WO2022161364A1 - 通路调节剂、含其的药物组合物、其用途和采用其的治疗方法 - Google Patents

通路调节剂、含其的药物组合物、其用途和采用其的治疗方法 Download PDF

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WO2022161364A1
WO2022161364A1 PCT/CN2022/073849 CN2022073849W WO2022161364A1 WO 2022161364 A1 WO2022161364 A1 WO 2022161364A1 CN 2022073849 W CN2022073849 W CN 2022073849W WO 2022161364 A1 WO2022161364 A1 WO 2022161364A1
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cells
disease
group
nepicastat
pharmaceutically acceptable
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PCT/CN2022/073849
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English (en)
French (fr)
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孙巧玲
潘柯
邓一军
陈峪
吕静
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江苏亚虹医药科技股份有限公司
上海亚虹医药科技有限公司
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Priority to CN202280007360.7A priority Critical patent/CN116490177A/zh
Priority to CA3205182A priority patent/CA3205182A1/en
Priority to MX2023008573A priority patent/MX2023008573A/es
Priority to KR1020237028368A priority patent/KR20230137374A/ko
Priority to EP22745238.0A priority patent/EP4285898A1/en
Priority to JP2023544376A priority patent/JP2024503906A/ja
Priority to AU2022213938A priority patent/AU2022213938A1/en
Publication of WO2022161364A1 publication Critical patent/WO2022161364A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/417—Imidazole-alkylamines, e.g. histamine, phentolamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to pathway modulators, pharmaceutical compositions containing the same, uses thereof, and methods of treatment employing the same.
  • Dopamine beta-hydroxylase also known as dopamine beta-monooxygenase, is an enzyme encoded by the DBH gene in humans (EC 1.14.17.1). DBH catalyzes the oxidation of dopamine by oxygen to norepinephrine and epinephrine as follows:
  • DBH is an approximately 290 kDa copper-containing oxygenase composed of four identical subunits whose activity requires ascorbic acid as a cofactor [1].
  • 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 endings in the central and peripheral nervous system and in chromium cells in the adrenal medulla.
  • DBH primarily contributes to the biosynthesis of trace amines and catecholamines, including epinephrine (epinephrine or adrenaline), norepinephrine (norepinephrine or noradrenaline), and dopamine. In addition, it is also involved in the metabolism of exogenous biomass associated with these substances. For example, human DBH catalyzes the ⁇ -hydroxylation of amphetamine and para-hydroxyamphetamine to yield norephedrine and para-hydroxynorephedrine, respectively [2-4].
  • DBH is thought to be associated with mental decision-making and drug addiction disorders, such as alcoholism [5] and smoking [6], attention deficit hyperactivity disorder [7], schizophrenia [8], and Alzheimer’s disease [9].
  • Deficiency of DBH is called dopamine beta-hydroxylase deficiency.
  • Nepicastat [10], Etamicastat [11], and Zamicastat [12] were successively developed as highly selective DBH inhibitors. It has also been further studied for its potential use in hypertension (Pulmonary Arterial Hypertension), congestive heart failure, cocaine dependence or post-traumatic stress disorder (PTSD).
  • hypertension Pulmonary Arterial Hypertension
  • congestive heart failure congestive heart failure
  • cocaine dependence post-traumatic stress disorder
  • Nepicastat may be used for the treatment of congestive heart failure and has been well tolerated in patients [13]. Further clinical research data show that nepicastat has no effect on the treatment of heart failure, but it is safe.
  • Nepicastat and its analogs have potential use in the treatment of hypertension.
  • Clinical trials evaluating nepicastat for the treatment of PTSD and cocaine dependence have been completed. The study found that when nepicastat was combined with cocaine, nepicastat was safe and inhibited the positive effects of cocaine. This result suggests that nepicastat can be used as a pharmacotherapy for cocaine dependence [14].
  • etaministat and etaministat have lower brain-blood-barrier penetration levels and can reduce norepinephrine levels in peripheral sympathetically innervated tissues, while spontaneously high levels of norepinephrine.
  • the brain tissue of hypertensive rats had no effect [15].
  • DBH inhibitors such as etaministat and etaministat can reduce adverse reactions or complications in the central nervous system in the treatment of peripheral-related diseases.
  • etaministat 200 mg was observed to reduce systolic and diastolic blood pressure in a dose-dependent manner after 10 days of treatment, with good tolerability and safety [16] .
  • Eminostat (1200 mg) also showed good safety when administered for 10 consecutive days (NCT02151994).
  • a clinical trial of eminostat for pulmonary hypertension is currently underway (NCT04316143).
  • DBH directly and indirectly runs through the entire brain source, tissues, blood vessels and peripheral blood through noradrenergic fibers. Except for noradrenergic fibers, its protein expression and localization also have certain tissue specificity. For example, DBH is present in chromaffin cells of adrenal medulla and noradrenergic cells of locus coeruleus, liver tissue, and intestinal tissue. A certain level of expression, of which DBH is the highest expression in the adrenal medulla.
  • DBH expression in tissues or high levels of DBH secretion in peripheral blood may be associated with some neurological or endocrine diseases [18]; disease, hypothyroidism, and PTSD.
  • DBH levels in peripheral blood may be related to neurological diseases
  • DBH inhibitors in clinical trials have not achieved good efficacy in Alzheimer's disease and PTSD (see the database of CLINICALTRIALS).
  • DBH expression was up-regulated in inflammatory tissue in patients with autoimmune enteritis, but this study did not clarify the necessary connection between the two [19].
  • autoimmune diseases there are more than 100 types of autoimmune diseases.
  • non-targeted drugs such as hormones and/or immunosuppressive drugs are mainly used to control symptoms, and at the same time bring great and irreversible adverse reactions and damages to patients.
  • the technical problems to be solved by the present disclosure include providing a pathway modulator, a pharmaceutical composition containing the same, its use and a treatment method using the same.
  • the prior art pathway modulators including one or more of dopamine beta-hydroxylase inhibitors (abbreviated as DBH inhibitors), receptor agonists and receptor antagonists, can Through immunomodulation, the occurrence and development of autoimmune diseases can be inhibited (for example, embodied in the improvement of weight loss, improvement of DAI score, and normalization of colon density), which can provide potential therapeutic drugs for the treatment of autoimmune diseases .
  • DBH inhibitors dopamine beta-hydroxylase inhibitors
  • receptor agonists and receptor antagonists receptor agonists and receptor antagonists
  • the first aspect of the present disclosure relates to the use of a pathway regulator in the preparation of a medicine for treating autoimmune diseases; wherein the pathway regulator is one of a DBH inhibitor, a receptor agonist and a receptor antagonist or more.
  • the pathway modulator is preferably a DBH inhibitor.
  • the DBH inhibitor can be the DBH inhibitor that has been disclosed in the prior art or later, as an example, can be nepicastat, etaminista, imistat, fusaric acid , disulfiram, cysteamine, pantethine, copper chelator, fumaric acid, hydralazine, 2-thiophen-2-ylallylamine, its pharmaceutically acceptable salts and its prodrugs one or more of.
  • the DBH inhibitor is preferably selected from the group consisting of nepicastat, epiminostat, eminostat, pharmaceutically acceptable salts thereof and prodrugs thereof.
  • the DBH inhibitor is more preferably selected from the group consisting of nepicastat, etaminostat and etaminostat.
  • the use of one or more of nepicastat, etaminostat, and etaminostat to treat autoimmune diseases will not cause the patient to suffer from non-targeted drugs of hormones and/or immunosuppressants. Great and irreversible adverse reactions and damage, it is a safe therapeutic agent that can treat autoimmune diseases.
  • the DBH inhibitor is preferably selected from nepicastat, its pharmaceutically acceptable salts and its prodrugs.
  • the DBH inhibitor is more preferably nepicastat. Treatment of autoimmune diseases with nepicastat significantly reduces weight loss and colon density in patients.
  • the pathway modulator is nepicastat or a pharmaceutically acceptable salt thereof, and the unit dose is: 10-100 mg/kg; 60, 65, 70, 75, 80, 85, 90, 95, 100 mg/kg, or a range between any two of the foregoing values; preferably 20-50 mg/kg; or,
  • the pathway modulator is etaministat 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 any two of the foregoing range between values; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is eminostat 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or
  • 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 any two of the foregoing range between values; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is disulfiram 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is fumaric acid 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg.
  • unit doses can be whole numbers or fractions. It should be understood that, for example, "10 to 100" is a shorthand way of writing, and although not every point value within this range is given, it is considered to have been expressly disclosed herein.
  • the above technical solution is used for the treatment of autoimmune diseases, and can significantly reduce the weight loss of the patient and the colon density of the patient.
  • the autoimmune disease can be one or more of the autoimmune diseases disclosed in the prior art, for example, can be selected from achalasia (Achalasia), Addison's disease (Addison's disease) , Adult Still's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/anti-TBM nephritis (Anti-GBM/Anti-TBM nephritis), Antiphospholipid syndrome, Autoimmune angioedema, Autoimmune dysautonomia, Autoimmune encephalomyelitis encephalomyelitis), autoimmune hepatitis (Autoimmune hepatitis), autoimmune inner ear disease (AIED), autoimmune myocarditis (Autoimmune myocarditis), autoimmune oophoritis (Autoimmune oophoritis), autoimmune testis Autoimmune orchitis, Autoimmune
  • a second aspect of the present disclosure relates to a pathway modulator for the treatment of autoimmune diseases; wherein the pathway modulator is one or more of a DBH inhibitor, a receptor agonist, and a receptor antagonist .
  • the pathway regulator is preferably a DBH inhibitor.
  • the DBH inhibitor may be a DBH inhibitor that has been disclosed in the prior art, for example, nepicastat, etaminostat, etaminostat, fusaric acid, bismuth Sulfuram, cysteamine, pantethine, copper chelators, fumaric acid, hydralazine, 2-thiophen-2-ylallylamine, pharmaceutically acceptable salts thereof and prodrugs thereof one or more.
  • the DBH inhibitor is preferably selected from the group consisting of nepicastat, epiminostat, eminostat, pharmaceutically acceptable salts thereof and prodrugs thereof.
  • the DBH inhibitor is more preferably selected from the group consisting of nepicastat, etaminostat and etaminostat.
  • the DBH inhibitor is preferably selected from nepicastat, its pharmaceutically acceptable salts and its prodrugs.
  • the DBH inhibitor is more preferably nepicastat.
  • the pathway modulator is nepicastat or a pharmaceutically acceptable salt thereof, and the unit dose is: 10-100 mg/kg; 60, 65, 70, 75, 80, 85, 90, 95, 100 mg/kg, or a range between any two of the foregoing values; preferably 20-50 mg/kg; or,
  • the pathway modulator is etaministat 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 any two of the foregoing range between values; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is eminostat 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or
  • the pathway modulator is Fusaric acid 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is disulfiram 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is fumaric acid 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg.
  • the definition of the autoimmune disease is as defined above.
  • a third aspect of the present disclosure relates to a method for treating an autoimmune disease, comprising the steps of: administering to a subject a therapeutically effective amount of a pathway modulator; wherein the pathway modulator is a DBH inhibitor, a receptor One or more of agonists and receptor antagonists.
  • the pathway modulator is preferably a DBH inhibitor.
  • the DBH inhibitor can be a DBH inhibitor that has been disclosed in the prior art, for example, can be nepicastat, etamistat, eminostat, fusaric acid, disulfiram Alan, cysteamine, panthionine, copper chelator, fumaric acid, hydralazine, 2-thiophen-2-ylallylamine, one of its pharmaceutically acceptable salts and its prodrugs one or more.
  • the DBH inhibitor is preferably selected from the group consisting of nepicastat, epiminostat, eminostat, pharmaceutically acceptable salts thereof and prodrugs thereof.
  • the DBH inhibitor is more preferably selected from the group consisting of nepicastat, etaminostat and etaminostat.
  • the DBH inhibitor is preferably selected from nepicastat, its pharmaceutically acceptable salts and its prodrugs.
  • the DBH inhibitor is more preferably nepicastat.
  • the DBH inhibitor may be administered in combination with one or more selected from the group consisting of chemotherapeutic agents, targeted therapeutic agents, immunotherapeutic agents and anti-inflammatory agents.
  • the pathway modulator is nepicastat or a pharmaceutically acceptable salt thereof, and the unit dose is: 10-100 mg/kg; 60, 65, 70, 75, 80, 85, 90, 95, 100 mg/kg, or a range between any two of the foregoing values; preferably 20-50 mg/kg; or,
  • the pathway modulator is etaministat 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 any two of the foregoing range between values; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is eminostat 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or
  • the pathway modulator is Fusaric acid 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is disulfiram 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is fumaric acid 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg.
  • the definition of the autoimmune disease is as previously defined.
  • a fourth aspect of the present disclosure relates to a pharmaceutical composition for treating an autoimmune disease, comprising:
  • the pathway modulator is one or more of DBH inhibitors, receptor agonists and receptor antagonists.
  • the pathway modulator is preferably a DBH inhibitor.
  • the DBH inhibitor can be a DBH inhibitor that has been disclosed in the prior art, such as nepicastat, etamistat, eminostat, fusaric acid, bismuth Sulfuram, cysteamine, pantethine, copper chelators, fumaric acid, hydralazine, 2-thiophen-2-ylallylamine, pharmaceutically acceptable salts thereof and prodrugs thereof one or more.
  • the DBH inhibitor is preferably selected from the group consisting of nepicastat, epiminostat, eminostat, pharmaceutically acceptable salts thereof and prodrugs thereof.
  • the DBH inhibitor is more preferably selected from the group consisting of nepicastat, etaminostat and etaminostat.
  • the DBH inhibitor is preferably selected from nepicastat, its pharmaceutically acceptable salts and its prodrugs.
  • the DBH inhibitor is more preferably nepicastat.
  • the pathway modulator is nepicastat or a pharmaceutically acceptable salt thereof, and the unit dose is: 10-100 mg/kg; 60, 65, 70, 75, 80, 85, 90, 95, 100 mg/kg, or a range between any two of the foregoing values; preferably 20-50 mg/kg; or,
  • the pathway modulator is etaministat 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 any two of the foregoing range between values; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is eminostat 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or
  • the pathway modulator is Fusaric acid 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is disulfiram 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is fumaric acid 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg.
  • the definition of the autoimmune disease is as defined above.
  • the fifth aspect of the present disclosure relates to the use of a pathway regulator in the preparation of a medicament; the medicament is used 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 The proportion of CD8+ T cells, reducing the secretion of pro-inflammatory factors of CD4+ T cells, reducing the secretion of pro-inflammatory factors of CD8+ T cells, inhibiting the activation of B cells and inhibiting the activation of NK cells; reducing the levels of lymphocytes, neutrophils in peripheral blood Cell and monocyte content; reduced inflammatory cell infiltration in the dermis and subdermal capillary hyperplasia; improved skin fibrosis; reduced incidence of uveitis; improved skin inflammation; improved stool formability score, improved CW/CL , Improve CW/BW, improve CW/CL/BW, inhibit colon ulcer area increase, improve colonic inflammatory cell infiltration score, improve tissue damage score; improve disease activity score, improve blood in the stool or
  • the pathway modulator is preferably a DBH inhibitor.
  • the DBH inhibitor can be a DBH inhibitor that has been disclosed in the prior art, such as nepicastat, etamistat, eminostat, fusaric acid, disulfiram One of , cysteamine, pantethine, copper chelating agent, fumaric acid, hydralazine, 2-thiophen-2-ylallylamine, its pharmaceutically acceptable salt and its prodrug or more.
  • the DBH inhibitor is preferably selected from the group consisting of nepicastat, epiminostat, eminostat, pharmaceutically acceptable salts thereof and prodrugs thereof.
  • the DBH inhibitor is more preferably selected from the group consisting of nepicastat, etaminostat and etaminostat.
  • the DBH inhibitor is preferably selected from nepicastat, its pharmaceutically acceptable salts and its prodrugs.
  • the DBH inhibitor is more preferably nepicastat.
  • the pathway modulator is nepicastat or a pharmaceutically acceptable salt thereof, and the unit dose is: 10-100 mg/kg; 60, 65, 70, 75, 80, 85, 90, 95, 100 mg/kg, or a range between any two of the foregoing values; preferably 20-50 mg/kg; or,
  • the pathway modulator is etaministat 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 any two of the foregoing range between values; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is eminostat 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or
  • the pathway modulator is Fusaric acid 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is disulfiram 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is fumaric acid 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg.
  • the medicine is preferably used for one or more purposes selected from the following: reducing the proportion of CD4+ T cells, increasing the proportion of regulatory T cells, reducing the secretion of pro-inflammatory factors of CD4+ T cells, inhibiting the secretion of B cells. Activation and inhibition of NK cell activation.
  • the pro-inflammatory factor of CD4+ T cells is preferably one or more of IL-17A, IFN- ⁇ and TNF- ⁇ .
  • the pro-inflammatory factor of CD8+ T cells is preferably IL-17A and/or TNF- ⁇ .
  • the regulatory T cells are preferably CD25+FOXP3+Treg cells.
  • the B cells are preferably B220+ cells, more preferably CD69+B220+B cells.
  • the NK cells are preferably NK1.1+ cells, more preferably NK1.1+CD107a+NK cells.
  • a 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 proportion of CD4+ T cells +T cells secrete pro-inflammatory factors, reduce CD8+ T cells' pro-inflammatory factor secretion, inhibit the activation of B cells and inhibit the activation of NK cells; reduce the content of lymphocytes, neutrophils and monocytes in peripheral blood; Reduced inflammatory cell infiltration in the dermis and subdermal capillary hyperplasia; improved skin fibrosis; reduced incidence of uveitis; improved skin inflammation; improved stool formability score, improved CW/CL, improved CW/BW, improved CW /CL/BW, inhibit the increase of colon ulcer area, improve the score of colonic inflammatory cell infiltration, improve the score of tissue damage; improve the score of disease activity, improve blood in the stool or occult blood.
  • the pathway modulator is
  • the pathway regulator is preferably a DBH inhibitor.
  • the DBH inhibitor may be a DBH inhibitor that has been disclosed in the prior art, for example, nepicastat, etaminostat, etaminostat, fusaric acid, bismuth Sulfuram, cysteamine, pantethine, copper chelators, fumaric acid, hydralazine, 2-thiophen-2-ylallylamine, pharmaceutically acceptable salts thereof and prodrugs thereof one or more.
  • the DBH inhibitor is preferably selected from the group consisting of nepicastat, epiminostat, eminostat, pharmaceutically acceptable salts thereof and prodrugs thereof.
  • the DBH inhibitor is more preferably selected from the group consisting of nepicastat, etaminostat and etaminostat.
  • the DBH inhibitor is preferably selected from nepicastat, its pharmaceutically acceptable salts and its prodrugs.
  • the DBH inhibitor is more preferably nepicastat.
  • the pathway modulator is nepicastat or a pharmaceutically acceptable salt thereof, and the unit dose is: 10-100 mg/kg; 60, 65, 70, 75, 80, 85, 90, 95, 100 mg/kg, or a range between any two of the foregoing values; preferably 20-50 mg/kg; or,
  • the pathway modulator is etaministat 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 any two of the foregoing range between values; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is eminostat 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or
  • the pathway modulator is Fusaric acid 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is disulfiram 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is fumaric acid 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg.
  • the drug is preferably used 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, reducing the secretion of pro-inflammatory factors from CD4+ T cells, inhibiting B Activation of cells and inhibition of NK cell activation.
  • the pro-inflammatory factor of CD4+ T cells is preferably one or more of IL-17A, IFN- ⁇ and TNF- ⁇ .
  • the pro-inflammatory factors of CD8+ T cells are preferably IL-17A and/or TNF- ⁇ .
  • the regulatory T cells are preferably CD25+FOXP3+Treg cells.
  • the B cells are preferably B220+ cells, more preferably CD69+B220+B cells.
  • the NK cells are preferably NK1.1+ cells, more preferably NK1.1+CD107a+NK cells.
  • a seventh aspect of the present disclosure relates to a method of modulating immune cell function in vivo or in vitro, comprising the steps of contacting an effective amount of a pathway modulator in vivo or in vitro with immune cells, the immune cells from a subject; wherein , the pathway modulator is one or more of DBH inhibitors, receptor agonists and receptor antagonists;
  • the regulating immune cell function refers to 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, and reducing the pro-inflammatory factors of CD4+ T cells It secretes, reduces the secretion of pro-inflammatory factors of CD8+ T cells, inhibits the activation of B cells and inhibits the activation of NK cells.
  • the pathway modulator is preferably a DBH inhibitor.
  • the DBH inhibitor can be a DBH inhibitor that has been disclosed in the prior art, for example, can be nepicastat, etamistat, eminostat, fusaric acid, disulfiram One of , cysteamine, pantethine, copper chelating agent, fumaric acid, hydralazine, 2-thiophen-2-ylallylamine, its pharmaceutically acceptable salt and its prodrug or more.
  • the DBH inhibitor is preferably selected from the group consisting of nepicastat, epiminostat, eminostat, pharmaceutically acceptable salts thereof and prodrugs thereof.
  • the DBH inhibitor is more preferably selected from the group consisting of nepicastat, etaminostat and etaminostat.
  • the DBH inhibitor is preferably selected from nepicastat, its pharmaceutically acceptable salts and its prodrugs.
  • the DBH inhibitor is more preferably nepicastat.
  • the pathway modulator is nepicastat or a pharmaceutically acceptable salt thereof, and the unit dose is: 10-100 mg/kg; 60, 65, 70, 75, 80, 85, 90, 95, 100 mg/kg, or a range between any two of the foregoing values; preferably 20-50 mg/kg; or,
  • the pathway modulator is etaministat 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 any two of the foregoing range between values; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is eminostat 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or
  • the pathway modulator is Fusaric acid 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is disulfiram 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is fumaric acid 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg.
  • the DBH inhibitor may be administered in combination with one or more selected from the group consisting of chemotherapeutic agents, targeted therapeutic agents, immunotherapeutic agents and anti-inflammatory agents.
  • the medicament is preferably used 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, reducing the secretion of pro-inflammatory factors of CD4+ T cells, inhibiting the proliferation of B cells. Activation and inhibition of NK cell activation.
  • the pro-inflammatory factor of CD4+ T cells is preferably one or more of IL-17A, IFN- ⁇ and TNF- ⁇ .
  • the pro-inflammatory factor of CD8+ T cells is preferably IL-17A and/or TNF- ⁇ .
  • the regulatory T cells are preferably CD25+FOXP3+Treg cells.
  • the B cells are preferably B220+ cells, more preferably CD69+B220+B cells.
  • the NK cells are preferably NK1.1+ cells, more preferably NK1.1+CD107a+NK cells.
  • the eighth aspect of the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a pathway modulator and a pharmaceutically acceptable carrier; wherein the pathway modulator is one of a DBH inhibitor, a receptor agonist and a receptor antagonist one or more;
  • 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 pro-inflammatory factors of CD4+ T cells, reducing Pro-inflammatory factor secretion of CD8+ T cells, inhibition of B cell activation and inhibition of NK cell activation; reduction of lymphocytes, neutrophils and monocytes in peripheral blood; reduction of inflammatory cell infiltration in dermis and dermis lower capillary hyperplasia; improve skin fibrosis; reduce uveitis incidence; improve skin inflammation; , Improve the score of colonic inflammatory cell infiltration, improve the score of tissue damage; improve the score of disease activity, improve blood in the stool or occult blood.
  • the pathway modulator is preferably a DBH inhibitor.
  • the DBH inhibitor can be a DBH inhibitor that has been disclosed in the prior art, such as nepicastat, etamistat, eminostat, fusaric acid, bismuth Sulfuram, cysteamine, pantethine, copper chelators, fumaric acid, hydralazine, 2-thiophen-2-ylallylamine, pharmaceutically acceptable salts thereof and prodrugs thereof one or more.
  • the DBH inhibitor is preferably selected from the group consisting of nepicastat, epiminostat, eminostat, pharmaceutically acceptable salts thereof and prodrugs thereof.
  • the DBH inhibitor is more preferably selected from the group consisting of nepicastat, etaminostat and etaminostat.
  • the DBH inhibitor is preferably selected from nepicastat, its pharmaceutically acceptable salts and its prodrugs.
  • the DBH inhibitor is more preferably nepicastat.
  • the pathway modulator is nepicastat or a pharmaceutically acceptable salt thereof, and the unit dose is: 10-100 mg/kg; 60, 65, 70, 75, 80, 85, 90, 95, 100 mg/kg, or a range between any two of the foregoing values; preferably 20-50 mg/kg; or,
  • the pathway modulator is etaministat 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 any two of the foregoing range between values; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is eminostat 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or
  • the pathway modulator is Fusaric acid 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 any two of the foregoing values range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is disulfiram 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg; or,
  • the pathway modulator is fumaric acid 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 any two of the foregoing values. range between; preferably 90-110 mg/kg, more preferably 100 mg/kg.
  • the pharmaceutical composition may further contain one or more substances selected from the group consisting of chemotherapeutic agents, targeted therapy agents, immunotherapeutic agents and anti-inflammatory agents.
  • the medicine is preferably used for one or more purposes selected from the following: reducing the proportion of CD4+ T cells, increasing the proportion of regulatory T cells, reducing the secretion of pro-inflammatory factors of CD4+ T cells, inhibiting B Activation of cells and inhibition of NK cell activation.
  • the pro-inflammatory factor of CD4+ T cells is preferably one or more of IL-17A, IFN- ⁇ and TNF- ⁇ .
  • the pro-inflammatory factor of CD8+ T cells is preferably IL-17A and/or TNF- ⁇ .
  • the regulatory T cells are preferably CD25+FOXP3+Treg cells.
  • the B cells are preferably B220+ cells, more preferably CD69+B220+B cells.
  • the NK cells are preferably NK1.1+ cells, more preferably NK1.1+CD107a+NK cells.
  • DBH inhibitors can inhibit the occurrence and development of autoimmune diseases through immunomodulation (as examples, the improvement of weight loss, the improvement of DAI score and the normalization of colon density ), which in turn may provide new options for the treatment of autoimmune diseases.
  • FIG. 1A shows the results of the body weight assessment of each group of mice after the experiment was performed according to the experimental procedure described in Example 1.
  • FIG. 1A shows the results of the body weight assessment of each group of mice after the experiment was performed according to the experimental procedure described in Example 1.
  • FIG. 1B shows the DAI score results of mice in each group after the experiment was performed according to the experimental procedure described in Example 1.
  • FIG. 1C shows the results of colonic density of mice in each group after the experiment was performed according to the experimental procedure described in Example 1.
  • Figures 2A to 2E show CD4+T cell subsets (Figure 2A), CD25+FOXP3+Treg cell subsets ( Figure 2B), CD4+T cell subsets from the mouse mesenteric lymph nodes of each group in Example 4 Effects of IL-17A (Fig. 2C), IFN- ⁇ from CD4+ T cell subsets (Fig. 2D), and TNF- ⁇ from CD4+ T cell subsets (Fig. 2E).
  • Figures 3A to 3D show CD8+ T cell subsets (Fig. 3A), IFN- ⁇ from CD8+ T cell subsets (Fig. 3B), CD8+ T cell subsets in mouse mesenteric lymph nodes of each group as in Example 4 Effects of IL-17A on cell subsets (FIG. 3C) and TNF-[alpha] from CD8+ T cell subsets (FIG. 3D).
  • Figures 4A-4B show the effect of CD69+B220+B cell subsets (Figure 4A) and NK1.1+CD107a+NK cell subsets (Figure 4B) in mouse mesenteric lymph nodes of each group as in Example 4.
  • * means p ⁇ 0.05
  • ** means p ⁇ 0.01
  • *** means p ⁇ 0.001
  • **** means p ⁇ 0.0001.
  • 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.
  • FIG. 6 shows the body weight change rate in each group of Example 5.
  • 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.
  • Figure 8 shows the number of viscera in each group of Example 5; One-way ANOVA: # p ⁇ 0.05, ## p ⁇ 0.01, ### p ⁇ 0.001 vs. vehicle group.
  • Figure 9 shows peripheral blood red blood cell (RBC) counts 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.
  • Figure 10 shows the content of peripheral blood hemoglobin (HGB) in each group of Example 5; One-way ANOVA: ***p ⁇ 0.001vs normal group, ## p ⁇ 0.01, ### p ⁇ 0.001vs vehicle group, &&& p ⁇ 0.001 vs imatinib group.
  • FIG 11 shows peripheral blood white blood cell (WBC) counts in each group of Example 5; One-way ANOVA: *p ⁇ 0.05, ***p ⁇ 0.001 vs normal group, ## p ⁇ 0.01 vs vehicle group.
  • Figure 12 shows peripheral blood lymphocyte (LYMPH) counts in each group of Example 5; One-way ANOVA: # p ⁇ 0.05, ### p ⁇ 0.001 vs vehicle group.
  • Figure 13 shows peripheral blood mononuclear cell (MONO) counts in each group of Example 5; One-way ANOVA: ## p ⁇ 0.01 vs vehicle group.
  • Figure 14 shows peripheral blood neutrophil (NEUT) counts in each group of Example 5; One-way ANOVA: ***p ⁇ 0.001 vs normal group.
  • Figure 15 shows the peripheral blood platelet content in each group of Example 5; One-way ANOVA: ***p ⁇ 0.001vs normal group, ### p ⁇ 0.001vs vehicle group, && p ⁇ 0.01, &&& p ⁇ 0.001vs. nintedanib group, $$ p ⁇ 0.01 vs. imatinib group.
  • Figure 16 shows the dermal inflammatory cell infiltration scores in each group of Example 5; One-way ANOVA: # p ⁇ 0.05 vs. vehicle group, $ p ⁇ 0.05 vs. Nepicastat-25 mg/kg, qd, po.
  • Figure 17 shows the intradermal capillary density scores in each group of Example 5; One-way ANOVA: # p ⁇ 0.05, ## p ⁇ 0.01 vs. vehicle group.
  • Figure 18 shows dermal thickness in each group of Example 5; One-way ANOVA: *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 vs. normal group.
  • Figure 19 shows the clinical scores in each group of Example 6.
  • Figure 21 shows the body weights of animals in each group of Example 8.
  • Figure 22 shows the AUC of the total score of erythema + scale + thickness in each group of Example 8; mean ⁇ standard error, Two way ANOVA, p* ⁇ 0.05, p** ⁇ 0.01, p** compared to the corresponding vehicle group * ⁇ 0.001, p**** ⁇ 0.0001 (Note: the total score AUC of the normal group is 0, not shown in the figure).
  • FIG. 23 shows the spleen weight in each group of Example 8.
  • Figure 24 shows the body weights of animals in each group of Example 9.
  • Figure 25 shows the clinical scores in each group of Example 9.
  • Figure 26 shows the AUC in each group of Example 9.
  • Figure 27 shows the incidence in each group of Example 9.
  • Figure 28 shows the body weights of animals in each group of Example 10.
  • FIG. 29 shows the change rate of animal body weight in each group of Example 10.
  • Figure 30 shows stool scores in each group of Example 10.
  • Figure 31 shows stool score AUC in each group of Example 10.
  • Figure 32 shows changes in body weight in each group of Example 11.
  • Figure 33 shows DAI in each group of Example 11.
  • the term "receptor agonist” refers to a substance capable of acting on the receptor of the substrate catalyzed by dopamine beta-hydroxylase (ie, dopamine), and exerting the same mechanism and effect as the increase of dopamine.
  • the receptor agonist is preferably a dopamine receptor agonist.
  • receptor antagonist refers to a receptor capable of acting on a product catalyzed by dopamine beta-hydroxylase (ie, norepinephrine and/or epinephrine) and interacting with norepinephrine and/or adrenaline Or substances that reduce the same mechanism and effect of epinephrine.
  • the receptor antagonist is preferably a noradrenergic receptor antagonist and/or adrenergic receptor antagonist.
  • autoimmune disease refers to an attack by the own immune system of the body, characterized by the breakdown of adaptive immune tolerance mechanisms that recognize self/non-self, and the abnormal response of adaptive immune cells leading to inflammatory damage to self tissues.
  • dopamine beta-hydroxylase is intended to encompass dopamine beta-hydroxylase of human origin, and fragments, variants, precursors and functional domains thereof.
  • DBH inhibitor refers to the ability to affect (meaning to reduce, reduce, inhibit, block, repress, inactivate, or prevent activation) the structure, expression, or activation of dopamine beta-hydroxylase at the nucleic acid or protein level. Active any natural or artificial compound. DBH inhibitors include those known in the art, as well as those provided in the future. It includes the DBH inhibitors disclosed in CN87103323A and WO9529165, as well as Nepicastat, Etamicastat, Zamicastat, Fusaric acid, disulfiram, cysteamine , cysteamine derivatives, pantethine and pantethine derivatives.
  • pharmaceutically acceptable carrier refers to pharmaceutically acceptable excipients.
  • salts which are pharmaceutically acceptable salts and which possess the intended pharmacological activity of the parent compound. This salt includes:
  • an acid addition salt formed with an inorganic acid, or an acid addition salt formed with an organic acid wherein, the inorganic acid can be one or more of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid ;
  • the organic acid can be formic acid, oxalic acid, succinic acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamate acid, glycolic acid, xinafoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid one or more of acid, dibenzoyl-L-tartaric acid, tarta
  • compositions of the present disclosure can be in various conventional dosage forms, such as tablets, aqueous suspensions, oily suspensions, dispersible powders, dispersible granules, emulsions, hard capsules, soft capsules, sterile injectable aqueous solutions, sterile Injectable oil-in-water microemulsion, or suppository.
  • dosage forms can be prepared by conventional preparation methods.
  • the adjuvants in the tablets of the present disclosure may be one or more of fillers, binders, lubricants, glidants and disintegrants.
  • the filler can be one or more of microcrystalline cellulose, starch, lactose monohydrate and calcium hydrogen phosphate.
  • the binder may be one or more of starch, gelatin, polyvinylpyrrolidone, and acacia.
  • the lubricant may be one or more of magnesium stearate, stearic acid and sodium lauryl sulfate.
  • the glidant may be one or both of micropowder silica gel and talc.
  • the disintegrant can be one or more of crospovidone, sodium starch glycolate, low-substituted hydroxypropyl cellulose and croscarmellose sodium.
  • the tablets may also contain a coating.
  • the tablet can also be made into a sustained-release preparation, and the sustained-release material in the sustained-release preparation can be one or both of hydroxypropyl methylcellulose and xanthan gum.
  • the adjuvants in the aqueous suspension of the present disclosure can be one or more of suspending agents, dispersing agents, preservatives and flavoring agents.
  • the suspending agent can be one or more of sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone and acacia.
  • the dispersing agent can be a naturally occurring phospholipid (eg, lecithin), a condensation product of an alkylene oxide with a fatty acid (eg, polyoxyethylene stearate), a condensation product of ethylene oxide with a long-chain fatty alcohol (eg, ten Heptacarbon ethyleneoxycetyl alcohol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols (eg polyethylene oxide sorbitan monooleate), ethylene oxide with One or more of the condensation products of fatty acids and hexitol-derived partial esters (eg, polyethylene oxide sorbitan monooleate).
  • the preservative may be ethylparaben and/or n-propylparaben.
  • the flavoring agent may be one or more of sucrose, saccharin and aspartame.
  • the adjuvants in the oil suspensions of the present disclosure can be one or more of suspending agents, thickening agents, flavoring agents and antioxidants.
  • the suspending agent may be vegetable oil and/or mineral oil, the vegetable oil may be one or more of peanut oil, olive oil, sesame oil and coconut oil, and the mineral oil may be liquid paraffin.
  • the thickening agent may be one or more of beeswax, hard paraffin, and cetyl alcohol.
  • the flavoring agent may be one or more of sucrose, saccharin and aspartame.
  • the antioxidant may be one or more of butylated hydroxyanisole, alpha-tocopherol and ascorbic acid.
  • the adjuvants in the dispersible powders and dispersible granules of the present disclosure may be one or more of suspending agents, dispersing agents, preservatives, flavoring agents and antioxidants.
  • suspending agents dispersing agents, preservatives, flavoring agents and antioxidants.
  • preservatives flavoring agents and antioxidants.
  • antioxidants antioxidants.
  • the adjuvants in the emulsions of the present disclosure can be one or more of suspending agents, emulsifiers, flavoring agents, preservatives and antioxidants.
  • the suspending agent may be vegetable oil and/or mineral oil, the vegetable oil may be olive oil and/or peanut oil, and the mineral oil may be liquid paraffin.
  • the emulsifier can be a naturally occurring phospholipid (such as soybean lecithin), an ester or partial ester derived from fatty acids and hexitol anhydrides (such as sorbitan monooleate) and the condensation of the partial ester with ethylene oxide One or more of the products such as polyethylene oxide sorbitan monooleate.
  • the flavoring agent may be one or more of glycerol, propylene glycol, sorbitol and sucrose.
  • the preservative may be ethylparaben and/or n-propylparaben.
  • the antioxidant may be one or more of butylated hydroxyanisole, alpha-tocopherol and ascorbic acid.
  • the adjuvants 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.
  • the adjuvants 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 glycol, peanut oil, liquid paraffin and olive oil.
  • 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.
  • the adjuvant in the sterile injectable oil-in-water microemulsion of the present disclosure can be an oil phase adjuvant and an aqueous phase adjuvant
  • the oil phase adjuvant can be a mixture of soybean oil and lecithin
  • the water phase adjuvant can be a mixture of water and glycerol mixture.
  • the adjuvants in the suppositories of the present disclosure may be one or more of cocoa butter, glycerin, gelatin, hydrogenated vegetable oils, polyethylene glycols and fatty acid esters of polyethylene glycols.
  • the term "subject" refers to an animal, preferably a mammal.
  • the subject is a mammal, including, for example, camels, donkeys, zebras, cows, pigs, horses, goats, sheep, cats, dogs, rats, rabbits, guinea pigs, mice, primates (e.g. people).
  • the subject is a human.
  • the subject is a human susceptible, suspected of having, or already having an autoimmune disease.
  • treating means eliminating the disease, arresting the progression of the disease, slowing the progression of the disease, reducing the duration of one or more symptoms associated with the disease, improving or reversing at least one measurable parameter associated with the disease, or increasing the prevalence of the disease. Survival of subjects with disease.
  • an effective amount refers to that amount of a pharmaceutically active ingredient that elicits the desired effect in a subject.
  • selection of an effective amount can be determined by those skilled in the art based on consideration of a variety of factors (eg, via clinical trials), including the disease to be treated, the symptoms involved, the route of administration, the disease the severity of the disease, the weight of the patient, the immune status of the patient, and other factors known to those of skill in the art.
  • Effective amounts in particular embodiments can be derived from dose-response curves derived from animal model test systems, and are allowed to be determined according to the judgment of the physician and each patient's circumstances.
  • An effective amount of a pharmaceutical compound of the present disclosure may 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.
  • the same pharmaceutical active ingredient referring to a single pharmaceutical compound
  • different pharmaceutical active ingredients referring to more than two pharmaceutical compounds
  • the administration may comprise a single administration, or two or more administrations spaced at appropriate time intervals.
  • two adjacent administrations 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 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.
  • Each pharmaceutically active ingredient (each drug compound) mentioned herein may be used as the sole active compound or may be administered in combination with other active compounds (meaning compounds other than those described herein), as long as they do not produce Other adverse effects, such as allergic reactions, etc.
  • Co-administration includes simultaneous or sequential administration of the active compounds.
  • co-administration refers to a method of simultaneously or sequentially providing two or more active compounds to a subject for therapeutic purposes.
  • time interval between each administration is sufficient to achieve a synergistic effect between the active compounds administered.
  • Two or more active compounds are in the same different container.
  • Example 1 Inhibitory effect of DBH inhibitors on dextran sulfate sodium (DSS)-induced colitis
  • composition of the vehicle was as follows: 20 v% PEG400 + 10 v% (30% Solutol HS15) + 70 v% normal saline.
  • Test substance-nepicastat liquid 27 mg of nepicastat was completely dissolved in 9 mL of solvent (this concentration corresponds to test group 1#), prepared once every 3 days, and stored at 4°C to maintain the effect.
  • Test substance - etaminista liquid 9mg, 27mg, 90mg of etaminista were completely dissolved in 9mL solvent respectively (the three concentrations correspond to test group 2#, test group 3#, test group 4 respectively. #), prepared every 3 days and stored at 4°C to maintain the effect.
  • Test substance-eminostat liquid Dissolve 9mg, 27mg, 90mg of eminostat in 9mL solvent respectively (the three concentrations correspond to detection group 5#, detection group 6#, and detection group 7# respectively) , prepared once every 3 days and stored at 4°C to maintain the effect.
  • Positive drug control-cyclosporin A drug solution Dissolve 200 mg of cyclosporin A in 10 mL of normal saline to prepare a 20 mg/mL solution (corresponding to the positive drug control group), once every 3 days, at 4°C Save to keep the effect.
  • mice in each group Treatment of mice in each group:
  • mice 8-10 week old female C57BL/6J mice purchased from Charles River Laboratories were used. In a constant temperature (20 ⁇ 2°C) room (5 per cage), a 12-hour light-dark cycle was performed. All experimental protocols were approved by the Wuxiapptec Institutional Animal Care and Use Committee. The mice were acclimated for at least three days in WuXi AppTec's animal testing center before the experiment.
  • 3% DSS aqueous solution (the aqueous solution was prepared by adding 3 g of DSS to 100 mL of water, wherein the molecular weight of DSS was 36000-50000) was freely drunk for 7 consecutive days to induce colitis (the colon Inflammation belongs to autoimmune colitis), and is divided into 9 groups for administration respectively (see the vehicle group, positive drug control group and detection group 1# to detection group 7# in Table 3), wherein 3% DSS aqueous solution is freshly prepared every day . Euthanasia and end-point samples were obtained at the end of the experiment (see below).
  • mice in the normal group did not undergo colitis induced by 3% DSS aqueous solution, but drank blank water freely. Euthanasia and end-point samples were obtained at the end of the experiment (see below).
  • the animal body weight and DAI score were carried out under double-blind conditions, that is, when the researchers did not know the group and drug administration, the body weight of the mice was collected, and the stool characteristics and bloody stool were observed and scored every day.
  • the DAI score is the sum of the weight loss score, defecation score, and bleeding score. The scoring criteria are shown in Table 4.
  • Example 2 Effects of DBH inhibitors on immune cell activation and cytokines
  • the positive drug control group and detection group 1# of Example 1 to the detection group 7#, 24 hours after the administration on the 8th day is the experimental end point; the experimental end point of the normal group and the vehicle group is the same as the positive drug control group and the detection group 1# to 7#. Detection group 7#.
  • mesenteric lymph nodes were collected and processed as follows:
  • the mesenteric lymph nodes were lightly triturated and filtered through a 70 ⁇ M filter (BD bioscience, Cat. No. 352350) to obtain a cell suspension and count the cells.
  • Suspension (this suspension is prepared by the following method: using a mixture of 90v% 1640 medium and 10v% fetal bovine serum to dilute the cell activation mixture to a concentration of 1 ⁇ ) to suspend the cells, adjust the cells The density was 2.5 ⁇ 10 7 /mL and incubated at 37°C for 5 hours.
  • the cell subsets analyzed by flow detection include CD4+T cell subsets, IL-17A+CD4+T cell subsets, IFN- ⁇ +CD4+T cell subsets, TNF- ⁇ +CD4+T cell subsets population, CD25+FOXP3+Treg cell subsets; CD8+T cell subsets, IL-17A+CD8+T cell subsets, IFN- ⁇ +CD8+T cell subsets, TNF- ⁇ +CD8+T cell subsets ; CD69+B220+B cell subsets and NK1.1+CD107a+NK cell subsets.
  • mice in test group 1#, test group 4# and test group 7# all had significantly reduced body weight loss on the 7th day of treatment (Fig. 1A, Table 7).
  • test group 1# the DSS-induced colitis in test group 1#, test group 4# and test group 7# was milder than that in the vehicle group, suggesting that nepicastat, etaminista and etaminastat have adverse effects on the colon. inflammation has a potential therapeutic effect.
  • Example 2 According to the method of Example 2, the effects of nepicastat and eminostat on immune cells and cytokines were analyzed, and the results obtained are shown in FIGS. 2A to 2E , 3A to 3D , and 4A to 4B .
  • cytokines secreted by CD4+ T cell subsets were further analyzed.
  • the results showed that, compared with the vehicle group, both the test group 1# and the test group 7# could reduce the secretion of pro-inflammatory factors IL-17A (Fig. 2C), IFN- ⁇ (Fig. 2D) and TNF- ⁇ (Fig. 2E). , which suggests that DBH inhibitors play a role in inhibiting inflammation and immunity by reducing the secretion of pro-inflammatory factors by CD4+ T cell subsets.
  • Example 5 Inhibitory effect of DBH inhibitors on bleomycin-induced skin fibrosis in male C57 mice
  • Composition of vehicle 20% PEG400 + 10% (30% Solutol HS15) + 70% saline. Measure 100 mL of PEG400 and 50 mL of 30% (V/V) solutol, add them to 350 mL of normal saline; place on a magnetic stirrer, stir until they are evenly mixed, and store at 4°C for later use.
  • Test substance-nepicastat liquid Weigh 40 mg of test substance into a brown sample bottle, add 0.8 mL of PEG400, vortex on a vortex machine, sonicate for 15 minutes, and heat in a 40°C water bath for 15 minutes to form a suspension , and then add 0.4 mL of 30% Solutol HS15, vortex and mix on a vortex, and then add 2.8 mL of normal saline, and vortex and mix on a vortex to form a solution with a compound concentration of 10 mg/mL. Prepare once every three days and store at 4°C for later use.
  • Reference substance - nintedanib liquid accurately weigh 25mg nintedanib (BIBF1120), add 5mL solvent (0.5%MC+0.2%Tween80), mix well until the solution is clear and transparent, the drug concentration is 5mg /mL (administration dose of 10mL/kg), formulated every seven days.
  • Reference substance - imatinib liquid accurately weigh 15 mg of imatinib mesylate powder into a sample bottle, add 3 mL of normal saline, and vortex on a vortexer until it is completely dissolved, and the drug concentration is 5 mg/mL. Ready to use, ready to use within half an hour.
  • mice weight 20 to 22 g were housed in the SPF barrier system of Case Air Biotechnology (Suzhou) Co., Ltd., and the animal use certificate number: SYXK (Su) 2017-0041, in accordance with international standards Standard temperature, humidity and light control system.
  • the animals were anesthetized with isoflurane (2.0 to 2.5%), the back coat was removed, and an area of 1 cm2 was selected for intradermal injection of bleomycin (0.3 mg/kg, 100 ⁇ L) every two days to establish skin fibers ization model.
  • mice were divided into 7 groups according to their body weight, with 10 animals in each group, namely: normal group, vehicle group, nintedanib group (positive control drug), imatinib (positive control drug), test group 1#, test group 2 #, detection group 3#, see Table 12 for details.
  • the starting time of administration was the day of modeling, and the body weight of each animal was weighed before administration to calculate the administration volume, wherein the administration period was 28 days.
  • Mode of administration The imatinib group was intraperitoneal injection, and the other groups were intragastric administration.
  • Dosing frequency twice a day in the nintedanib group and once a day in the remaining groups.
  • the body weight changes of animals were recorded twice a week from the day of modeling; the clinical symptoms of animals were closely observed, such as shortness of breath, difficulty, abdominal suction, decreased activity, and lethargy.
  • the animals were euthanized by intraperitoneal injection of an overdose of sodium pentobarbital (100 mg/kg), and then photographs of local skin were taken.
  • the animals were then anesthetized by intraperitoneal injection of sodium pentobarbital for euthanasia.
  • the contents and order of dissection and sampling were as follows: take the spleen, weigh it and record it, take the bilateral inguinal lymph nodes and store them in a -80°C refrigerator after quick freezing, and take the lesioned skin tissue first.
  • the skin images were collected, then immersed in 10% formalin fixative for fixation, and the ratio of tissue to formalin was 1:10. After 48 hours of fixation, the histopathological examination was performed. The details are shown in Table 13 below.
  • the lesioned skin was dehydrated, embedded and sectioned, and the tissue was stained with HE and Masson. Histopathological analysis was performed as follows:
  • Masson-stained sections were scanned by Nanozomer S210, and the scanned images were quantitatively analyzed by Visiopharm VIS6.0 software. The degree of fibrosis is currently expressed by the thickness of the dermis after Masson staining.
  • the proportion of capillary angiogenesis is less than 25%
  • the body weight of the animals in the normal group showed an upward trend, and the body weight of the test group 1#, the test group 2#, and the test group 3# remained stable and fluctuated within the normal range. Only the nintedanib group and the imatinib group showed a slow decreasing trend after dosing and remained significantly different from the vehicle group during the period from 25 to 28 days (see Figure 5, Image 6). One animal in the imatinib group died on day 28, and the body weight on the day of death decreased by 23.7% compared with the body weight before modeling.
  • the spleen was taken at the end of the experiment, the weight of the spleen was weighed and the number of viscera (spleen/body weight ⁇ 100%) was calculated. The results are shown in FIG. 7 . Compared with the normal group, the weight of the spleen in the vehicle group was significantly decreased. Compared with the vehicle group, the weight of the spleen in the nintedanib group, the imatinib group, the test group 1#, the test group 2#, and the test group 3# was also significantly decreased.
  • peripheral blood was collected for routine blood test.
  • imatinib significantly reduced peripheral blood red blood cell and hemoglobin content, which was also consistent with the symptoms of anemia in this group of animals at the end of the experiment.
  • imatinib can significantly reduce the content of peripheral blood leukocytes (including lymphocytes and neutrophils); there is a trend to reduce monocytes, but there is no significant difference.
  • the detection group 3# Compared with the vehicle group, the detection group 3# has significant differences in reducing lymphocytes, neutrophils and monocytes, indicating that the treatment group 3# dose (100mg/kg) has the effect of reducing inflammation, This is consistent with the pathological findings ( Figures 9 to 15).
  • SSc Systemic sclerosis
  • bleomycin was used to establish a mouse fibrosis model.
  • the results of pathological HE staining showed that compared with the vehicle group and the normal group, significant inflammatory cell infiltration and subdermal capillary proliferation were observed; Masson staining results Animals in the vehicle group were shown to have a stronger degree of skin fibrosis, and their dermal thickness was also significantly increased.
  • the treatment of nintedanib and imatinib can significantly reduce the infiltration of inflammatory cells in the lesion skin and the proliferation of subdermal capillaries.
  • the test group 2# and the test group 3# can significantly reduce the infiltration of inflammatory cells in the dermis and the proliferation of subdermal capillaries.
  • test group 3# had better effect of improving skin fibrosis (Fig. 16 to Fig. 18).
  • test group 2# and test group 3# can reduce the number of inflammatory cells in peripheral blood after stimulation with bleomycin, and reduce the infiltration of inflammatory cells and the proliferation of capillaries in the dermis of the diseased skin, especially
  • the dose of 100 mg/kg corresponding to test group 3# had the best effect, and 100 mg/kg could also reduce the degree of fibrosis of the diseased skin.
  • Example 6 The effect of DBH inhibitor on bovine IRBP R16-induced Lewis rat experimental autoimmune uveoretinitis (EAU)
  • Test substance - Nepicastat liquid Take the preparation of 10mg/mL Nepicastat liquid as an example: Weigh 40mg Nepicastat into a brown sample bottle, add 0.8mL PEG400, vortex on a vortexer, Sonicate for 15 minutes, heat in a water bath at 40°C for 15 minutes to form a suspension, then add 0.4 mL of 30% Solutol HS15, vortex the suspension, then add 2.8 mL of normal saline, and vortex completely to the solution. Ready to use every day.
  • Reference substance - dexamethasone liquid suspend dexamethasone in sodium carboxymethyl cellulose with a mass concentration of 0.5%, and the concentration is 0.04 mg/mL. Ready to use every day.
  • IRBP R16 solution Dissolve bovine IRBP R16 polypeptide in physiological saline to a final concentration of 300 ⁇ g/mL.
  • CFA Complete Freund's Adjuvant
  • Emulsions are prepared by hand mixing. First, take 2 10 mL syringes, one of which aspirates 4 mL of the 300 ⁇ g/mL IRBP R16 solution and connect to the 3-way stopcock, making sure all air bubbles are removed. Then attach a syringe containing 4 mL of CFA and start mixing quickly. Mix manually by pushing the plunger back and forth for 5 minutes. Finally, use another 10mL syringe to inhale 8mL of the emulsion, then connect a large-bore needle (such as 18g), insert the needle into the end of the 1mL syringe, and dispense the emulsion into 10 1mL syringes. Push the plunger back and forth after the syringe and make sure there are no air bubbles. Emulsion should be used within 3 hours after preparation.
  • test rats were 6- to 8-week-old female Lewis rats, about 180 to 220 g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and did not carry specific pathogens at the beginning of the experiment.
  • a 12/12 hour light-dark cycle was performed in a room (2 to 4 per cage) at room temperature (20 to 26°C, 40 to 70% relative humidity). All experimental protocols were approved by Pengli Bio IACUC (Experimental Animal Care Committee). Before the experiment, the test rats were acclimated in the Pengli Biological Laboratory for 7 days.
  • day 0 refers to the day of immunization.
  • the body weight of each rat was monitored twice a week after immunization.
  • Dosing began on day 0 and was precisely dosed according to the animal's body weight for a total of 16 days.
  • the dosing regimen for the test rats is shown in Table 18.
  • Results are presented as "mean ⁇ standard error”. Statistical analysis was performed using Graphpad Prism or SPSS and p ⁇ 0.05 was considered statistically significant.
  • the body weight changes of the test rats are specifically shown in Table 19 below, which are shown as the mean and standard error, respectively.
  • Inhibition rate [AUC(model control)-AUC(test group)]/AUC(model control), test group includes dexamethasone group and nepicastat group.
  • Rat uveitis model was successfully established by the increase of EAU clinical score. Treatment with nepicastat slightly improved symptoms of uveitis and reduced EAU clinical score and clinical score AUC, but this effect did not reach statistical significance.
  • the inhibition rates of nepicastat on clinical score AUC at doses of 25, 50 and 100 mg/kg were 8.67%, 20.06% and 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 of clinical score AUC was 84.53%.
  • Example 7 Effects of DBH inhibitors on NMO-IgG-induced mouse neuromyelitis optica (NMO)
  • Test substance - Nepicastat liquid Take the preparation of 5mg/mL Nepicastat liquid as an example: Weigh 50mg Nepicastat into a brown sample bottle, add 1.98mL PEG400, vortex on a vortexer, Sonicate for 15 minutes, heat in a water bath at 40°C for 15 minutes to form a suspension, then add 0.99 mL of 30% Solutol HS15, vortex 2.97 mL of the suspension, then add 6.93 mL of normal saline, and vortex completely to the solution. Ready to use every day.
  • Control substance - dexamethasone liquid Disperse dexamethasone in normal saline with a concentration of 0.1 mg/mL. Ready to use every day.
  • Reference substance-tanshinone IIa liquid dissolve tanshinone IIa in PBS containing 2% DMSO at a concentration of 0.0736 mg/mL.
  • test mice were female C57 mice purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., 20 to 22 g, and did not carry specific pathogens at the beginning of the experiment.
  • the drug was administered 3 days before modeling, and administered for 7 days after modeling, and was administered continuously for 10 days.
  • the specific dosing schedule is shown in Table 25 below.
  • mice On the 8th day of the model (that is, the day after the last administration), the experimental mice were euthanized, and the brain tissues were taken for frozen sections. Then, the immune response area of the mouse astrocyte markers in each group was measured and evaluated (for the experimental method, please refer to the literature: Ye Gong et al., Journal of Neuroinflammation, 17(1).doi:10.1186/s12974-020-01874-6) .
  • Figures 20A to 20D show that, from the point of view of the lesion ratio of AQP4 and GFAP, compared with the control group, the nepicastat group can significantly reduce the lesion ratio, and the effect is lower than that of the positive drug group.
  • the immune parameters CD45 and IBA1 there was no significant effect on the immune parameters CD45 and IBA1. Therefore, nepicastat has a certain inhibitory effect on NMO-IgG-induced mouse neuromyelitis optica.
  • Example 8 Therapeutic effect of DBH inhibitors on imiquimod (IMQ)-induced psoriasis
  • Test substance-nepicastat oral medication solution prepare 9.9mL of nepicastat solution with a concentration of 5mg/mL for oral administration: weigh 50mg of nepicastat, add 1.98mL of PEG400, sonicate for 15 minutes, 40 Heat in a water bath for 15 min and vortex to suspension. To 1.98 mL of Nepicastat suspension was added 0.99 mL of 30% Solutol HS15 and vortexed to suspension. Add 6.93 mL of normal saline to 2.97 mL of the Nepicastat suspension and vortex to solution.
  • Test substance-nepicastat topical solution prepare 1 mL of nepicastat (50 mg/mL) for external use: weigh 50.13 mg of nepicastat, add 100 ⁇ L of PG, and stir at 45°C for 10 minutes to obtain a uniform opaque suspension; add another 200 ⁇ L of ethanol and stir at 45 °C for 5 min to obtain a homogeneous opaque suspension; then add another 200 ⁇ L of Remophor EL and stir at 45 °C for 5 min to obtain a homogeneous opaque suspension. An additional 500 ⁇ L of 5% Poloxamer 188 was added to the suspension and stirred at 45°C for 30 minutes to obtain a clear solution.
  • Reference substance - tofacitinib citrate solution dissolve tofacitinib citrate in dimethyl sulfoxide to obtain a solution with a concentration of 5 mg/mL, and add 50 ⁇ L of the above solution to IMQ ointment for topical application.
  • mice One day before the experiment, the backs of all mice were shaved with a pet hair clipper in an area of 2 ⁇ 3 cm.
  • Vehicle A 20% PEG400+10% (30% Solutol HS15)+70% saline;
  • Vehicle B 10% PG/20% EtOH/20% Cremophor EL/50% (5% Poloxamer 188 in water).
  • Imiquimod (IMQ) cream from G2 to G7, the corresponding dose per mouse is 62.5mg IMQ cream;
  • Dexamethasone (DEX) ointment the corresponding dose for each mouse in G4 is 70 mg ointment;
  • Tofacitinib Dissolve tofacitinib in dimethyl sulfoxide to 5 mg/mL and add 50 ⁇ L to IMQ cream for daily treatment.
  • the back skin was scored according to Table 30. Erythema, scaling, and thickening were independently scored on a scale of 0 to 3, with 0, none; 1, mild; 2, moderate; 3, marked/severe.
  • the nepicastat oral group suppressed erythema and thickness from day 5 to day 7.
  • the nepicastat topical group suppressed erythema from day 4 to day 7.
  • Tofacitinib showed efficacy after doubling the dose.
  • Nepicastat treatment did not affect disease progression and morbidity, but showed efficacy later in the course of the disease (days 5 to 7).
  • the area under the curve (AUC) was calculated from the total clinical score curve for each group of animals ( Figure 22).
  • inhibition rate [AUC(vehicle)-AUC(treatment)]/AUC(vehicle) ⁇ 100%.
  • vehicle refers to the vehicle group corresponding to the mode of administration.
  • the inhibition rate of dexamethasone treatment group was 96.96%.
  • the 50mpk group treated with nepicastat had a significant inhibitory effect on the skin clinical score, and the inhibition rate was 23.64%.
  • the 100mpk group treated with nepicastat had no significant effect.
  • the purpose of this example is to study the efficacy of the test compounds on the IMQ-induced psoriasis model. The results showed that application of IMQ resulted in severe skin inflammation. Compared with the vehicle group and the control group, the PO treatment group of nepicastat had a slight therapeutic effect on clinical symptoms.
  • Bovine collagen type II CII, Sichuan University
  • Acetic acid Sigma (St. Louis, MO, USA), Cat. No. A8976;
  • Anesthesia machine Raymain, RM-HSIV-u;
  • Lighting Fluorescent light, bright (08:00-20:00), dark (20:00-08:00) for 12 hours each
  • Drinking water free drinking water (prepared by molar (ultra) water purifier).
  • acetic acid dilute 2N acetic acid to 100 mM, filter with a 0.22-micron filter, and store at 4°C.
  • Bovine type II collagen solution Dissolve bovine type II collagen (CII) in 100 mM acetic acid solution and store at 4°C overnight. The final concentration of collagen was 8 mg/mL.
  • Emulsion preparation Mix the CII solution kept overnight with an equal volume of complete Freund's adjuvant and homogenize using a high-speed homogenizer at 30,000 rpm for approximately 60 minutes on ice until the solution forms a stable emulsion.
  • mice When the model mice developed clinical symptoms, the average score was about 0.5 (around the 28th day), and they were re-randomized into 5 experimental groups according to body weight and score, with 8 mice in each group. Dosing for 14 consecutive days. Compounds were prepared every 3 days and stored at 4°C.
  • mice were weighed three times a week and clinical scores were recorded until the end of the experiment.
  • Clinical scoring According to the different degrees of lesions (redness, swelling, joint deformation), it is scored according to the standard of 0-4 points, the maximum score of each limb is 4 points, and the maximum score of each animal is 16 points.
  • mice were collected and fixed in PFA for H&E staining and pathological scoring;
  • the experimental data were expressed as the mean ⁇ standard error, and the weight and clinical scores were analyzed by Two-way ANOVA, and p ⁇ 0.05 was considered to be significantly different.
  • nepicastat has a known effect on collagen-induced arthritis in mice, but not as pronounced as the positive control.
  • Example 10 Therapeutic effect of DBH inhibitor on 2,4-dinitrobenzenesulfonic acid (DNBS)-induced enteritis in rats
  • Test substance-nepicastat liquid weigh 12 mg of nepicastat into a brown sample bottle, add 0.8 mL of polyethylene glycol 400, vortex and sonicate for 15 minutes, and heat in a water bath at 40°C for 15 minutes to form a suspension solution, then add 0.4 mL of 30% polyethylene glycol-15 hydroxystearate, vortex, then add 2.8 mL of normal saline, and vortex to dissolve completely.
  • Control substance - prednisone liquid the concentration of prednisone is 0.9 mg/mL, and 0.5% sodium carboxymethyl cellulose is used to prepare a suspension, which is prepared twice a week.
  • DNBS liquid DNBS powder was dissolved in 30% ethanol, and the final concentration was 50 mg/mL.
  • Administration record no administration history
  • Gender, age, weight male, 5 to 6 weeks, 160 to 180g;
  • Room common area room
  • Lighting fluorescent lighting, 12 hours lighting and 12 hours no lighting
  • Animal feeding 2 to 4 rats per cage (same administration group);
  • Water Free access to drinking water (reverse osmosis treated or autoclaved).
  • a total of 90 male Wistar rats were purchased from Shanghai Slack Laboratory Animal Co., Ltd., and none of the animals carried specific pathogens. When they arrived at the Pengli Biological Animal Room, they were about 4 to 5 weeks old (140 to 150 g).
  • the animal room personnel will transfer the animals from the transport packaging to the rat cages and inspect each animal.
  • the scope of inspection includes appearance, limbs and cavities, etc., and whether there is any abnormal performance when the animal is stationary or moving.
  • the acclimation period is 7 days.
  • Group G2-G6 the flexible tube was inserted from the anus to the left flexure of the colon (about 8 cm from the anus), and DNBS enema (0.5 mL/rat) was used to induce colitis in rats.
  • the normal control group (G1) was enema with 30% ethanol in the same way. The enema animal was kept head down for 15 min and then kept in Trendelenburg position until the animal awoke to avoid enema fluid reflux.
  • Animal body weight Measure and record the body weight of the animal every day, observe the daily activities of the animal, and record the abnormal situation. The percent body weight was calculated according to the following formula: (day x body weight - starting body weight)/starting body weight] x 100%.
  • proximal, ulcer if there is no ulcer, take the approximate site
  • distal part of neutral formalin-fixed colon tissue were embedded in paraffin, sectioned (thickness 5 ⁇ m), and stained with H&E for histopathological scoring.
  • 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)] ⁇ 100%;
  • IR2 (inhibition rate 2) ⁇ [CW/BW (model group)-CW/BW (test drug group)]/[CW/BW (model)-CW/BW (normal group)] ⁇ 100%;
  • IR3 (inhibition rate 3) ⁇ [CW/CL (model group)-CW/CL (test drug group)]/[CW/CL (model group)-CW/CL (normal group)] ⁇ 100% .
  • Inflammatory colitis was induced in Wistar rats by intracolonic perfusion with DNBS. Colitis is manifested by a significant decrease in animal body weight, a significant increase in stool formability score, a significant reduction in colon length, 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 (ie CW/CL/BW) was significantly increased, colon ulcer area was significantly increased, colonic inflammatory cell infiltration score and tissue damage score were significantly increased.
  • the positive control drug prednisone can significantly reduce the fecal formability score AUC, intestinal weight, CW/CL, CW/BW, CW/CL/BW and inflammatory cell infiltration score of colon ulcer in model rats, Among them, the inhibition rates of CW/CL/BW, CW/BW and CW/CL reached 44.20%, 50.13% and 46.51%, respectively.
  • the inhibition rates of CW/CL/BW, CW/BW and CW/CL in test group 1# (3 mg/kg) were 32.98%, 40.24% and 30.82%, respectively.
  • Test group 2# (Nepicastat 10mg/kg can significantly reduce the total score of CW/CL, CW/CL/BW and colon tissue damage, the inhibition rates of CW/CL/BW, CW/BW and CW/CL were 44.95 %, 47.06% and 43.80%.
  • the inhibition rates of CW/CL/BW, CW/BW and CW/CL in detection group 3# (30 mg/kg) were 27.12%, 39.85% and 26.68%, respectively.
  • nepicastat has a certain preventive effect on DNBS-induced colitis in rats.
  • Example 11 Inhibitory effect of DBH inhibitors on dextran sulfate sodium (DSS)-induced colitis
  • Experimental animal strain C57BL/6 mouse; source: Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.; gender: female.
  • Main instrument Electronic balance: Sartorius, QUINTIX35-1CN.
  • CsA cyclosporin A
  • nepicastat weigh an appropriate amount of nepicastat into a brown sample bottle, add a certain proportion of 20% PEG400, 10% (30% Solutol HS15) and 70% normal saline in sequence, mix well, dissolve by ultrasonic, and mix every 3 days to maintain its stability.
  • mice 70, 8-week-old, about 20 g, female C57BL/6 mice were housed in individually ventilated cages (IVC, 5 per cage), temperature-controlled (20 ⁇ 2°C), and light/dark alternated for 12/12 hours. Before the experiment, the mice were adaptively fed in the SPF animal room for three days, during which time, sufficient water and food were ensured.
  • IVC individually ventilated cages
  • mice were adaptively fed for three days in the SPF animal room and randomly divided into 7 groups (8 model groups and 1 control group) with 10 mice in each group. Mice dosing started on day 0 and ended on day 7.
  • the mouse enteritis model was induced by DSS: the model group mice drank 3.1% DSS (dextran sulfate sodium, molecular weight 36000-50000) aqueous solution from the 0th day to the 7th day, and the DSS aqueous solution was replaced every day. prevent degradation.
  • the mice in the normal control group drank normal water.
  • the experimental period was 9 days. At the end of the experiment, mice were euthanized and samples were collected.
  • the DAI score was performed every day, and the DAI score consisted of 3 parts: weight change, fecal characteristics and blood in the stool (or occult blood) score.
  • the specific scoring standards are shown in Table 49. DAI scoring of all mice was performed by the same person throughout the experiment.
  • mice were euthanized, the colons of the mice were dissected to take pictures, the length was measured, and the contents were removed and weighed.
  • the disease activity score (DAI) of the positive control CsA (50 mg/kg, QD) group and nepicastat (50 mg/kg, QD) mice was significantly lower than that of the vehicle group.
  • the fusaric acid (100 mg/kg, bid) group, disulfiram (100 mg/kg, qd) and fumaric acid (100 mg/kg, qd) group had a DAI can be slightly improved, and the performance is more obvious on the 4th and 5th days, but the effect is lower than that of nepicastat.
  • Phenylisopropylamine stimulants amphetamine-related agents.Principles of Medicinal Chemistry(7th ed.).Philadelphia,USA:Wolters Kluwer Health/Lippincott Williams&Wilkins.2013,pp.646–648.ISBN 9781609133450.

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Abstract

一种通路调节剂、含其的药物组合物、其用途和采用其的治疗方法。该通路调节剂(包括多巴胺β-羟化酶抑制剂、受体激动剂和受体拮抗剂中的一种或多种)可通过免疫调节，而抑制自身免疫性疾病的发生发展，进而可为自身免疫性疾病的治疗提供潜在治疗药物。

Description

通路调节剂、含其的药物组合物、其用途和采用其的治疗方法
本公开要求2021年01月26日提交的中国专利申请(申请号2021101034359)的优先权。
技术领域
本公开涉及通路调节剂、含其的药物组合物、其用途和采用其的治疗方法。
背景技术
多巴胺β-羟化酶(Dopamine beta-hydroxylase，以下简称DBH)，也被称为多巴胺β-单加氧酶，是一种在人体中由DBH基因编码的酶(EC 1.14.17.1)。DBH催化多巴胺被氧气氧化为去甲肾上腺素和肾上腺素，如下所示：
Figure PCTCN2022073849-appb-000001
DBH是一种约290kDa的含铜加氧酶，其由四个相同的亚基组成，其活性需要抗坏血酸作为辅助因子[1]。
DBH是唯一参与小分子膜结合的神经递质合成的酶，其使得去甲肾上腺素成为在囊泡内合成的唯一递质。去甲肾上腺素在中枢和外周神经系统的嗜去甲肾上腺素能神经末梢以及肾上腺髓质的铬细胞中表达。
DBH主要有助于痕量胺和儿茶酚胺的生物合成，包括肾上腺素(epinephrine或adrenaline)、去甲肾上腺素(norepinephrine或noradrenaline)和多巴胺。此外，其还参与与这些物质有关的外源性生物质的代谢。例如，人的DBH催化苯丙胺和对羟基苯丙胺的β-羟基化，分别产生去甲麻黄碱和对羟基去甲麻黄碱[2-4]。
DBH被认为是与思维决策和药物成瘾的病症相关，例如，酒精中毒[5]和吸烟[6]、注意力缺陷多动障碍[7]、精神分裂症[8]和阿尔茨海默病[9]。而DBH不足被称为多巴胺β-羟化酶缺乏症。
在20世纪末21世纪初，相继开发了内匹司他(Nepicastat)[10]、依他米司他(Etamicastat)[11]、依米司他(Zamicastat)[12]这些高选择性的DBH抑制剂，也进一步研究了其对高血压(Pulmonary Arterial Hypertension)、充血性心衰、可卡因依赖或创伤后应激障碍(PTSD)等潜在用途。
内匹司他可能用于充血性心力衰竭的治疗，并且在患者中显示良好的耐受性[13]。进一步的临床研究数据表明，内匹司他在治疗心衰上没有效果，但具有安全性。
内匹司他及其类似物(如依他米司他、依米司他)在治疗高血压方面具有潜 在用途。评估内匹司他治疗PTSD和可卡因依赖的临床试验已经完成。研究发现，当内匹司他与可卡因联合用药时，内匹司他是安全的，并可以抑制可卡因的正向效果。这一结果提示内匹司他可作为治疗可卡因依赖的药物疗法[14]。
与内匹司他相比，依他米司他及依米司他具有较低的脑-血-屏障渗透水平，能够降低外周交感神经支配组织中的去甲肾上腺素水平，而对自发性高血压大鼠的脑组织没有作用[15]。这一结果提示依他米司他及依米司他这类DBH抑制剂在治疗外周相关疾病时，可以减少中枢神经系统不良反应或并发症。在一项II期临床研究中，观察到依他米司他(200mg)在10天的治疗后，剂量依赖性地降低收缩压和舒张压，且显示良好的耐受性及安全性[16]。依米司他(1200mg)连续10天用药时也显示较好的安全性(NCT02151994)。目前，依米司他用于肺动脉高压疗效的临床试验正在开展中(NCT04316143)。
最早在1998年的研究中，报道了DBH对免疫调节的作用。研究发现，在无病原体条件下，dbh-/-小鼠的白细胞数量正常、T、B细胞发育正常，体外功能也都正常，但dbh-/-小鼠更容易感染病原体(如单核增生李斯特菌或肺结核分枝杆菌)；同时，动物表现出严重的胸腺退化，T细胞受损功能，包括产生Th1细胞因子。这些结果提示生理上的正常发育不需要产生儿茶酚胺，但在感染的免疫调节中儿茶酚胺起着重要作用[17]。
DBH作为自主神经的一部分，通过去甲肾上腺素能纤维直接和间接的贯穿整个脑源、组织、血管及外周血。除去甲肾上腺素能纤维外，其蛋白的表达和定位也具有一定的组织特异性，如DBH在肾上腺髓质的嗜铬细胞和蓝斑的去甲肾上腺能细胞、肝组织、肠组织中均有一定水平表达，其中DBH在肾上腺髓质中的表达最高。
某项研究发现，组织中DBH高表达或外周血高水平DBH分泌可能与一些神经或内分泌疾病相关[18]；比如，外周血高水平DBH可能与阿尔茨海默病、双相情感障碍、亨廷顿病、甲状腺功能减退、PTSD相关。尽管外周血DBH水平可能与神经疾病相关，但在临床实验中DBH抑制剂对于阿尔茨海默病及PTSD都没有获得很好的疗效(见CLINICALTRIALS的数据库)。类似地，还有研究发现，在自身免疫性肠炎患者中，炎症组织中DBH表达上调，但该研究并没有阐明二者的必然联系[19]。
据Autoimmune Association协会统计，有100种以上自身免疫性疾病类型。在临床中，对于自身免疫性疾病的治疗，主要以激素和/或免疫抑制剂非靶向性的药物控制症状，与此同时给患者带来极大且不可逆的不良反应和损伤。
由此可见，寻求一种安全的能够治疗自身免疫性疾病的治疗剂是该领域亟需解决的技术问题。
发明内容
本公开所要解决的技术问题包括提供一种通路调节剂、含其的药物组合物、其用途和采用其的治疗方法。
发明人意外地发现，现有技术中的通路调节剂，包括多巴胺β-羟化酶抑制剂(简称为DBH抑制剂)、受体激动剂和受体拮抗剂中的一种或多种，可通过免疫调节，而抑制自身免疫性疾病的发生发展(例如，具体体现在对体重减轻的改善、DAI评分的改善及使结肠密度正常化)，进而可为自身免疫性疾病的治疗提供潜在治疗药物。
本公开通过以下技术方案解决上述技术问题：
本公开的第一方面涉及一种通路调节剂在制备治疗自身免疫性疾病的药物中的用途；其中，所述通路调节剂为DBH抑制剂、受体激动剂和受体拮抗剂中的一种或多种。
上述用途中，所述通路调节剂优选为DBH抑制剂。
上述用途中，所述DBH抑制剂可为现有技术中的已经公开的或之后的DBH抑制剂，作为示例可为内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、半胱胺、泛硫乙胺、铜螯合剂、富马酸、肼苯哒嗪、2-噻吩-2-基烯丙基胺、其药学上可接受的盐和其前药中的一种或多种。所述DBH抑制剂优选地选自内匹司他、依他米司他、依米司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选地选自内匹司他、依他米司他和依米司他。采用内匹司他、依他米司他和依米司他中的一种或多种治疗自身免疫性疾病，不会存在激素和/或免疫抑制剂非靶向性的药物给患者带来的极大且不可逆的不良反应和损伤，是一种安全的能够治疗自身免疫性疾病的治疗剂。
上述用途中，所述DBH抑制剂优选地选自内匹司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选为内匹司他。采用内匹司他治疗自身免疫性疾病，可以显著减少患者的体重降低以及显著降低患者的结肠密度。
上述用途中，作为示例：
所述通路调节剂为内匹司他或其药学上可接受的盐，单位剂量为：10-100mg/kg；例如10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100mg/kg，或前述任意两个数值之间的范围；优选20-50mg/kg；或者，
所述通路调节剂为依他米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为依米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者
所述通路调节剂为镰孢菌酸或其药学上可接受的盐，单位剂量为80-120 mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为双硫仑或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为富马酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg。
上下文中，单位剂量可以是整数或分数。应当理解，例如，“10至100”是一种简洁的书写方式，尽管没有给出该范围内的每一个点值，但视为已经在文中明确得以披露。
上述技术方案用于治疗自身免疫性疾病，可以显著减少患者的体重降低以及显著降低患者的结肠密度。
上述用途中，所述自身免疫性疾病可为现有技术中公开的自身免疫性疾病中的一种或多种，例如可选自贲门失弛缓症(Achalasia)、艾迪生氏病(Addison’s disease)、成人斯蒂尔氏病(Adult Still's disease)、血球蛋白血症(Agammaglobulinemia)、斑秃(Alopecia areata)、淀粉样变性(Amyloidosis)、强直性脊柱炎(Ankylosing spondylitis)、抗GBM/抗TBM肾炎(Anti-GBM/Anti-TBM nephritis)、抗磷脂综合征(Antiphospholipid syndrome)、自身免疫性血管性水肿(Autoimmune angioedema)、自身免疫性自主神经异常(Autoimmune dysautonomia)、自身免疫性脑脊髓炎(Autoimmune encephalomyelitis)、自身免疫性肝炎(Autoimmune hepatitis)、自身免疫性内耳疾病(Autoimmune inner ear disease，缩写AIED)、自身免疫性心肌炎(Autoimmune myocarditis)、自身免疫性卵巢炎(Autoimmune oophoritis)、自身免疫性睾丸炎(Autoimmune orchitis)、自身免疫性胰腺炎(Autoimmune pancreatitis)、自身免疫性视网膜病变(Autoimmune retinopathy)、自身免疫性荨麻疹(Autoimmune urticaria)、轴突神经病(Axonal&neuronal neuropathy，缩写AMAN)、巴洛病(Baló disease)、白塞氏病(Behcet’s disease)、良性粘膜类天疱疮(Benign mucosal pemphigoid)、大疱性类天疱疮(Bullous pemphigoid)、卡斯尔曼病(Castleman disease，缩写CD)、麸质过敏症(Celiac disease)、恰加斯病(Chagas disease)、慢性炎性脱髓鞘性多神经病(Chronic inflammatory demyelinating polyneuropathy，缩写CIDP)、慢性复发多灶性骨髓炎(Chronic recurrent multifocal osteomyelitis，缩写CRMO)、嗜酸性肉芽肿(Eosinophilic Granulomatosis，缩写EGPA)、瘢痕性类天疱疮(Cicatricial pemphigoid)、科根综合症(Cogan’s syndrome)、冷凝集素病(Cold agglutinin disease)、先天性心脏传导阻滞(Congenital heart block)、柯萨奇心肌炎(Coxsackie myocarditis)、肢端硬皮综合征(CREST syndrome)、克罗恩病(Crohn’s disease)、 疱疹样皮炎(Dermatitis herpetiformis)、皮肌炎(Dermatomyositis)、视神经脊髓炎(Devic’s disease(neuromyelitis optica))、盘状狼疮(Discoid lupus)、德莱勒综合征(Dressler’s syndrome)、子宫内膜异位症(Endometriosis)、嗜酸性食管炎(Eosinophilic esophagitis，缩写EoE)、嗜酸性筋膜炎(Eosinophilic fasciitis)、结节性红斑(Erythema nodosum)、基本混合性冷球蛋白血症(Essential mixed cryoglobulinemia)、埃文斯综合症(Evans syndrome)、纤维肌痛症(Fibromyalgia)、纤维化肺泡炎(Fibrosing alveolitis)、巨细胞动脉炎(Giant cell arteritis)、巨细胞心肌炎(Giant cell myocarditis)、肾小球肾炎(Glomerulonephritis)、肺出血肾炎综合征(Goodpasture’s syndrome)、肉芽肿合并多血管炎(Granulomatosis with Polyangiitis)、甲状腺机能亢进(Graves’disease)、格林-巴利综合征(Guillain-Barre syndrome)、桥本氏甲状腺炎(Hashimoto’s thyroiditis)、溶血性贫血(Hemolytic anemia)、过敏性紫癜(Henoch-Schonlein purpura，缩写HSP)、妊娠期疱疹或类天疱疮(Herpes gestationis or pemphigoid gestationis，缩写PG)、化脓性汗腺炎(Hidradenitis Suppurativa，缩写HS)、低丙球蛋白血症(Hypogammalglobulinemia)、IgA肾病(IgA Nephropathy)、IgG4相关性硬化症(IgG4-related sclerosing disease)、免疫性血小板减少性紫癜(Immune thrombocytopenic purpura，缩写ITP)、包涵体肌炎(Inclusion body myositis，缩写IBM)、间质性膀胱炎(Interstitial cystitis，缩写IC)、青少年关节炎(Juvenile arthritis)、青少年I型糖尿病(Juvenile diabetes Type 1)、青少年肌炎(Juvenile myositis，缩写JM)、川崎病(Kawasaki disease)、朗伯-伊顿综合征(Lambert-Eaton syndrome)、白细胞碎裂性血管炎(Leukocytoclastic vasculitis)、扁平苔癣(Lichen planus)、萎缩性硬化性苔藓(Lichen sclerosus)、木样结膜炎(Ligneous conjunctivitis)、线性IgA病(Linear IgA disease，缩写LAD)、红斑狼疮(Lupus)、慢性莱姆病(Lyme disease chronic)、梅尼埃病(Meniere’s disease)、显微镜下多血管炎(Microscopic polyangiitis，缩写MPA)、混合性结缔组织病(Mixed connective tissue disease，缩写MCTD)、蚕蚀性角膜溃疡(Mooren’s ulcer)、穆-哈二氏病(急性痘疮样苔藓状糠疹)(Mucha-Habermann disease)、多灶性运动神经病(Multifocal Motor Neuropathy，缩写MMN或MMNCB)、多发性硬化症(Multiple sclerosis)、重症肌无力(Myasthenia gravis)、肌炎(Myositis)、嗜睡症(Narcolepsy)、新生儿狼疮(Neonatal Lupus)、中性粒细胞减少症(Neutropenia)、眼瘢痕性类天疱疮(Ocular cicatricial pemphigoid)、视神经炎(Optic neuritis)、回文风湿病(Palindromic rheumatism，缩写PR)、潘达斯症(PANDAS)、副肿瘤性小脑变性(Paraneoplastic cerebellar degeneration，缩写PCD)、阵发性夜间血红蛋白尿(Paroxysmal nocturnal hemoglobinuria，缩写PNH)、帕里伯格综合症(Parry Romberg syndrome)、睫状体平坦部炎(Pars planitis)、帕森纳-特纳综合征(Parsonage-Turner syndrome)、天疱疮(Pemphigus)、周围神经病变(Peripheral neuropathy)、静脉周脑脊髓炎(Perivenous encephalomyelitis)、恶性贫血(Pernicious  anemia，缩写PA)、POEMS综合症(POEMS syndrome)、结节性多动脉炎(Polyarteritis nodosa)、I型多腺综合征(Polyglandular syndromes type I)、II型多腺综合征(Polyglandular syndromes type II)、III型多腺综合征(Polyglandular syndromes type III)、风湿性多肌痛(Polymyalgia rheumatica)、多肌炎(Polymyositis)、心肌梗塞后综合症(Postmyocardial infarction syndrome)、心包切开术后综合征(Postpericardiotomy syndrome)、原发性胆汁性肝硬化(Primary biliary cirrhosis)、原发性硬化性胆管炎(Primary sclerosing cholangitis)、孕激素性皮炎(Progesterone dermatitis)、银屑病(Psoriasis)、银屑病关节炎(Psoriatic arthritis)、纯红细胞再生障碍性贫血(Pure red cell aplasia，缩写PRCA)、坏疽性脓皮病(Pyoderma gangrenosum)、雷诺现象(Raynaud’s phenomenon)、反应性关节炎(Reactive Arthritis)、反射交感性营养不良(Reflex sympathetic dystrophy)、复发性多发性软骨炎(Relapsing polychondritis)、不宁腿综合征(Restless legs syndrome，缩写RLS)、腹膜后纤维化(Retroperitoneal fibrosis)、风湿热(Rheumatic fever)、类风湿性关节炎(Rheumatoid arthritis)、结节病(Sarcoidosis)、施密特综合症(Schmidt syndrome)、巩膜炎(Scleritis)、硬皮病(Scleroderma)、干燥综合征(
Figure PCTCN2022073849-appb-000002
syndrome)、精子和睾丸自身免疫(Sperm&testicular autoimmunity)、僵硬者综合征(Stiff person syndrome，缩写SPS)、亚急性细菌性心内膜炎(Subacute bacterial endocarditis，缩写SBE)、苏萨克综合征(Susac’s syndrome)、交感性眼炎(Sympathetic ophthalmia，缩写SO)、大动脉炎(Takayasu’s arteritis)、颞动脉炎/巨细胞动脉炎(Temporal arteritis/Giant cell arteritis)、血小板减少性紫癜(Thrombocytopenic purpura，缩写TTP)、甲状腺眼病(Thyroid eye disease，缩写TED)、痛性眼肌麻痹综合征(Tolosa-Hunt syndrome，缩写THS)、横向脊髓炎(Transverse myelitis)、I型糖尿病(Type 1diabetes)、溃疡性结肠炎(Ulcerative colitis，缩写UC)、未分化结缔组织病(Undifferentiated connective tissue disease，缩写UCTD)、葡萄膜炎(Uveitis)、血管炎(Vasculitis)、白癜风(Vitiligo)和沃格特-小柳-原田病(Vogt-Koyanagi-Harada Disease)；优选为自身免疫性结肠炎、视神经脊髓炎、类风湿关节炎、硬皮病、银屑病、或葡萄膜炎。
本公开的第二方面涉及一种通路调节剂，其用于治疗自身免疫性疾病；其中，所述通路调节剂为DBH抑制剂、受体激动剂和受体拮抗剂中的一种或多种。
上述通路调节剂中，所述通路调节剂优选为DBH抑制剂。
上述通路调节剂中，所述DBH抑制剂可为现有技术中的已经公开的DBH抑制剂，例如可为内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、半胱胺、泛硫乙胺、铜螯合剂、富马酸、肼苯哒嗪、2-噻吩-2-基烯丙基胺、其药学上可接受的盐和其前药中的一种或多种。所述DBH抑制剂优选地选自内匹司他、依他米司他、依米司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选地选自内匹司他、依他米司他和依米司他。
上述通路调节剂中，所述DBH抑制剂优选地选自内匹司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选为内匹司他。
上述通路调节剂中，作为示例：
所述通路调节剂为内匹司他或其药学上可接受的盐，单位剂量为：10-100mg/kg；例如10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100mg/kg，或前述任意两个数值之间的范围；优选20-50mg/kg；或者，
所述通路调节剂为依他米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为依米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者
所述通路调节剂为镰孢菌酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为双硫仑或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为富马酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg。
上述通路调节剂中，所述自身免疫性疾病的定义如前所定义。
本公开的第三方面涉及一种用于治疗自身免疫性疾病的方法，其包括步骤：向受试者施用治疗有效量的通路调节剂；其中，所述通路调节剂为DBH抑制剂、受体激动剂和受体拮抗剂中的一种或多种。
上述治疗方法中，所述通路调节剂优选为DBH抑制剂。
上述治疗方法中，所述DBH抑制剂可为现有技术中的已经公开的DBH抑制剂，例如可为内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、半胱胺、泛硫乙胺、铜螯合剂、富马酸、肼苯哒嗪、2-噻吩-2-基烯丙基胺、其药学上可接受的盐和其前药中的一种或多种。所述DBH抑制剂优选地选自内匹司他、依他米司他、依米司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选地选自内匹司他、依他米司他和依米司他。
上述治疗方法中，所述DBH抑制剂优选地选自内匹司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选为内匹司他。
上述治疗方法中，所述DBH抑制剂可与选自化学治疗剂、靶向治疗剂、免疫 治疗剂和抗炎剂中的一种或多种联合施用。
上述治疗方法中，作为示例：
所述通路调节剂为内匹司他或其药学上可接受的盐，单位剂量为：10-100mg/kg；例如10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100mg/kg，或前述任意两个数值之间的范围；优选20-50mg/kg；或者，
所述通路调节剂为依他米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为依米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者
所述通路调节剂为镰孢菌酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为双硫仑或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为富马酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg。
上述治疗方法中，所述自身免疫性疾病的定义如前所定义。
本公开的第四方面涉及一种治疗自身免疫性疾病的药物组合物，其包含：
-通路调节剂、和
-药学上可接受的载体；
其中，所述通路调节剂为DBH抑制剂、受体激动剂和受体拮抗剂中的一种或多种。
上述药物组合物中，所述通路调节剂优选为DBH抑制剂。
上述药物组合物中，所述DBH抑制剂可为现有技术中的已经公开的DBH抑制剂，例如可为内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、半胱胺、泛硫乙胺、铜螯合剂、富马酸、肼苯哒嗪、2-噻吩-2-基烯丙基胺、其药学上可接受的盐和其前药中的一种或多种。所述DBH抑制剂优选地选自内匹司他、依他米司他、依米司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选地选自内匹司他、依他米司他和依米司他。
上述药物组合物中，所述DBH抑制剂优选地选自内匹司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选为内匹司他。
上述药物组合物中，作为示例：
所述通路调节剂为内匹司他或其药学上可接受的盐，单位剂量为：10-100mg/kg；例如10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100mg/kg，或前述任意两个数值之间的范围；优选20-50mg/kg；或者，
所述通路调节剂为依他米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为依米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者
所述通路调节剂为镰孢菌酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为双硫仑或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为富马酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg。
上述药物组合物中，所述自身免疫性疾病的定义如前所定义。
本公开的第五方面涉及一种通路调节剂在制备药物中的用途；所述药物用于选自以下的一种或多种用途：降低CD4+T细胞比例、增加调节性T细胞比例、增加CD8+T细胞比例、降低CD4+T细胞的促炎因子分泌、降低CD8+T细胞的促炎因子分泌、抑制B细胞的活化和抑制NK细胞的活化；降低外周血中淋巴细胞、中性粒细胞和单核细胞的含量；减轻真皮层中炎症性细胞浸润和真皮下的毛细血管增生；改善皮肤纤维化；降低葡萄膜炎发病率；改善皮肤炎症；改善粪便成形性评分、改善CW/CL、改善CW/BW、改善CW/CL/BW、抑制结肠溃疡面积增加、改善结肠炎性细胞浸润评分、改善组织损伤评分；改善疾病活动度评分、改善便血或隐血。其中，所述通路调节剂为DBH抑制剂、受体激动剂和受体拮抗剂中的一种或多种。
上述用途中，所述通路调节剂优选为DBH抑制剂。
上述用途中，所述DBH抑制剂可为现有技术中的已经公开的DBH抑制剂，例如可为内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、半胱胺、泛硫乙胺、铜螯合剂、富马酸、肼苯哒嗪、2-噻吩-2-基烯丙基胺、其药学上可接受的盐和其前药中的一种或多种。所述DBH抑制剂优选地选自内匹司他、依他米司他、 依米司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选地选自内匹司他、依他米司他和依米司他。
上述用途中，所述DBH抑制剂优选地选自内匹司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选为内匹司他。
上述用途中，作为示例：
所述通路调节剂为内匹司他或其药学上可接受的盐，单位剂量为：10-100mg/kg；例如10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100mg/kg，或前述任意两个数值之间的范围；优选20-50mg/kg；或者，
所述通路调节剂为依他米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为依米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者
所述通路调节剂为镰孢菌酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为双硫仑或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为富马酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg。
上述用途中，所述药物优选用于选自以下的一种或多种用途：降低CD4+T细胞比例、增加调节性T细胞比例、降低CD4+T细胞的促炎因子分泌、抑制B细胞的活化和抑制NK细胞的活化。
上述用途中，所述CD4+T细胞的促炎因子优选为IL-17A、IFN-γ和TNF-α中的一种或多种。
上述用途中，所述CD8+T细胞的促炎因子优选为IL-17A和/或TNF-α。
上述用途中，所述调节性T细胞优选为CD25+FOXP3+Treg细胞。
上述用途中，所述B细胞优选为B220+细胞，更优选为CD69+B220+B细胞。
上述用途中，所述NK细胞优选为NK1.1+细胞，更优选为NK1.1+CD107a+NK细胞。
本公开的第六方面涉及一种通路调节剂，其用于选自以下的一种或多种用途：降低CD4+T细胞比例、增加调节性T细胞比例、增加CD8+T细胞比例、降低CD4+T 细胞的促炎因子分泌、降低CD8+T细胞的促炎因子分泌、抑制B细胞的活化和抑制NK细胞的活化；降低外周血中淋巴细胞、中性粒细胞和单核细胞的含量；减轻真皮层中炎症性细胞浸润和真皮下的毛细血管增生；改善皮肤纤维化；降低葡萄膜炎发病率；改善皮肤炎症；改善粪便成形性评分、改善CW/CL、改善CW/BW、改善CW/CL/BW、抑制结肠溃疡面积增加、改善结肠炎性细胞浸润评分、改善组织损伤评分；改善疾病活动度评分、改善便血或隐血。其中，所述通路调节剂为DBH抑制剂、受体激动剂和受体拮抗剂中的一种或多种。
上述通路调节剂中，所述通路调节剂优选为DBH抑制剂。
上述通路调节剂中，所述DBH抑制剂可为现有技术中的已经公开的DBH抑制剂，例如可为内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、半胱胺、泛硫乙胺、铜螯合剂、富马酸、肼苯哒嗪、2-噻吩-2-基烯丙基胺、其药学上可接受的盐和其前药中的一种或多种。所述DBH抑制剂优选地选自内匹司他、依他米司他、依米司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选地选自内匹司他、依他米司他和依米司他。
上述通路调节剂中，所述DBH抑制剂优选地选自内匹司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选为内匹司他。
上述通路调节剂中，作为示例：
所述通路调节剂为内匹司他或其药学上可接受的盐，单位剂量为：10-100mg/kg；例如10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100mg/kg，或前述任意两个数值之间的范围；优选20-50mg/kg；或者，
所述通路调节剂为依他米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为依米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者
所述通路调节剂为镰孢菌酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为双硫仑或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为富马酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg。
上述通路调节剂中，所述药物优选用于选自以下的一种或多种用途：降低CD4+T细胞比例、增加调节性T细胞比例、降低CD4+T细胞的促炎因子分泌、抑制B细胞的活化和抑制NK细胞的活化。
上述通路调节剂中，所述CD4+T细胞的促炎因子优选为IL-17A、IFN-γ和TNF-α中的一种或多种。
上述通路调节剂中，所述CD8+T细胞的促炎因子优选为IL-17A和/或TNF-α。
上述通路调节剂中，所述调节性T细胞优选为CD25+FOXP3+Treg细胞。
上述通路调节剂中，所述B细胞优选为B220+细胞，更优选为CD69+B220+B细胞。
上述通路调节剂中，所述NK细胞优选为NK1.1+细胞，更优选为NK1.1+CD107a+NK细胞。
本公开的第七方面涉及一种在体内或体外调节免疫细胞功能的方法，其包括如下步骤：使有效量的通路调节剂在体内或体外接触免疫细胞，所述免疫细胞来自受试者；其中，所述通路调节剂为DBH抑制剂、受体激动剂和受体拮抗剂中的一种或多种；
所述调节免疫细胞功能指的是选自以下的一种或多种功能：降低CD4+T细胞比例、增加调节性T细胞比例、增加CD8+T细胞比例、降低CD4+T细胞的促炎因子分泌、降低CD8+T细胞的促炎因子分泌、抑制B细胞的活化和抑制NK细胞的活化。
上述方法中，所述通路调节剂优选为DBH抑制剂。
上述方法中，所述DBH抑制剂可为现有技术中的已经公开的DBH抑制剂，例如可为内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、半胱胺、泛硫乙胺、铜螯合剂、富马酸、肼苯哒嗪、2-噻吩-2-基烯丙基胺、其药学上可接受的盐和其前药中的一种或多种。所述DBH抑制剂优选地选自内匹司他、依他米司他、依米司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选地选自内匹司他、依他米司他和依米司他。
上述方法中，所述DBH抑制剂优选地选自内匹司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选为内匹司他。
上述方法中，作为示例：
所述通路调节剂为内匹司他或其药学上可接受的盐，单位剂量为：10-100mg/kg；例如10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100mg/kg，或前述任意两个数值之间的范围；优选20-50mg/kg；或者，
所述通路调节剂为依他米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为依米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者
所述通路调节剂为镰孢菌酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为双硫仑或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为富马酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg。
上述方法中，所述DBH抑制剂可与选自化学治疗剂、靶向治疗剂、免疫治疗剂和抗炎剂中的一种或多种联合施用。
上述方法中，所述药物优选用于选自以下的一种或多种用途：降低CD4+T细胞比例、增加调节性T细胞比例、降低CD4+T细胞的促炎因子分泌、抑制B细胞的活化和抑制NK细胞的活化。
上述方法中，所述CD4+T细胞的促炎因子优选为IL-17A、IFN-γ和TNF-α中的一种或多种。
上述方法中，所述CD8+T细胞的促炎因子优选为IL-17A和/或TNF-α。
上述方法中，所述调节性T细胞优选为CD25+FOXP3+Treg细胞。
上述方法中，所述B细胞优选为B220+细胞，更优选为CD69+B220+B细胞。
上述方法中，所述NK细胞优选为NK1.1+细胞，更优选为NK1.1+CD107a+NK细胞。
本公开的第八方面涉及一种药物组合物，其包含通路调节剂和药学上可接受的载体；其中，所述通路调节剂为DBH抑制剂、受体激动剂和受体拮抗剂中的一种或多种；
所述药物组合物具有选自以下的一种或多种功能：降低CD4+T细胞比例、增加调节性T细胞比例、增加CD8+T细胞比例、降低CD4+T细胞的促炎因子分泌、降低CD8+T细胞的促炎因子分泌、抑制B细胞的活化和抑制NK细胞的活化；降低外周血中淋巴细胞、中性粒细胞和单核细胞的含量；减轻真皮层中炎症性细胞浸润和真皮下的毛细血管增生；改善皮肤纤维化；降低葡萄膜炎发病率；改善皮肤炎症；改善粪便成形性评分、改善CW/CL、改善CW/BW、改善CW/CL/BW、抑制结肠溃疡面积增加、改善结肠炎性细胞浸润评分、改善组织损伤评分；改善疾病活动度评分、改善便血或隐血。
上述药物组合物中，所述通路调节剂优选为DBH抑制剂。
上述药物组合物中，所述DBH抑制剂可为现有技术中的已经公开的DBH抑制剂，例如可为内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、半胱胺、泛硫乙胺、铜螯合剂、富马酸、肼苯哒嗪、2-噻吩-2-基烯丙基胺、其药学上可接受的盐和其前药中的一种或多种。所述DBH抑制剂优选地选自内匹司他、依他米司他、依米司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选地选自内匹司他、依他米司他和依米司他。
上述药物组合物中，所述DBH抑制剂优选地选自内匹司他、其药学上可接受的盐和其前药。所述DBH抑制剂更优选为内匹司他。
上述药物组合物中，作为示例：
所述通路调节剂为内匹司他或其药学上可接受的盐，单位剂量为：10-100mg/kg；例如10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100mg/kg，或前述任意两个数值之间的范围；优选20-50mg/kg；或者，
所述通路调节剂为依他米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为依米司他或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者
所述通路调节剂为镰孢菌酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为双硫仑或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg；或者，
所述通路调节剂为富马酸或其药学上可接受的盐，单位剂量为80-120mg/kg；例如，80、85、90、95、100、110、120，或前述任意两个数值之间的范围；优选90-110mg/kg，更优选100mg/kg。
上述药物组合物中，所述药物组合物还可含有选自化学治疗剂、靶向治疗剂、免疫治疗剂和抗炎剂中的一种或多种物质。
上述药物组合物中，所述药物优选用于选自以下的一种或多种用途：降低CD4+T细胞比例、增加调节性T细胞比例、降低CD4+T细胞的促炎因子分泌、抑制B细胞的活化和抑制NK细胞的活化。
上述药物组合物中，所述CD4+T细胞的促炎因子优选为IL-17A、IFN-γ和TNF-α中的一种或多种。
上述药物组合物中，所述CD8+T细胞的促炎因子优选为IL-17A和/或TNF-α。
上述药物组合物中，所述调节性T细胞优选为CD25+FOXP3+Treg细胞。
上述药物组合物中，所述B细胞优选为B220+细胞，更优选为CD69+B220+B细胞。
上述药物组合物中，所述NK细胞优选为NK1.1+细胞，更优选为NK1.1+CD107a+NK细胞。
本公开的积极效果在于：发明人发现，DBH抑制剂可通过免疫调节，而抑制自身免疫性疾病的发生发展(作为示例，具体体现在对体重减轻的改善、DAI评分的改善及结肠密度正常化)，进而可为自身免疫性疾病的治疗提供新的选择。
附图说明
图1A显示根据实施例1中描述的实验程序进行实验后，各组小鼠体重评估结果。
图1B显示根据实施例1中描述的实验程序进行实验后，各组小鼠DAI评分结果。
图1C显示根据实施例1中描述的实验程序进行实验后，各组小鼠结肠密度结果。
图2A至图2E显示实施例4中各组的小鼠肠系膜淋巴结中CD4+T细胞亚群(图2A)、CD25+FOXP3+Treg细胞亚群(图2B)、来自CD4+T细胞亚群的IL-17A(图2C)、来自CD4+T细胞亚群的IFN-γ(图2D)及来自CD4+T细胞亚群的TNF-α(图2E)的影响。
图3A至图3D显示如实施例4中各组的小鼠肠系膜淋巴结中CD8+T细胞亚群(图3A)、来自CD8+T细胞亚群的IFN-γ(图3B)、来自CD8+T细胞亚群的IL-17A(图3C)及来自CD8+T细胞亚群的TNF-α(图3D)的影响。
图4A至图4B显示如实施例4中各组的小鼠肠系膜淋巴结中CD69+B220+B细胞亚群(图4A)及NK1.1+CD107a+NK细胞亚群(图4B)的影响。上述各图中*指的是p<0.05，**指的是p<0.01，***指的是p<0.001，****指的是p<0.0001。
图5显示实施例5各组中的体重变化；Two-way ANOVA： #p<0.05， ###p<0.001vs.溶媒组。
图6显示实施例5各组中的体重变化率。
图7显示实施例5各组中的脾脏重量；One-way ANOVA:***p<0.001vs.正常组,#p<0.05,##p<0.01vs.溶媒组。
图8显示实施例5各组中的脏体系数；One-way ANOVA: #p<0.05, ##p<0.01, ###p<0.001vs.溶媒组。
图9显示实施例5各组中外周血红细胞(RBC)计数；One-way ANOVA：***p<0.001vs正常组, ##p<0.01, ###p<0.001vs溶媒组, &&&p<0.001vs伊马替尼组。
图10显示实施例5各组中外周血血红蛋白(HGB)含量；One-way ANOVA： ***p<0.001vs正常组, ##p<0.01, ###p<0.001vs溶媒组, &&&p<0.001vs伊马替尼组。
图11显示实施例5各组中外周血白细胞(WBC)计数；One-way ANOVA：*p<0.05,***p<0.001vs正常组, ##p<0.01vs溶媒组。
图12显示实施例5各组中外周血淋巴细胞(LYMPH)计数；One-way ANOVA： #p<0.05, ###p<0.001vs溶媒组。
图13显示实施例5各组中外周血单核细胞(MONO)计数；One-way ANOVA： ##p<0.01vs溶媒组。
图14显示实施例5各组中外周血中性粒细胞(NEUT)计数；One-way ANOVA：***p<0.001vs正常组。
图15显示实施例5各组中外周血血小板含量；One-way ANOVA：***p<0.001vs正常组, ###p<0.001vs溶媒组, &&p<0.01, &&&p<0.001vs.尼达尼布组, $$p<0.01vs.伊马替尼组。
图16显示实施例5各组中真皮层炎细胞浸润评分；One-way ANOVA： #p<0.05vs.溶媒组, $p<0.05vs.内匹司他-25mg/kg,qd,po。
图17显示实施例5各组中真皮层内毛细血管密度评分；One-way ANOVA： #p<0.05, ##p<0.01vs.溶媒组。
图18显示实施例5各组中真皮厚度；One-way ANOVA：*p<0.05,**p<0.01,***p<0.001vs.正常组。
图19显示实施例6各组中的临床评分。
图20A至图20D：实施例7各组中AQP4、GFAP、IBA1、CD45阳性细胞比例。
图21显示实施例8各组中动物的体重。
图22显示实施例8各组中红斑+鳞屑+厚度总评分的AUC；均值±标准误差，Two way ANOVA，与相应的溶媒组相比，p*<0.05，p**<0.01，p***<0.001，p****<0.0001(注：正常组的总评分AUC为0，图中未显示)。
图23显示实施例8各组中脾脏重量。
图24显示实施例9各组中的动物体重。
图25显示实施例9各组中的临床评分。
图26显示实施例9各组中的AUC。
图27显示实施例9各组中的发病率。
图28显示实施例10各组中动物体重。
图29显示实施例10各组中动物体重变化率。
图30显示实施例10各组中粪便评分。
图31显示实施例10各组中粪便评分AUC。
图32显示实施例11各组中体重变化。
图33显示实施例11各组中DAI。
具体实施方式
在背景技术和整个说明书中引用了各种出版物、专利申请；这些参考文献中的每一篇均通过引用整体并入本文。除非另外定义，本文所使用的所有技术和科学术语具有与本公开所属领域的普通技术人员通常所理解的相同含义。
术语
本文中，术语“受体激动剂”指的是能够作用于多巴胺β-羟化酶催化的底物(即多巴胺)的受体，并发挥与多巴胺升高相同的机制及效应的物质。所述受体激动剂优选为多巴胺受体激动剂。
本文中，术语“受体拮抗剂”指的是能够作用于多巴胺β-羟化酶催化的产物(即去甲肾上腺素和/或肾上腺素)的受体，并发挥与去甲肾上腺素和/或肾上腺素降低相同的机制及效应的物质。所述受体拮抗剂优选为去甲肾上腺素受体拮抗剂和/或肾上腺素受体拮抗剂。
术语“自身免疫性疾病”是指自身的免疫系统攻击机体，以识别自身/非己的适应性免疫耐受机制破坏为特征，适应性免疫细胞异常反应导致自身组织的炎症损害。
术语“多巴胺β-羟化酶”旨在涵盖人源的多巴胺β-羟化酶、及其片段、变体、前体和功能结构域。
术语“DBH抑制剂”指的是能够在核酸或蛋白水平影响(该影响指的是降低、减少、抑制、阻断、阻遏、失活或防止激活)多巴胺β-羟化酶的结构、表达或活性的任何天然化合物或人工化合物。DBH抑制剂包括现有技术中已知的，以及未来提供的DBH抑制剂。其包括CN87103323A和WO9529165中公开的DBH抑制剂，以及内匹司他(Nepicastat)、依他米司他(Etamicastat)、依米司他(Zamicastat)、镰孢菌酸、双硫仑、半胱胺、半胱胺衍生物、泛硫乙胺和泛硫乙胺衍生物。
术语“药学上可接受的载体”指的是药学上可接受的辅料。
术语“药学上可接受的盐”应该被理解为指代下列盐，其是药学上可接受的盐并且其具备预期的母体化合物的药理活性。这种盐包括：
(1)与无机酸形成的酸加成盐，或与有机酸形成的酸加成盐；其中，所述无机酸可为盐酸、氢溴酸、硫酸、硝酸和磷酸中的一种或多种；所述有机酸可为甲酸、草酸、丁二酸、乙酸、苯磺酸、苯甲酸、樟脑磺酸、柠檬酸、乙磺酸、富马酸、葡庚糖酸、葡糖酸、谷氨酸、乙醇酸、羟萘酸、2-羟基乙磺酸、乳酸、马来酸、苹果酸、扁桃酸、甲磺酸、粘康酸、2-萘磺酸、丙酸、水杨酸、琥珀酸、二苯甲酰基-L-酒石酸、酒石酸、对甲苯磺酸、三甲基乙酸和三氟乙酸中的一种或多种；和(2)母体化合物中存在的酸质子被金属离子，例如，碱金属离子(例如，Na +、K +或Li +)、碱土金属离子(如Ca 2+或Mg 2+)或铝离子代替；或者，与有机碱或 无机碱配位时形成的盐；其中，所述有机碱可为吡啶类、咪唑类、吡嗪类、吲哚类、嘌啉类、叔胺类和苯胺类有机碱中的一种或多种，优选为吡啶、甲基吡啶、4-二甲氨基吡啶、2-甲基-5-乙基吡啶、三乙胺、N,N-二异丙基乙胺、N,N-二甲基苯胺、二乙醇胺、乙醇胺、N-甲基葡糖胺、三乙醇胺和氨丁三醇中的一种或多种；所述无机碱可为氢氧化铝、氢氧化钙、氢氧化钾、碳酸钠和氢氧化钠中的一种或多种。
本公开的药物组合物可以是各种常规剂型，例如片剂，水混悬液，油混悬液，可分散粉末，可分散颗粒，乳剂，硬胶囊、软胶囊，无菌注射水溶液，无菌注射水包油微乳，或栓剂。上述各剂型均可通过常规的制备方法制得。
本公开的片剂中的辅料可为填充剂、粘合剂、润滑剂、助流剂和崩解剂中的一种或多种。其中，所述填充剂可为微晶纤维素、淀粉、一水乳糖和磷酸氢钙中的一种或多种。所述粘合剂可为淀粉、明胶、聚乙烯吡咯烷酮和阿拉伯胶中的一种或多种。所述润滑剂可为硬脂酸镁、硬脂酸和十二烷基硫酸钠中的一种或多种。所述助流剂可为微粉硅胶和滑石粉中的一种或两种。所述崩解剂可为交联聚维酮、羧甲淀粉钠、低取代羟丙纤维素和交联羧甲基纤维素钠中的一种或多种。所述片剂还可含有包衣。所述片剂还可制成缓释制剂，所述缓释制剂中的缓释材料可为羟丙基甲基纤维素和黄原胶中的一种或两种。
本公开的水混悬液中的辅料可为悬浮剂、分散剂、防腐剂和矫味剂中的一种或多种。其中，所述悬浮剂可为羧基甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、藻酸钠、聚乙烯吡咯烷酮和阿拉伯胶中的一种或多种。所述分散剂可为天然产生的磷脂(例如卵磷脂)、烯化氧与脂肪酸的缩合产物(例如聚氧乙烯硬脂酸酯)、环氧乙烷与长链脂肪醇的缩合产物(例如十七碳亚乙基氧基鲸蜡醇)、环氧乙烷与由脂肪酸和己糖醇衍生的部分酯的缩合产物(例如聚环氧乙烷山梨醇单油酸酯)、环氧乙烷与由脂肪酸和己糖醇酐衍生的偏酯的缩合产物(例如聚环氧乙烷脱水山梨醇单油酸酯)中的一种或多种。所述防腐剂可为尼泊金乙酯和/或尼泊金正丙酯。所述矫味剂可为蔗糖、糖精和阿司帕坦中的一种或多种。
本公开的油混悬液中的辅料可为悬浮剂、增稠剂、矫味剂和抗氧化剂中的一种或多种。所述悬浮剂可为植物油和/或矿物油，所述植物油可为花生油、橄榄油、芝麻油和椰子油中的一种或多种，所述矿物油可为液体石蜡。所述增稠剂可为蜂蜡、硬石蜡和鲸蜡醇中的一种或多种。所述矫味剂可为蔗糖、糖精和阿司帕坦中的一种或多种。所述抗氧化剂可为丁羟茴醚、α-生育酚和抗坏血酸中的一种或多种。
本公开的可分散粉末和可分散颗粒中的辅料可为悬浮剂、分散剂、防腐剂、矫味剂和抗氧化剂中的一种或多种。上述各组分的具体选择同水混悬液中的辅料。
本公开的乳剂中的辅料可为悬浮剂、乳化剂、矫味剂、防腐剂和抗氧剂中的一种或多种。所述悬浮剂可为植物油和/或矿物油，所述植物油可为橄榄油和/或花生油，所述矿物油可为液体石蜡。所述乳化剂可为天然产生的磷脂(例如大豆卵 磷脂)、由脂肪酸和己糖醇酐衍生的酯或偏酯(例如山梨坦单油酸酯)和所述偏酯和环氧乙烷的缩合产物(例如聚环氧乙烷山梨醇单油酸酯)中的一种或多种。所述矫味剂可为甘油、丙二醇、山梨醇和蔗糖中的一种或多种。所述防腐剂可为尼泊金乙酯和/或尼泊金正丙酯。所述抗氧化剂可为丁羟茴醚、α-生育酚和抗坏血酸中的一种或多种。
本公开的硬胶囊中辅料可为常规的惰性固体稀释剂，例如可为碳酸钙、磷酸钙和高岭土中的一种或多种。
本公开的软胶囊中的辅料可为常规的水溶性载体和/或常规的油溶媒，例如可为聚乙二醇、花生油、液体石蜡和橄榄油中的一种或多种。
本公开的无菌注射水溶液中的辅料可为药学上可接受的溶媒，例如水、林格氏液或等渗氯化钠溶液。
本公开的无菌注射水包油微乳中的辅料可为油相辅料和水相辅料，所述油相辅料可为大豆油和卵磷脂的混合物，所述水相辅料可为水和甘油的混合物。
本公开的栓剂中的辅料可为可可脂、甘油、明胶、氢化植物油、聚乙二醇和聚乙二醇的脂肪酸酯中的一种或多种。
术语“受试者”是指动物，优选哺乳动物。根据具体的实施方案，受试者为哺乳动物，包括例如骆驼、驴、斑马、牛、猪、马、山羊、绵羊、猫、狗、大鼠、兔、豚鼠、小鼠、灵长动物(例如人)。在具体的实施方案中，受试者为人。在具体的实施方案中，受试者为易感于、疑似患有、已患有自身免疫性疾病的人。
术语“治疗”指的是消除疾病，阻止疾病进展，减缓疾病进展，减少与疾病相关的一种或多种症状的持续时间，至少一个与疾病相关的可测量参数的改善或逆转，或者增加患有疾病的受试者的存活率。
术语“有效量”是指药物活性成分在受试者中引发期望效果的量。在具体的实施方案中，本领域技术人员可以基于对多种因素的考虑(例如经由临床试验)来确定有效量的选择，所述因素包括待治疗的疾病、所涉及的症状、施用途径、疾病的严重程度、患者的体重、患者的免疫状态和本领域技术人员已知的其他因素。具体实施方案中的有效量，可以从来源于动物模型测试系统的剂量-应答曲线而得到，并允许根据医生的判断和每位患者的情况来决定。其中，动物和人的用药量的相互关系描述于Freireich等人1966，Cancer Chemother Rep 50：219，且人体表面积可以近似地由患者的身高和体重确定。本公开的药物化合物的有效量可为0.5mg/kg至500mg/kg，优选为1mg/kg至200mg/kg，更优选为10mg/kg至100mg/kg。
本文中，同一药物活性成分(指的是单一的药物化合物)或不同药物活性成分(指的是两个以上的药物化合物)可一次性施用，或者可分成许多更小的单位剂量，以一定时间间隔施用。应理解治疗的确切剂量、持续时间、间隔时间是所治疾病的函数，并且可使用动物或临床试验数据推断而确定。所述施用可包括单次施用，或者间隔适当时间间隔的两次或更多次施用。其中，相邻两次的施用可 间隔30分钟、40分钟、50分钟、60分钟、2小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、12小时、14小时、16小时、18小时、20小时、22小时、24小时、一天半、2天、3天、4天、5天、6天、7天、8天、9天、10天、1周、2周、3周、4周、5周、6周、1个月、2个月、3个月、4个月、5个月、6个月、7个月、8个月、9个月、10个月、11个月或12个月。
本文中提及的各药物活性成分(各药物化合物)可作为唯一的活性化合物使用，也可以与其他活性化合物(指的是本文所述的药物化合物之外的化合物)联合施用，只要它们不产生其他不利的作用，例如过敏反应等。联合施用包括将各活性化合物同时或先后施用。
术语“联合施用”是指为了达到治疗目的，而将两种或两种以上活性化合物同时或先后提供给受试者的方法。当涉及“联合施用”时，每次施用之间的时间间隔，足以实现施用的各活性化合物之间的协同作用。两种或两种以上活性化合物在相同的不同容器中。
缩写：QD：每天一次；BID：每天两次；PO：口服；IP：腹腔注射；IM：肌肉注射。
实施例1：DBH抑制剂对硫酸葡聚糖钠(DSS)诱导的结肠炎的抑制作用
1.材料和设备：
表1.试剂
试剂 供应商 货号
杜氏磷酸缓冲液(1×DPBS) Corning Incorporated 21-031-CVR
硫酸葡聚糖钠(DSS) MP Biomedicals SR01606
环孢菌素A(CsA) Novartis /
生理盐水 Ke Lun Pharmaceutical Co.,Ltd /
PEG400 Sigma Aldrich 25322-68-3
30％Solutol HS15 Sigma Aldrich 70142-34-6
内匹司他 Selleckchem S2695
依他米司他 南京药石 677773-32-9
依米司他 MCE 1080028-80-3
表2.仪器
仪器 供应商 型号
电子天平 常州天之平 YH-2000
电子分析天平 Mettle Toledo 585310
2.药液的配制及保存：
溶媒的组成如下：20v％PEG400+10v％(30％Solutol HS15)+70v％生理盐水。
待测物-内匹司他药液：将内匹司他27mg完全溶解于9mL溶媒中(该浓度对应检测组1#)，每3天配制一次，4℃保存，以保持效果。
待测物-依他米司他药液：分别将9mg，27mg，90mg的依他米司他完全溶解于9mL溶媒中(三个浓度分别对应检测组2#、检测组3#、检测组4#)，每3天配制一次，4℃保存，以保持效果。
待测物-依米司他药液：分别将9mg，27mg，90mg的依米司他完全溶解于9mL溶媒中(三个浓度分别对应检测组5#、检测组6#、检测组7#)，每3天配制一次，4℃保存，以保持效果。
阳性药对照-环孢菌素A药液：将200mg环孢菌素A完全溶解于10mL生理盐水中，配制成20mg/mL的溶液(对应阳性药对照组)，每3天配制一次，4℃保存，以保持效果。
3.各组小鼠的处理：
使用从维通利华实验动物技术有限公司(Charles River Laboratories)购买的8-10周龄雌性C57BL/6J小鼠。在恒温(20±2℃)的房间里(每个笼子5只)，进行12小时的明暗循环。所有的实验方案都得到了药明康德(Wuxiapptec)机构动物保护和使用委员会的批准。实验前，这些小鼠在药明康德的动物实验中心至少适应三天。
(1)溶媒组、阳性药对照组和检测组
为了建立硫酸葡聚糖钠诱导的结肠炎模型，连续7天自由饮用3％DSS水溶液(于100mL水中加入3g DSS制得该水溶液，其中DSS的分子量为36000-50000)以诱发结肠炎(该结肠炎属于自身免疫性结肠炎)，并分为9组分别给药(见表3中的溶媒组、阳性药对照组和检测组1#至检测组7#)，其中3％DSS水溶液每天新鲜配制。在实验终点时(见下文)被实施安乐死并获得终点样品。
(2)正常组
正常组的小鼠没有经过3％DSS水溶液诱发结肠炎，而是自由饮用空白水。在实验终点时(见下文)被实施安乐死并获得终点样品。
表3.各组别及给药方案
Figure PCTCN2022073849-appb-000003
Figure PCTCN2022073849-appb-000004
4.体重评估及疾病活动指数(disease activity Index，DAI)评分：
对于动物体重及DAI评分均在双盲的条件下进行，即科研人员未知组别及给药情况下，每天收集小鼠体重、观察大便性状及血便及进行评分。DAI评分为体重减轻评分、排便评分和出血评分之和。评分标准如表4所示。
表4.DAI评分体系
Figure PCTCN2022073849-appb-000005
5.统计分析：
数据分析采用方差分析，具体采用的方法如下：使用Graph Pad Prism 6.0软件，采用post-hoc Dunnett's Dunnett多重比较检验方法。将其余组(指的是正常组、阳性药对照组和检测组1#至7#)与溶媒组比较，分析其余组与溶媒组相比是否具有显著性。若p值<0.05，则具有统计学意义上的差异，具有显著性。数据以均数±S.E.M.表示。
实施例2：DBH抑制剂对免疫细胞活化及细胞因子影响
1.材料：
表5.流式抗体试剂
Figure PCTCN2022073849-appb-000006
Figure PCTCN2022073849-appb-000007
表6.其他试剂
Figure PCTCN2022073849-appb-000008
2.方法：
实施例1的阳性药对照组和检测组1#至检测组7#，第8天给药后24小时为实验终点；正常组和溶媒组的实验终点同阳性药对照组和检测组1#至检测组7#。
实验终点时，采集肠系膜淋巴节，并进行如下处理：
1)轻轻研碎肠系膜淋巴结，用70μM滤网(BD bioscience，货号352350)过滤，获得细胞悬液，计数细胞。
2)采用悬浮液(该悬浮液采用如下方法制得：采用90v％1640培养基与10v％胎牛血清组成的混合液将细胞活化混合液稀释至浓度为1×即得)悬浮细胞，调整细胞密度为2.5×10 7/mL，在37℃下孵育5小时。
3)采用杜氏磷酸缓冲液清洗细胞，利用死/活(viable/dead)染料(表5中编号为12染料)常温染色15min，区分活细胞及死细胞；用染色缓冲液(胎牛血清与杜氏磷酸缓冲液按2:98的体积比混合后制得)清洗细胞，加入Fc阻断剂工作液(Fc阻断剂与染色缓冲液按1:200的体积比混合后制得)，室温孵育15分钟。
4)无需清洗细胞，用100μL流式抗体染色液表面标记细胞，4℃孵育30分钟。
5)采用染色缓冲液洗涤细胞1次，加入100μL细胞固定液，4℃孵育过夜。
6)加入200μL细胞破膜液，4℃孵育30分钟。
7)用250μL细胞破膜洗液清洗细胞后，用100μL染色缓冲液悬浮细胞，用BD LSRFortessa仪器检测荧光。
8)进行流式检测分析的细胞亚群包括CD4+T细胞亚群、IL-17A+CD4+T细胞亚群、IFN-γ+CD4+T细胞亚群、TNF-α+CD4+T细胞亚群、CD25+FOXP3+Treg细胞亚群；CD8+T细胞亚群、IL-17A+CD8+T细胞亚群、IFN-γ+CD8+T细胞亚群、TNF-α+CD8+T细胞亚群；CD69+B220+B细胞亚群及NK1.1+CD107a+NK细胞亚群。
实施例3：DBH抑制剂改善了DSS诱导的结肠损伤
根据实施例1中描述的实验程序进行实验后，各组小鼠体重评估结果见表7及图1A；各组小鼠DAI评分结果见表8及图1B；各组小鼠结肠密度结果见表9及图1C。
Figure PCTCN2022073849-appb-000009
Figure PCTCN2022073849-appb-000010
与溶媒组相比，检测组1#、检测组4#和检测组7#的小鼠在治疗第7天时的体重降低均明显减少(图1A，表7)。
与溶媒组相比，检测组1#，检测组2#，检测组3#，检测组4#，检测组5#，检测组6#和检测组7#的小鼠在治疗第7天的DAI评分均显著降低(图1B，表8)。
小鼠给予DSS后，通常会导致结肠缩短，结肠密度提高，上述这两个指标能够反映出DSS所致结肠炎的严重程度。与溶媒组相比，检测组1#、检测组4#和检测组7#的结肠密度显著降低(图1C，表9)。这些数据表明，内匹司他、依他米司他及依米司他改善了DSS诱导的结肠炎的严重程度，降低了疾病的活动性。
综上所述，检测组1#、检测组4#组和检测组7#的DSS诱导的结肠炎较溶媒组更轻，提示内匹司他、依他米司他及依米司他对结肠炎有潜在的治疗作用。
实施例4：内匹司他及依米司他对免疫细胞及细胞因子的影响
按照实施例2方法，分析内匹司他及依米司他对免疫细胞及细胞因子的影响，所得结果见图2A至图2E，图3A至图3D，图4A至图4B。
首先，对来自CD3+CD45+细胞亚群的CD4+T细胞亚群的变化进行了分析，也对来自CD4+细胞亚群的CD25+FOXP3+Treg细胞亚群的变化进行了分析。结果表明，与溶媒组相比，检测组1#和检测组7#，可以降低CD4+T细胞亚群比例(图2A)，同时增加CD25+FOXP3+Treg细胞亚群比例(图2B)。上述结果提示，DBH抑制剂可显著抑制炎症及免疫。
然后，对CD4+T细胞亚群所分泌的细胞因子进一步分析。结果表明，与溶媒组相比，检测组1#和检测组7#，均可降低促炎因子IL-17A(图2C)、IFN-γ(图2D)及TNF-α(图2E)的分泌，进而提示DBH抑制剂通过降低CD4+T细胞亚群分泌促炎因子而发挥抑制炎症及免疫的作用。
此外，对来自CD3+CD45+细胞亚群的CD8+T细胞亚群及其分泌的细胞因子进行了类似分析。结果表明，与溶媒组相比，DBH抑制剂可增加CD8+T细胞亚群比例(图3A)，对IFN-γ(图3B)分泌没有显著影响，但可抑制IL-17A(图3C)和TNF-α(图3D)分泌。上述结果提示，DBH抑制剂通过降低CD8+T细胞亚群分泌IL-17A和TNF-α从而抑制炎症及免疫。
最后，对B细胞(指的是来自CD3-细胞亚群的CD69+B220+B细胞亚群)及NK细胞(指的是来自CD3-B220-细胞亚群的NK1.1+CD107a+NK细胞亚群)的活化情况进行了评估。结果表明，与溶媒组相比，DBH抑制剂均可抑制B细胞(图4A)及NK细胞(图4B)的活化。上述结果提示，DBH抑制剂可以降低B细胞产生自身抗体的风险及NK细胞介导的非特性对粘膜组织的杀伤，从而缓解炎症及抑制免疫。
实施例5：DBH抑制剂对博来霉素诱导的雄性C57小鼠皮肤纤维化的抑制作 用
1.材料和设备
表10.试剂
试剂 供应商 货号
生理盐水 浙江都邦药业股份有限公司 2009240101
Solutol HS15 Sigma BCCF2234
PEG400 国药集团化学试剂有限公司 20181205
MC Sigma SLBT4343
Tween 80 Sigma WXBD2914v
异氟烷 RWD瑞沃徳 20120101
盐酸博莱霉素 日本化药株式会社 600700
表11.仪器
名称 型号和生产商
呼吸麻醉机 AMS(Gene&I)北京吉安得尔科技有限公司(吉安得尔)
自动血液分析仪 Sysmex XS-800i
组织脱水机 LEICA HistoCore Pearl
包埋机 Leica Histocore Arcadia C&H
切片机 LEICA,RM2235
自动染色机 LEICA,ST5020
切片扫描仪 HAMAMATSU Nano Zoomer S210
数控超声波清洗器 KQ-100DE，昆山市超声仪器有限公司
2.药液的配制及保存
溶媒的组成：20％PEG400+10％(30％Solutol HS15)+70％生理盐水。量取100mL的PEG400和50mL的30％(V/V)solutol，加入至350mL的生理盐水中；置于磁力搅拌器上，搅拌至其混合均匀，4℃保存备用。
待测物-内匹司他药液：称取40mg待测物至棕色样品瓶中，加入0.8mL PEG400，于漩涡仪上涡旋，超声15分钟，40℃水浴加热15分钟，成悬浊液，然后在加入0.4mL 30％Solutol HS15，于漩涡仪上涡旋混匀，然后再加入2.8mL生理盐水，于漩涡仪上涡旋混匀，至成溶液，化合物浓度为10mg/mL。每三天配制一次，保存于4℃备用。
对照品-尼达尼布药液：精确称取25mg尼达尼布(BIBF1120)，加入5mL溶媒(0.5％MC+0.2％Tween80)中，充分混匀至溶液呈澄清透明状态，药物浓度为5mg/mL(给药剂量为10mL/kg)，每七天配制一次。
对照品-伊马替尼药液：精确称取15mg甲磺酸伊马替尼粉末至样品瓶中，加入3mL生理盐水，于旋涡仪上涡旋至完全溶解，药物浓度为5mg/mL。现配现用，半小时内用完。
3.试验方法
3.1动物饲养
雄性C57BL/6小鼠70只(体重20至22g)，动物饲养于凯斯艾生物科技(苏州)有限公司SPF级屏障系统内，动物使用合格证号：SYXK(苏)2017-0041，遵循国际标准温、湿、光控制系统。
本试验动物操作方案经由IACUC委员会联合审批确认。严格遵循凯斯艾生物科技(苏州)有限公司的SOP实行操作和管理。
3.2模型建立
选用异氟烷(2.0至2.5％)将动物进行麻醉，去除背部被毛，选择大小为1cm 2面积进行每两天一次的博莱霉素(0.3mg/kg，100μL)皮内注射建立皮肤纤维化模型。
3.3试验分组
根据动物体重分成7组，每组10只，分别是：正常组、溶媒组、尼达尼布组(阳性对照药)、伊马替尼(阳性对照药)、检测组1#、检测组2#、检测组3#，具体情况见表12。
表12.试验分组和给药计划
Figure PCTCN2022073849-appb-000011
备注：a：20％PEG400+10％(30％Solutol HS15)+70％生理盐水。
3.4试验给药
给药开始时间为建模当天，每只动物给药前称量动物体重计算给药体积，其中给药周期为28天。给药方式：伊马替尼组为腹腔注射，其余组为灌胃给药。给药频率：尼达尼布组为一天两次，其余组为每天一次。
3.5试验动物生理观察
建模当日起每周2次记录动物体重变化；密切观察动物临床表征，如呼吸急促、困难，吸腹，活动量减少，精神萎靡等。
3.6局部皮肤观察
试验终点，动物经腹腔注射过量的戊巴比妥钠(100mg/kg)进行安乐死，然后拍摄皮肤局部照片。
3.7试验终点
试验终点称重并记录，计算给药体积。给药后6h，首先从小鼠眶静脉丛取全血，使用自动血液分析仪(Sysmex XS-800i)进行血常规检测。具体检测步骤如下： 检测前，将试管中的血液样品充分混匀；将样品置于吸样针下，然后按开始键吸样，待仪器指示灯灭后移走样品；仪器开始自动检测并输出结果。
然后动物腹腔注射戊巴比妥钠麻醉动物进行安乐死，解剖取材内容和顺序依次是：取脾胀称重并记录，取双侧腹股沟淋巴结速冻后保存于-80℃冰箱，取病变皮肤组织先进行皮肤图像采集，再浸泡于10％福尔马林固定液固定，组织与福尔马林比例1:10，固定48h后进行组织病理学检测。具体内容如下表13所示。
表13.实验终点
Figure PCTCN2022073849-appb-000012
3.8组织病理学检测
按照病理学SOP，对病变皮肤进行皮肤组织脱水，包埋和切片，组织进行HE和Masson染色。按照以下方法进行组织病理学分析：
3.8.1真皮厚度
Masson染色的切片，经过Nanozomer S210进行全景扫描，扫描图像利用Visiopharm VIS6.0软件进行定量分析。纤维化程度目前用Masson染色后的真皮厚度来表示。
3.8.2真皮层内毛细血管密度评分
在病灶区的真皮层内选择观察视野(根绝病灶区大小决定选择多少个视野)按照毛细血管密度在该区域所占面积进行评分：
0：未见毛细血管生成
1：毛细血管生成占比小于25％；
2：毛细血管生成占比25-50％；
3：毛细血管生成占比50-75％；
4：毛细血管生成占比大于75％。
3.8.3真皮层炎细胞浸润评分
在病灶区的真皮层内选择观察视野(根绝病灶区大小决定选择多少个视野)按照炎细胞浸润程度进行评分：
0：未见炎细胞浸润：
1：少量局灶性的炎细胞浸润；
2：散在弥漫性的炎细胞浸润：
3：大量弥漫性的炎细胞浸润
4：大量聚聚成团的炎细胞浸润。
3.9数据分析
使用Graphpad prism6.0软件，计算均值±SEM，差异显著性检验使用t-检验,one-way ANOVA检验或two-way ANOVA检验，p<0.05时认为两组之间具有显著性差异。
4试验结果
4.1体重变化
试验期间，正常组动物体重呈上升趋势，检测组1#、检测组2#、检测组3#体重保持稳定，在正常范围内波动。只有尼达尼布组和伊马替尼组，在给药后，呈缓慢降低的趋势，并且在第25至28天期间内，与溶媒组相比，一直有显著差异(见下图5、图6)。其中伊马替尼组有一只动物在第28天出现死亡，死亡当天的体重与建模前的体重相比下降了23.7％。
4.2脾脏和脏体系数
在实验终点取脾脏，称量脾脏重量并计算脏体系数(脾脏/体重×100％)，结果如图7所示。溶媒组脾脏重量对比于正常组显著下降，尼达尼布组、伊马替尼组、检测组1#、检测组2#、检测组3#与溶媒组相比，脾脏重量也显著降低。
在脾脏脏体系数方面，溶媒组和正常组相比，没有明显差异，表示造模后并不能改变小鼠的脾脏脏体系数，只有尼达尼布组、检测组1#、检测组2#、检测组3#，与溶媒组相比，有显著性差异(表14，图8)。
表14.脾脏重量和脏体系数
Figure PCTCN2022073849-appb-000013
***p<0.001vs.正常组， #p<0.05, ##p<0.01vs.溶媒组。
4.3血常规
实验终点采集外周血进行血常规检测。结果显示，伊马替尼显著减少外周血红细胞和血红蛋白含量，这也与该组动物在实验终点时出现贫血症状相符。此外，伊马替尼能显著减少外周血白细胞的含量(包括淋巴细胞和中性粒细胞)；对单核细胞有降低的趋势，但无显著性差异。
与溶媒组相比，检测组3#在减少淋巴细胞、中性粒细胞和单核细胞方面，均 具有显著差异，表明检测组3#的剂量(100mg/kg)的治疗具有减轻炎症的作用，这与病理结果相一致(图9至图15)。
4.4皮肤病理染色结果
系统性硬化症(SSc)是一种自身免疫性疾病，表现为炎症、血管病变、皮肤和脏器纤维化。
本实施例选用博来霉素(BLM)建立小鼠纤维化模型，病理HE染色结果显示，与溶媒组和正常组相比，表现出显著的炎细胞浸润、真皮下毛细血管增生；Masson染色结果显示溶媒组动物具有更强的皮肤纤维化程度，其真皮厚度也明显增加。尼达尼布和伊马替尼的治疗可以明显减轻病变皮肤炎细胞浸润和真皮下毛细血管增生，检测组2#、检测组3#能显著减轻真皮层炎细胞浸润和真皮下毛细血管增生。根据真皮厚度的统计结果来看，待测物内匹司他的三个不同剂量降低真皮厚度的效果呈递进式的，剂量越高降低结果越明显，但与模型组相比均无显著性差异。与检测组1#、检测组2#相比，检测组3#具有更好的改善皮肤纤维化的效果(图16至图18)。
由以上试验结果可以看出，检测组2#、检测组3#能够降低博莱霉素刺激后外周血炎细胞反应性增加的数量，减轻病变皮肤真皮层内炎细胞浸润和毛细血管增生，尤其以检测组3#对应的100mg/kg剂量的效果最佳，而且100mg/kg亦能减轻病变皮肤的纤维化程度。
实施例6：DBH抑制剂对牛IRBP R16诱导的Lewis大鼠实验性自身免疫性葡萄膜视网膜炎(EAU)的影响
1.材料和设备
表15.试剂
Figure PCTCN2022073849-appb-000014
表16.设备
设备 厂家 型号
组织处理仪 Fisher/Thermo A78400006
组织包埋机 Fisher/Thermo B64100010
2.药液的配制及保存
待测物-内匹司他药液：以10mg/mL内匹司他药液的配制为例：称取40mg内匹司他至棕色样品瓶中，添加0.8mL PEG400，在旋涡仪上旋涡，超声15分钟，于40℃的水浴中加热15分钟，形成悬浊液，然后添加0.4mL 30％Solutol HS15，旋涡悬浊液，然后添加2.8mL生理盐水，完全旋涡至溶液。每天现配现用。
对照品-地塞米松药液：将地塞米松悬浮在质量浓度为0.5％的羧甲基纤维素钠中，浓度为0.04mg/mL。每天现配现用。
IRBP R16溶液：将牛IRBP R16多肽溶解于生理盐水中至终浓度为300μg/mL。
完全弗氏佐剂(CFA)：将15mg结核分枝杆菌H37Ra与10mL CFA混合，最终浓度为2.5mg/mL(CFA本身含1.5mg/mL的H37Ra，10mL CFA中加入15mg的H37Ra，终浓度为2.5mg/mL)。
乳化液制备：乳化液是用手工混合的方法制备的。首先，取2个10mL的注射器，其中一个注射器吸入4mL的300μg/mL IRBP R16溶液并连接到三通旋塞阀上，确保所有气泡均已清除。然后连接一个含有4mL CFA的注射器并快速开始混合。通过来回推柱塞手动混合5分钟。最后使用另一个10mL的注射器吸入8mL乳化液，然后连接一个大口径针头(例如18g)，针头插入1毫升注射器的端部，将乳化液分装入10个1mL的注射器中，同时当针头套上注射器后可来回推动柱塞，并确保没有气泡。乳化液在配制好后3小时以内使用。
3.试验方法
测试大鼠为购自北京维通利华实验动物技术有限公司的6至8周龄雌性Lewis大鼠，约180至220g，实验开始时无携带特定病原体。在室温(20至26℃，相对湿度40至70％)的房间里(每个笼子2至4只)进行12/12小时的明暗循环。所有实验方案均得到澎立生物IACUC(实验动物管理委员会)的批准。实验前，测试大鼠在澎立生物实验室中适应7天。
(1)IRBP R16乳化液免疫大鼠
在第-1天(即免疫接种的前一天，下同)，将60只大鼠根据体重随机分为6组(n＝10)(见表18)。第0天，第2至6组大鼠用异氟烷麻醉，然后皮下注射共200mL的IRBP R16与CFA按1:1v/v比例乳化所得的乳化液，在两条大腿(每个部位50μL)和尾部根部(100μL)分别进行。
其中第0天是指免疫接种的当天。每只大鼠的体重在免疫后每周监测两次。
(2)大鼠的EAU临床评价
从免疫后第0天开始，测试大鼠的眼睛的上下眼睑被轻轻地拨开，每天用手 电筒检查大鼠的眼睛，记录疾病的发生率。根据表17中列出的标准，从疾病开始到研究结束，以盲法对临床症状进行评分。
表17.大鼠EAU临床评分
评分 标准
0 无疾病；眼睛半透明并反射光线(红色反射)
0.5(痕量) 虹膜血管扩张
1 虹膜血管充盈；瞳孔收缩异常
2 模糊性前房；红色反射减弱
3 中度不透明前房，但瞳孔仍可见；暗红色反射
4 前房混浊，瞳孔模糊；红色反射缺失；眼球突出
(3)给药方案
从第0天开始给药，按动物体重精确给药，一共16天。测试大鼠的给药方案见表18。
表18.给药方案
Figure PCTCN2022073849-appb-000015
a：20％PEG400+10％(30％Solutol HS15)+70％生理盐水
b：0.5％羧甲基纤维素钠。
(4)实验结果的统计分析
结果以“均值±标准误差”表示。使用Graphpad Prism或SPSS进行统计分析，p<0.05被认为具有统计学意义。
4.试验结果
(1)大鼠体重
试验大鼠的体重变化具体如下表19所示，分别以均值和标准误差示出。
表19.大鼠体重(g)
均值 第-1天 第2天 第6天 第9天 第13天 第16天
空白对照 195.89 198.20 209.00 215.42 216.80 221.80
模型对照 186.99 192.94 200.05 204.24 209.85 210.04
地塞米松 188.46 174.65 166.65 164.05 162.10 161.29
内匹司他(25mpk) 188.31 193.95 204.67 207.81 214.28 219.00
内匹司他(50mpk) 189.34 194.04 202.28 206.07 216.20 218.42
内匹司他(100mpk) 189.92 194.02 205.97 214.36 223.98 227.40
标准误差 第-1天 第2天 第6天 第9天 第13天 第16天
空白对照 2.77 2.32 1.97 2.84 2.62 3.28
模型对照 2.49 1.87 2.47 2.33 2.98 2.84
地塞米松 2.41 1.66 2.19 2.29 3.13 3.27
内匹司他(25mpk) 2.55 2.13 2.94 2.31 2.50 3.23
内匹司他(50mpk) 2.61 2.21 2.14 3.36 3.39 2.81
内匹司他(100mpk) 2.72 3.82 3.38 3.85 3.28 3.86
(2)重量变化率
表20.重量变化率(％)
均值 第-1天 第2天 第6天 第9天 第13天 第16天
空白对照 0.00％ 3.47％ 7.84％ 9.71％ 11.59％ 13.89％
模型对照 0.00％ 3.25％ 7.06％ 9.26％ 12.23％ 12.36％
地塞米松 0.00％ -7.26％ -11.53％ -12.92％ -13.99％ -14.44％
内匹司他(25mpk) 0.00％ 3.25％ 7.06％ 9.26％ 12.23％ 12.36％
内匹司他(50mpk) 0.00％ 2.53％ 6.90％ 8.83％ 14.24％ 15.41％
内匹司他(100mpk) 0.00％ 2.11％ 8.45％ 12.84％ 17.96％ 19.77％
标准误差 第-1天 第2天 第6天 第9天 第13天 第16天
空白对照 0.00％ 0.61％ 0.88％ 0.78％ 0.76％ 0.81％
模型对照 0.00％ 0.72％ 1.24％ 0.60％ 0.75％ 0.95％
地塞米松 0.00％ 0.92％ 0.91％ 0.91％ 1.28％ 1.18％
内匹司他(25mpk) 0.00％ 0.72％ 0.84％ 0.87％ 1.20％ 1.06％
内匹司他(50mpk) 0.00％ 0.61％ 0.81％ 0.87％ 1.48％ 1.05％
内匹司他(100mpk) 0.00％ 0.97％ 0.88％ 0.84％ 0.89％ 1.46％
(3)临床评分
参见图19和表21。
表21.临床评分的曲线下面积AUC
Figure PCTCN2022073849-appb-000016
a：抑制率＝[AUC(模型对照)-AUC(测试组)]/AUC(模型对照)，测试组包括地塞米松组和内匹司他组。
(4)发病率
表22.发病率(％)
Figure PCTCN2022073849-appb-000017
通过EAU临床评分的增加可证明大鼠葡萄膜炎模型造模成功。内匹司他的治疗可轻度改善葡萄膜炎的症状，降低了EAU临床评分和临床评分AUC，但这种效果没有达到统计学意义。
内匹司他在25、50和100mg/kg剂量下对临床评分AUC的抑制率分别为8.67％、20.06％、24.09％。作为阳性对照，通过地塞米松治疗，葡萄膜炎的临床评分和临床评分AUC显著降低，对临床评分AUC的抑制率为84.53％。
实施例7：DBH抑制剂对NMO-IgG诱导的小鼠视神经脊髓炎(NMO)的影响
1.材料和设备
表23.试剂
Figure PCTCN2022073849-appb-000018
表24.设备
设备 厂家 型号
RWD脑立体定位仪 瑞沃德生命科技有限公司 68025
Kd Scientific注射泵 Kd Scientific legato130
Hamilton微量进样针 Hamilton Gastight#1702
一次性带线缝合针 宁波医用缝针有限公司 角针3/0单针
2.药液的配制及保存
待测物-内匹司他药液：以5mg/mL内匹司他药液的配制为例：称取50mg内匹司他至棕色样品瓶中，添加1.98mL PEG400，在旋涡仪上旋涡，超声15分钟，于40℃的水浴中加热15分钟，形成悬浊液，然后添加0.99mL 30％Solutol HS15，旋涡2.97mL的悬浊液，然后添加6.93mL生理盐水，完全旋涡至溶液。每天现配现用。
对照品-地塞米松药液：将地塞米松分散于生理盐水中，浓度为0.1mg/mL。每天现配现用。
对照品-丹参酮Ⅱa药液：将丹参酮Ⅱa溶解于含有2％DMSO的PBS中，浓度为0.0736mg/mL。
3.试验方法
测试小鼠为购自北京维通利华实验动物技术有限公司的雌性C57小鼠，20至22g，实验开始时无携带特定病原体。
(1)造模步骤
1)5％水合氯醛腹腔注射麻醉C57雌性小鼠(6至8周)(70μL/10g)。
2)头顶剪毛漏出头皮，将小鼠头部固定于脑立体定位仪，使其头顶颅骨保持大致水平。碘伏消毒。
3)纵向剪开头皮约0.5cm，寻找前囟和后囟区域。旋转立体定位仪卡尺，将微量进样针针尖移动至前囟及后囟区域，通过观察针尖距前后囟点高度使老鼠颅骨保持水平。调至水平后，将针尖移到前囟点，然后向右移2mm，进行定位。
4)在定位后的小鼠颅骨表面进行标记，用颅骨钻在该点进行钻孔。钻孔过程中，注意避开血管，小心深入，避免损伤硬膜以及脑组织。
5)用立体定位仪垂直将微量进样针插入小鼠脑组织中，垂直进针深度3mm，使针保持原位，停针等待5min。
6)开启注射泵，选择Infuse Only模式，速度为1μL/min，进行注射(2μL NMO-IgG+3μL人补体+5μL PBS/药品，提前混合吹匀，置于冰上)。
7)注射完成后，停针等待10min。旋转卡尺缓慢向上拔针，向上1mm后停针5min，直至进样针全部拔出，缝合皮肤，用碘伏再次消毒。
(2)给药方案
在造模前3天开始给药，造模后给药7天，连续给药10天。具体给药方案如 下表25所示。
表25.给药方案
Figure PCTCN2022073849-appb-000019
(3)样品采集
在模型第8天(即最后一次给药的后一天)，安乐死处死实验小鼠，取脑部组织作冰冻切片。然后测定各组小鼠星形胶质细胞标志物的免疫反应面积并评价(实验方法参见文献：Ye Gong等，Journal of Neuroinflammation,17(1).doi:10.1186/s12974-020-01874-6)。
4.试验结果
图20A至图20D显示，从AQP4和GFAP的病灶比来看，与对照组相比，内匹司他组可显著减少病灶比，效果低于阳性药组。但对于免疫参数CD45和IBA1无显著影响。因此，内匹司他对NMO-IgG诱导的小鼠视神经脊髓炎具有一定的抑制作用。
实施例8：DBH抑制剂对咪喹莫特(IMQ)诱导的银屑病的治疗作用
1.材料和设备
表26.试剂
试剂 供应商 货号
5％咪喹莫特乳膏 Aldara,3M Pharmaceuticals /
地塞米松乳膏 三军医药有限公司 H44024170
枸橼酸托法替尼 大连美仑生物科技有限公司 MB3358
PEG400 西格玛 202398
Solutol HS15 西格玛 42966
EtOH 上海阿拉丁生化科技有限公司 A500737-0500
Cremophor EL Sigma C5135
Poloxamer 188 Sigma 9003-11-6
表27.设备
设备 型号
电子天平 MFC：9092250
分析天平 SECURA225D-1CN+YDP20-0CEV1
离心机 Eppendorf_5424
表28.试验动物
动物种类和品系： Balb/c小鼠
饲养者/供应商： 北京维通利华实验动物技术有限公司
性别、年龄： 雌性，7-8周
实验机构： 药明康德医药科技有限公司(南通)
适应期： 7天
房间： SPF级
室温： 20-26℃
相对湿度： 40-70％
光照周期： 12/12小时光照/黑暗交替
饲养密度： 5只/笼
食物和水： 自由获取食物和水
本方案中进行的所有实验均经药明康德(Wuxiapptec)机构动物保护和使用委员会IACUC的批准。
2.药液的配制和保存
待测物-内匹司他口服用药溶液：制备9.9mL浓度为5mg/mL的内匹司他溶液用于口服给药：称取50mg内匹司他，加入1.98mL PEG400，超声15分钟，40℃水浴加热15分钟，旋涡至悬浮液。向1.98mL内匹司他悬浮液中添加0.99mL30％Solutol HS15并旋涡至悬浮液。将6.93mL生理盐水加入2.97mL内匹司他悬浮液中，并旋涡到溶液。
待测物-内匹司他局部用药溶液：制备1mL内匹司他(50mg/mL)外用：称取50.13mg内匹司他，加入100μL PG，在45℃下搅拌10分钟，得到均匀的不透明悬浮液；再添加200μL乙醇，并在45℃下搅拌5分钟，以获得均匀的不透明悬浮液；然后再加入200μLCremophor EL，在45℃下搅拌5分钟，以获得均匀的不透明悬浮液。再向悬浮液中加入500μL 5％Poloxamer 188，在45℃下搅拌30分钟，得到澄清溶液。
对照品-枸橼酸托法替尼溶液：将枸橼酸托法替尼溶于二甲基亚砜中得浓度为5mg/mL的溶液，在IMQ软膏中加入50μL上述溶液用于局部应用。
3.试验方法
(1)剃须毛发
在实验开始前一天，所有老鼠的背部都用宠物理发器剃毛，面积为2×3厘米。
(2)分组
将65只动物按体重随机分组，给药方案如下。
表29.分组和给药方案
Figure PCTCN2022073849-appb-000020
溶媒A：20％PEG400+10％(30％Solutol HS15)+70％生理盐水；
溶媒B：10％PG/20％EtOH/20％Cremophor EL/50％(5％Poloxamer 188水溶液)。
(3)IMQ和阳性对照制剂
咪喹莫特(IMQ)乳膏：从G2到G7，每只小鼠对应剂量为62.5mg IMQ乳膏；
地塞米松(DEX)软膏：G4中每只小鼠对应剂量为70mg药膏；
托法替尼：将托法替尼溶解在二甲基亚砜中至5mg/mL，加入50μL到IMQ乳膏中进行日常治疗。
(4)IMQ致敏和给药
从第0天到第7天，第2组到第7组的小鼠在剃光的背部接受每日局部剂量62.5mg的IMQ乳膏。
试验化合物和对照品的给药按表29进行，连续7天。
(5)疾病评估
为了评估背部皮肤炎症的严重程度，对背部皮肤按表30进行评分。红斑、鳞屑和增厚在0到3范围内独立评分，0、无；1、轻度；2、中度；3、明显/严重。
表30.临床评分参数
Figure PCTCN2022073849-appb-000021
(6)试验终止与取样
实验终止时，通过CO 2吸入安乐死小鼠：
1)通过心脏穿刺采集血液，并对血浆进行处理，各3份，1份在保护溶液中，2份快速冷冻；
2)每组收取5个背部皮肤样品放入保护液中；5个背部皮肤样品放入PFA中；5个背部皮肤样品快速冷冻；
3)收集淋巴结，快速冷冻；
4)收集脾脏样品并称重(图23)。
(7)数据统计
使用Graph Pad Prism6.0软件，通过单因素方差分析对数据进行统计分析。
4.研究结果
(1)体重
在整个研究过程中，每天监测体重。连续使用IMQ乳膏可降低溶媒组的平均体重。除此之外，地塞米松治疗组(G4)的体重下降更为明显。但是治疗组和溶媒组之间没有统计学显著差异(图21)。
(2)临床评分
每天对背部皮肤的红斑、鳞屑和厚度进行评分。此外，三者相加(红斑+厚度+鳞屑)表示为总分。地塞米松治疗组对炎症有明显的抑制作用，提示IMQ乳膏诱导的银屑病模型是成功的。
内匹司他口服组从第5天到第7天抑制红斑和厚度。内匹司他局部组从第4天到第7天抑制红斑。托法替尼在剂量加倍后，显示出疗效。内匹司他治疗不影响疾病的发展和发病率，但在病程后期(第5天至第7天)显示出疗效。根据各组动物的总临床评分曲线，计算曲线下面积(AUC)(图22)。
按以下公式计算抑制率：抑制率＝[AUC(溶媒)-AUC(治疗)]/AUC(溶媒)×100％。其中：溶媒是指对应给药方式的溶媒组。
地塞米松治疗组抑制率为96.96％。内匹司他经口服治疗的50mpk组对皮肤临床评分有显著抑制作用，抑制率为23.64％。然而，内匹司他经局部治疗的100mpk组无明显疗效。
表31.红斑评分结果
Figure PCTCN2022073849-appb-000022
Figure PCTCN2022073849-appb-000023
表32.鳞屑评分结果
Figure PCTCN2022073849-appb-000024
表33.厚度评分结果
Figure PCTCN2022073849-appb-000025
表34.红斑+鳞屑+厚度总评分结果
Figure PCTCN2022073849-appb-000026
Figure PCTCN2022073849-appb-000027
5.本实施例的目的是研究试验化合物对IMQ诱导的银屑病模型的疗效。结果表明，应用IMQ会导致严重的皮肤炎症。与溶媒组对照组相比，内匹司他的PO治疗组对临床症状的治疗效果轻微。
实施例9：DBH抑制剂对胶原诱导的小鼠关节炎(CIA)中的治疗作用
1.实验材料
牛二型胶原，CII，四川大学；
乙酸，Sigma(St.Louis，MO，USA)，货号：A8976；
完全弗氏佐剂，Sigma，货号：F5881；
20％PEG400+10％(30％Solutol HS15)+70％生理盐水；
托法替尼，>98％，大连美仑生物技术有限公司MB3358。
2.受试化合物
表35
Figure PCTCN2022073849-appb-000028
称取40mg待测物至棕色样品瓶中，加入0.8ml PEG400，于漩涡仪上涡旋，超声15分钟，40℃水浴加热15分钟，成悬浊液；然后在加入0.4ml 30％Solutol HS15，于漩涡仪上涡旋混匀；然后再加入2.8ml生理盐水，于漩涡仪上涡旋混匀，至成溶液；化合物浓度为10mg/ml。每三天配置一次，保存于4℃备用。
3.实验仪器
麻醉机：Raymain，RM-HSIV-u；
高速匀浆机：IKA，T10basic；
化合物称量天平：Sartorius，CPA225D；
动物称量天平：常州天之平电子天平，YH2000。
4.实验动物及饲养环境
动物种系:DBA/1小鼠
供应商:北京维通利华实验动物技术有限公司
性别体重：雄性，14-20g，无特定病原体(SPF)
适应期：3-7天
环境：SPF动物饲养室，药明康德上海外高桥动物饲养中心
温度：20-26℃
湿度：40-70％
光照：荧光灯，明(08:00-20:00)、暗(20:00-08:00)各12小时
饲养密度：5只/笼
食物：自由进食(辐射灭菌饲料)
饮水：自由饮水(摩尔(超)纯水器制备)。
本实施例报告描述的动物操作通过了药明康德公司实验动物使用及管理(伦理)委员会(IACUC)的审核及批准。
5.实验方法
1)二型胶原/完全弗氏佐剂免疫
乙酸的配制：稀释2N的乙酸至100mM，用0.22微米滤膜过滤后，4℃保存。
牛二型胶原溶液：将牛二型胶原(CII)溶解于100mM的乙酸溶液中，并置于4℃过夜保存。胶原蛋白的终浓度为8mg/mL。
乳剂的制备：将过夜保存的CII溶液与等体积的完全弗氏佐剂混合，使用高速匀浆机，在冰上以30000转每分钟匀浆大约60分钟，直至溶液形成稳定的乳剂。
2)关节炎的诱导
DBA/1小鼠经异氟烷麻醉后，在尾部皮下注射50微升的制备好的胶原乳剂(包含200微克CII)。第一次免疫当天记为第0天，随后的天数依序标注。第21天，尾部注射相同体积胶原乳剂。
3)给药和剂量设计
当模型小鼠出现临床症状，平均分约为0.5(第28天左右)，按照体重和评分重新随机分组到5个实验组，每组8只小鼠。连续给药14天。化合物每3天配置一次，4℃保存。
表36.分组及剂量设计
Figure PCTCN2022073849-appb-000029
4)关节炎发病指标测定
从第28天后，一周三次对小鼠进行称重并记录临床评分，直至实验结束。
临床评分：根据病变的不同程度(红肿，关节变形)按照0-4分的标准进行评分，每个肢体的最高评分为4分，每只动物最高评分为16分。
表37.关节炎临床评分标准
Figure PCTCN2022073849-appb-000030
5)关节炎发病指标测定
a.收集小鼠后爪，固定于PFA，用于H&E染色和病理评分；
b.收集小鼠脾脏，进行FACS染色。
6)统计学处理
实验数据用平均数±标准误表示，体重和临床评分用双因素方差分析(Two-way ANOVA)，p<0.05认为有显著性差异。
6.实验结果
参见图24至图27，内匹司他对胶原诱导的小鼠关节炎有一定的已知作用，但不如阳性对照药明显。
实施例10：DBH抑制剂对2,4-二硝基苯磺酸(DNBS)诱导大鼠肠炎的治疗作用
1.材料和设备
表38.试剂
Figure PCTCN2022073849-appb-000031
表39.仪器
仪器 供应商 型号
灌肠软管 法国Vygon ACL 7157348
2.药液的配制及保存
待测物-内匹司他药液：称取12mg内匹司他至棕色样品瓶，加入0.8mL聚乙二醇400，涡旋振荡并超声15分钟，40℃水浴加热15分钟，形成混悬液，然后加入0.4mL 30％聚乙二醇-15羟基硬脂酸酯，涡旋，然后加入2.8mL生理盐水，涡旋使其完全溶解。
对照品-泼尼松药液：泼尼松的使用浓度为0.9mg/mL，使用0.5％羧甲基纤维素钠制备成混悬液，一周配制两次。
DNBS药液的制备：将DNBS粉末用30％乙醇溶解，终浓度为50mg/mL。
3.大鼠的处理
动物种属及品系：Wistar大鼠；
给药记录：无给药史；
性别、年龄、体重：雄性，5至6周，160至180g；
养殖方/供应商：上海斯莱克实验动物有限公司；
实验机构：澎立生物动物房；
适应期：7天；
房间：普通区房间；
室内温度：20至26C；
室内相对湿度：40至70％；
灯光：日光灯照明，12小时照明及12小时无照明；
动物饲养：每笼2至4只大鼠(同一给药组)；
食物：自由获取饲料(经辐照消毒，江苏省协同医药生物工程有限公司，中国)；
水：自由获取饮用水(经反渗透处理或高压灭菌)。
从上海斯莱克实验动物有限公司购入共计90只雄性Wistar大鼠，动物均无携带特定病原，到达澎立生物动物房时约为4至5周(140至150g)。
动物到达澎立生物后，动物房人员将动物从运输包装中转移至鼠笼并对每只动物进行检查。检查范围包括外观、四肢和孔腔等，以及动物静止或运动时是否有异常表现。适应期为7天。
设计应用于动物的实验操作方案由澎立生物IACUC(实验动物管理和使用委员会)进行审批。
根据动物体重，在第-1天(即正式实验前的一天)，将90只动物随机分组，确保每组动物的体重值相似，减少偏差。实验前大鼠禁食40小时，并且在禁食期间给大鼠皮下注射5％葡萄糖盐水(10mL/kg，每天一次)。
实验分组情况：
实验第1天，腹腔注射舒泰(25mg/kg替来他明和25mg/kg唑拉西泮)和5 mg/kg甲苯噻嗪麻醉禁食大鼠。
G2组-G6组，将软管从肛门伸入到结肠左曲(约肛门8cm处)，用DNBS灌肠(0.5mL/只)诱导大鼠结肠炎。正常对照组(G1)以同样的方法用30％乙醇灌肠。灌肠的动物使其头下低15min，然后保持特伦德伦伯卧位姿势直到动物苏醒，以避免灌肠液回流。
表40.分组及给药方案
Figure PCTCN2022073849-appb-000032
4.检测指标
(1)动物体重：每天测量记录动物的体重，并对动物的日常活动进行观察，记录异常情况。体重百分比按照以下公式计算：(第x天体重-起始体重)/起始体重]×100％。
(2)粪便评分：实验过程中，每天对大鼠粪便状态进行评分(0＝正常，1＝潮湿/粘，2＝松软，3＝液体)。
(3)结肠观察：实验结束后，所有动物用舒泰(静脉注射，25mg/kg)麻醉，打开腹腔，腹主动脉采血，EDTA抗凝(离心条件：4℃，2000g，10min)动物放血处死后，取出结肠(从盲肠到肛门)，立即测量结肠长度。将结肠纵向刨开，冲洗干净后记录结肠重量、溃疡面积并整体拍照，将结肠分为三部分，两部分液氮速冻后-80℃保存，用于MPO和细胞因子检测。另一部分结肠用10％中性福尔马林固定。如果溃疡为不规则的形状,它可以被认为是矩形，然后其测量长度和宽度进行溃疡面积评估。溃疡面积(cm 2)＝溃疡长度(cm)×溃疡宽度(cm)。
(4)结肠组织进行病理分析
将中性福尔马林固定结肠组织的近端、溃疡(如无溃疡，取近似部位)和远端部分用石蜡包埋，进行切片(厚度5μm)，用H&E染色进行组织病理学评分。
表41
Figure PCTCN2022073849-appb-000033
5.统计分析
实验数据用均数±标准误表示。数据由Graphpad Prism采用相应的统计方法分析。p<0.05认为有显著性差异。
6.实验结果
(1)体重和粪便评分参见图28至图31。
(2)第7天结肠宏观评价
表42.第7天的结肠宏观评价
Figure PCTCN2022073849-appb-000034
表43.第7天的结肠宏观评价
Figure PCTCN2022073849-appb-000035
Figure PCTCN2022073849-appb-000036
BW：体重；
CL：结肠长度；
CW：结肠重量；
a：IR1(抑制率1)＝{[CW/CL/BW(模型组)-CW/CL/BW(测试药组)]/[CW/CL/BW(模型组)-CW/CL/BW(正常组)]}×100％；
b：IR2(抑制率2)＝{[CW/BW(模型组)-CW/BW(测试药组)]/[CW/BW(模型)-CW/BW(正常组)]}×100％；
c：IR3(抑制率3)＝{[CW/CL(模型组)-CW/CL(测试药组)]/[CW/CL(模型组)-CW/CL(正常组)]}×100％。
(3)病理评分
表44.病理评分
Figure PCTCN2022073849-appb-000037
7.结论
Wistar大鼠用DNBS结肠内灌注诱导其产生炎症性结肠炎。结肠炎表现为动物体重显著下降，粪便成形性评分显著增加，结肠长度显著缩短，结肠重量、结 肠重量：结肠长度(即CW/CL)、结肠重量：体重比率(即CW/BW)和结肠重量：结肠长度：体重比率(即CW/CL/BW)显著增加，结肠溃疡面积显著增大，结肠炎性细胞浸润评分和组织损伤评分显著增加。
在本实施例中，阳性对照药泼尼松能够显著降低模型大鼠粪便成形性评分AUC、肠重、CW/CL、CW/BW、CW/CL/BW和结肠溃疡端炎性细胞浸润评分，其中CW/CL/BW、CW/BW、CW/CL的抑制率分别达44.20％、50.13％和46.51％。
检测组1#(3mg/kg)对CW/CL/BW、CW/BW和CW/CL的抑制率分别为32.98％、40.24％和30.82％。
检测组2#(内匹司他10mg/kg可显著降低CW/CL、CW/CL/BW和结肠组织损伤总评分，CW/CL/BW、CW/BW和CW/CL的抑制率分别为44.95％、47.06％和43.80％。
检测组3#(30mg/kg)对CW/CL/BW、CW/BW和CW/CL的抑制率分别为27.12％、39.85％和26.68％。
综上所述，内匹司他对DNBS诱导大鼠结肠炎有一定的防治作用。
实施例11：DBH抑制剂对硫酸葡聚糖钠(DSS)诱导的结肠炎的抑制作用
1.实验材料
表45.试剂
Figure PCTCN2022073849-appb-000038
表46.待测物
Figure PCTCN2022073849-appb-000039
实验动物：品系C57BL/6小鼠；来源：浙江维通利华实验动物技术有限公司；性别：雌。主要仪器：电子天平：赛多利斯，QUINTIX35-1CN。
2.实验方法
2.1化合物的配制与贮存
称取适量环孢菌素A(CsA)粉末放置于棕色样品瓶中，依次加入适量比例的2％DMSO、30％PEG300、5％Tween 80和63％Saline，振荡溶解，每天配制一次。
称取适量内匹司他至棕色样品瓶中，依次加入适量一定比例的20％PEG400、10％(30％Solutol HS15)和70％生理盐水混匀，超声溶解，每3天一配，以维持其稳定性。
称取适量的镰孢菌酸放置于棕色样品瓶中，加入适量生理盐水，超声溶解，每七天配置一次。
称取适量的双硫仑放置于棕色样品瓶中，依次加入适量一定比例的50％PEG300和50％生理盐水，超声溶解成悬浮液，给药前应先混匀，每七天配置一次。
称取适量的富马酸放置于棕色样品瓶中，加入适量的生理盐水，水浴加热溶解，每天配置一次。
表47.给药体积和药物贮存浓度
Figure PCTCN2022073849-appb-000040
2.2动物饲养
70只，8周龄，20g左右，雌性C57BL/6小鼠，饲养于单独通风笼子(IVC，每个笼子5只)，温度控制(20±2℃)，光照/黑暗交替12/12小时。开展实验之前，小鼠需在SPF级动物房适应性喂养三天，期间，保证充足的水跟食物。
2.3分组与给药方案
实验小鼠在SPF级动物房适应性喂养三天，随机分成7组(8个模型组和1个对照组)每组10只。小鼠给药从第0天开始，第7天结束。
表48.动物分组及给药方案
Figure PCTCN2022073849-appb-000041
2.4模型的建立
本实施例小鼠肠炎模型采用DSS诱导建模：模型组小鼠从第0天到第7天饮用3.1％DSS(硫酸葡聚糖纳，分子量36000-50000)水溶液，DSS水溶液每天现配更换，防止降解。正常对照组小鼠饮用正常水。实验周期为9天，实验终点将小鼠安乐死并进行收样。
2.5疾病活动度(DAI)评分
每天进行DAI评分，DAI评分由3部分组成：体重变化、粪便性状和便血(或隐血)评分，具体的评分标准如表49所示。整个实验过程中，所有小鼠的DAI评分由同一人完成。
表49.DAI评分标准
评分 体重降低百分比 粪便性状 便血或隐血
0 0 正常 隐血阴性
1 1至5％ 软便 隐血弱阳性
2 6至10％ 松散便 隐血阳性
3 11至20％ 稀便 少量血迹
4 >20％ 水样便 大量血迹
2.6结肠组织的采集
实验终点安乐死小鼠，剖取小鼠结肠进行拍照，测量长度，去除内容物后进行称重。
2.7统计分析
数据以均值±标准误
Figure PCTCN2022073849-appb-000042
表示，体重变化与疾病活动度评分采用双因素方法分析(two-way ANOVA)方法进行统计学分析并应用Dunnett’s进行组间比较，其余数据采用单因素方法分析(one-way ANOVA)方法进行统计学分析并应用Dunnett’s进行组间比较。所有分析使用GraphPad Prism软件进行。*P<0.05，**P<0.01，***P<0.005，****P<0.0001vs.溶媒组。
3.实验结果与分析
收集实验期间各组小鼠体重数据并进行相应的DAI评分，数据的分析采用Two-way ANOVA的方法。
如图32所示，与溶媒组相比，阳性对照CsA(50mg/kg，QD)和内匹司他(50mg/kg，QD)有效缓解了DSS诱导的小鼠体重下降。相比于溶媒组，镰孢菌酸(100mg/kg，bid)和双硫仑(100mg/kg，qd)未能得到有效缓解DSS诱导的小鼠体重下降。富马酸(100mg/kg，qd)有缓解DSS诱导的小鼠体重下降的趋势。
如图33所示，阳性对照CsA(50mg/kg，QD)组与内匹司他(50mg/kg，QD)小鼠的疾病活动度评分(DAI)显著低于溶媒组。在给药期间，相比于溶媒组组，镰孢菌酸(100mg/kg，bid)组、双硫仑(100mg/kg，qd)和富马酸(100mg/kg， qd)组小鼠的DAI能得到轻微的改善，其中第4天，第5天表现较为明显，但效果低于内匹司他。
综合体重变化和DAI评分可知，阳性对照CsA(50mg/kg，QD)和内匹司他(50mg/kg，QD)有效缓解DSS诱导结肠炎造成的小鼠体重降低及拉稀和便血的症状。镰孢菌酸(100mg/kg，bid)组、双硫仑(100mg/kg，qd)和富马酸(100mg/kg，qd)能轻微改善小鼠的DSS诱导结肠炎造成的小鼠拉稀和便血的症状，但未能有效缓解DSS诱导结肠炎造成的小鼠体重降低。
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Claims (17)

  1. 通路调节剂在制备治疗自身免疫性疾病的药物中的用途；其中，
    所述通路调节剂为多巴胺β-羟化酶抑制剂、受体激动剂和受体拮抗剂中的一种或多种。
  2. 根据权利要求1所述的用途，其中，
    所述通路调节剂为多巴胺β-羟化酶抑制剂，所述多巴胺β-羟化酶抑制剂为内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、半胱胺、泛硫乙胺、铜螯合剂、富马酸、肼苯哒嗪、2-噻吩-2-基烯丙基胺、其药学上可接受的盐和其前药中的一种或多种；
    优选地，所述多巴胺β-羟化酶抑制剂选自：内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、富马酸、其药学上可接受的盐和其前药。
  3. 根据权利要求1所述的用途，其中，
    所述通路调节剂为内匹司他或其药学上可接受的盐，单位剂量为：10-100mg/kg、优选20-50mg/kg；或者，
    所述通路调节剂为依他米司他或其药学上可接受的盐，单位剂量为80-120mg/kg，优选90-110mg/kg，更优选100mg/kg；或者，
    所述通路调节剂为依米司他或其药学上可接受的盐，单位剂量为80-120mg/kg，优选90-110mg/kg，更优选100mg/kg；或者
    所述通路调节剂为镰孢菌酸或其药学上可接受的盐，单位剂量为80-120mg/kg，优选90-110mg/kg，更优选100mg/kg；或者，
    所述通路调节剂为双硫仑或其药学上可接受的盐，单位剂量为80-120mg/kg，优选90-110mg/kg，更优选100mg/kg；或者，
    所述通路调节剂为富马酸或其药学上可接受的盐，单位剂量为80-120mg/kg，优选90-110mg/kg，更优选100mg/kg。
  4. 根据权利要求1-3中任一项所述的用途，其中，
    所述自身免疫性疾病选自：贲门失弛缓症、艾迪生氏病、成人斯蒂尔氏病、血球蛋白血症、斑秃、淀粉样变性、强直性脊柱炎、抗GBM/抗TBM肾炎、抗磷脂综合征、自身免疫性血管性水肿、自身免疫性自主神经异常、自身免疫性脑脊髓炎、自身免疫性肝炎、自身免疫性内耳疾病、自身免疫性心肌炎、自身免疫性卵巢炎、自身免疫性睾丸炎、自身免疫性胰腺炎、自身免疫性视网膜病变、自身免疫性荨麻疹、轴突神经病、巴洛病、白塞氏病、良性粘膜类天疱疮、大疱性类天疱疮、卡斯尔曼病、麸质过敏症、恰加斯病、慢性炎性脱髓鞘性多发性神经病、慢性复发性多灶性骨髓炎、嗜酸性肉芽肿、瘢痕性类天疱疮、科根综合症、冷凝 集素病、先天性心脏传导阻滞、柯萨奇心肌炎、肢端硬皮综合征、克罗恩病、疱疹样皮炎、皮肌炎、视神经脊髓炎、盘状狼疮、德莱勒综合症、子宫内膜异位症、嗜酸性食管炎、嗜酸性筋膜炎、结节性红斑、基本混合性冷球蛋白血症、埃文斯综合症、纤维肌痛、纤维化性肺泡炎、巨细胞动脉炎、巨细胞心肌炎、肾小球肾炎、肺出血肾炎综合征、肉芽肿合并多血管炎、甲状腺机能亢进、格林巴利综合征、桥本甲状腺炎、溶血性贫血、过敏性紫癜、妊娠期疱疹或类天疱疮妊娠、化脓性汗腺炎、低丙球蛋白血症、IgA肾病、IgG4相关性硬化症、免疫性血小板减少性紫癜、包涵体肌炎、间质性膀胱炎、青少年关节炎、青少年I型糖尿病、青少年肌炎、川崎病、朗伯-伊顿综合征、白细胞碎裂性血管炎、扁平苔藓、萎缩性硬化性苔藓、木样结膜炎、线性IgA病、红斑狼疮、慢性莱姆病、梅尼埃病、显微镜下多血管炎、混合性结缔组织病、蚕蚀性角膜溃疡、穆-哈二氏病、多灶性运动神经病、多发性硬化、重症肌无力、肌炎、嗜睡症、新生儿狼疮、中性粒细胞减少症、眼瘢痕性类天疱疮、视神经炎、回文风湿病、潘达斯症、副肿瘤性小脑变性、阵发性夜间血红蛋白尿、帕里伯格综合症、睫状体平坦部炎、帕森纳-特纳综合征、天疱疮、周围神经病变、静脉周脑脊髓炎、恶性贫血、POEMS综合征、结节性多动脉炎、I型多腺综合征、II型多腺综合征、III型多腺综合征、风湿性多肌痛、多肌炎、心肌梗塞后综合征、心包切开术后综合征、原发性胆汁性肝硬化、原发性硬化性胆管炎、孕激素性皮炎、银屑病、银屑病关节炎、纯红细胞再生障碍性贫血、坏疽性脓皮病、雷诺现象、反应性关节炎、反射性交感神经营养不良、复发性多发性软骨炎、不宁腿综合征、腹膜后纤维化、风湿热、类风湿关节炎、结节病、施密特综合征、巩膜炎、硬皮病、干燥综合征、精子和睾丸自身免疫、僵硬者综合征、亚急性细菌性心内膜炎、苏萨克综合症、交感性眼炎、大动脉炎、颞动脉炎/巨细胞动脉炎、血小板减少性紫癜、甲状腺眼病、痛性眼肌麻痹综合征、横向脊髓炎、I型糖尿病、溃疡性结肠炎、未分化结缔组织病、葡萄膜炎、血管炎、白癜风和沃格特-小柳-原田病；
    优选地，选自以下任一项：自身免疫性结肠炎、视神经脊髓炎、类风湿关节炎、硬皮病、银屑病、葡萄膜炎；
    所述自身免疫性结肠炎选自以下任一项：克罗恩病、溃疡性结肠炎。
  5. 一种用于治疗自身免疫性疾病的方法，其包括步骤：
    向受试者施用治疗有效量的通路调节剂；
    其中，所述通路调节剂为多巴胺β-羟化酶抑制剂、受体激动剂和受体拮抗剂中的一种或多种。
  6. 根据权利要求5所述的方法，其中，
    所述通路调节剂为多巴胺β-羟化酶抑制剂，所述多巴胺β-羟化酶抑制剂为内 匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、半胱胺、泛硫乙胺、铜螯合剂、富马酸、肼苯哒嗪、2-噻吩-2-基烯丙基胺、其药学上可接受的盐和其前药中的一种或多种；
    优选地，所述多巴胺β-羟化酶抑制剂选自：内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、富马酸、其药学上可接受的盐和其前药。
  7. 根据权利要求5或6所述的方法，其中，
    所述自身免疫性疾病如权利要求4中所定义。
  8. 一种治疗自身免疫性疾病的药物组合物，其包含：
    通路调节剂、和
    药学上可接受的载体；
    其中，所述通路调节剂为多巴胺β-羟化酶抑制剂、受体激动剂和受体拮抗剂中的一种或多种。
  9. 根据权利要求8所述的药物组合物，其中，
    所述通路调节剂为多巴胺β-羟化酶抑制剂，所述多巴胺β-羟化酶抑制剂为内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、半胱胺、泛硫乙胺、铜螯合剂、富马酸、肼苯哒嗪、2-噻吩-2-基烯丙基胺、其药学上可接受的盐和其前药中的一种或多种；
    优选地，所述多巴胺β-羟化酶抑制剂选自内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、富马酸、其药学上可接受的盐和其前药。
  10. 根据权利要求8所述的药物组合物，其中，所述自身免疫性疾病如权利要求4中所定义。
  11. 根据权利要求1所述的用途，其中所述药物用于选自以下的一种或多种：
    降低CD4+T细胞比例、增加调节性T细胞比例、增加CD8+T细胞比例、降低CD4+T细胞的促炎因子分泌、降低CD8+T细胞的促炎因子分泌、抑制B细胞的活化和抑制NK细胞的活化；减少外周血中淋巴细胞、中性粒细胞和单核细胞的含量；减轻真皮层中炎症性细胞浸润和真皮下的毛细血管增生；改善皮肤纤维化；降低葡萄膜炎的发病率；改善皮肤炎症；改善粪便成形性评分、改善CW/CL、改善CW/BW、改善CW/CL/BW、抑制结肠溃疡面积增加、改善结肠炎性细胞浸润评分、改善组织损伤评分；改善疾病活动度评分、改善便血或隐血。
  12. 根据权利要求11所述的用途，其中：
    所述CD4+T细胞的促炎因子为IL-17A、IFN-γ和TNF-α中的一种或多种；
    所述CD8+T细胞的促炎因子为IL-17A和/或TNF-α。
  13. 根据权利要求11所述的用途，其中，所述调节性T细胞为CD25+FOXP3+Treg细胞；
    和/或，所述B细胞为B220+细胞，优选为CD69+B220+B细胞；
    和/或，所述NK细胞为NK1.1+细胞，优选为NK1.1+CD107a+NK细胞。
  14. 一种在体内或体外调节免疫细胞功能的方法，其包括如下步骤：
    使有效量的通路调节剂在体内或体外接触免疫细胞，
    其中，
    所述免疫细胞来自受试者；
    所述通路调节剂为多巴胺β-羟化酶抑制剂、受体激动剂和受体拮抗剂中的一种或多种；
    所述调节免疫细胞功能指的是选自以下的一种或多种功能：降低CD4+T细胞比例、增加调节性T细胞比例、增加CD8+T细胞比例、降低CD4+T细胞的促炎因子分泌、降低CD8+T细胞的促炎因子分泌、抑制B细胞的活化和抑制NK细胞的活化。
  15. 根据权利要求14所述的方法，其中，所述多巴胺β-羟化酶抑制剂为内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、半胱胺、泛硫乙胺、铜螯合剂、富马酸、肼苯哒嗪、2-噻吩-2-基烯丙基胺、其药学上可接受的盐和其前药中的一种或多种；
    优选地，所述多巴胺β-羟化酶抑制剂选自：内匹司他、依他米司他、依米司他、镰孢菌酸、双硫仑、富马酸、其药学上可接受的盐和其前药。
  16. 根据权利要求14所述的方法，其中：
    所述CD4+T细胞的促炎因子为IL-17A、IFN-γ和TNF-α中的一种或多种；
    所述CD8+T细胞的促炎因子为IL-17A和/或TNF-α。
  17. 根据权利要求14所述的方法，其中，所述调节性T细胞为CD25+FOXP3+Treg细胞；
    和/或，所述B细胞为B220+细胞，优选为CD69+B220+B细胞；
    和/或，所述NK细胞为NK1.1+细胞，优选为NK1.1+CD107a+NK细胞。
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CN116490177A (zh) 2023-07-25
EP4285898A1 (en) 2023-12-06
JP2024503906A (ja) 2024-01-29
AU2022213938A1 (en) 2023-09-07
KR20230137374A (ko) 2023-10-04
MX2023008573A (es) 2023-08-08

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