyl)-2-methoxycyclohexyl stearate.

[0154] Specifically, rapamycin (100 mg), vinyl stearate (300 mg) and Novozym435 enzyme (5 mg) were weighed and dissolved in 20 ml of toluene, and condensed and refluxed at 45°C for 36 hours under nitrogen protection. Then, the mixture was filtered, the solvent was rotary evaporated, and silica gel column chromatography was performed to purify the mixture to obtain a white powder. The yield was 70.2%. HRMS: C 69 H 113 NO 14 Na for [M+Na] + ,calculated,1202.805328,found 1202.80425

[0155]

[0156] Example 12

[0157] (1R,2R,4S)-4-((R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a- Synthesis of tetrahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azepan-3-yl)propyl)-2-methoxycyclohexyl 4-(hexadecylamino)-4-(2-(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxycarbonyl)amino)ethyl)amino)-4-oxobutanoate

[0158] Weigh cholesteryl chloroformate (0.449 g, 1 mmol) and dissolve it in dichloromethane (5 mL). Anhydrous ethylenediamine (0.93 mL, 14 mmol) is dissolved in dichloromethane (5 mL), added dropwise to the cholesteryl chloroformate solution at 0-5°C, and stirred overnight. The reaction solution is washed three times with a saturated NaCl solution and once with pure water. The system is layered, the lower layer is dried over anhydrous sodium sulfate, filtered, and the solvent is removed on a rotary evaporator to obtain (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]phenanthrene-3-yl(2-aminoethyl)carbamate (Compound 3)

[0159] Referring to the synthesis method of Example 1, Compound 2 and Compound 3 were used as starting materials and obtained by the same synthesis method with a yield of (45.5%). HRMS: C 85 .H 133 N3O 17 Na for [M+Na] + ,calculated,1490.952721,found1490.952291.

[0160]

[0161] Example 13

[0162] Self-assembled nanoparticles were prepared by a one-step nanoprecipitation method. 5 mg of the prodrug in Example 1 and DSPE-PEG 2K 1 mg was dissolved in 1 mL of methanol. Under stirring, the mixed solution of prodrug and stabilizer was slowly added dropwise to 5 mL of deionized water to self-assemble into nanoparticles. Stirring was continued for 10 min to make it evenly dispersed. The organic solvent was removed by rotary evaporation at 37 ° C, and the volume was fixed to 5 mL with deionized water to obtain rapamycin prodrug self-assembled nanoparticles with a concentration of 1 mg / mL.

[0163] Embodiment 14

[0164] POPC (2.5 mg), PHPC (1 mg), cholesterol (0.4 mg), tricaprylin (1 ml) and rapamycin stearic acid prodrug, i.e., Example 11 (0.5 mg), were dissolved in chloroform and combined in a 100 ml flask for rotary evaporation and drying. After being spun dry, the obtained film was redissolved in an acetonitrile / methanol solution (95:5 (v / v)), and a PBS solution was taken for standby use. Using a microfluidic device, the two solutions were injected into a mixer at the same time, the flow rate of the lipid solution was 1 ml / min, and the flow rate of PBS was 2 ml / min, washed and ultrafiltered, and filtered through a 0.22 μm microporous membrane filter to obtain a nanoemulsion. By adjusting the mixing flow rate and the ratio of the lipid solution to PBS, the particle size can be controlled at 20 to 300 nm.

[0165] Embodiment 15

[0166] 2 mg of the rapamycin prodrug derivative synthesized in Example 12, 10 mg of gliadin, and 30 mg of soybean lecithin were weighed and dissolved in 1 mL of 60% (v / v) ethanol solution. Subsequently, the mixture was slowly injected into deionized water at a volume ratio of 1:5 and stirred for 30 minutes to form nanoparticles. The residual ethanol in the suspension was removed by a rotary evaporator under vacuum at 37°C to obtain the finished nanobiological preparation (RCL@NPs).

[0167] The RCL@NPs obtained above were stored at 4°C, and samples were taken at 0, 1, 2, 3, 4, 5, 6, and 7 days, and the particle size distribution was measured using a Malvern particle size analyzer. Fig.18 The stability of the obtained nanoparticles has a great guiding significance for the effectiveness of the preparation. As can be seen from the figure, the nanoparticles have good stability when placed at 4°C.

[0168] Example 16

[0169] Nanoparticles were obtained by self-assembly of the prodrugs of Example 1, Example 2, Example 4, and Example 7 in the manner described in Example 13. The different nanoparticles were stored at 4°C and samples were taken at 0, 5, 10, 15, 20, and 25 days, and the particle size distribution was measured using a Malvern particle size analyzer. The physical stability of the nanoparticles is shown in the attached figure. Fig.13 , 14 , 15, 16. The stability of the obtained nanoparticles has a great guiding significance for the effectiveness of the preparation. It can be seen from the figure that the nanoparticles have good stability when placed at 4°C.

[0170] Embodiment 17

[0171] The optimal formulation of RCL@NPs prepared in Example 15 was screened by changing the amount of gliadin added. The formulation screening was:

[0172] According to the method for preparing RCL@NPs in Example 15, preparations with different amounts of gliadin were prepared, wherein the amounts of gliadin added were 2.5, 5.0, 7.5, 10, 12.5, and 15.0 mg, respectively. The particle sizes of RCL@NPs containing different amounts of gliadin were then measured. The results of the prescription screening are shown in the attached Fig.17 According to the changes in particle size and PDI, the optimal formulation was finally selected with a gliadin addition amount of 10 mg. The particle size result was 181.4±6.69nm and the PDI was 0.153±0.043.

[0173] Embodiment 18

[0174] In order to evaluate the ability of the prodrug compounds (Lipid-Rapa NPs) of different general formulae prepared in the above examples to induce immune tolerance to free antigens administered simultaneously, BALB / c mice were administered once a week, and were intravenously injected with highly immunogenic hemocyanin (KLH) (dosage of 200 μg) and Lipid-Rapa NPs (rapamycin equivalent dose of 50 μg) three times, and then stimulated repeatedly with KLH, administered once a week, and stimulated three times in total. The anti-KLH antibody levels in mice after administration were investigated, and the ability of nanoparticles based on different Lipid-Rapa derivatives to inhibit ADA formation was analyzed, and Lipid-Rapa NPs with stronger tolerance induction ability were screened out (see Fig.19 ). As shown in the figure, compared with the positive control group, Lipid-Rapa NPs had the effect of inhibiting the production of anti-KLH antibodies.

[0175] The groups were as follows: KLH group (each mouse was given 200 μg KLH); RAPA-C 10：0 NPs group (each mouse was given 50 μg of rapamycin equivalent dose of RAPA-C 10：0NPs); RAPA-C 12：0 NPs group (each mouse was given 50 μg of rapamycin equivalent dose of RAPA-C 12：0 NPs); RAPA-C 16：0 NPs group (each mouse was given 50 μg of rapamycin equivalent dose of RAPA-C 16：0 NPs); RAPA-C 18：0 NPs group (each mouse was given 50 μg of rapamycin equivalent dose of RAPA-C 18：0 NPs); RAPA-C 18：1 NPs group (each mouse was given 50 μg rapamycin equivalent dose; RAPA-C 18：1 NPs); RAPA-C 15：0 -COOH NPs group (each mouse was given 50 μg of RAPA-C equivalent to rapamycin) 15：0 -COOH NPs).

[0176] RAPA-C 10：0 NPs were prepared as follows: 5.0 mg RAPA-C was accurately weighed 10：0 The compound of Example 1 was dissolved in 5 mL of methanol. 10：0 and DSPE-PEG 2000 The mixed solution was slowly added dropwise to 5 mL of deionized water to self-assemble into nanoparticles. Stirring was continued for 10 min to make them dispersed evenly. The organic solvent was removed by rotary evaporation at 37 °C and the volume was fixed to 5 mL with deionized water to obtain RAPA-C with a concentration of 1 mg / mL. 10：0 NPs.

[0177] RAPA-C 12：0 NPs were prepared as follows: 5.0 mg RAPA-C was accurately weighed 12：0 The compound of Example 2 was dissolved in 5 mL of methanol. 12：0 and DSPE-PEG 2000 The mixed solution was slowly added dropwise to 5 mL of deionized water to self-assemble into nanoparticles. Stirring was continued for 10 min to make them dispersed evenly. The organic solvent was removed by rotary evaporation at 37 °C and the volume was fixed to 5 mL with deionized water to obtain RAPA-C with a concentration of 1 mg / mL. 12：0 NPs.

[0178] RAPA-C 16：0 NPs were prepared as follows: 5.0 mg RAPA-C was accurately weighed 16：0 The compound of Example 5 was dissolved in 5 mL of methanol.16：0 and DSPE-PEG 2000 The mixed solution was slowly added dropwise to 5 mL of deionized water to self-assemble into nanoparticles. Stirring was continued for 10 min to make them dispersed evenly. The organic solvent was removed by rotary evaporation at 37 °C and the volume was fixed to 5 mL with deionized water to obtain RAPA-C with a concentration of 1 mg / mL. 16：0 NPs.

[0179] RAPA-C 18：0 NPs were prepared as follows: 5.0 mg RAPA-C was accurately weighed 18：0 The compound of Example 7 was dissolved in 5 mL of methanol. 18：0 and DSPE-PEG 2000 The mixed solution was slowly added dropwise to 5 mL of deionized water to self-assemble into nanoparticles. Stirring was continued for 10 min to make them dispersed evenly. The organic solvent was removed by rotary evaporation at 37 °C and the volume was fixed to 5 mL with deionized water to obtain RAPA-C with a concentration of 1 mg / mL. 18：0 NPs.

[0180] RAPA-C 18：1 NPs were prepared as follows: 5.0 mg RAPA-C was accurately weighed 18：1 The compound of Example 8 was dissolved in 5 mL of methanol. 18：1 and DSPE-PEG 2000 The mixed solution was slowly added dropwise to 5 mL of deionized water to self-assemble into nanoparticles. Stirring was continued for 10 min to make them dispersed evenly. The organic solvent was removed by rotary evaporation at 37 °C and the volume was fixed to 5 mL with deionized water to obtain RAPA-C with a concentration of 1 mg / mL. 18：1 NPs.

[0181] RAPA-C 15：0 The preparation of -COOH NPs was as follows: 5.0 mg RAPA-C 15：0 -COOH, the compound of Example 10, was dissolved in 5 mL of methanol. 15：0 -COOH and DSPE-PEG 2000 The mixed solution was slowly added dropwise to 5 mL of deionized water to self-assemble into nanoparticles. Stirring was continued for 10 min to make them dispersed evenly. The organic solvent was removed by rotary evaporation at 37 °C and the volume was fixed to 5 mL with deionized water to obtain RAPA-C with a concentration of 1 mg / mL. 15：0 -COOH NPs.

[0182] Embodiment 19

[0183] To investigate the dose-effect relationship of Lipid-Rapa NPs in reducing the immunogenicity of KLH, four different doses were injected and administered to BALB / c mice on days 0, 7, and 14, respectively. The mice were stimulated with free KLH and the anti-KLH antibody titer in the mice was measured by ELISA to investigate the dose-effect relationship of Lipid-Rapa NPs (see Fig. 20 ). The results showed that the therapeutic effect of Lipid-RapaNPs was dose-dependent. Compared with the low-dose treatment group, the high-dose treatment group had a significant advantage and showed a stronger inhibitory effect on the production of anti-KLH antibodies.

[0184] The groups were as follows: KLH group (each mouse was given 200 μg KLH); C18-15 μg group (each mouse was given 15 μg rapamycin equivalent dose of RAPA-C 18：0 NPs); C18-50μg group (each mouse was given 50μg rapamycin-equivalent dose of RAPA-C 18：0 NPs); C18-100μg group (each mouse was given 100μg rapamycin-equivalent dose of RAPA-C 18：0 NPs); C18-200 μg group (each mouse was given 200 μg rapamycin equivalent dose of RAPA-C 18：0 NPs); C12-15μg group (each mouse was given 15μg rapamycin equivalent dose of RAPA-C 12：0 NPs); C12-50μg group (each mouse was given 50μg rapamycin-equivalent dose of RAPA-C 12：0 NPs); C12-100 μg group (each mouse was given 100 μg rapamycin-equivalent dose of RAPA-C 12：0 NPs); C12-200 μg group (each mouse was given 200 μg rapamycin-equivalent dose of RAPA-C 12：0 NPs).

[0185] RAPA-C 12：0 NPs were prepared as follows: 5.0 mg RAPA-C was accurately weighed 12：0 The compound of Example 2 was dissolved in 5 mL of methanol. 12：0 and DSPE-PEG 2000 The mixed solution was slowly added dropwise to 5 mL of deionized water to self-assemble into nanoparticles. Stirring was continued for 10 min to make them dispersed evenly. The organic solvent was removed by rotary evaporation at 37 °C and the volume was fixed to 5 mL with deionized water to obtain RAPA-C with a concentration of 1 mg / mL. 12：0 NPs.

[0186] RAPA-C 18：0 NPs were prepared as follows: 5.0 mg RAPA-C was accurately weighed 18：0 The compound of Example 7 was dissolved in 5 mL of methanol. 18：0 and DSPE-PEG 2000 The mixed solution was slowly added dropwise to 5 mL of deionized water to self-assemble into nanoparticles. Stirring was continued for 10 min to make them dispersed evenly. The organic solvent was removed by rotary evaporation at 37 °C and the volume was fixed to 5 mL with deionized water to obtain RAPA-C with a concentration of 1 mg / mL. 18：0 NPs.

[0187] Embodiment 20

[0188] Thirty healthy female C57BL / 6 mice aged 8.5 weeks were selected, weighing 18-20g. At this time, the hair of the mice is in the resting phase of the hair growth cycle. On the 0th day of the experiment, the hair on the back of the mice was removed with a pet shaver. On the 1st, 3rd, 5th and 7th days after the hair on the back of the mice was shaved, the therapeutic drug was injected subcutaneously every other day for a total of 4 times.

[0189] The treatment experimental groups are as follows: low dose (LD), medium dose (MD), and high dose (HD) of the rapamycin prodrug and diphenylcyclopropenone co-assembled nanoparticle groups of Example 1 and Example 2 (the rapamycin equivalent dosage is 0.05 mg / kg (LD), 0.5 mg / kg (MD), and 2.5 mg / kg (HD), respectively).

[0190] Co-assembly of rapamycin prodrug and diphenylcyclopropenone (DPCP) into nanoparticles DPCP / RAPA-C 10 The preparation of @NPs was as follows: 5.0 mg of rapamycin prodrug C was accurately weighed 10 The compound of Example 1 was dissolved in 5 mL of methanol. 5 mg of diphenylcyclopropenone DPCP was dissolved in 1 mL of acetone. The DPCP solution and the prodrug, DSPE-mPEG 2000 The mixed solution was added dropwise to 10 mL of deionized water to self-assemble into nanoparticles. Stirring was continued for 10 min to make it dispersed evenly. The organic solvent was removed by rotary evaporation at 37 °C and the volume was fixed to 10 mL with deionized water to obtain rapamycin prodrug and diphenylcyclopropenone co-assembled nanoparticles DPCP / RAPA-C with a concentration of 0.5 mg / mL. 10 @NPs. Rapamycin prodrug and diphenylcyclopropenone co-assembled nanoparticles DPCP / RAPA-C 12 The preparation of @NPs was as follows: 5.0 mg of rapamycin prodrug C was accurately weighed 12The compound of Example 2 was dissolved in 5 mL of methanol. 5 mg of diphenylcyclopropenone DPCP was dissolved in 1 mL of acetone. The DPCP solution and the prodrug, DSPE-mPEG 2000 The mixed solution was added dropwise to 10 mL of deionized water to self-assemble into nanoparticles. Stirring was continued for 10 min to make it dispersed evenly. The organic solvent was removed by rotary evaporation at 37 °C and the volume was fixed to 10 mL with deionized water to obtain rapamycin prodrug and diphenylcyclopropenone co-assembled nanoparticles DPCP / RAPA-C with a concentration of 0.5 mg / mL. 12 @NPs.

[0191] From the beginning of drug administration, the treated side of the mice was compared with the non-treated side, and photos were taken regularly to record the hair growth and skin color changes of the mice. On the last day of observation, hair was randomly plucked from the shaved back area of ​​the mice. After 10 hairs were plucked from each mouse, the length of the hair was measured with a vernier caliper, and the results were expressed as mean ± SD. Tissue sections were used to observe the tissue thickness of the drug-treated area. Hair growth is shown in the attached Figure 21-1 , hair growth length see attached Figure 21-2 , the changes in dermal thickness are shown in the attached Figure 21-3 .

[0192] The results are as follows Figure 21-1 and Figure 21-2 As shown in the figure, under the treatment of each preparation, the low-dose rapamycin prodrug (0.05 mg / kg) of Example 2 and the prodrug nanoparticles co-assembled with diphenylcyclopropenone have obvious advantages in hair growth length and grayscale, and have shown obvious hair growth on the 18th day, which has a unique therapeutic advantage. Figure 21-3 As shown, the skin thickness under low-dose rapamycin prodrug (0.05 mg / kg) and diphenylcyclopropenone treatment was generally higher than the skin thickness when other treatment groups were applied. Taken together, these data imply that low-dose rapamycin prodrug (0.05 mg / kg) and diphenylcyclopropenone co-assembled nanoparticles effectively enhance hair regeneration by accelerating the transition from telogen to growth phase without damaging the hair shaft or epidermis.

[0193] Embodiment 21

[0194] Thirty healthy female C57BL / 6 mice aged 8.5 weeks were selected, weighing 18-20g. At this time, the hair of the mice is in the resting phase of the hair growth cycle. On the 0th day of the experiment, the hair on the back of the mice was removed with a pet shaver. On the 1st, 3rd, 5th and 7th days after the hair on the back of the mice was shaved, the therapeutic drug was injected subcutaneously every other day for a total of 4 times.

[0195] The treatment experimental groups are as follows: low dose (LD), medium dose (MD), high dose (HD) of the rapamycin prodrug self-assembled nanoparticle group of Example 2 and the rapamycin prodrug and diphenylcyclopropenone co-assembled nanoparticle group of Example 2 (the rapamycin equivalent dosages are 0.0005 mg / kg, 0.005 mg / kg and 0.05 mg / kg, respectively).

[0196] Rapamycin prodrug C 12 The preparation of @NPs was as follows: 5.0 mg of rapamycin prodrug C was accurately weighed 12 The compound of Example 2 was dissolved in 5 mL of methanol. 2K The mixed solution was slowly added dropwise to 10 mL of deionized water to self-assemble into nanoparticles. Stirring was continued for 10 min to make them dispersed evenly. The organic solvent was removed by rotary evaporation at 37 °C and the volume was fixed to 10 mL with deionized water to obtain rapamycin C with a concentration of 0.5 mg / mL. 12 @NPs. Rapamycin prodrug and diphenylcyclopropenone co-assembled nanoparticles DPCP / RAPA-C 12 The preparation of @NPs was as follows: 5.0 mg of rapamycin prodrug C was accurately weighed 12 The compound of Example 2 was dissolved in 5 mL of methanol. 5 mg of diphenylcyclopropenone DPCP was dissolved in 1 mL of acetone. The DPCP solution and the prodrug, DSPE-mPEG 2K The mixed solution was added dropwise to 10 mL of deionized water to self-assemble into nanoparticles. Stirring was continued for 10 min to make it dispersed evenly. The organic solvent was removed by rotary evaporation at 37 °C and the volume was fixed to 10 mL with deionized water to obtain rapamycin prodrug and diphenylcyclopropenone co-assembled nanoparticles DPCP / RAPA-C with a concentration of 0.5 mg / mL. 12 @NPs.

[0197] From the beginning of drug administration, the treated side of the mice was compared with the non-treated side, and photos were taken regularly to record the hair growth and skin color changes of the mice. On the last day of observation, hair was randomly plucked from the shaved back area of ​​the mice. After 10 hairs were plucked from each mouse, the length of the hair was measured with a vernier caliper, and the results were expressed as mean ± SD. Tissue sections were used to observe the tissue thickness of the drug-treated area. Hair growth is shown in the attached Figure 22-1 , hair growth length see attached Figure 22-2 , the changes in dermal thickness are shown in the attached Figure 22-3 .

[0198] The results are as follows Figure 22-1 and Figure 22-2As shown in the figure, under the treatment of each preparation, the high-dose rapamycin prodrug (0.05 mg / kg) self-assembled nanoparticle group of Example 2 and the prodrug nanoparticles co-assembled with rapamycin prodrug (0.05 mg / kg) and diphenylcyclopropenone have obvious advantages in terms of hair growth length. Compared with the prodrug nanoparticles co-assembled with rapamycin prodrug and diphenylcyclopropenone and the rapamycin prodrug self-assembled nanoparticle group, the co-assembled nanoparticles can promote hair growth better. The results are shown in Figure 22-3 As shown, the skin thickness treated with high-dose rapamycin prodrug (0.05 mg / kg) of Example 2 and diphenylcyclopropenone co-assembled prodrug nanoparticles was significantly higher than that of other groups, indicating that the co-assembled nanoparticles can effectively enhance hair regeneration by accelerating the transition from the resting phase to the growth phase.

[0199] Embodiment 22

[0200] Healthy C57BL / 6 mice (female, 6 weeks old, weighing 18-20 g) were selected for modeling. The modeling method was as follows: MOG 35-55 Dilute with 0.9% saline to 5 mg / mL, take 0.5 mL MOG 35-55 The solution was thoroughly mixed and emulsified with 0.5 mL of complete Freund's adjuvant (CFA, H37Ra concentration was 5 mg / mL). The specific operation was as follows: under stirring conditions, the polypeptide solution was slowly added drop by drop into CFA, and stirred at a low speed in the dark at 4°C for 4 hours to obtain a viscous milky white emulsion. If it did not disperse after being dropped into water for 10 minutes, it was considered to be an oil-in-water emulsion. Pertussis toxin (PTX) was diluted with water to a mother liquor with a concentration of 100 μg / mL, and 25 μL was taken and diluted to 2.5 μg / mL with normal saline. After C57BL / 6 mice were anesthetized with 4% chloral hydrate intraperitoneally (0.1 mL / 10 g), MOG was subcutaneously injected at 4 points in the neck, tail, axilla, and groin of the mice. 35-55 A total of 0.1 mL of the emulsion was used, and 0.2 mL of PTX solution (500 ng) was injected intraperitoneally twice on the day of immunization (0 h) and the second day (48 h). On the 7th day, the mice were immunized for the second time according to the same method. Kono's 5-point method was used to clinically score the mice: 0 points for no disease; 1 point for tail weakness; 2 points for mild hind limb weakness; 3 points for severe hind limb paralysis; 4 points for quadriplegia; 5 points for dying or death. Symptoms between the two were calculated as 0.5 points. The behavioral state of the mice was observed and the weight of the mice was recorded every day, and the neurological function was scored.

[0201] The treatment experimental groups were as follows: control group (healthy mice), EAE model group (mice were subjected to EAE model), rapamycin prodrug C 12 @NPs group, rapamycin prodrug C 12 / MOG 35-55@NPs group, rapamycin prodrug C 12 / Cys-MOG 35-55 @NPs group, free MOG 35-55 Group and free MOG 35-55 Co-administration with free rapamycin.

[0202] Each mouse in the control group was not given any medication; each mouse in the EAE model group was subjected to EAE modeling; rapamycin prodrug C 12 @NPs group, the administration volume was 129 μl per mouse, and the dosage of rapamycin was 100 ug / mouse (2.5 mg / kg); rapamycin prodrug C 12 / MOG 35-55 @NPs was administered in a volume of 129ul per mouse, rapamycin was administered in a dose of 100ug / mouse (2.5mg / kg), MOG 35-55 The dosage is 3-5 μg / mouse; rapamycin prodrug C 12 / Cys-MOG 35-55 @NPs group, the administration volume for each mouse was 129 μl, the dosage of rapamycin was 100 ug / mouse (2.5 mg / kg), MOG 35-55 The dosage of MOG is 3-5 μg / mouse. 35-55 The dosage was 12.9 μg / mouse: free MOG 35-55 Each mouse in the group was given 129ul of MOG. 35-55 The dosage was 12.9 μg / mouse; free MOG 35-55 The volume of rapamycin administered to each mouse was 129ul, and the dose of rapamycin was 100ug / mouse (2.5mg / kg). 35-55 The dosage was 12.9 μg / mouse.

[0203] Rapamycin prodrug C 12 The preparation of @NPs was as follows: 5.0 mg of rapamycin prodrug C was accurately weighed 12 The compound of Example 2 was dissolved in 5 mL of methanol. 2K The mixed solution was slowly added dropwise to 5 mL of deionized water to self-assemble into nanoparticles. Stirring was continued for 10 min to make them dispersed evenly. The organic solvent was removed by rotary evaporation at 37 °C and the volume was fixed to 5 mL with deionized water to obtain rapamycin C with a concentration of 1 mg / mL. 12 @NPs. Rapamycin prodrug C 12 / MOG 35-55 The preparation of @NPs was as follows: 5.0 mg of rapamycin prodrug C was accurately weighed 12The compound of Example 2 was dissolved in 5 mL of methanol. The antigen peptide MOG was added to deionized water. 35-55 , under stirring, the prodrug and DSPE-PEG 2K The mixed solution was slowly added dropwise to 5 mL of deionized water containing antigenic peptides to self-assemble into nanoparticles. Stirring was continued for 10 min to make them dispersed evenly. The organic solvent was removed by rotary evaporation at 37 °C, free antigenic peptides were removed by ultrafiltration centrifugation, and the volume was fixed to 5 mL with deionized water to obtain rapamycin prodrug C with a concentration of 1 mg / mL. 12 @NPs. Rapamycin prodrug C 12 / Cys-MOG 35-55 The preparation of @NPs was as follows: 5.0 mg of rapamycin prodrug C was accurately weighed 12 The compound of Example 2 was dissolved in 5 mL of methanol. 2K , DSPE-PEG 2K -MAL (distearoylphosphatidylethanolamine-polyethylene glycol-maleimide) mixed solution (DSPE-PEG 2K :DSPE-PEG 2K -MAL mass ratio of 1:1) was slowly added to 5 mL of PBS solution to self-assemble into nanoparticles, and stirred for 10 min to make them evenly dispersed. The organic solvent was removed by rotary evaporation at 37 °C, and then Cys-MOG 35-55 (the antigen peptide sequence is Cys-Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys) was slowly added dropwise to the above nanoparticles, stirred at room temperature for 2 hours to make it evenly dispersed, and then fixed to 5 ml with PBS solution to obtain rapamycin prodrug C with a concentration of 1 mg / mL. 12 / Cys-MOG 35-55 @NPs. Starting from the first day of modeling, subcutaneous injection was performed on the 11th, 14th, 17th, and 19th days after modeling, for a total of 4 times. The body weight and clinical scores of mice in different groups were recorded every day after modeling. Clinical scores are shown in the attached Figure 23-1 , see attached for weight Figure 23-2 .

[0204] Embodiment 24

[0205] Female C57BL / 6 mice (6-7 weeks old) were subcutaneously inoculated with 100 μL of an emulsion containing 200 μg Mycobacterium tuberculosis H37Ra and 100 μg of gliadin, distributed at three locations on the lateral wall. Gliadin was reconstituted from lyophilized powder in 50 mM acetic acid (5 mg / ml). This gliadin solution was then further diluted with PBS solution. Mice were intravenously injected with different nanoparticle formulations on days 0 and 7, respectively. Delayed-type hypersensitivity reactions were detected on day 14 after initiation. Mice were anesthetized by inhalation of isoflurane, and gliadin or negative control OVA solution (10 μL, 1 mg / mL) in PBS was immediately injected intradermally into the left and right ears after the auricle thickness was assessed with a caliper. The increase in auricle thickness was measured 24 hours later, and the change in auricle thickness (ΔT) was calculated as follows: ΔT = (auricle thickness 24 hours after induction) - (auricle thickness before induction) (see Fig.24 ).

[0206] The experimental groups are as follows: mice were given three preparations after immunization with gliadin: (1) free protein and rapamycin (Free Rapa+Glia), (2) nanoformulations without rapamycin prodrug (GL@NPs), and (3) RGL@NPs. The preparation method of Free Rapa+Glia is as follows: weigh appropriate amounts of rapamycin and gliadin, dissolve them in DMSO to 2 mg / mL, and further dilute them with PBS solution to the corresponding concentration to obtain the final Free Rapa+Glia group. The preparation method of GL@NPs is as follows: dissolve a mixture of gliadin and soybean lecithin in a 60% (v / v) ethanol solution at a mass ratio of 1:3. Then, the mixture is slowly injected into deionized water at a volume ratio of 1:5 and stirred for 30 minutes to form a particle suspension. The residual ethanol in the suspension is removed by a rotary evaporator under vacuum conditions at 37°C to finally obtain the finished nanobiological preparation CL@NPs. The preparation method of RGL@NPs is shown in Example 15.

[0207] Fig.24 It can be seen that the three preparations all alleviated the swelling of the auricle to varying degrees. Among them, there was no significant change in the swelling of the ear in the group of restimulated auricle with OVA solution, which proved that RGL@NPs specifically induced immunity to gliadin. Secondly, the nanoparticle group containing rapamycin and gliadin had the most obvious inhibitory effect on the swelling of the auricle after restimulation. Compared with the free drug group and the rapamycin nanoformulation group, it was proved that the rapamycin-gliadin-phosphatidylcholine composite nanoformulation prepared in this example performed best in inducing the body's immune tolerance to gliadin, which has certain guiding significance. In the future, it can be further considered to verify the efficacy of this nanodrug system in a systemic model of celiac disease and explore its potential physiological and pharmacological mechanisms, laying a theoretical foundation for the development of new strategies for the clinical diagnosis and treatment of celiac disease.

Claims

1. A rapamycin prodrug, characterized in that: Rapamycin prodrugs are 2. A pharmaceutical preparation, characterized in that: The pharmaceutical preparation contains the rapamycin prodrug according to claim 1; the preparation is nanoparticles or emulsions.

3. The pharmaceutical preparation according to claim 2, characterized in that: The pharmaceutical preparation is self-assembled nanoparticles prepared from the rapamycin prodrug according to claim 1.

4. The pharmaceutical preparation according to claim 3, characterized in that: The prodrug according to claim 1 is prepared by using an auxiliary agent in the presence of a stabilizer or in the absence of a stabilizer to form nanoparticles with a particle size ranging from 10 to 700 nm.

5. The pharmaceutical preparation according to claim 4, characterized in that: The stabilizer is a compound with an amphiphilic structure having surface activity, and the added amount thereof is 0.1%-50% of the mass of the prodrug according to claim 1.

6. A use of the rapamycin prodrug according to claim 1, characterized in that: Use of the rapamycin prodrug according to claim 1 in the preparation of drugs for preventing or treating transplant rejection, autoimmune diseases, allergies, and immune responses to therapeutic agents.

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