Pathway modulator, pharmaceutical composition having same, use thereof, and therapeutic method using same

Abstract

A pathway modulator, a pharmaceutical composition containing the same, uses thereof and a treatment method using the same. The pathway modulator (including one or more of dopamine beta-hydroxylase inhibitors, receptor agonists and receptor antagonists) can inhibit the occurrence and development of autoimmune diseases through immunomodulation, and thus can provide a potential therapeutic drug for the treatment of autoimmune diseases.

Description

[0001] The present disclosure claims priority to Chinese patent application (application number 2021101034359) filed on January 26, 2021. TECHNICAL FIELD

[0002] The present disclosure relates to pathway modulators, pharmaceutical compositions containing the same, uses thereof, and therapeutic methods employing the same. BACKGROUND

[0003] Dopamine beta-hydroxylase (hereinafter DBH), also known as dopamine beta-monooxygenase, is an enzyme (EC 1.14.17.1) encoded in humans by the DBH gene. DBH catalyzes the oxidation of dopamine by oxygen to norepinephrine and epinephrine, as shown below:

[0004]

[0005] DBH is a copper-containing oxygenase of about 290 kDa, composed of four identical subunits, whose activity requires ascorbic acid as a cofactor [1].

[0006] DBH is the only enzyme involved in the synthesis of small-molecule membrane-bound neurotransmitters, which makes norepinephrine the only transmitter synthesized within vesicles. Norepinephrine is expressed in noradrenergic nerve terminals of the central and peripheral nervous system, as well as in chromaffin cells of the adrenal medulla.

[0007] DBH mainly contributes to the biosynthesis of trace amines and catecholamines, including epinephrine (adrenaline), norepinephrine (noradrenaline), and dopamine. In addition, it is involved in the metabolism of exogenous biological substances related to these substances. For example, human DBH catalyzes the beta-hydroxylation of amphetamines and para-hydroxyamphetamines, resulting in norpseudoephedrine and para-hydroxynorsedrine, respectively [2-4].

[0008] DBH is considered to be associated with conditions related to thought decision and drug addiction, such as alcoholism [5] and smoking [6], attention deficit hyperactivity disorder [7], schizophrenia [8], and Alzheimer's disease [9]. While DBH deficiency is known as dopamine beta-hydroxylase deficiency.

[0009] In the late 20th and early 21st centuries, Nepicastat

[10] , Etamicastat

[11] , and Zamicastat

[12] were developed as highly selective DBH inhibitors. Further studies were conducted on their potential use in Pulmonary Arterial Hypertension, congestive heart failure, cocaine dependence, and post-traumatic stress disorder (PTSD).

[0010] Nepicastat was potentially used for the treatment of congestive heart failure and showed good tolerability in patients

[13] . Further clinical study data showed that Nepicastat was not effective in treating heart failure but was safe.

[0011] Nepicastat and its analogs (e.g., Etamicastat and Zamicastat) have potential use in treating hypertension. Clinical trials evaluating Nepicastat for treating PTSD and cocaine dependence have been completed. The studies found that Nepicastat was safe and could inhibit the positive effects of cocaine when used in combination with cocaine, suggesting that Nepicastat could be used as a pharmacotherapy for treating cocaine dependence

[14] .

[0012] Compared with Nepicastat, Etamicastat and Zamicastat have lower brain-blood barrier penetration levels and can reduce norepinephrine levels in peripheral sympathetic innervated tissues without affecting the brain tissue of spontaneously hypertensive rats

[15] . This result suggests that DBH inhibitors such as Etamicastat and Zamicastat can reduce adverse reactions or complications in the central nervous system when used to treat peripheral-related diseases. In a phase II clinical study, Etamicastat (200 mg) was observed to dose-dependently reduce systolic and diastolic blood pressure after 10 days of treatment and showed good tolerability and safety

[16] . Zamicastat (1200 mg) also showed good safety when used for 10 consecutive days (NCT02151994). Currently, a clinical trial of Zamicastat for treating Pulmonary Arterial Hypertension is being conducted (NCT04316143).

[0013] The role of DBH in immune regulation was first reported in a study in 1998. The study found that dbh- / - mice had normal white blood cell counts, normal T and B cell development, and normal in vitro functions under pathogen-free conditions, but were more susceptible to pathogenic infections (e.g., Listeria monocytogenes or Mycobacterium tuberculosis); at the same time, the animals showed severe thymic involution and impaired T cell function, including the production of Th1 cytokines. These results suggest that physiological development does not require the production of catecholamines, but catecholamines play an important role in the immune regulation of infections

[17] .

[0014] DBH is a part of the autonomic nervous system, and directly and indirectly penetrates the whole brain source, tissue, blood vessels and peripheral blood through noradrenergic fibers. In addition to noradrenergic fibers, the expression and localization of its protein also have certain tissue specificity. For example, DBH has a certain level of expression in chromaffin cells of adrenal medulla and noradrenergic cells of locus coeruleus, liver tissue and intestinal tissue, among which the expression of DBH in adrenal medulla is the highest.

[0015] A study found that high expression of DBH in tissues or high level of DBH secretion in peripheral blood may be related to some neurological or endocrine diseases

[18] ; for example, high level of DBH in peripheral blood may be related to Alzheimer's disease, bipolar disorder, Huntington's disease, hypothyroidism and PTSD. Although the level of DBH in peripheral blood may be related to neurological diseases, DBH inhibitors have not achieved good efficacy in clinical trials for Alzheimer's disease and PTSD (see the database of CLINICAL TRIALS). Similarly, another study found that the expression of DBH in inflammatory tissues was up-regulated in patients with autoimmune enteritis, but the study did not elucidate the inevitable relationship between the two

[19] .

[0016] According to statistics of the Autoimmune Association, there are more than 100 types of autoimmune diseases. In clinical practice, the main treatment for autoimmune diseases is to control symptoms with hormone and / or immunosuppressant non-targeted drugs, which at the same time brings great and irreversible adverse reactions and damage to patients.

[0017] Therefore, it is a technical problem to be solved in the field to seek a safe therapeutic agent for treating autoimmune diseases. SUMMARY

[0018] The technical problem to be solved by the present disclosure includes providing a pathway modulator, a pharmaceutical composition containing the same, uses thereof and a treatment method using the same.

[0019] The inventors have unexpectedly found that one or more of the pathway modulators in the prior art, including dopamine β-hydroxylase inhibitors (referred to as DBH inhibitors), receptor agonists and receptor antagonists, can inhibit the occurrence and development of autoimmune diseases (for example, embodied in the improvement of body weight loss, the improvement of DAI score and the normalization of colon density) through immunomodulation, and thus can provide potential therapeutic drugs for the treatment of autoimmune diseases.

[0020] The present disclosure solves the above technical problems by the following technical solutions:

[0021] The first aspect of the present disclosure relates to use of a pathway modulator in the manufacture of a medicament for treating an autoimmune disease; wherein the pathway modulator is one or more of a DBH inhibitor, a receptor agonist and a receptor antagonist.

[0022] In the above use, the pathway modulator is preferably a DBH inhibitor.

[0023] In the above use, the DBH inhibitor can be a DBH inhibitor disclosed in the prior art or thereafter, which can be exemplified by one or more of neresultat, ethamsuccinam, emixustat, fusaric acid, disulfiram, cysteamine, pantethine, a copper chelator, fumaric acid, hydralazine, 2-thien-2-ylallylamine, a pharmaceutically acceptable salt thereof and a prodrug thereof. The DBH inhibitor is preferably selected from neresultat, ethamsuccinam, emixustat, a pharmaceutically acceptable salt thereof and a prodrug thereof. The DBH inhibitor is more preferably selected from neresultat, ethamsuccinam and emixustat. Treating autoimmune diseases with one or more of neresultat, ethamsuccinam and emixustat can significantly reduce the great and irreversible adverse reactions and damage to patients caused by non-targeted drugs of hormones and / or immunosuppressants, and is a safe therapeutic agent for treating autoimmune diseases.

[0024] In the above use, the DBH inhibitor is preferably selected from neresultat, a pharmaceutically acceptable salt thereof and a prodrug thereof. The DBH inhibitor is more preferably neresultat. Treating autoimmune diseases with neresultat can significantly reduce weight loss and colon density in patients.

[0025] In the above use, as an example:

[0026] The pathway modulator is neresultat or a pharmaceutically acceptable salt thereof, and the unit dose is 10-100 mg / kg; for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg, or a range between any two of the foregoing; preferably 20-50 mg / kg; or,

[0027] The pathway modulator is ethamsuccinam or a pharmaceutically acceptable salt thereof, and the unit dose is 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0028] The pathway modulator is emicsilat or a pharmaceutically acceptable salt thereof, in a unit dosage of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or

[0029] The pathway modulator is fusaric acid or a pharmaceutically acceptable salt thereof, in a unit dosage of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0030] The pathway modulator is disulfiram or a pharmaceutically acceptable salt thereof, in a unit dosage of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0031] The pathway modulator is fumaric acid or a pharmaceutically acceptable salt thereof, in a unit dosage of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg.

[0032] In the above and below context, a unit dosage can be an integer or a fraction. It is understood that, for example, "10 to 100" is a shorthand way of writing, and although every point value within the range is not given, it is considered to have been expressly disclosed in the text.

[0033] The above technical solution is used for treating autoimmune diseases, which can significantly reduce the weight loss of patients and significantly reduce the colon density of patients.

[0034] In the above uses, the autoimmune disease can be one or more of the autoimmune diseases disclosed in the prior art, for example can be selected from Achalasia, Addison’s disease, Adult Still’s disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM / Anti-TBM nephritis, Antiphospholipid syndrome, Autoimmune angioedema, Autoimmune dysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune urticaria, Axonal & neuronal neuropathy (AMAN), Baló disease, Behcet’s disease, Benign mucosal pemphigoid, Bullous pemphigoid, Castleman disease (CD), Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIPD), Churg-Strauss syndrome (CSS), Cicatricial pemphigoid, CREST syndrome, Crohn’s disease, Discoid lupus, Epidermolysis bullosa acquisita (EBA), Essential mixed cryoglobulinemia, Eosinophilic esophagitis (EoE), Eosinophilic fasciitis, Eosinophilic granulomatosis with polyangiitis (EGPA), Evans syndrome, Felty’s syndrome, Giant cell arteritis, Goodpasture’s disease, Granulomatosis with polyangiitis (GPA), Graves’ disease, Guillain-Barre syndrome (GBS), Hashimoto’s disease, Hashimoto’s encephalopathy, Hashimoto’s meningitis, Henoch-Schonlein purpura (HSP), Idiopathic thrombocytopenic purpura (ITP), IgG4-related disease, Immune thrombocytopenia (ITP), Inclusion body myositis (IBM), Interstitial cystitis (IC), Lambert-Eaton syndrome, Latent autoimmune diabetes in adults (LADA), Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Livedoid vasculitis, Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multiple sclerosis (MS), Myasthenia gravis (MG), Myositis, Narcolepsy, Neuromyelitis optica (NMO), Ord’s syndrome, Paraneoplastic cerebellar degeneration (PCD), Paraneoplastic encephalomyelitis (PEM), Parotitis, Pars planitis, Pemphigus, Post-myocardial infarction syndrome, Primary biliary cholangitis (PBC), Primary Sjogren’s syndrome, Psoriasis, Psoriatic arthritis, Raynaud’s phenomenon, ReactiveAbbreviations include CIDP, Chronic recurrent multifocal osteomyelitis (CRMO), Eosinophilic granulomatosis (EGPA), Cicatricial pemphigoid, Cogan's syndrome, Cold agglutinin disease, Congenital heart block, Coxsackiemyocarditis, CREST syndrome, Crohn's disease, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus, and Dressler's syndrome. Evans syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis, giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatous polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis thyroiditis, hemolytic anemia, and allergic purpura.abbreviated HSP), Herpes gestationis or pemphigoid gestationis (abbreviated PG), Hidradenitis Suppurativa (abbreviated HS), Hypogammalglobulinemia, IgA Nephropathy, IgG4-related sclerosing disease, Immunethrombocytopenic purpura (abbreviated ITP), Inclusion body myositis (abbreviated IBM), Interstitial cystitis (abbreviated IC), Juvenile arthritis, Juvenile diabetes Type 1, Juvenile myositis (abbreviated JM), Kawasaki disease, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (abbreviated LAD), Lupus, Lyme disease chronic, Meniere’s disease, Microscopic polyangiitis (abbreviated MPA), Mixed connective tissue disease (abbreviated MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multifocal Motor Neuropathy, Myasthenia Gravis, Mycosis fungoides, Myelodysplastic syndrome, Myopathies, Narcolepsy, Neuromyelitis Optica, Neuropathies, Neuromyelitis Optica, Neuropathies, Non-Small Cell Lung Cancer, Ocular Cicatricial Pemphigoid, Ocular Inflammatory Disease, Osteoarthritis, Osteoporosis, Paraneoplastic Syndromes, Paraplegia, Paraplegia, Paroxysmal Kinesigenic Dyskinesia, Parry Romberg Syndrome, Parsonage-Turner Syndrome, Pemphigus, Pemphigus Vulgaris, Pernicious Anaemia, Peripheral Neuropathy, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pernicious Anaemia, Pabbreviated MMN or MMNCB), Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neonatal Lupus, Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism (abbreviated PR), PANDAS, Paraneoplastic cerebellar degeneration (abbreviated PCD), Paroxysmal nocturnal hemoglobinuria (abbreviated PNH), Parry Romberg syndrome, Pars planitis, Parsonage-Turner syndrome, Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, Peripheral neuropathy, Pernicious anemia, PeripheralThe abbreviations for polyarteritis (PA) include: POEMS syndrome, polyarteritis nodosa, polyglandular syndromes type I, polyglandular syndromes type II, polyglandular syndromes type III, polymyalgia rheumatica, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, and pure red cell aplasia. aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, syndrome), Sperm & testicular autoimmunity, Stiff person syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac’s syndrome, Sympathetic ophthalmia (SO), Takayasu’s arteritis, Temporal arteritis / Giant cell arteritis, Thrombocytopenic purpura (TTP), Thyroid eye disease (TED), Tolosa-Hunt syndrome (THS), Transverse myelitis, Type 1 diabetes, Ulcerative colitis (UC), Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vitiligo, and Vogt-Koyanagi-Harada Disease; preferably autoimmune colitis, neuromyelitis optica, rheumatoid arthritis, scleroderma, psoriasis, or uveitis.

[0035] A second aspect of the present disclosure relates to a pathway modulator for use in the treatment of an autoimmune disease; wherein the pathway modulator is one or more of a DBH inhibitor, a receptor agonist, and a receptor antagonist.

[0036] Among the pathway modulators described above, the pathway modulator is preferably a DBH inhibitor.

[0037] Among the pathway modulators described above, the DBH inhibitor can be one or more of the DBH inhibitors already disclosed in the prior art, such as one or more of intepidine, ethamsylate, emoxymate, fusaric acid, disulfiram, cysteamine, pantethine, copper chelators, fumaric acid, hydralazine, 2-thiophene-2-ylallylamine, pharmaceutically acceptable salts thereof, and prodrugs thereof. The DBH inhibitor is preferably selected from the group consisting of intepidine, ethamsylate, emoxymate, pharmaceutically acceptable salts thereof, and prodrugs thereof. The DBH inhibitor is more preferably selected from the group consisting of intepidine, ethamsylate, and emoxymate.

[0038] Among the pathway modulators described above, the DBH inhibitor is preferably selected from the group consisting of intepidine, pharmaceutically acceptable salts thereof, and prodrugs thereof. The DBH inhibitor is more preferably intepidine.

[0039] Among the pathway modulators described above, as an example:

[0040] The pathway modulator is intepidine or a pharmaceutically acceptable salt thereof, in a unit dose of: 10-100 mg / kg; such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg, or a range between any two of the foregoing; preferably 20-50 mg / kg; or,

[0041] The pathway modulator is ethamsylate or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; such as 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0042] The pathway modulator is emoxymate or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; such as 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or

[0043] The pathway modulator is fusaric acid or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; such as 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0044] The pathway modulator is disulfiram or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0045] The pathway modulator is fumaric acid or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg.

[0046] In the aforementioned pathway modulator, the autoimmune disease is as defined above.

[0047] A third aspect of the present disclosure relates to a method for treating an autoimmune disease, comprising the step of: administering to a subject a therapeutically effective amount of a pathway modulator; wherein the pathway modulator is one or more of a DBH inhibitor, a receptor agonist, and a receptor antagonist.

[0048] In the aforementioned method of treatment, the pathway modulator is preferably a DBH inhibitor.

[0049] In the aforementioned method of treatment, the DBH inhibitor can be one or more of the DBH inhibitors already disclosed in the prior art, for example, can be one or more of neresiftame, epostimetame, emimetame, fusaric acid, disulfiram, cysteamine, pantethine, a copper chelator, fumaric acid, hydralazine, 2-thiophene-2-ylallylamine, a pharmaceutically acceptable salt thereof, and a prodrug thereof. The DBH inhibitor is preferably selected from neresiftame, epostimetame, emimetame, a pharmaceutically acceptable salt thereof, and a prodrug thereof. The DBH inhibitor is more preferably selected from neresiftame, epostimetame, and emimetame.

[0050] In the aforementioned method of treatment, the DBH inhibitor is preferably selected from neresiftame, a pharmaceutically acceptable salt thereof, and a prodrug thereof. The DBH inhibitor is more preferably neresiftame.

[0051] In the aforementioned method of treatment, the DBH inhibitor can be administered in combination with one or more of a chemotherapeutic agent, a targeted therapeutic agent, an immunotherapeutic agent, and an anti-inflammatory agent.

[0052] In the aforementioned method of treatment, as an example:

[0053] The pathway modulator is endoximstat or a pharmaceutically acceptable salt thereof, in a unit dose of 10-100 mg / kg; for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg, or a range between any two of the foregoing; preferably 20-50 mg / kg; or,

[0054] The pathway modulator is endoximstat or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0055] The pathway modulator is endoximstat or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or

[0056] The pathway modulator is endoximstat or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0057] The pathway modulator is endoximstat or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0058] The pathway modulator is endoximstat or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg.

[0059] In the above method of treatment, the autoimmune disease is as defined above.

[0060] A fourth aspect of the present disclosure relates to a pharmaceutical composition for treating an autoimmune disease, comprising:

[0061] - a pathway modulator, and

[0062] - a pharmaceutically acceptable carrier.

[0063] wherein the pathway modulator is one or more of a DBH inhibitor, a receptor agonist, and a receptor antagonist.

[0064] In the above pharmaceutical composition, the pathway modulator is preferably a DBH inhibitor.

[0065] In the above pharmaceutical composition, the DBH inhibitor can be one or more of the DBH inhibitors already disclosed in the prior art, such as one or more of neresultat, ethamsuccinamate, emixstat, fusidic acid, disulfiram, cysteamine, pantethine, a copper chelator, fumaric acid, hydralazine, 2-thiophene-2-ylallylamine, a pharmaceutically acceptable salt thereof, and a prodrug thereof. The DBH inhibitor is preferably selected from neresultat, ethamsuccinamate, emixstat, a pharmaceutically acceptable salt thereof, and a prodrug thereof. The DBH inhibitor is more preferably selected from neresultat, ethamsuccinamate, and emixstat.

[0066] In the above pharmaceutical composition, the DBH inhibitor is preferably selected from neresultat, a pharmaceutically acceptable salt thereof, and a prodrug thereof. The DBH inhibitor is more preferably neresultat.

[0067] In the above pharmaceutical composition, as an example:

[0068] The pathway modulator is neresultat or a pharmaceutically acceptable salt thereof, and the unit dose is 10-100 mg / kg; such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg, or a range between any two of the aforementioned values; preferably 20-50 mg / kg; or,

[0069] The pathway modulator is ethamsuccinamate or a pharmaceutically acceptable salt thereof, and the unit dose is 80-120 mg / kg; such as 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0070] The pathway modulator is emixstat or a pharmaceutically acceptable salt thereof, and the unit dose is 80-120 mg / kg; such as 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg; or

[0071] The pathway modulator is fusaric acid or a pharmaceutically acceptable salt thereof, at a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0072] The pathway modulator is disulfiram or a pharmaceutically acceptable salt thereof, at a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0073] The pathway modulator is fumaric acid or a pharmaceutically acceptable salt thereof, at a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg.

[0074] In the above pharmaceutical composition, the autoimmune disease is as defined above.

[0075] A fifth aspect of the present disclosure relates to a use of a pathway modulator in the preparation of a medicament; the medicament is for one or more purposes selected from the group consisting of: reducing the proportion of CD4+ T cells, increasing the proportion of regulatory T cells, increasing the proportion of CD8+ T cells, reducing the secretion of pro-inflammatory factors by CD4+ T cells, reducing the secretion of pro-inflammatory factors by CD8+ T cells, inhibiting the activation of B cells and inhibiting the activation of NK cells; reducing the content of lymphocytes, neutrophils and monocytes in peripheral blood; reducing inflammatory cell infiltration in the dermis and capillary proliferation under the dermis; improving skin fibrosis; reducing the incidence of uveitis; improving skin inflammation; improving stool form score, improving CW / CL, improving CW / BW, improving CW / CL / BW, inhibiting the increase in colonic ulcer area, improving colonic inflammatory cell infiltration score, improving tissue damage score; improving disease activity score, improving hematochezia or occult blood. The pathway modulator is one or more of a DBH inhibitor, a receptor agonist and a receptor antagonist.

[0076] In the above use, the pathway modulator is preferably a DBH inhibitor.

[0077] In the above uses, the DBH inhibitor can be one or more of the DBH inhibitors already disclosed in the prior art, for example can be one or more of neresidiline, ethamsylate, emixidiline, fusaric acid, disulfiram, cysteamine, pantethine, copper chelators, fumaric acid, hydralazine, 2-thiophene-2-ylallylamine, pharmaceutically acceptable salts thereof, and prodrugs thereof. The DBH inhibitor is preferably selected from neresidiline, ethamsylate, emixidiline, pharmaceutically acceptable salts thereof, and prodrugs thereof. The DBH inhibitor is more preferably selected from neresidiline, ethamsylate, and emixidiline.

[0078] In the above uses, the DBH inhibitor is preferably selected from neresidiline, pharmaceutically acceptable salts thereof, and prodrugs thereof. The DBH inhibitor is more preferably neresidiline.

[0079] In the above uses, as examples:

[0080] The pathway modulator is neresidiline or a pharmaceutically acceptable salt thereof, in a unit dose of: 10-100 mg / kg; for example 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg, or a range between any two of the foregoing; preferably 20-50 mg / kg; or,

[0081] The pathway modulator is ethamsylate or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0082] The pathway modulator is emixidiline or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or

[0083] The pathway modulator is fusaric acid or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0084] The pathway modulator is disulfiram or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0085] The pathway modulator is fumaric acid or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the foregoing; preferably 90-110 mg / kg, more preferably 100 mg / kg.

[0086] In the above use, the medicament is preferably for one or more uses selected from the group consisting of: reducing the proportion of CD4+ T cells, increasing the proportion of regulatory T cells, reducing the secretion of proinflammatory factors by CD4+ T cells, inhibiting the activation of B cells, and inhibiting the activation of NK cells.

[0087] In the above use, the proinflammatory factor of the CD4+ T cells is preferably one or more of IL-17A, IFN-γ, and TNF-α.

[0088] In the above use, the proinflammatory factor of the CD8+ T cells is preferably IL-17A and / or TNF-α.

[0089] In the above use, the regulatory T cells are preferably CD25+ FOXP3+ Treg cells.

[0090] In the above use, the B cells are preferably B220+ cells, more preferably CD69+ B220+ B cells.

[0091] In the above use, the NK cells are preferably NK1.1+ cells, more preferably NK1.1+ CD107a+ NK cells.

[0092] The sixth aspect of the present disclosure relates to a pathway modulator for one or more uses selected from the group consisting of: reducing the proportion of CD4+ T cells, increasing the proportion of regulatory T cells, increasing the proportion of CD8+ T cells, reducing the secretion of pro-inflammatory factors by CD4+ T cells, reducing the secretion of pro-inflammatory factors by CD8+ T cells, inhibiting the activation of B cells and inhibiting the activation of NK cells; reducing the content of lymphocytes, neutrophils and monocytes in peripheral blood; reducing inflammatory cell infiltration in the dermis and capillary proliferation under the dermis; improving skin fibrosis; reducing the incidence of uveitis; improving skin inflammation; improving stool formability score, improving CW / CL, improving CW / BW, improving CW / CL / BW, inhibiting the increase in colonic ulcer area, improving colonic inflammatory cell infiltration score, improving tissue damage score; improving disease activity score, improving blood or occult blood. Wherein the pathway modulator is one or more of a DBH inhibitor, a receptor agonist and a receptor antagonist.

[0093] In the above pathway modulator, the pathway modulator is preferably a DBH inhibitor.

[0094] In the above pathway modulator, the DBH inhibitor can be a DBH inhibitor already disclosed in the prior art, for example, can be one or more of iniprine, ethamsylate, emilsylate, fusaric acid, disulfiram, cysteamine, pantethine, copper chelators, fumaric acid, hydralazine, 2-thiophene-2-ylallylamine, pharmaceutically acceptable salts thereof and prodrugs thereof. The DBH inhibitor is preferably selected from iniprine, ethamsylate, emilsylate, pharmaceutically acceptable salts thereof and prodrugs thereof. The DBH inhibitor is more preferably selected from iniprine, ethamsylate and emilsylate.

[0095] In the above pathway modulator, the DBH inhibitor is preferably selected from iniprine, pharmaceutically acceptable salts thereof and prodrugs thereof. The DBH inhibitor is more preferably iniprine.

[0096] In the above pathway modulator, as an example:

[0097] The pathway modulator is iniprine or a pharmaceutically acceptable salt thereof, the unit dose is: 10-100 mg / kg; for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg, or a range between any two of the foregoing; preferably 20-50 mg / kg; or,

[0098] The pathway modulator is emicamptast or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0099] The pathway modulator is emicamptast or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg; or

[0100] The pathway modulator is fusarinic acid or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0101] The pathway modulator is disulfiram or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0102] The pathway modulator is fumaric acid or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg.

[0103] In the aforementioned pathway modulator, the medicament is preferably for one or more uses selected from the group consisting of: reducing the proportion of CD4+ T cells, increasing the proportion of regulatory T cells, reducing the secretion of pro-inflammatory factors by CD4+ T cells, inhibiting the activation of B cells, and inhibiting the activation of NK cells.

[0104] In the aforementioned pathway modulator, the pro-inflammatory factor of the CD4+ T cells is preferably one or more of IL-17A, IFN-gamma, and TNF-alpha.

[0105] In the aforementioned pathway modulator, the pro-inflammatory factor of the CD8+ T cells is preferably IL-17A and / or TNF-alpha.

[0106] In the aforementioned pathway modulator, the regulatory T cells are preferably CD25+ FOXP3+ Treg cells.

[0107] In the pathway modulator, the B cells are preferably B220+ cells, and more preferably CD69+ B220+ B cells.

[0108] In the pathway modulator, the NK cells are preferably NK1.1+ cells, and more preferably NK1.1+ CD107a+ NK cells.

[0109] A seventh aspect of the present disclosure relates to a method of modulating immune cell function in vivo or in vitro, comprising the step of: contacting an effective amount of a pathway modulator with an immune cell in vivo or in vitro, wherein the immune cell is from a subject; and wherein the pathway modulator is one or more of a DBH inhibitor, a receptor agonist, and a receptor antagonist.

[0110] The modulating immune cell function refers to one or more functions selected from the group consisting of: decreasing the proportion of CD4+ T cells, increasing the proportion of regulatory T cells, increasing the proportion of CD8+ T cells, decreasing the secretion of proinflammatory factors by CD4+ T cells, decreasing the secretion of proinflammatory factors by CD8+ T cells, inhibiting the activation of B cells, and inhibiting the activation of NK cells.

[0111] In the method, the pathway modulator is preferably a DBH inhibitor.

[0112] In the method, the DBH inhibitor can be one or more of the DBH inhibitors disclosed in the prior art, such as one or more of neresultat, ethamsuccinamate, emimastat, fumaramic acid, disulfiram, cysteamine, pantethine, a copper chelator, hydralazine, 2-thiophene-2-ylallylamine, a pharmaceutically acceptable salt thereof, and a prodrug thereof. The DBH inhibitor is preferably selected from the group consisting of neresultat, ethamsuccinamate, emimastat, a pharmaceutically acceptable salt thereof, and a prodrug thereof. The DBH inhibitor is more preferably selected from the group consisting of neresultat, ethamsuccinamate, and emimastat.

[0113] In the method, the DBH inhibitor is preferably selected from the group consisting of neresultat, a pharmaceutically acceptable salt thereof, and a prodrug thereof. The DBH inhibitor is more preferably neresultat.

[0114] In the method, as an example:

[0115] The pathway modulator is neresultat or a pharmaceutically acceptable salt thereof, and the unit dose is: 10-100 mg / kg; such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg, or a range between any two of the foregoing; preferably 20-50 mg / kg; or,

[0116] The pathway modulator is emicashift or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0117] The pathway modulator is emicashift or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg; or

[0118] The pathway modulator is fusarinic acid or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0119] The pathway modulator is disulfiram or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg; or,

[0120] The pathway modulator is fumaric acid or a pharmaceutically acceptable salt thereof, in a unit dose of 80-120 mg / kg; for example, 80, 85, 90, 95, 100, 110, 120, or a range between any two of the aforementioned values; preferably 90-110 mg / kg, more preferably 100 mg / kg.

[0121] In the above method, the DBH inhibitor can be administered in combination with one or more selected from a chemotherapeutic agent, a targeted therapy agent, an immunotherapeutic agent, and an anti-inflammatory agent.

[0122] In the above method, the medicament is preferably for one or more uses selected from reducing the proportion of CD4+ T cells, increasing the proportion of regulatory T cells, reducing the secretion of pro-inflammatory factors by CD4+ T cells, inhibiting the activation of B cells, and inhibiting the activation of NK cells.

[0123] In the above method, the pro-inflammatory factor of the CD4+ T cells is preferably one or more of IL-17A, IFN-gamma, and TNF-alpha.

[0124] In the above method, the pro-inflammatory factor of the CD8+ T cells is preferably IL-17A and / or TNF-alpha.

[0125] In the above method, the regulatory T cells are preferably CD25+FOXP3+Treg cells.

[0126] In the above method, the B cells are preferably B220+cells, more preferably CD69+B220+B cells.

[0127] In the above method, the NK cells are preferably NK1.1+cells, more preferably NK1.1+CD107a+NK cells.

[0128] An eighth aspect of the present disclosure relates to a pharmaceutical composition comprising a pathway modulator and a pharmaceutically acceptable carrier; wherein the pathway modulator is one or more of a DBH inhibitor, a receptor agonist, and a receptor antagonist;

[0129] The pharmaceutical composition has one or more functions selected from the group consisting of: reducing the proportion of CD4+T cells, increasing the proportion of regulatory T cells, increasing the proportion of CD8+T cells, reducing the secretion of proinflammatory factors by CD4+T cells, reducing the secretion of proinflammatory factors by CD8+T cells, inhibiting the activation of B cells and inhibiting the activation of NK cells; reducing the content of lymphocytes, neutrophils and monocytes in peripheral blood; reducing inflammatory cell infiltration in the dermis and capillary proliferation under the dermis; improving skin fibrosis; reducing the incidence of uveitis; improving skin inflammation; improving stool form score, improving CW / CL, improving CW / BW, improving CW / CL / BW, inhibiting the increase in colonic ulcer area, improving colonic inflammatory cell infiltration score, improving tissue damage score; improving disease activity score, improving hematochezia or occult blood.

[0130] In the above pharmaceutical composition, the pathway modulator is preferably a DBH inhibitor.

[0131] In the above pharmaceutical composition, the DBH inhibitor can be a DBH inhibitor already disclosed in the prior art, for example, one or more of nimesulide, etamicast, emicast, fusidic acid, disulfiram, cysteamine, pantethine, copper chelators, fumaric acid, hydralazine, 2-thiophene-2-ylallylamine, pharmaceutically acceptable salts thereof, and prodrugs thereof. The DBH inhibitor is preferably selected from nimesulide, etamicast, emicast, pharmaceutically acceptable salts thereof, and prodrugs thereof. The DBH inhibitor is more preferably selected from nimesulide, etamicast, and emicast.

[0132] In the above pharmaceutical composition, the DBH inhibitor is preferably selected from nimesulide, pharmaceutically acceptable salts thereof, and prodrugs thereof. The DBH inhibitor is more preferably nimesulide.

[0133] In the above pharmaceutical composition, as an example:

[0134] The pathway modulator is endoximstat or a pharmaceutically acceptable salt thereof, in a unit dose of: 10-100 mg / kg; such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg, or a range between any two of the aforementioned values; preferably 20-50 mg / kg; or,

[0135] The pathway modulator is endoximstat or a pharmaceutically acceptable salt thereof, in a unit dose of: 10-100 mg / kg; such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg, or a range between any two of the aforementioned values; preferably 20-50 mg / kg; or,

[0136] The pathway modulator is endoximstat or a pharmaceutically acceptable salt thereof, in a unit dose of: 10-100 mg / kg; such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg, or a range between any two of the aforementioned values; preferably 20-50 mg / kg; or,

[0137] The pathway modulator is endoximstat or a pharmaceutically acceptable salt thereof, in a unit dose of: 10-100 mg / kg; such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg, or a range between any two of the aforementioned values; preferably 20-50 mg / kg; or,

[0138] The pathway modulator is endoximstat or a pharmaceutically acceptable salt thereof, in a unit dose of: 10-100 mg / kg; such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg, or a range between any two of the aforementioned values; preferably 20-50 mg / kg; or,

[0139] The pathway modulator is endoximstat or a pharmaceutically acceptable salt thereof, in a unit dose of: 10-100 mg / kg; such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg, or a range between any two of the aforementioned values; preferably 20-50 mg / kg; or,

[0140] The above pharmaceutical composition can further comprise one or more substances selected from the group consisting of a chemotherapeutic agent, a targeted therapy agent, an immunotherapy agent, and an anti-inflammatory agent.

[0141] In the above pharmaceutical composition, the agent is preferably for one or more uses selected from the group consisting of reducing the proportion of CD4+ T cells, increasing the proportion of regulatory T cells, reducing the secretion of proinflammatory factors from CD4+ T cells, inhibiting the activation of B cells, and inhibiting the activation of NK cells.

[0142] In the above pharmaceutical composition, the proinflammatory factor of the CD4+ T cells is preferably one or more of IL-17A, IFN-γ, and TNF-α.

[0143] In the above pharmaceutical composition, the proinflammatory factor of the CD8+ T cells is preferably IL-17A and / or TNF-α.

[0144] In the above pharmaceutical composition, the regulatory T cells are preferably CD25+ FOXP3+ Treg cells.

[0145] In the above pharmaceutical composition, the B cells are preferably B220+ cells, and more preferably CD69+ B220+ B cells.

[0146] In the above pharmaceutical composition, the NK cells are preferably NK1.1+ cells, and more preferably NK1.1+ CD107a+ NK cells.

[0147] The positive effects of the present disclosure are that the inventors have found that DBH inhibitors can inhibit the development of autoimmune diseases through immunomodulation (as exemplified by improvements in body weight loss, DAI scores, and normalization of colon density), and thus can provide new options for the treatment of autoimmune diseases. BRIEF DESCRIPTION OF DRAWINGS

[0148] Figure 1A The results of the body weight evaluation of the mice in each group after the experiment according to the experimental procedure described in Example 1 are shown.

[0149] Figure 1B The results of the DAI scores of the mice in each group after the experiment according to the experimental procedure described in Example 1 are shown.

[0150] Figure 1C The results of the colon density of the mice in each group after the experiment according to the experimental procedure described in Example 1 are shown.

[0151] Figures 2A-2E The results of the CD4+ T cell subpopulation in the mesenteric lymph nodes of the mice in each group in Example 4 are shown. Figure 2A The results of the CD25+ FOXP3+ Treg cell subpopulation in the mesenteric lymph nodes of the mice in each group in Example 4 are shown. Figure 2B The results of IL-17A from the CD4+ T cell subpopulation in the mesenteric lymph nodes of the mice in each group in Example 4 are shown. Figure 2C The results of IFN-γ from the CD4+ T cell subpopulation in the mesenteric lymph nodes of the mice in each group in Example 4 are shown. Figure 2DTNF-α from CD8+ T cell subsets Figure 2E

[0152] Figures 3A-3D shows the effect of CD69+ B220+ B cell subsets Figure 3A IFN-γ from CD8+ T cell subsets Figure 3B IL-17A from CD8+ T cell subsets Figure 3C TNF-α from CD8+ T cell subsets Figure 3D

[0153] Figures 4A-4B shows the effect of CD69+ B220+ B cell subsets Figure 4A NK1.1+ CD107a+ NK cell subsets Figure 4B in the mesenteric lymph nodes of mice in each group of Example 4. * in each figure above means p < 0.05, ** means p < 0.01, *** means p < 0.001, **** means p < 0.0001.

[0154] Figure 5 shows the body weight change in each group of Example 5; Two-way ANOVA: # p < 0.05, ### p < 0.001 vs. vehicle group.

[0155] Figure 6 shows the body weight change rate in each group of Example 5.

[0156] Figure 7 shows the spleen weight in each group of Example 5; One-way ANOVA: *** p < 0.001 vs. normal group, # p < 0.05, ## p < 0.01 vs. vehicle group.

[0157] Figure 8 shows the splanchnic index in each group of Example 5; One-way ANOVA: # p < 0.05, ## p < 0.01, ### p < 0.001 vs. vehicle group.

[0158] Figure 9 shows the peripheral red blood cell (RBC) count in each group of Example 5; One-way ANOVA: *** p < 0.001 vs. normal group, ## p < 0.01, ### p < 0.001 vs. vehicle group, &&& p < 0.001 vs. imatinib group. ​​

[0159] Figure 10 Hemoglobin (HGB) levels in peripheral blood in each group of Example 5 are shown; One-way ANOVA: ***p<0.001 vs normal group, ## p<0.01, ### p<0.001 vs vehicle group, &&& p<0.001 vs imatinib group.

[0160] Figure 11 White blood cell (WBC) counts in peripheral blood in each group of Example 5 are shown; One-way ANOVA: *p<0.05, ***p<0.001 vs normal group, ## p<0.01 vs vehicle group.

[0161] Figure 12 Lymphocyte (LYMPH) counts in peripheral blood in each group of Example 5 are shown; One-way ANOVA: # p<0.05, ### p<0.001 vs vehicle group.

[0162] Figure 13 Monocyte (MONO) counts in peripheral blood in each group of Example 5 are shown; One-way ANOVA: ## p<0.01 vs vehicle group.

[0163] Figure 14 Neutrophil (NEUT) counts in peripheral blood in each group of Example 5 are shown; One-way ANOVA: ***p<0.001 vs normal group.

[0164] Figure 15 Platelet levels in peripheral blood in each group of Example 5 are shown; One-way ANOVA: ***p<0.001 vs normal group, ### p<0.001 vs vehicle group, && p<0.01, &&& p<0.001 vs. nintedanib group, $$ p<0.01 vs. imatinib group.

[0165] Figure 16 Dermal layer inflammatory cell infiltration scores in each group of Example 5 are shown; One-way ANOVA: # p<0.05 vs. vehicle group, $ p<0.05 vs. endostar-25 mg / kg, qd, po.

[0166] Figure 17Dermis layer intracapillary density score in each group of Example 5; One-way ANOVA: # p<0.05, ## p<0.01 vs. vehicle group.

[0167] Figure 18 Dermis thickness in each group of Example 5; One-way ANOVA: *p<0.05, **p<0.01, ***p<0.001 vs. normal group.

[0168] Figure 19 Clinical score in each group of Example 6.

[0169] Figures 20A-20D Proportion of AQP4, GFAP, IBA1, CD45 positive cells in each group of Example 7.

[0170] Figure 21 Animal body weight in each group of Example 8.

[0171] Figure 22 AUC of erythema + scaling + thickness total score in each group of Example 8; Mean ± standard error, Two way ANOVA, p*<0.05, p**<0.01, p***<0.001, p****<0.0001 vs. corresponding vehicle group (Note: AUC of total score of normal group is 0, not shown in the figure).

[0172] Figure 23 Spleen weight in each group of Example 8.

[0173] Figure 24 Animal body weight in each group of Example 9.

[0174] Figure 25 Clinical score in each group of Example 9.

[0175] Figure 26 AUC in each group of Example 9.

[0176] Figure 27 Incidence rate in each group of Example 9.

[0177] Figure 28 Animal body weight in each group of Example 10.

[0178] Figure 29 Animal body weight change rate in each group of Example 10.

[0179] Figure 30 Fecal score in each group of Example 10.

[0180] Figure 31AUC of stool score in each group of Example 10 is shown.

[0181] Figure 32 Weight change in each group of Example 11 is shown.

[0182] Figure 33 DAI in each group of Example 11 is shown. DETAILED DESCRIPTION

[0183] Throughout this application various publications, patents and patent applications; are referenced; each of these references is herein incorporated by reference in its entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0184] TERMINOLOGY

[0185] The term "receptor agonist" as used herein refers to a substance which is capable of acting on the receptors of the product of the catalysis of dopamine beta-hydroxylase (i.e. norepinephrine and / or epinephrine) and exerting the same mechanism and effect as the reduction of norepinephrine and / or epinephrine. The receptor agonist is preferably a norepinephrine receptor agonist and / or an epinephrine receptor agonist.

[0186] The term "receptor antagonist" as used herein refers to a substance which is capable of acting on the receptors of the substrate of the catalysis of dopamine beta-hydroxylase (i.e. dopamine) and exerting the same mechanism and effect as the elevation of dopamine. The receptor antagonist is preferably a dopamine receptor antagonist.

[0187] The term "autoimmune disease" refers to a condition characterized by the immune system attacking the body, the breakdown of adaptive immune tolerance mechanisms to recognize self / non-self, and the abnormal response of adaptive immune cells leading to inflammatory damage of self-tissues.

[0188] The term "dopamine beta-hydroxylase" is intended to encompass human dopamine beta-hydroxylase, fragments, variants, precursors and functional domains thereof.

[0189] The term "DBH inhibitor" refers to any natural or artificial compound which is capable of affecting (the affecting refers to reducing, decreasing, inhibiting, blocking, repressing, inactivating or preventing the activation of) the structure, expression or activity of dopamine beta-hydroxylase at the nucleic acid or protein level. DBH inhibitors include those known in the prior art, as well as those to be provided in the future. They include the DBH inhibitors disclosed in CN87103323A and WO9529165, as well as Nepicastat, Etamicastat, Zamicastat, Fusaric acid, disulfiram, cysteamine, cysteamine derivatives, pantethine and pantethine derivatives.

[0190] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable excipient.

[0191] The term "pharmaceutically acceptable salt" should be understood to mean a salt which is pharmaceutically acceptable and which possesses the desired pharmacological activity of the parent compound. Such salts include:

[0192] (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid, or formed with organic acids such as formic acid, oxalic acid, succinic acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzoyl-L-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid and trifluoroacetic acid; and (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion (e.g., Na + , K + or Li + ), an alkaline earth metal ion (e.g., Ca 2+ or Mg 2+ ), an aluminum ion or coordinates with an organic or inorganic base; wherein the organic base can be one or more of pyridine, imidazole, pyrazine, indole, quinoline, tertiary amines and aniline organic bases, preferably one or more of pyridine, collidine, 4-dimethylaminopyridine, 2-methyl-5-ethylpyridine, triethylamine, N,N-diisopropylethylamine, N,N-dimethylaniline, diethanolamine, ethanolamine, N-methylglucamine, triethanolamine and tromethamine; and the inorganic base can be one or more of aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.

[0193] The pharmaceutical compositions of the present disclosure can be in a variety of conventional dosage forms, such as tablets, aqueous suspensions, oily suspensions, dispersible powders, dispersible granules, emulsions, hard gelatin capsules, soft gelatin capsules, sterile aqueous injection solutions, sterile oil-in-water microemulsions for injection, or suppositories. Each of the above dosage forms can be prepared by conventional methods.

[0194] The excipients in the tablet of the present disclosure can be one or more of a filler, a binder, a lubricant, a glidant, and a disintegrant. Among them, the filler can be one or more of microcrystalline cellulose, starch, lactose monohydrate, and calcium hydrogen phosphate. The binder can be one or more of starch, gelatin, polyvinylpyrrolidone, and gum arabic. The lubricant can be one or more of magnesium stearate, stearic acid, and sodium lauryl sulfate. The glidant can be one or both of microfine silica and talc. The disintegrant can be one or more of crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and croscarmellose sodium. The tablet can further contain a coating. The tablet can also be prepared as a sustained-release formulation, and the sustained-release material in the sustained-release formulation can be one or both of hydroxypropyl methylcellulose and xanthan gum.

[0195] The excipients in the aqueous suspension of the present disclosure can be one or more of a suspending agent, a dispersing agent, a preservative, and a flavoring agent. Among them, the suspending agent can be one or more of sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinylpyrrolidone, and gum arabic. The dispersing agent can be one or more of a naturally occurring phospholipid (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long-chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol monooleate), and a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The preservative can be nipagin ethyl and / or nipagin n-propyl. The flavoring agent can be one or more of sucrose, saccharin, and aspartame.

[0196] The excipients in the oil suspension of the present disclosure can be one or more of a suspending agent, a thickening agent, a flavoring agent, and an antioxidant. The suspending agent can be a vegetable oil and / or a mineral oil, the vegetable oil can be one or more of peanut oil, olive oil, sesame oil, and coconut oil, and the mineral oil can be liquid paraffin. The thickening agent can be one or more of beeswax, hard paraffin, and cetyl alcohol. The flavoring agent can be one or more of sucrose, saccharin, and aspartame. The antioxidant can be one or more of butylated hydroxyanisole, α-tocopherol, and ascorbic acid.

[0197] The excipients in the dispersible powder and dispersible granules of the present disclosure can be one or more of a suspending agent, a dispersing agent, a preservative, a flavoring agent, and an antioxidant. The specific selection of each component is the same as the excipients in the aqueous suspension.

[0198] The excipients in the emulsions of the present disclosure can be one or more of suspending agents, emulsifying agents, flavoring agents, preservatives, and antioxidants. The suspending agents can be vegetable oils and / or mineral oils, the vegetable oils can be olive oil and / or peanut oil, and the mineral oils can be liquid paraffin. The emulsifying agents can be one or more of naturally occurring phosphatides (e.g., soybean lecithin), esters derived from fatty acids and hexitol anhydrides (e.g., sorbitan monooleate), and condensation products of said partial esters and ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). The flavoring agents can be one or more of glycerin, propylene glycol, sorbitol, and sucrose. The preservatives can be nipagin and / or nipasol. The antioxidants can be one or more of butylated hydroxyanisole, alpha-tocopherol, and ascorbic acid.

[0199] The excipients in the hard capsules of the present disclosure can be conventional inert solid diluents, such as one or more of calcium carbonate, calcium phosphate, and kaolin.

[0200] The excipients in the soft capsules of the present disclosure can be conventional water-soluble carriers and / or conventional oil vehicles, such as one or more of polyethylene glycols, peanut oil, liquid paraffin, and olive oil.

[0201] The excipients in the sterile injectable aqueous solutions of the present disclosure can be pharmaceutically acceptable vehicles, such as water, Ringer's solution, or isotonic sodium chloride solution.

[0202] The excipients in the sterile injectable oil-in-water microemulsions of the present disclosure can be oil phase excipients and water phase excipients, the oil phase excipients can be a mixture of soybean oil and lecithin, and the water phase excipients can be a mixture of water and glycerol.

[0203] The excipients in the suppositories of the present disclosure can be one or more of cacao butter, glycerin, gelatin, hydrogenated vegetable oil, polyethylene glycol, and fatty acid esters of polyethylene glycol.

[0204] The term "subject" refers to an animal, preferably a mammal. According to specific embodiments, the subject is a mammal, including, for example, a camel, a donkey, a zebra, a cow, a pig, a horse, a goat, a sheep, a cat, a dog, a rat, a rabbit, a guinea pig, a mouse, a primate (e.g., a human). In specific embodiments, the subject is a human. In specific embodiments, the subject is a human susceptible to, suspected of having, or having an autoimmune disease.

[0205] The term "treatment" refers to eliminating the disease, preventing the disease from progressing, slowing the progression of the disease, reducing the duration of one or more symptoms associated with the disease, an improvement or reversal of at least one measurable parameter associated with the disease, or increasing the survival rate of a subject having the disease.

[0206] The term "effective amount" refers to the amount of a pharmaceutically active ingredient that elicits the desired effect in a subject. In particular embodiments, the selection of an effective amount can be determined by one skilled in the art based on considerations of a variety of factors, such as via clinical trials, including the disease to be treated, the symptoms involved, the route of administration, the severity of the disease, the weight of the patient, the immune status of the patient, and other factors known to one skilled in the art. The effective amount in a particular embodiment can be derived from dose-response curves obtained in animal model test systems and allows for the determination of the appropriate amount depending on the judgment of the physician and the situation of each patient. The interrelationship of dosages for use in animals and humans is described by Freireich et al. 1966, Cancer Chemother Rep 50:219, and the table of body surface area of patients can be used as an appropriate guide to the dosage for humans. An effective amount of a pharmaceutical compound of the present disclosure can be 0.5 mg / kg to 500 mg / kg, preferably 1 mg / kg to 200 mg / kg, more preferably 10 mg / kg to 100 mg / kg.

[0207] Herein, the same pharmaceutically active ingredient (referring to a single pharmaceutical compound) or different pharmaceutically active ingredients (referring to two or more pharmaceutical compounds) can be administered at once or can be divided into a plurality of smaller unit doses and administered at a time interval. It should be understood that the exact dosage, duration, and interval of treatment are a function of the disease being treated and can be determined using animal or clinical trial data. The administration can include a single administration or two or more administrations at appropriate time intervals. Among them, the administration of adjacent two times can be separated by 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, one and a half days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

[0208] Each pharmaceutically active ingredient (each pharmaceutical compound) mentioned herein can be used as the only active compound or can be administered in combination with other active compounds (referring to compounds other than the pharmaceutical compounds described herein) as long as they do not produce other adverse effects, such as an allergic reaction, etc. The combination administration includes simultaneous or sequential administration of each active compound.

[0209] The term "co-administration" refers to a method wherein two or more active compounds are provided to a subject at the same time or sequentially for the purpose of achieving a therapeutic outcome. When "co-administration" is involved, the time interval between each administration is sufficient to achieve synergy between the active compounds being administered. The two or more active compounds are in the same or different containers.

[0210] Abbreviations: QD: once a day; BID: twice a day; PO: per oral; IP: intraperitoneal injection; IM: intramuscular injection.

[0211] Example 1: Inhibitory effect of DBH inhibitor on sodium dextran sulfate (DSS)-induced colitis

[0212] 1. Materials and equipment:

[0213] Table 1. Reagents

[0214] Reagent Supplier Catalogue Number Dulbecco's Phosphate Buffered Saline (1x DPBS) Corning Incorporated 21-031-CVR Dextran sulfate sodium (DSS) MP Biomedicals SR01606 Cyclosporine A (CsA) Novartis / Physiological saline Ke Lun Pharmaceutical Co., Ltd / PEG400 Sigma Aldrich 25322-68-3 30% Solutol HS15 Sigma Aldrich 70142-34-6 Enipristin Selleckchem S2695 Etaracilast Nanjing Yabang 677773-32-9 Emicizimast MCE 1080028-80-3

[0215] Table 2. Instruments

[0216] Instrument Supplier Model Electronic balance Changzhou Tianzhiping YH-2000 Electronic analytical balance Mettle Toledo 585310

[0217] 2. Preparation and storage of liquid medicine:

[0218] The composition of the solvent is as follows: 20v% PEG400 + 10v% (30% Solutol HS15) + 70v% physiological saline.

[0219] Test substance - nimodipine liquid medicine: 27 mg of nimodipine was completely dissolved in 9 mL of solvent (this concentration corresponds to test group 1#), and prepared once every 3 days, and stored at 4°C to maintain the effect.

[0220] Test substance - etami- mide liquid medicine: 9 mg, 27 mg, and 90 mg of etamivide were completely dissolved in 9 mL of solvent, respectively (three concentrations correspond to test group 2#, test group 3#, and test group 4#, respectively), and prepared once every 3 days, and stored at 4°C to maintain the effect.

[0221] Test substance - emiviride liquid medicine: 9 mg, 27 mg, and 90 mg of emiviride were completely dissolved in 9 mL of solvent, respectively (three concentrations correspond to test group 5#, test group 6#, and test group 7#, respectively), and prepared once every 3 days, and stored at 4°C to maintain the effect.

[0222] Positive drug control - cyclosporine A liquid medicine: 200 mg of cyclosporine A was completely dissolved in 10 mL of physiological saline to prepare a solution of 20 mg / mL (corresponding to the positive drug control group), and prepared once every 3 days, and stored at 4°C to maintain the effect.

[0223] 3. Treatment of mice in each group:

[0224] 8-10 week old female C57BL / 6J mice were purchased from Charles River Laboratories. The mice were housed in a room with a constant temperature (20±2℃) and a 12-hour light-dark cycle (5 mice per cage). All experimental protocols were approved by the Wuxiapptec Institutional Animal Care and Use Committee. Prior to the experiment, the mice were acclimated for at least three days in the animal experiment center of Wuxiapptec.

[0225] (1) Vehicle group, positive drug control group and test group

[0226] To establish a sodium dextran sulfate-induced colitis model, the mice were allowed to freely drink a 3% DSS aqueous solution (3 g of DSS was added to 100 mL of water to prepare the aqueous solution, and the molecular weight of DSS was 36000-50000) for 7 consecutive days to induce colitis (which belongs to autoimmune colitis), and the mice were divided into 9 groups for administration (see Table 3, vehicle group, positive drug control group and test groups 1# to 7#), wherein the 3% DSS aqueous solution was freshly prepared every day. At the end of the experiment (see below), the mice were euthanized and end-point samples were obtained.

[0227] (2) Normal group

[0228] The mice in the normal group were not induced with a 3% DSS aqueous solution to develop colitis, but were allowed to freely drink blank water. At the end of the experiment (see below), the mice were euthanized and end-point samples were obtained.

[0229] Table 3. Groups and administration regimens

[0230]

[0231]

[0232] 4. Body weight evaluation and disease activity index (DAI) score:

[0233] The animal body weight and DAI score were both performed under double-blind conditions, i.e., the researchers did not know the groups and administration regimens, and the body weight of the mice was collected every day, the stool characteristics, bloody stool and scores were observed. The DAI score was the sum of the body weight loss score, stool frequency score and bleeding score. The scoring criteria are shown in Table 4.

[0234] Table 4. DAI scoring system

[0235]

[0236] 5. Statistical analysis:

[0237] Data analysis used ANOVA, and the method used was as follows: using Graph Pad Prism 6.0 software, post-hoc Dunnett's Dunnett multiple comparison test method was used. The remaining groups (referring to the normal group, positive drug control group and detection groups 1# to 7#) were compared with the solvent group, and whether the remaining groups were significantly different from the solvent group was analyzed. If the p value <0.05, it has a statistically significant difference, and has significance. The data is expressed as mean ± S.E.M.

[0238] Example 2: Effect of DBH inhibitor on immune cell activation and cytokines

[0239] 1. Materials:

[0240] Table 5. Flow cytometry antibody reagents

[0241]

[0242]

[0243] Table 6. Other reagents

[0244]

[0245] 2. Methods:

[0246] The positive drug control group and detection groups 1# to 7# of Example 1, 24 hours after administration on the 8th day were the experimental endpoint; the experimental endpoint of the normal group and the solvent group was the same as that of the positive drug control group and detection groups 1# to 7#.

[0247] At the experimental endpoint, the mesenteric lymph nodes were collected and treated as follows:

[0248] 1) Gently crush the mesenteric lymph nodes, filter with a 70μM filter (BD bioscience, item number 352350) to obtain a cell suspension, and count the cells.

[0249] 2) The cells were suspended in suspension (the suspension was prepared as follows: the cell activation mixture was diluted to a concentration of 1x using a mixture of 90v% 1640 medium and 10v% fetal bovine serum) to adjust the cell density to 2.5x10 7 / mL, and incubated at 37°C for 5 hours.

[0250] 3) Wash the cells with Dulbecco's PBS, and stain with dead / viable dye (No. 12 in Table 5) at room temperature for 15 min to distinguish live and dead cells; wash the cells with staining buffer (fetal bovine serum mixed with Dulbecco's PBS at a volume ratio of 2:98), and add Fc blocker working solution (prepared by mixing Fc blocker with staining buffer at a volume ratio of 1:200) to incubate at room temperature for 15 min.

[0251] 4) Without washing the cells, label the cells with 100 μL of flow antibody staining solution, and incubate at 4°C for 30 min.

[0252] 5) Wash the cells with staining buffer once, add 100 μL of cell fixation solution, and incubate at 4°C overnight.

[0253] 6) Add 200 μL of cell membrane breaking solution, and incubate at 4°C for 30 min.

[0254] 7) Wash the cells with 250 μL of cell membrane breaking washing solution, suspend the cells with 100 μL of staining buffer, and detect fluorescence with a BD LSR Fortessa instrument.

[0255] 8) The cell subgroups for flow detection analysis include CD4+T cell subgroups, IL-17A+CD4+T cell subgroups, IFN-γ+CD4+T cell subgroups, TNF-α+CD4+T cell subgroups, CD25+FOXP3+Treg cell subgroups; CD8+T cell subgroups, IL-17A+CD8+T cell subgroups, IFN-γ+CD8+T cell subgroups, TNF-α+CD8+T cell subgroups; CD69+B220+B cell subgroups, and NK1.1+CD107a+NK cell subgroups.

[0256] Example 3: DBH inhibitors improve DSS-induced colon injury

[0257] After the experiment was performed according to the experimental procedure described in Example 1, the body weight evaluation results of the mice in each group are shown in Table 7 and Figure 1A ; the DAI score results of the mice in each group are shown in Table 8 and Figure 1B ; and the colon density results of the mice in each group are shown in Table 9 and Figure 1C .

[0258]

[0259]

[0260] Compared with the vehicle group, the body weight reduction of the mice in test group 1#, test group 4#, and test group 7# on the 7th day of treatment was significantly reduced Figure 1A (Table 7).

[0261] Compared with the solvent group, mice in test groups 1#, 2#, 3#, 4#, 5#, 6# and 7# all showed significantly lower DAI scores on day 7 of treatment. Figure 1B (Table 8).

[0262] After mice were administered DSS, colonic shortening and increased colonic density were typically observed; these two indicators reflect the severity of DSS-induced colitis. Compared to the solvent group, colonic density was significantly reduced in test groups 1#, 4#, and 7#. Figure 1C (Table 9). These data indicate that nipiclone, ethamistat, and ethamistat improved the severity of DSS-induced colitis and reduced disease activity.

[0263] In conclusion, the DSS-induced colitis in test groups 1#, 4#, and 7# was milder than that in the solvent group, suggesting that nipiclone, ethamistat, and ethamistat have potential therapeutic effects on colitis.

[0264] Example 4: Effects of nipiclone and emistril on immune cells and cytokines

[0265] Following the method in Example 2, the effects of nipiclone and emistrhein on immune cells and cytokines were analyzed, and the results are shown in [Figure 2]. Figures 2A-2E , Figures 3A-3D , Figures 4A-4B .

[0266] First, changes in the CD4+ T cell subset derived from the CD3+CD45+ cell subset were analyzed, as were changes in the CD25+FOXP3+ Treg cell subset derived from the CD4+ cell subset. The results showed that, compared to the solvent group, detection groups 1# and 7# reduced the proportion of CD4+ T cell subsets. Figure 2A ), while increasing the proportion of CD25+FOXP3+Treg cell subsets ( Figure 2B The above results suggest that DBH inhibitors can significantly suppress inflammation and immunity.

[0267] Then, the cytokines secreted by CD4+ T cell subsets were further analyzed. The results showed that, compared with the solvent group, both test groups 1# and 7# reduced the pro-inflammatory factor IL-17A (IL-17A). Figure 2C ), IFN-γ Figure 2D ) and TNF-α Figure 2E The secretion of pro-inflammatory factors by CD4+ T cells suggests that DBH inhibitors exert their anti-inflammatory and immune effects by reducing the secretion of pro-inflammatory factors by CD4+ T cell subsets.

[0268] In addition, similar analysis was performed on CD8+T cell subpopulation from CD3+CD45+cell subpopulation and cytokines secreted by the same. The results showed that, compared with the vehicle group, DBH inhibitors could increase the proportion of CD8+T cell subpopulation Figure 3A ), had no significant effect on IFN-γ Figure 3B secretion, but could inhibit IL-17A Figure 3C and TNF-α Figure 3D secretion. The above results suggest that DBH inhibitors inhibit inflammation and immunity by reducing IL-17A and TNF-α secretion of CD8+T cell subpopulation.

[0269] Finally, the activation of B cells (referring to CD69+B220+B cell subpopulation from CD3-cell subpopulation) and NK cells (referring to NK1.1+CD107a+NK cell subpopulation from CD3-B220-cell subpopulation) was evaluated. The results showed that, compared with the vehicle group, DBH inhibitors could inhibit the activation of B cells Figure 4A and NK cells Figure 4B . The above results suggest that DBH inhibitors can reduce the risk of B cell producing autoantibodies and NK cell mediated non-specific killing of mucosal tissue, thereby alleviating inflammation and inhibiting immunity.

[0270] Example 5: Inhibition of bleomycin-induced skin fibrosis in male C57 mice by DBH inhibitors

[0271] 1. Materials and equipment

[0272] Table 10. Reagents

[0273] Reagent Supplier Catalogue Number Physiological saline Zhejiang Dobu Pharmaceutical Co., Ltd 2009240101 Solutol HS15 Sigma BCCF2234 PEG400 National Pharmaceutical Group Chemical Reagent Co., Ltd 20181205 MC Sigma SLBT4343 Tween 80 Sigma WXBD2914v Isoflurane RWD Life Science 20120101 Bleomycin hydrochloride Japan Chemicals 600700

[0274] Table 11. Instruments

[0275] Name Model and manufacturer Respiratory anesthesia machine AMS (Gene & I) Beijing Ji'andele Science and Technology Co., Ltd (Gene & I) Automatic blood analyzer Sysmex XS-800i Tissue dehydrator LEICA HistoCore Pearl Embedding machine Leica Histocore Arcadia C&H Microtome LEICA, RM2235 Automatic staining machine LEICA, ST5020 Section scanner HAMAMATSU Nano Zoomer S210 Digital ultrasonic cleaner KQ-100DE, Kunshan Ultrasonic Instrument Co., Ltd

[0276] 2. Preparation and storage of solutions

[0277] Composition of the vehicle: 20% PEG400 + 10% (30% Solutol HS15) + 70% physiological saline. Measure 100 mL of PEG400 and 50 mL of 30% (V / V) solutol, add to 350 mL of physiological saline; place on a magnetic stirrer and stir until it is mixed evenly, store at 4°C for standby.

[0278] Test substance - Intepirdine stock solution: 40 mg of test substance was weighed into a brown sample bottle, 0.8 mL of PEG400 was added, vortexed on a vortexer, sonicated for 15 minutes, heated in a 40°C water bath for 15 minutes, and then 0.4 mL of 30% Solutol HS15 was added, vortexed on a vortexer, and then 2.8 mL of normal saline was added, vortexed on a vortexer, until a solution was formed, and the compound concentration was 10 mg / mL. It was prepared once every three days and stored at 4°C for standby.

[0279] Control - Nintedanib stock solution: 25 mg of nintedanib (BIBF1120) was accurately weighed into 5 mL of solvent (0.5% MC + 0.2% Tween 80), mixed thoroughly until the solution was clear and transparent, and the drug concentration was 5 mg / mL (dose 10 mL / kg). It was prepared once every seven days.

[0280] Control - Imatinib stock solution: 15 mg of imatinib mesylate powder was accurately weighed into a sample bottle, 3 mL of normal saline was added, and vortexed on a vortexer until completely dissolved, and the drug concentration was 5 mg / mL. It was prepared and used within half an hour.

[0281] 3. Test method

[0282] 3.1 Animal feeding

[0283] Seventy male C57BL / 6 mice (body weight 20 to 22 g) were fed in the SPF barrier system of Kaisai Biotechnology (Suzhou) Co., Ltd. The animal use license number was SYXK (Su) 2017-0041, and the international standard temperature, humidity, and light control system was followed.

[0284] The test animal operation plan was jointly approved by the IACUC committee. The operation and management were strictly in accordance with the SOP of Kaisai Biotechnology (Suzhou) Co., Ltd.

[0285] 3.2 Model establishment

[0286] The animals were anesthetized with isoflurane (2.0 to 2.5%), the back hair was removed, and a 1 cm 2 The skin fibrosis model was established by intradermal injection of bleomycin (0.3 mg / kg, 100 μL) every two days.

[0287] 3.3 Test grouping

[0288] According to the body weight of the animals, they were divided into 7 groups, 10 in each group, namely: normal group, solvent group, nintedanib group (positive control drug), imatinib (positive control drug), test group 1#, test group 2#, and test group 3#, as shown in Table 12.

[0289] Table 12. Trial Groups and Dosing Schedule

[0290]

[0291] Note: a: 20% PEG400 + 10% (30% Solutol HS15) + 70% physiological saline.

[0292] 3.4 Test drug administration

[0293] Dosing began on the day of modeling. Each animal was weighed before administration to calculate the volume of medication. The dosing period was 28 days. Administration method: Imatinib was administered intraperitoneally, while the other groups were administered by gavage. Dosing frequency: Nintedanib was administered twice daily, while the other groups were administered once daily.

[0294] 3.5 Physiological observation of experimental animals

[0295] Record changes in animal weight twice a week from the start of modeling; closely observe clinical signs in animals, such as rapid and difficult breathing, abdominal sucking, reduced activity, and lethargy.

[0296] 3.6 Local skin observation

[0297] At the end of the experiment, the animals were euthanized by intraperitoneal injection of an excessive amount of sodium pentobarbital (100 mg / kg), and then local skin photographs were taken.

[0298] 3.7 Test endpoint

[0299] At the endpoint of the experiment, the weight was weighed and recorded, and the volume of medication administered was calculated. Six hours after administration, whole blood was collected from the orbital venous plexus of mice and a complete blood count was performed using an automated blood analyzer (Sysmex XS-800i). The specific testing steps were as follows: Before testing, the blood sample in the test tube was thoroughly mixed; the sample was placed under the sampling needle, and then the start button was pressed to aspirate the sample. After the instrument indicator light went out, the sample was removed; the instrument began automatic testing and output the results.

[0300] The animals were then anesthetized by intraperitoneal injection of sodium pentobarbital and euthanized. The dissection and sampling procedures were as follows: the spleen was distended, weighed, and its weight recorded; bilateral inguinal lymph nodes were collected, frozen, and stored at -80°C; diseased skin tissue was taken, images were acquired, and then it was fixed in 10% formalin (tissue to formalin ratio 1:10) for 48 hours before histopathological examination. Specific details are shown in Table 13 below.

[0301] Table 13. Experimental endpoints

[0302]

[0303] 3.8 Histopathological Examination

[0304] According to the pathology SOP, the lesion skin was dehydrated, embedded and sectioned, and the tissues were HE and Masson stained. Histopathological analysis was performed according to the following method:

[0305] 3.8.1 Dermis thickness

[0306] Masson-stained sections were scanned by Nanozomer S210, and the scanned images were quantitatively analyzed by Visiopharm VIS6.0 software. The degree of fibrosis was currently represented by the thickness of the dermis after Masson staining.

[0307] 3.8.2 Score of capillary density in dermis layer

[0308] Select the observation field in the dermis layer of the lesion area (determine how many fields to select according to the size of the lesion area), and score according to the area occupied by capillary density in the area:

[0309] 0: No capillary formation

[0310] 1: Capillary formation accounts for less than 25%;

[0311] 2: Capillary formation accounts for 25-50%;

[0312] 3: Capillary formation accounts for 50-75%;

[0313] 4: Capillary formation accounts for more than 75%.

[0314] 3.8.3 Score of inflammatory cell infiltration in dermis layer

[0315] Select the observation field in the dermis layer of the lesion area (determine how many fields to select according to the size of the lesion area), and score according to the degree of inflammatory cell infiltration:

[0316] 0: No inflammatory cell infiltration:

[0317] 1: Small amount of local inflammatory cell infiltration;

[0318] 2: Scattered and diffuse inflammatory cell infiltration:

[0319] 3: Large amount of diffuse inflammatory cell infiltration

[0320] 4: Large amount of inflammatory cell infiltration in clusters.

[0321] 3.9 Data analysis

[0322] The mean ± SEM was calculated using Graphpad prism 6.0 software, and the significant difference test was performed using t-test, one-way ANOVA test or two-way ANOVA test, and it was considered that there was a significant difference between two groups when p < 0.05.

[0323] 4 Test results

[0324] 4.1 Body weight change

[0325] During the experiment, the body weight of the normal group showed an upward trend, and the body weight of the test group 1#, test group 2# and test group 3# remained stable and fluctuated within the normal range. Only the nintedanib group and the imatinib group showed a slow downward trend after administration, and there was a significant difference compared with the vehicle group during the period from the 25th to the 28th day (see Table 3 below Figure 5 , Figure 6 ). Among them, one animal in the imatinib group died on the 28th day, and the body weight on the day of death decreased by 23.7% compared with the body weight before modeling.

[0326] 4.2 Spleen and viscera index

[0327] The spleen was taken at the end of the experiment, the weight of the spleen was weighed and the viscera index (spleen / weight x 100%) was calculated, and the results are shown in Table 14 Figure 7 . The spleen weight of the vehicle group was significantly decreased compared with the normal group, and the spleen weight of the nintedanib group, imatinib group, test group 1#, test group 2# and test group 3# was also significantly decreased compared with the vehicle group.

[0328] In terms of spleen viscera index, there was no significant difference between the vehicle group and the normal group, indicating that the modeling did not change the spleen viscera index of the mice, and only the nintedanib group, test group 1#, test group 2# and test group 3# had a significant difference compared with the vehicle group (Table 14, Figure 8 ).

[0329] Table 14. Spleen weight and viscera index

[0330]

[0331] *** p < 0.001 vs. normal group, # p < 0.05, ## p < 0.01 vs. vehicle group.

[0332] 4.3 Blood routine

[0333] At the end of the experiment, peripheral blood was collected for blood routine test. The results showed that imatinib significantly reduced the content of red blood cells and hemoglobin in peripheral blood, which was also consistent with the anemia symptoms of the animals in this group at the end of the experiment. In addition, imatinib could significantly reduce the content of white blood cells (including lymphocytes and neutrophils) in peripheral blood; it had a decreasing trend for monocytes, but there was no significant difference.

[0334] Compared with the vehicle group, test group 3# had significant differences in reducing lymphocytes, neutrophils and monocytes, indicating that the dose (100 mg / kg) of test group 3# had the effect of reducing inflammation, which was consistent with the pathological results Figures 9-15 ).

[0335] 4.4 Skin pathological staining results

[0336] Systemic sclerosis (SSc) is an autoimmune disease characterized by inflammation, vasculopathy, and fibrosis of the skin and organs.

[0337] In this example, bleomycin (BLM) was used to establish a mouse fibrosis model. The pathological HE staining results showed that compared with the vehicle group and the normal group, there was significant inflammatory cell infiltration and dermal subcutaneous capillary proliferation. The Masson staining results showed that the vehicle group animals had a stronger degree of skin fibrosis, and the dermal thickness was significantly increased. The treatment of nintedanib and imatinib could significantly reduce the inflammatory cell infiltration and dermal subcutaneous capillary proliferation of the diseased skin. Test group 2# and test group 3# could significantly reduce the inflammatory cell infiltration and dermal subcutaneous capillary proliferation of the dermal layer. According to the statistical results of the dermal thickness, the three different doses of the test substance, intedanib, showed a progressive effect in reducing the dermal thickness, and the higher the dose, the more obvious the result, but compared with the model group, there was no significant difference. Compared with test group 1# and test group 2#, test group 3# had a better effect on improving skin fibrosis Figures 16-18 ).

[0338] From the above test results, it can be seen that test group 2# and test group 3# can reduce the number of peripheral blood inflammatory cells that increase in response to bleomycin stimulation, reduce the inflammatory cell infiltration and capillary proliferation in the dermal layer of the diseased skin, especially test group 3# with a dose of 100 mg / kg, which has the best effect, and 100 mg / kg can also reduce the degree of fibrosis of the diseased skin.

[0339] Example 6: Effect of DBH inhibitors on bovine IRBP R16-induced experimental autoimmune uveoretinitis (EAU) in Lewis rats

[0340] 1. Materials and equipment

[0341] Table 15. Reagents

[0342]

[0343] Table 16. Equipment

[0344] Equipment Manufacturer Model Tissue processor Fisher / Thermo A78400006 Tissue embedding machine Fisher / Thermo B64100010

[0345] 2. Preparation and storage of solutions

[0346] Test article - Intepirdine solution: For the preparation of a 10 mg / mL intepirdine solution, weigh 40 mg of intepirdine into a brown sample bottle, add 0.8 mL of PEG 400, vortex on a vortexer, sonicate for 15 minutes, heat in a 40 °C water bath for 15 minutes, form a suspension, then add 0.4 mL of 30% Solutol HS 15, vortex the suspension, then add 2.8 mL of normal saline, vortex completely to solution. Prepare fresh daily.

[0347] Control article - dexamethasone solution: Dexamethasone is suspended in 0.5% sodium carboxymethylcellulose to a concentration of 0.04 mg / mL. Prepare fresh daily.

[0348] IRBP R16 solution: Dissolve bovine IRBP R16 polypeptide in normal saline to a final concentration of 300 μg / mL.

[0349] Complete Freund's Adjuvant (CFA): Mix 15 mg of Mycobacterium tuberculosis H37Ra with 10 mL of CFA to a final concentration of 2.5 mg / mL (CFA itself contains 1.5 mg / mL of H37Ra, add 15 mg of H37Ra to 10 mL of CFA to a final concentration of 2.5 mg / mL).

[0350] Emulsion preparation: The emulsion is prepared by hand mixing. First, take two 10 mL syringes, one of which is filled with 4 mL of the 300 μg / mL IRBP R16 solution and attached to a three-way stopcock valve, ensuring that all air bubbles are removed. Then attach a syringe containing 4 mL of CFA and begin mixing rapidly. Mix by hand by pushing the plunger back and forth for 5 minutes. Finally, use the other 10 mL syringe to draw up 8 mL of the emulsion, then attach a large gauge needle (e.g., 18 g), insert the needle into the end of a 1 mL syringe, and divide the emulsion into 10 1 mL syringes, ensuring that there are no air bubbles by pushing the plunger back and forth while the needle is attached to the syringe. The emulsion should be used within 3 hours of preparation.

[0351] 3. Test method

[0352] The test rats were 6- to 8-week-old female Lewis rats, about 180 to 220 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and were free of specific pathogens at the start of the experiment. The test rats were kept in a room (2 to 4 rats per cage) under a 12 / 12-hour light-dark cycle at room temperature (20 to 26°C, relative humidity 40 to 70%). All experimental protocols were approved by the Pingli Biological IACUC (Institutional Animal Care and Use Committee). Before the experiment, the test rats were acclimated in the Pingli Biological laboratory for 7 days.

[0353] (1) Immunization of rats with IRBP R16 emulsion

[0354] On day -1 (i.e., the day before immunization, the same below), 60 rats were randomly divided into 6 groups (n = 10) according to body weight (see Table 18). On day 0, rats in groups 2 to 6 were anesthetized with isoflurane, and then 200 mL of emulsion obtained by emulsifying IRBP R16 with CFA at a ratio of 1:1 v / v was injected subcutaneously, respectively, in both thighs (50 μL per site) and the base of the tail (100 μL).

[0355] where day 0 refers to the day of immunization. The body weight of each rat was monitored twice a week after immunization.

[0356] (2) Clinical evaluation of EAU in rats

[0357] Starting from day 0 after immunization, the upper and lower eyelids of the eyes of the test rats were gently probed, and the eyes of the rats were examined every day with a flashlight, and the incidence of disease was recorded. According to the criteria listed in Table 17, the clinical symptoms were scored in a blinded manner from the onset of the disease to the end of the study.

[0358] Table 17. Clinical score of EAU in rats

[0359] Score Standard 0 No disease; eye translucent and reflecting light (red reflection) 0.5 (trace) Iris blood vessels dilated 1 Iris blood vessels filled; abnormal pupil contraction 2 Blurred anterior chamber; red reflection weakened 3 Moderate opaque anterior chamber, but pupil still visible; dark red reflection 4 Anterior chamber turbidity, pupil blurred; red reflection missing; eyeball protrusion

[0360] (3) Dosing regimen

[0361] Dosing started on day 0 and was precise according to the body weight of the animals, for a total of 16 days. The dosing regimen of the test rats is shown in Table 18.

[0362] Table 18. Dosing regimen

[0363]

[0364] a: 20% PEG400 + 10% (30% Solutol HS15) + 70% physiological saline

[0365] b: 0.5% sodium carboxymethyl cellulose.

[0366] (4) Statistical analysis of experimental results

[0367] Results are expressed as "mean ± standard error". Statistical analysis was performed using Graphpad Prism or SPSS, and p<0.05 was considered statistically significant.

[0368] 4. Test results

[0369] (1) Body weight of rats

[0370] The body weight changes of the test rats are shown in Table 19 below, and are shown as mean and standard error, respectively.

[0371] Table 19. Body weight of rats (g)

[0372] Mean Day -1 Day 2 Day 6 Day 9 Day 13 Day 16 Vehicle 195.89 198.20 209.00 215.42 216.80 221.80 Model 186.99 192.94 200.05 204.24 209.85 210.04

[0373] Dexamethasone 188.46 174.65 166.65 164.05 162.10 161.29 Inopidem (25 mpk) 188.31 193.95 204.67 207.81 214.28 219.00 Inopidem (50 mpk) 189.34 194.04 202.28 206.07 216.20 218.42 Inopidem (100 mpk) 189.92 194.02 205.97 214.36 223.98 227.40 Standard error Day -1 Day 2 Day 6 Day 9 Day 13 Day 16 Vehicle 2.77 2.32 1.97 2.84 2.62 3.28 Model 2.49 1.87 2.47 2.33 2.98 2.84 Dexamethasone 2.41 1.66 2.19 2.29 3.13 3.27 Inopidem (25 mpk) 2.55 2.13 2.94 2.31 2.50 3.23 Inopidem (50 mpk) 2.61 2.21 2.14 3.36 3.39 2.81 Inopidem (100 mpk) 2.72 3.82 3.38 3.85 3.28 3.86

[0374] (2) Rate of weight change

[0375] Table 20. Rate of weight change (%)

[0376] Mean Day -1 Day 2 Day 6 Day 9 Day 13 Day 16 Vehicle 0.00％ 3.47％ 7.84％ 9.71％ 11.59％ 13.89％ Model 0.00％ 3.25％ 7.06％ 9.26％ 12.23％ 12.36％ Dexamethasone 0.00％ -7.26％ -11.53％ -12.92％ -13.99％ -14.44％ Inopidem (25 mpk) 0.00％ 3.25％ 7.06％ 9.26％ 12.23％ 12.36％ Inopidem (50 mpk) 0.00％ 2.53％ 6.90％ 8.83％ 14.24％ 15.41％ Inopidem (100 mpk) 0.00％ 2.11％ 8.45％ 12.84％ 17.96％ 19.77％ Standard error Day -1 Day 2 Day 6 Day 9 Day 13 Day 16 Vehicle 0.00％ 0.61％ 0.88％ 0.78％ 0.76％ 0.81％ Model 0.00％ 0.72％ 1.24％ 0.60％ 0.75％ 0.95％ Dexamethasone 0.00％ 0.92％ 0.91％ 0.91％ 1.28％ 1.18％ Inopidem (25 mpk) 0.00％ 0.72％ 0.84％ 0.87％ 1.20％ 1.06％ Inopidem (50 mpk) 0.00％ 0.61％ 0.81％ 0.87％ 1.48％ 1.05％ Inopidem (100 mpk) 0.00％ 0.97％ 0.88％ 0.84％ 0.89％ 1.46％

[0377] (3) Clinical score

[0378] See Figure 19 and Table 21.

[0379] Table 21. Area under the curve (AUC) of clinical score

[0380]

[0381] a: Inhibition rate = [AUC (model control) - AUC (test group)] / AUC (model control), and the test group includes the dexamethasone group and the intepicet group.

[0382] (4) Incidence

[0383] Table 22. Incidence (%)

[0384]

[0385] The success of the rat uveitis model was confirmed by the increase in the EAU clinical score. The treatment of intepicet slightly improved the symptoms of uveitis, reduced the EAU clinical score and the clinical score AUC, but this effect did not reach statistical significance.

[0386] The inhibition rates of nepicastat on clinical score AUC at 25, 50 and 100 mg / kg doses were 8.67%, 20.06%, 24.09%, respectively. As a positive control, by dexamethasone treatment, the clinical score of uveitis and clinical score AUC were significantly reduced, and the inhibition rate on clinical score AUC was 84.53%.

[0387] Example 7: Effect of DBH inhibitors on NMO-IgG-induced mouse opticospinal encephalomyelitis (NMO)

[0388] 1. Materials and equipment

[0389] Table 23. Reagents

[0390]

[0391] Table 24. Equipment

[0392] Equipment Manufacturer Model RWD Stereotaxic Instrument RWD Life Science Co., Ltd. 68025 Kd Scientific syringe pump Kd Scientific legato 130 Hamilton microsyringe needle Hamilton Gastight #1702 Disposable suture needle with wire Ningbo Medical Suture Needle Co., Ltd. Angled needle 3 / 0 single needle

[0393] 2. Preparation and storage of liquid medicine

[0394] Test substance-nepicastat liquid medicine: for example, 5 mg / mL nepicastat liquid medicine was prepared by weighing 50 mg of nepicastat into a brown sample bottle, adding 1.98 mL of PEG400, vortexing on a vortex instrument, ultrasonicating for 15 minutes, heating in a 40°C water bath for 15 minutes to form a suspension, then adding 0.99 mL of 30% Solutol HS15, vortexing 2.97 mL of the suspension, then adding 6.93 mL of normal saline, and completely vortexing to a solution. Freshly prepared daily.

[0395] Control-dexamethasone liquid medicine: dexamethasone was dispersed in normal saline at a concentration of 0.1 mg / mL. Freshly prepared daily.

[0396] Control-tanshinone IIa liquid medicine: tanshinone IIa was dissolved in PBS containing 2% DMSO at a concentration of 0.0736 mg / mL.

[0397] 3. Test method

[0398] Test mice were female C57 mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., weighing 20 to 22 g, and free of specific pathogens at the start of the experiment.

[0399] (1) Modeling step

[0400] 1) C57 female mice (6 to 8 weeks) were anesthetized by intraperitoneal injection of 5% chloral hydrate (70 μL / 10 g).

[0401] 2) Top of the head cut hair leak scalp, the mouse head fixed on the brain stereotaxic instrument, so that the top of the head skull remains roughly horizontal. Iodophor disinfection.

[0402] 3) Longitudinal cut the scalp about 0.5 cm, find the anterior fontanelle and posterior fontanelle area. Rotate the stereotaxic instrument caliper, move the microsyringe needle tip to the anterior fontanelle and posterior fontanelle area, keep the mouse skull horizontal by observing the height of the needle tip from the anterior and posterior fontanelle points. After adjusting to the horizontal, move the needle tip to the anterior fontanelle point, then move 2 mm to the right for positioning.

[0403] 4) Mark the mouse skull surface after positioning, drill a hole at the point with a skull drill. During drilling, pay attention to avoid blood vessels, carefully deepen, avoid damaging the dura mater and brain tissue.

[0404] 5) Use the stereotaxic instrument to vertically insert the microsyringe needle into the mouse brain tissue, vertically insert the needle to a depth of 3 mm, keep the needle in place, and stop the needle for 5 min.

[0405] 6) Turn on the infusion pump, select the Infuse Only mode, and the speed is 1 μL / min, for injection (2 μL NMO-IgG + 3 μL human complement + 5 μL PBS / drug, mixed and blown evenly in advance, placed on ice).

[0406] 7) After injection is completed, stop the needle and wait for 10 min. Rotate the caliper and slowly pull out the needle upward, stop the needle for 5 min after 1 mm upward, until the microsyringe needle is completely pulled out, suture the skin, and disinfect with iodophor again.

[0407] (2) Dosing regimen

[0408] Dosing starts 3 days before modeling, and dosing lasts for 7 days after modeling, for a total of 10 days of continuous dosing. The specific dosing regimen is shown in Table 25.

[0409] Table 25. Dosing regimen

[0410]

[0411] (3) Sample collection

[0412] On the 8th day of the model (i.e. the day after the last dose), the experimental mice were euthanized and the brain tissue was taken for frozen section. Then the immune response area of the astrocyte marker of each group of mice was determined and evaluated (for experimental methods, see the literature: Ye Gong et al., Journal of Neuroinflammation, 17(1). doi: 10.1186 / s12974-020-01874-6).

[0413] 4. Test results

[0414] Figures 20A-20DFrom the lesion ratio of AQP4 and GFAP, the group of Intepirdine can significantly reduce the lesion ratio compared with the control group, and the effect is lower than that of the positive drug group. But there is no significant effect on immune parameters CD45 and IBA1. Therefore, Intepirdine has a certain inhibitory effect on NMO-IgG induced mouse optic neuritis.

[0415] Example 8: Therapeutic effect of DBH inhibitor on imiquimod (IMQ) induced psoriasis

[0416] 1. Materials and equipment

[0417] Table 26. Reagents

[0418] Reagent Supplier Catalogue number 5% Imiquimod cream Aldara, 3M Pharmaceuticals / Dexamethasone cream Sanjiyiyao Co., Ltd. H44024170 Tofacitinib citrate Dalian Meilun Biotech Co., Ltd. MB3358 PEG400 Sigma 202398 Solutol HS15 Sigma 42966 EtOH Shanghai Aldrin Biochemical Technology Co., Ltd. A500737-0500 Cremophor EL Sigma C5135 Poloxamer 188 Sigma 9003-11-6

[0419] Table 27. Equipment

[0420] Equipment Model Electronic balance MFC: 9092250 Analytical balance SECURA 225D-1CN+YDP20-0CEV1 Centrifuge Eppendorf_5424

[0421] Table 28. Test animals

[0422] Animal species and strain: Balb / c mice Breeder / supplier: Beijing Vital River Laboratory Animal Technology Co., Ltd. Gender, age: Female, 7-8 weeks Experimental institution: Pharmaron (Nantong) Co., Ltd. Acclimation period: 7 days Room: SPF level Room temperature: 20-26℃ Relative humidity: 40-70％ Light cycle: 12 / 12 hours light / dark alternation Housing density: 5 / cage Food and water: Free access to food and water

[0423] All experiments conducted in this protocol were approved by the Institutional Animal Care and Use Committee (IACUC) of Wuxiapptec.

[0424] 2. Preparation and storage of drug solution

[0425] Test substance - Intepirdine oral administration solution: prepare 9.9 mL of 5 mg / mL Intepirdine solution for oral administration: weigh 50 mg of Intepirdine, add 1.98 mL of PEG400, ultrasonic for 15 minutes, 40℃ water bath heating for 15 minutes, vortex to suspension. Add 0.99 mL of 30% Solutol HS15 to the 1.98 mL Intepirdine suspension and vortex to suspension. Add 6.93 mL of normal saline to 2.97 mL of Intepirdine suspension and vortex to solution.

[0426] Test substance - Intepirdine topical administration solution: prepare 1 mL of Intepirdine (50 mg / mL) for external use: weigh 50.13 mg of Intepirdine, add 100 μL of PG, stir at 45℃ for 10 minutes to obtain a uniform opaque suspension; add another 200 μL of ethanol and stir at 45℃ for 5 minutes to obtain a uniform opaque suspension; then add another 200 μL of Cremophor EL and stir at 45℃ for 5 minutes to obtain a uniform opaque suspension. Add another 500 μL of 5% Poloxamer 188 to the suspension and stir at 45℃ for 30 minutes to obtain a clear solution.

[0427] Reference - tofacitinib citrate solution: tofacitinib citrate was dissolved in dimethyl sulfoxide to obtain a solution having a concentration of 5 mg / mL, and 50 μL of the solution was added to the IMQ ointment for topical application.

[0428] 3. Test method

[0429] (1) Shaving of the hair

[0430] One day before the start of the experiment, the backs of all the mice were shaved with a pet hair clipper, with an area of 2 x 3 cm.

[0431] (2) Grouping

[0432] The 65 animals were randomly grouped by weight, and the dosing regimen was as follows.

[0433] Table 29. Grouping and dosing regimen

[0434]

[0435] Solvent A: 20% PEG400 + 10% (30% Solutol HS15) + 70% physiological saline;

[0436] Solvent B: 10% PG / 20% EtOH / 20% Cremophor EL / 50% (5% Poloxamer 188 aqueous solution).

[0437] (3) IMQ and positive control preparations

[0438] Imiquimod (IMQ) ointment: 62.5 mg of IMQ ointment per mouse corresponding to the dose, from G2 to G7;

[0439] Dexamethasone (DEX) ointment: 70 mg of ointment per mouse corresponding to the dose in G4;

[0440] Tofacitinib: tofacitinib was dissolved in dimethyl sulfoxide to 5 mg / mL, and 50 μL was added to the IMQ ointment for daily treatment.

[0441] (4) IMQ sensitization and administration

[0442] From day 0 to day 7, the mice in groups 2 to 7 received a daily topical dose of 62.5 mg of IMQ ointment on the shaved backs.

[0443] The administration of the test compound and the reference was carried out as shown in Table 29, for 7 consecutive days.

[0444] (5) Disease assessment

[0445] To assess the severity of the inflammation of the dorsal skin, the dorsal skin was scored according to Table 30. Erythema, scaling and thickening were scored independently on a scale from 0 to 3, 0, none; 1, mild; 2, moderate; 3, marked / severe.

[0446] Table 30. Clinical scoring parameters

[0447]

[0448] (6) Termination of the experiment and sampling

[0449] At termination of the experiment, the mice were euthanized by CO2 inhalation:

[0450] 1) Blood was collected by cardiac puncture and plasma was processed in triplicate, one in a protective solution and two snap-frozen;

[0451] 2) Five dorsal skin samples were collected per group in a protective solution; five dorsal skin samples were collected in PFA; five dorsal skin samples were snap-frozen;

[0452] 3) Lymph nodes were collected and snap-frozen;

[0453] 4) Spleen samples were collected and weighed Figure 23 .

[0454] (7) Statistical analysis

[0455] Statistical analysis of the data was performed using Graph Pad Prism 6.0 software by one-way ANOVA.

[0456] 4. Results of the study

[0457] (1) Body weight

[0458] Body weight was monitored daily throughout the study. The continuous use of the IMQ cream reduced the average body weight in the vehicle group. In addition, the body weight decrease was more pronounced in the dexamethasone-treated group (G4). However, there was no statistically significant difference between the treated and vehicle groups Figure 21 .

[0459] (2) Clinical scores

[0460] The dorsal skin was scored daily for erythema, scaling and thickness. In addition, the sum of the three (erythema + thickness + scaling) was expressed as a total score. The dexamethasone-treated group had a clear inhibitory effect on the inflammation, suggesting that the psoriasis model induced by the IMQ cream was successful.

[0461] The oral group of Intoplicstat inhibited erythema and thickness from day 5 to day 7. The topical group of Intoplicstat inhibited erythema from day 4 to day 7. Tofacitinib showed efficacy after dose doubling. Intoplicstat treatment did not affect the development and incidence of the disease, but showed efficacy in the later stage of the disease (day 5 to day 7). The area under the curve (AUC) was calculated according to the total clinical score curve of each group of animals Figure 22

[0462] The inhibition rate was calculated according to the following formula: Inhibition rate = [AUC(solvent) - AUC(treatment)] / AUC(solvent) x 100%. Wherein: Solvent refers to the solvent group corresponding to the administration mode.

[0463] The inhibition rate of the dexamethasone treatment group was 96.96%. The 50mpk group of Intoplicstat treated orally had a significant inhibitory effect on the clinical score of the skin, with an inhibition rate of 23.64%. However, the 100mpk group of Intoplicstat treated topically had no significant effect.

[0464] Table 31. Erythema score results

[0465]

[0466]

[0467] Table 32. Scale score results

[0468]

[0469] Table 33. Thickness score results

[0470]

[0471] Table 34. Total score results of erythema + scale + thickness

[0472]

[0473]

[0474] 5. The purpose of this example is to study the efficacy of the test compound on the IMQ-induced psoriasis model. The results show that the application of IMQ will cause severe skin inflammation. Compared with the control group of the solvent group, the PO treatment group of Intoplicstat has a slight treatment effect on clinical symptoms.

[0475] Example 9: Therapeutic effect of DBH inhibitors on collagen-induced arthritis (CIA) in mice

[0476] 1. Experimental materials

[0477] Bovine type II collagen, CII, Sichuan University;

[0478] ​Acetic acid, Sigma (St. Louis, MO, USA), Cat. No. A8976;

[0479] Complete Freund's Adjuvant, Sigma, Cat. No. F5881;

[0480] 20% PEG400 + 10% (30% Solutol HS15) + 70% normal saline;

[0481] Tofacitinib, >98%, Dalian Milen Biotechnology Co., Ltd. MB3358.

[0482] 2. Test compound

[0483] Table 35

[0484]

[0485] Weigh 40 mg of the test substance into a brown sample bottle, add 0.8 ml of PEG400, vortex on a vortexer, ultrasonic for 15 minutes, 40°C water bath heating for 15 minutes, into a suspension; then add 0.4 ml of 30% Solutol HS15, vortex on a vortexer to mix evenly; then add 2.8 ml of normal saline, vortex on a vortexer to mix evenly, into a solution; compound concentration is 10 mg / ml. Prepare once every three days, stored at 4°C for standby.

[0486] 3. Experimental instruments

[0487] Anesthesia machine: Raymain, RM-HSIV-u;

[0488] High-speed homogenizer: IKA, T10basic;

[0489] Compound weighing balance: Sartorius, CPA225D;

[0490] Animal weighing balance: Changzhou Tianzhiping Electronic Balance, YH2000.

[0491] 4. Experimental animals and feeding environment

[0492] Animal pedigree: DBA / 1 mice

[0493] Supplier: Beijing Vito Lihua Experimental Animal Technology Co., Ltd.

[0494] Gender weight: male, 14-20g, specific pathogen free (SPF)

[0495] Adaptation period: 3-7 days

[0496] Environment: SPF animal feeding room, Pharm Link Shanghai Waigaoqiao Animal Feeding Center

[0497] Temperature: 20-26℃

[0498] Humidity: 40-70%

[0499] Lighting: Fluorescent lamps, 12 hours each of bright (08:00-20:00) and dark (20:00-08:00).

[0500] Stocking density: 5 birds / cage

[0501] Food: Free access to food (radiation-sterilized feed)

[0502] Drinking water: Free drinking water (prepared by a mole (ultra)pure water purifier).

[0503] The animal handling described in this embodiment report was reviewed and approved by the Laboratory Animal Use and Management (Ethics) Committee (IACUC) of WuXi AppTec.

[0504] 5. Experimental Methods

[0505] 1) Type II collagen / complete Freund's adjuvant immunization

[0506] Preparation of acetic acid: Dilute 2N acetic acid to 100mM, filter through a 0.22-micron filter membrane, and store at 4°C.

[0507] Bovine type 2 collagen solution: Dissolve bovine type 2 collagen (CII) in 100 mM acetic acid solution and store overnight at 4°C. The final collagen concentration is 8 mg / mL.

[0508] Emulsion preparation: The overnight CII solution was mixed with an equal volume of complete Freund's adjuvant and homogenized on ice at 30,000 rpm for about 60 minutes using a high-speed homogenizer until a stable emulsion was formed.

[0509] 2) Induction of arthritis

[0510] DBA / 1 mice were anesthetized with isoflurane and then subcutaneously injected with 50 μL of prepared collagen emulsion (containing 200 μg CII) into the tail. The day of the first immunization was marked as day 0, and subsequent days were numbered sequentially. On day 21, the same volume of collagen emulsion was injected into the tail.

[0511] 3) Dosage and administration design

[0512] When the model mice developed clinical symptoms, with an average score of approximately 0.5 (around day 28), they were re-randomized into 5 experimental groups of 8 mice each, based on body weight and score. The mice were administered the drug continuously for 14 days. The compound was prepared every 3 days and stored at 4°C.

[0513] Table 36. Grouping and Dosage Design

[0514]

[0515] 4) Arthritis onset marker determination

[0516] From day 28, mice were weighed and clinical scores were recorded three times a week until the end of the experiment.

[0517] Clinical score: The score was performed according to the different degrees of lesions (swelling, joint deformation) according to the 0-4 point standard, the highest score of each limb was 4 points, and the highest score of each animal was 16 points.

[0518] Table 37. Arthritis clinical score standard

[0519]

[0520] 5) Arthritis onset marker determination

[0521] a. Collect the mouse hind paws, fix them in PFA for H&E staining and pathological scoring;

[0522] b. Collect the mouse spleen for FACS staining.

[0523] 6) Statistical processing

[0524] The experimental data were expressed as mean ± standard error, the body weight and clinical score were analyzed by two-way ANOVA, and p < 0.05 was considered to be significantly different.

[0525] 6. Experimental results

[0526] See Figures 24-27 , the known effect of endostatin on collagen-induced arthritis in mice, but not as obvious as the positive control drug.

[0527] Example 10: Therapeutic effect of DBH inhibitor on 2,4-dinitrobenzenesulfonic acid (DNBS)-induced enteritis in rats

[0528] 1. Materials and equipment

[0529] Table 38. Reagents

[0530]

[0531] Table 39. Instruments

[0532] Instrument Supplier Model Enema hose France Vygon ACL 7157348

[0533] 2. Preparation and storage of liquid medicine

[0534] Test substance - Intramitox drug solution: 12 mg of intramitox was weighed into a brown sample bottle, 0.8 mL of polyethylene glycol 400 was added, vortexed and ultrasonicated for 15 minutes, heated in a 40°C water bath for 15 minutes to form a suspension, then 0.4 mL of 30% polyethylene glycol-15 hydroxystearate was added, vortexed, and then 2.8 mL of normal saline was added, vortexed to completely dissolve it.

[0535] Control - Prednisone drug solution: The use concentration of prednisone was 0.9 mg / mL, and a suspension was prepared using 0.5% sodium carboxymethylcellulose, prepared twice a week.

[0536] Preparation of DNBS drug solution: DNBS powder was dissolved with 30% ethanol to a final concentration of 50 mg / mL.

[0537] 3. Treatment of rats

[0538] Animal species and strain: Wistar rats;

[0539] Dosing history: No dosing history;

[0540] Gender, age, body weight: Male, 5 to 6 weeks, 160 to 180 g;

[0541] Breeding farm / supplier: Shanghai Slake Experimental Animal Co., Ltd;

[0542] Experimental institution: Penglai Biological Animal Room;

[0543] Acclimation period: 7 days;

[0544] Room: Ordinary area room;

[0545] Indoor temperature: 20 to 26 C;

[0546] Indoor relative humidity: 40 to 70%;

[0547] Light: Daylight lamp lighting, 12 hours of lighting and 12 hours of no lighting;

[0548] Animal feeding: 2 to 4 rats per cage (same dosing group);

[0549] Food: Free access to feed (irradiated and sterilized, Jiangsu Synergy Pharmaceutical Biological Engineering Co., Ltd., China);

[0550] Water: Free access to drinking water (treated by reverse osmosis or autoclaved).

[0551] A total of 90 male Wistar rats were purchased from Shanghai Slake Experimental Animal Co., Ltd., and the animals were free of specific pathogens, and were about 4 to 5 weeks old (140 to 150 g) when they arrived at the Penglai Biological Animal Room.

[0552] Upon arrival at PRL, the animals were transferred from their shipping containers to their home cages and each animal was examined by the animal room staff. The examination included the animal's general appearance, limbs, and orifices, and any abnormal behavior while the animal was at rest or in motion. The acclimation period was 7 days.

[0553] The experimental protocol for the animals was designed and approved by PRL's IACUC (Institutional Animal Care and Use Committee).

[0554] Based on the animals' body weights, 90 animals were randomly assigned to groups on Day -1 (the day before the official experiment) to ensure that the animals in each group were similar in body weight and to reduce bias. The rats were fasted for 40 hours prior to the experiment, and during the fasting period, the rats were given a subcutaneous injection of 5% dextrose saline (10 mL / kg once daily).

[0555] Experimental groupings:

[0556] On Day 1 of the experiment, the fasted rats were anesthetized with a combination of Xylazine (25 mg / kg) and Zolazepam (25 mg / kg) and 5 mg / kg of Telzol.

[0557] For Groups G2-G6, a flexible tube was inserted into the colon from the anus (about 8 cm from the anus) and the rats were given a DNBS enema (0.5 mL / rat) to induce colitis. The normal control group (G1) was given a 30% ethanol enema in the same manner. The enema animals were placed in a Trendelenburg position for 15 minutes with their heads down, and then they were kept in this position until they were awake to prevent reflux of the enema fluid.

[0558] Table 40. Grouping and dosing schedule

[0559]

[0560] 4. Detection index

[0561] (1) Animal body weight: The animals' body weights were measured and recorded daily, and the animals' daily activities were observed and abnormal conditions were recorded. The percentage of body weight was calculated according to the following formula: [(body weight on day x - initial body weight) / initial body weight] x 100%.

[0562] (2) Fecal score: During the experiment, the rats' fecal condition was scored daily (0 = normal, 1 = wet / sticky, 2 = soft, 3 = liquid).

[0563] (3) Colon observation: At the end of the experiment, all animals were anesthetized with Xylazine (i.v., 25 mg / kg), the abdominal cavity was opened, and blood was collected from the abdominal aorta into EDTA tubes (centrifugation conditions: 4°C, 2000 g, 10 min). After the animals were sacrificed by exsanguination, the colon (from the cecum to the anus) was removed and immediately measured for length. The colon was split longitudinally, rinsed clean, and then the weight, ulcer area, and whole colon were photographed. The colon was divided into three parts, two of which were snap-frozen in liquid nitrogen and stored at -80°C for MPO and cytokine detection. The third part of the colon was fixed in 10% neutral buffered formalin. If the ulcer was irregular in shape, it was considered rectangular, and then its length and width were measured for ulcer area assessment. Ulcer area (cm 2 ) = ulcer length (cm) x ulcer width (cm).

[0564] (4) Pathological analysis of colon tissue

[0565] The proximal, ulcer (if no ulcer, the approximate site was taken), and distal parts of the colon tissue fixed in neutral formalin were embedded in paraffin, sectioned (5 μm thickness), and stained with H&E for histopathological scoring.

[0566] Table 41

[0567]

[0568] 5. Statistical analysis

[0569] The experimental data are expressed as mean ± standard error. The data were analyzed by Graphpad Prism using the appropriate statistical method. A p value of <0.05 was considered to be statistically significant.

[0570] 6. Experimental results

[0571] (1) Body weight and fecal score See Figures 28-31 .

[0572] (2) Macroscopic evaluation of the colon on day 7

[0573] Table 42. Macroscopic evaluation of the colon on day 7

[0574]

[0575] Table 43. Macroscopic evaluation of the colon on day 7

[0576]

[0577]

[0578] BW: body weight;

[0579] CL: colon length;

[0580] CW: colon weight;

[0581] a IR1 (inhibition rate 1) = {[CW / CL / BW (model group) - CW / CL / BW (test drug group)] / [CW / CL / BW (model group) - CW / CL / BW (normal group)]} x 100%;

[0582] b IR2 (inhibition rate 2) = {[CW / BW (model group) - CW / BW (test drug group)] / [CW / BW (model) - CW / BW (normal group)]} x 100%;

[0583] c IR3 (inhibition rate 3) = {[CW / CL (model group) - CW / CL (test drug group)] / [CW / CL (model group) - CW / CL (normal group)]} x 100%.

[0584] (3) Pathological score

[0585] Table 44. Pathological score

[0586]

[0587] 7. Conclusion

[0588] Wistar rats were induced to develop inflammatory colitis by intracolonic instillation of DNBS. The colitis was manifested by significant decrease in body weight, significant increase in stool form score, significant decrease in colon length, significant increase in colon weight, colon weight: colon length (i.e. CW / CL), colon weight: body weight ratio (i.e. CW / BW) and colon weight: colon length: body weight ratio (i.e. CW / CL / BW), significant increase in colon ulcer area, significant increase in colon inflammatory cell infiltration score and histological damage score.

[0589] In this example, the positive control drug prednisone was able to significantly decrease the AUC of stool form score, intestine, CW / CL, CW / BW, CW / CL / BW and colon ulcer end inflammatory cell infiltration score of the model rats, with inhibition rates of 44.20%, 50.13% and 46.51% for CW / CL / BW, CW / BW and CW / CL, respectively.

[0590] The inhibition rates of test group 1# (3 mg / kg) for CW / CL / BW, CW / BW and CW / CL were 32.98%, 40.24% and 30.82%, respectively.

[0591] Detection group 2# (internal match 10 mg / kg can significantly reduce CW / CL, CW / CL / BW and colon tissue damage total score, CW / CL / BW, CW / BW and CW / CL inhibition rate was 44.95%, 47.06% and 43.80% respectively.

[0592] Detection group 3# (30 mg / kg) on CW / CL / BW, CW / BW and CW / CL inhibition rate was 27.12%, 39.85% and 26.68% respectively.

[0593] In summary, internal match has certain prevention and treatment effect on DNBS induced colitis in rats.

[0594] Example 11: Inhibition of DBH inhibitors on sodium dextran sulfate (DSS) induced colitis

[0595] 1. Experimental materials

[0596] Table 45. Reagents

[0597]

[0598] Table 46. Test substances

[0599]

[0600] Experimental animals: C57BL / 6 mice; source: Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.; gender: female. Main instruments: electronic balance: Sartorius, QUINTIX 35-1CN.

[0601] 2. Experimental method

[0602] 2.1 Preparation and storage of compounds

[0603] Take an appropriate amount of cyclosporine A (CsA) powder and place it in a brown sample bottle. Add 2% DMSO, 30% PEG300, 5% Tween 80 and 63% saline in appropriate proportions, shake to dissolve, and prepare once a day.

[0604] Take an appropriate amount of internal match and place it in a brown sample bottle. Add 20% PEG400, 10% (30% Solutol HS15) and 70% physiological saline in appropriate proportions, mix well, and ultrasonic dissolve, and prepare every 3 days to maintain its stability.

[0605] Take an appropriate amount of fusaric acid and place it in a brown sample bottle. Add an appropriate amount of physiological saline and ultrasonic dissolve, and prepare once every seven days.

[0606] Take the appropriate amount of disulfiram and place it in a brown sample bottle. Add the appropriate amount of 50% PEG300 and 50% normal saline in a certain proportion, and ultrasonically dissolve it into a suspension. Mix well before administration. Prepare once every seven days.

[0607] Take the appropriate amount of fumaric acid and place it in a brown sample bottle. Add the appropriate amount of normal saline, and dissolve it in a water bath. Prepare once a day.

[0608] Table 47. Drug storage concentration and administration volume

[0609]

[0610] 2.2 Animal feeding

[0611] 70, 8-week-old, 20g or so, female C57BL / 6 mice were raised in separate ventilated cages (IVC, 5 per cage), with temperature control (20±2℃), light / dark alternation 12 / 12 hours. Before the experiment, the mice were adaptively fed in the SPF animal room for three days, during which sufficient water and food were ensured.

[0612] 2.3 Grouping and administration scheme

[0613] The experimental mice were adaptively fed in the SPF animal room for three days, and randomly divided into 7 groups (8 model groups and 1 control group) with 10 mice in each group. The mice were administered from day 0 to day 7.

[0614] Table 48. Animal grouping and administration scheme

[0615]

[0616] 2.4 Model establishment

[0617] The mouse enteritis model in this example was induced by DSS: model mice drank 3.1% DSS (sulfated dextran sodium, molecular weight 36000-50000) aqueous solution from day 0 to day 7. The DSS aqueous solution was prepared fresh every day to prevent degradation. The normal control group of mice drank normal water. The experimental period was 9 days, and the mice were euthanized at the end of the experiment and the samples were collected.

[0618] 2.5 Disease activity (DAI) score

[0619] DAI score was performed daily, and DAI score was composed of three parts: weight change, fecal character and blood in stool (or occult blood) score. The specific scoring criteria are shown in Table 49. During the entire experimental process, the DAI score of all mice was completed by the same person.

[0620] Table 49. DAI score criteria

[0621] Score Percentage of body weight loss Fecal character Blood in stool or occult blood 0 0 Normal Occult blood negative 1 1-5% Soft stool Weakly positive occult blood 2 6-10% Loose stool Positive occult blood 3 11-20% Diarrhea Small amount of blood 4 >20％ Water stool Large amount of blood

[0622] 2.6 Collection of colon tissue

[0623] Mice were euthanized at the end of the experiment, and the colon was removed for photography, length measurement, and weight measurement after removal of the contents.

[0624] 2.7 Statistical analysis

[0625] Data are expressed as mean ± SEM Body weight change and disease activity index were analyzed by two-way ANOVA and Dunnett’s for intergroup comparison, and the rest of the data were analyzed by one-way ANOVA and Dunnett’s for intergroup comparison. All analyses were performed using GraphPad Prism software. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 vs. vehicle group.

[0626] 3. Experimental results and analysis

[0627] The body weight data of mice in each group during the experiment and the corresponding DAI scores were collected, and the data were analyzed by Two-way ANOVA.

[0628] As shown in Figure 32 , compared with the vehicle group, the positive control CsA (50 mg / kg, QD) and Intoplicin (50 mg / kg, QD) effectively alleviated the DSS-induced weight loss in mice. Compared with the vehicle group, fusaric acid (100 mg / kg, bid) and disulfiram (100 mg / kg, qd) failed to effectively alleviate the DSS-induced weight loss in mice. Fumaric acid (100 mg / kg, qd) had a trend of alleviating DSS-induced weight loss in mice.

[0629] As shown in Figure 33 , the disease activity index (DAI) of mice in the positive control CsA (50 mg / kg, QD) group and Intoplicin (50 mg / kg, QD) group was significantly lower than that in the vehicle group. During the administration period, compared with the vehicle group, the DAI of mice in the fusaric acid (100 mg / kg, bid) group, disulfiram (100 mg / kg, qd), and fumaric acid (100 mg / kg, qd) groups was slightly improved, among which the improvement was more obvious on the 4th day and the 5th day, but the effect was lower than that of Intoplicin.

[0630] Based on the combined changes in body weight and DAI scores, the positive controls CsA (50 mg / kg, QD) and nepicillin (50 mg / kg, QD) effectively alleviated the symptoms of weight loss, diarrhea, and bloody stools in mice induced by DSS-induced colitis. Fusarium oxysporin (100 mg / kg, bid), disulfiram (100 mg / kg, qd), and fumaric acid (100 mg / kg, qd) slightly improved the symptoms of diarrhea and bloody stools in mice induced by DSS-induced colitis, but failed to effectively alleviate the weight loss caused by DSS-induced colitis.

[0631] References

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[0638] 7. Bhaduri N, Sinha S, Chattopadhyay A, Gangopadhyay PK, Singh M, Mukhopadhyay KK. Analysis of polymorphisms in the dopamine beta hydroxylase gene: association with attention deficit hyperactivity disorder in Indian children. Indian Pediatrics. 2005, 42(2): 123-9.

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[0640] 9. Combarros O, Warden DR, Hammond N, Cortina-Borja M, Belbin O, Lehmann MG, et al. The dopamine beta-hydroxylase-1021C / T polymorphism is associated with the risk of Alzheimer's disease in the Epistasis Project. BMC Medical Genetics. 2010, 11(161): 162.

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Claims

1. The use of pathway modulators in the preparation of drugs for treating autoimmune diseases; among which, The pathway regulator is a dopamine β-hydroxylase inhibitor, and the dopamine β-hydroxylase inhibitor is nepicistat or a pharmaceutically acceptable salt thereof; The autoimmune disease is autoimmune colitis, which is selected from Crohn's disease and ulcerative colitis.

2. The use according to claim 1, wherein, The unit dose of the pathway modulator is 10-100 mg / kg.

3. The use according to claim 1, wherein, The unit dose of the pathway modulator is 20-50 mg / kg.

4. Use of a pharmaceutical composition in the preparation of a medicament for treating autoimmune diseases, wherein the pharmaceutical composition comprises: Dopamine β-hydroxylase inhibitors, and Pharmaceutically acceptable carriers; in, The dopamine β-hydroxylase inhibitor is nepicistat or a pharmaceutically acceptable salt thereof. The autoimmune disease is autoimmune colitis, which is selected from Crohn's disease and ulcerative colitis.

5. The use according to claim 1 or 4, wherein the drug is used for one or more of the following: It reduces the proportion of CD4+ T cells, increases the proportion of regulatory T cells, increases the proportion of CD8+ T cells, reduces the secretion of pro-inflammatory factors by CD4+ T cells, reduces the secretion of pro-inflammatory factors by CD8+ T cells, inhibits the activation of B cells and NK cells; improves stool formation score, improves colon weight / colon length, improves colon weight / body weight, improves colon weight / colon length / body weight, inhibits the increase of colonic ulcer area, improves colonic inflammatory cell infiltration score, improves tissue damage score; improves disease activity score, and improves hematochezia or occult blood in stool.

6. The use according to claim 5, wherein: The pro-inflammatory factors of the CD4+ T cells are one or more of IL-17A, IFN-γ, and TNF-α; The pro-inflammatory factors of the CD8+ T cells are IL-17A and / or TNF-α.

7. The use according to claim 5, wherein, The regulatory T cells are CD25+FOXP3+Treg cells; And / or, the B cells are B220+ cells; And / or, the NK cells are NK1.1+ cells.

8. The use according to claim 5, wherein, The regulatory T cells are CD25+FOXP3+Treg cells; And / or, the B cells are CD69+B220+B cells; And / or, the NK cells are NK1.1+CD107a+NK cells.

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