WO2018171406A1 - 选择性tnfr1拮抗肽在制备预防和治疗类风湿性关节炎的药物中的应用 - Google Patents

选择性tnfr1拮抗肽在制备预防和治疗类风湿性关节炎的药物中的应用 Download PDF

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WO2018171406A1
WO2018171406A1 PCT/CN2018/077862 CN2018077862W WO2018171406A1 WO 2018171406 A1 WO2018171406 A1 WO 2018171406A1 CN 2018077862 W CN2018077862 W CN 2018077862W WO 2018171406 A1 WO2018171406 A1 WO 2018171406A1
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hydrostatin
peg
rheumatoid arthritis
antagonist peptide
preventing
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PCT/CN2018/077862
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English (en)
French (fr)
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陆一鸣
王洁
李安
江海龙
卞莹莹
张川
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桂林八加一药业股份有限公司
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Priority to US16/496,866 priority Critical patent/US20200188471A1/en
Priority to EP18770219.6A priority patent/EP3617223A4/en
Priority to JP2020500945A priority patent/JP6858305B2/ja
Publication of WO2018171406A1 publication Critical patent/WO2018171406A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the field of biomedical technology, in particular to a selective TNFR1 antagonist peptide Hydrostatin-SN10 derived from the snake venom of Qinghai, and its application in rheumatoid arthritis.
  • RA Rheumatoid arthritis
  • RA is a chronic autoimmune disease characterized by joint synovitis.
  • the main clinical manifestations are joint swelling and pain caused by synovial synovium, synovial inflammation and hyperplasia leading to cartilage and bone erosion, joint space narrowing, and joint stiffness, deformity and dysfunction due to severe bone destruction and absorption in the late stage.
  • RA is considered to be one of the top five diseases (cardiovascular disease, Alzheimer's disease, cancer, AIDS, rheumatoid arthritis) that affect human health.
  • RA can occur at any age, with the highest number of 20-50 years old.
  • TNF- ⁇ tumor necrosis factor- ⁇
  • IL-6 interleukin-6
  • IL-17 interleukin-17
  • IL-1 interleukin-1
  • TNF- ⁇ overexpression of TNF- ⁇ in the body and abnormal activation of TNF- ⁇ /TNFRs signaling pathway are closely related to the development of autoimmune diseases such as rheumatoid arthritis.
  • RA rheumatoid arthritis
  • TNF- ⁇ monoclonal antibody compared with biological agents such as TNF- ⁇ monoclonal antibody, the basic and applied research of small molecule antagonists of TNFRs is progressing slowly.
  • TNFR1 mainly transmits pro-inflammatory and apoptotic signals, and blocking the biological function of TNF- ⁇ by selectively blocking the signaling pathway of TNFR1 transmission has become a hot spot in the development of such drugs.
  • Hydrostatin-SN10 binds directly to TNFR1 and does not bind to TNF- ⁇ and TNFR2. It is a target-specific TNFR1 antagonist peptide.
  • Hydrostatin-SN1 is capable of treating diseases associated with TNF- ⁇ , and discloses that the TNF- ⁇ -related disease is rheumatoid arthritis, which is not apparently derived according to the mechanism disclosed in the prior application.
  • the Hydrostatin-SN10 of the present invention can be used for the treatment of rheumatoid arthritis.
  • the object of the present invention is to provide a selective TNFR1 antagonist peptide Hydrostatin-SN10 derived from Qinghai snake venom and its application in rheumatoid arthritis, and another object of the present invention is to provide a mPEG based (monomethoxy) Polyethylene glycol 2000) modified selective TNFR1 antagonist peptide Hydrostatin-SN10, PEG-SN10 in rheumatoid arthritis.
  • the main technical solution of the present invention is to prove that Hydrostatin-SN10 and PEG-SN10 have the use of treating rheumatoid arthritis by establishing an animal model of rheumatoid arthritis (CIA) induced by bovine type II collagen.
  • CIA animal model of rheumatoid arthritis
  • a selective TNFR1 antagonist peptide Hydrostatin-SN10 is provided, the amino acid sequence of the selective TNFR1 antagonist peptide Hydrostatin-SN10 is set forth in SEQ ID NO: 2.
  • the selective TNFR1 antagonist peptide Hydrostatin-SN10 is synthesized by using solid phase synthesis technology to synthesize Hydrostatin-SN10, and its purity and molecular weight are analyzed by HPLC and MS, the molecular weight is 1250.29 Dalton, and the isoelectric point is 4.39. .
  • a gene encoding a selective TNFR1 antagonist peptide Hydrostatin-SN10 is provided, the nucleotide sequence of which is set forth in SEQ ID NO: 1.
  • the medicament for preventing or treating rheumatoid arthritis is a pharmaceutical composition comprising the selective TNFR1 antagonist peptide Hydrostatin-SN10 as the sole active ingredient or the selective TNFR1 antagonist peptide Hydrostatin-SN10.
  • a selective TNFR1 antagonist peptide Hydrostatin-SN10 modified based on mPEG2000 (monomethoxypolyethylene glycol 2000), ie, PEG-SN10, wherein the carboxyl group of mPEG2000 is covalently linked to Hydrostatin - The free amino group of the N-terminal aspartic acid of the SN10 peptide chain.
  • Modification of Hydrostatin-SN10 with mPEG (monomethoxypolyethylene glycol) having an average molecular weight of about 2000 Daltons increases the half-life and stability of Hydrostatin-SN10.
  • the use of the above-described mPEG2000-modified selective TNFR1 antagonist peptide PEG-SN10 for the preparation of a medicament for preventing and treating rheumatoid arthritis is provided.
  • the medicament for preventing and treating rheumatoid arthritis is: a pharmaceutical composition comprising PEG-SN10 as the sole active ingredient or PEG-SN10.
  • the pharmaceutical composition and the pharmaceutically acceptable conventional pharmaceutical excipient are formulated into a pharmaceutical preparation.
  • the pharmaceutical preparation is a tablet, a granule, a dispersing agent, a capsule, a soft capsule, a dropping pill, an injection, a powder injection or an aerosol.
  • the above-mentioned medicament for preventing and treating rheumatoid arthritis selectively antagonizes TNFR1.
  • the invention uses BIAcore (biomacromolecule interaction analysis technology based on surface plasmon resonance technology) and MST (micro thermophoresis) technology to analyze the interaction between Hydrostatin-SN10 and TNF- ⁇ , TNFR1 and TNFR2, and proves that Hydrostatin-SN10 It binds directly to TNFR1 and does not bind to TNF- ⁇ and TNFR2.
  • the present invention employs an animal model of rheumatoid arthritis induced by bovine type II collagen to observe the therapeutic effect of the polypeptide provided by the present invention.
  • Hydrostatatin-SN10 and PEG-SN10 have significant anti-inflammatory effects; can significantly improve joint damage, synovial hyperplasia and inflammatory cell infiltration in CIA mice and can significantly reduce joints
  • the expression level of TNF- ⁇ significantly reduced the number of osteoclasts, and had significant anti-inflammatory effects from the pathological observation of joints;
  • Hydrostatin-SN10 and PEG-SN10 could regulate the imbalance of Th1/Th2 and Th17/Treg ratios, namely Hydrostatin- SN10 and PEG-SN10 can exert anti-rheumatoid arthritis by immunomodulation.
  • the above results indicate that the selective TNFR1 antagonist peptides Hydrostatin-SN10 and PEG-SN10 have good anti-rheumatoid arthritis effects.
  • the present invention finds a drug effective for preventing and treating rheumatoid arthritis.
  • Figure 3 uses BIAcore (SPR technology) to analyze the binding ability of Hydrostatin-SN10 to TNFR1; among them, the interaction of A, SN10 and TNFR1, the dissociation constant K D value is about 2.8 ⁇ M; B, SN10 and TNF- ⁇ Role, no binding; C, SN10 competitive inhibition of TNF- ⁇ -TNFR1 binding (TNFR1 on the chip); D, SN10 competitive inhibition of TNF- ⁇ -TNFR1 binding (TNF- ⁇ on the chip) E, SN10 competitive inhibition of TNF- ⁇ -TNFR2 binding.
  • SPR technology SPR technology
  • Figure 4 shows the binding ability of Hydrostatin-SN10 to TNFR1 by MST technique; among them, the interaction of A, SN10 and TNFR1, the dissociation constant KD value is about 2.8 ⁇ M; the interaction between B, SN10 and TNF- ⁇ , Binding; C, SN10 and TNFR2 interaction, no binding; D, SN10 competitive inhibition of TNFR1-TNF- ⁇ binding, (TNFR1 fluorescent labeling); E, SN10 competitive inhibition of TNFR1-TNF- ⁇ binding Role, (TNF- ⁇ fluorescent labeling); F, SN10 competitive inhibition of TNFR2-TNF- ⁇ binding, (TNFR2 fluorescent labeling).
  • FIG. 11 Effect of Hydrostatin-SN10 and PEG-SN10 on ankle joint lesions in CIA mice.
  • A is the normal group
  • B is the model group
  • C is Hydrostatatin-SN10
  • D is PEG-SN10.
  • E is the infliximab group.
  • FIG. 12 Effect of Hydrostatin-SN10 and PEG-SN10 on the expression of TNF- ⁇ in CIA mice, where A is the normal group, B is the model group, C is Hydrostatin-SN10, D is PEG-SN10, and E is Infliximab. Monoclonal antibody group.
  • FIG. 13 Effect of Hydrostatin-SN10 and PEG-SN10 on the expression of TRAP in CIA mice, where A is the normal group, B is the model group, C is Hydrostatatin-SN10, D PEG-SN10, and E is the infliximab group. .
  • Example 2-5 The experiments of Examples 2-5 were carried out using Hydrostatin-SN10 prepared in Example 1.
  • the PEG-SN10 used in the following examples was synthesized by Qiang Yao Biotechnology Co., Ltd., and the purity was ⁇ 98% by HPLC.
  • Example 1 Synthesis of a selective TNFR1 antagonist peptide Hydrostatin-SN10 from Qinghai sea snake
  • Hydrostatin-SN10 was synthesized by solid phase peptide synthesis technique, and its purity and molecular weight were analyzed by HPLC (Fig. 1) and MS (Fig. 2). The results showed that the purity was >97% and the molecular weight was 1250.29 g/mol.
  • Example 2 BIAcore analysis of the binding ability of Hydrostatin-SN10 to TNFR1.
  • the running buffer flows through the channel set in the CM-5 sensor chip at a flow rate of 10 ⁇ l/min until the baseline level is reached.
  • Hydrostatin-SN10 directly interacts with TNFR1 and has a binding capacity of 2.8 ⁇ M to TNFR1; Hydrostatin-SN10 does not bind to TNF- ⁇ and competitively inhibits the interaction of TNFR1 with TNF- ⁇ .
  • Example 3 MST analysis of the binding ability of Hydrostatin-SN10 to TNFR1.
  • a series of gradient concentrations of Hydrostatin-SN10 were prepared in a 1:1 dilution ratio, and an equal volume of fluorescently labeled TNF- ⁇ /TNFR1/TNFR2 200nM was uniformly mixed with Hydrostatin-SN10, incubated for 30 min in the dark, and a suitable amount of sample was taken up with a capillary pipette. Detect, observe the time trajectory of relative fluorescence values and the dose-response curve of thermophoresis Thermophoresis, and calculate the affinity K D value by software NTAffinityAnalysis v2.0.2 to determine whether there is a specific binding tendency.
  • a series of gradient concentrations of TNF- ⁇ were prepared in a 1:1 dilution ratio.
  • the same volume of fluorescently labeled TNFR1/TNFR2 200nM was uniformly mixed with TNF- ⁇ , incubated for 30 min in the dark, and the appropriate amount of sample was taken up by capillary pipette.
  • the K D values of the positive control TNFR1/TNFR2 and TNF- ⁇ were determined; 400 nM TNFR1 and 400 ⁇ M Hydrostatin-SN10 were mixed in equal volume, and then incubated with a series of concentrations of TNF- ⁇ in an equal volume for 30 min, and the appropriate amount was taken with a capillary pipette.
  • the sample was tested on the machine and the K D value was obtained by software fitting.
  • the changes of TNF- ⁇ saturation concentration, response amplitude and affinity constant K D before and after the addition of Hydrostatin-SN10 were compared.
  • the results of MST assay showed that the Hydrostatin-SN10 target was specific and could directly interact with TNFR1, and its binding ability to TNFR1 was about 2.8 ⁇ M; it only binds to TNFR1 and is selective, but not TNF- The binding of ⁇ and TNFR2 competitively inhibits the interaction between TNFR1 and TNF- ⁇ .
  • Example 4 Detection of plasma half-life of Hydrostatin-SN10 and PEG-SN10 in SD rats.
  • the measurement was carried out according to the product specification using an ELISA kit purchased from Genzyme. Serum samples were collected from the posterior iliac crest with heparinized 50 ⁇ L capillaries, and blood samples were collected at 1 min, 2 min, 3 min, 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h after treatment. After standing for 2 h, the sample was centrifuged, and the resulting supernatant was stored at -20 ° C for testing.
  • Example 5 Therapeutic effect of Hydrostatin-SN10 and PEG-SN10 on rheumatoid arthritis
  • mice Male, 8 to 10 weeks, weighing 18 to 20 g, 30 rats.
  • Bovine Type II Collagen (Chondrex), Complete Freund's Adjuvant (Chondrex), Incomplete Freund's Adjuvant (Chondrex), Horseradish Peroxidase Labeled IgG Antibody, ( ebioscience), mouse IL-17ELISA detection kit (ebioscience), mouse LEGENDplex TM multiple factor (TNF- ⁇ , IL-6 , IL-10, IFN- ⁇ ) detected by flow cytometry kit (Biolegend, USA), True -Nuclear TM Mouse Treg Flow TM Kit (FOXP3Alexa 488/CD4APC/CD25PE) (Biolegend, USA), FITC anti-mouse CD3 (Biolegend, USA), PE/Cy7 anti-mouse CD4 (Biolegend, USA), PerCP/Cy5.5 anti-mouse IFN- ⁇ (Biolegend, USA) , PE/Dazzle TM 594anti-mouse IL-4 (Biolegend, USA), Alexa 647 anti-m
  • DBA/1 mice were weighed and began to model. Six were randomly selected as the normal group, and the rest were the experimental group.
  • Day 0 Take bovine type II collagen (4 mg/ml) and vortex and mix with an equal volume of CFA (4 mg/ml). It is better to not disperse on the water, and 100 ul/head is injected intradermally into the tail of the mouse.
  • the primary arthritis was increased for 29 days, and the arthritis index of each group gradually increased, that is, the inflammation gradually increased.
  • the degree of swelling of the hind limbs of the mice gradually subsided, but the joints appeared to be stiff or deformed. The highest rating.
  • the arthritis index of both the Hydrostatin-SN10 and PEG-SN10 in the drug-administered group was alleviated, and the disease progression was slowed down.
  • the IgG and multi-factor kits were used to determine the content of collagen-specific antibody IgG and pro-inflammatory factors IL-17, TNF- ⁇ , IL-6, IFN- ⁇ and anti-inflammatory factor IL-10 in the serum of each group of mice.
  • the content of each group is shown in Figure 7.
  • the serum levels of collagen-specific antibody IgG and proinflammatory factors IL-17, TNF- ⁇ , IL-6 and IFN- ⁇ in the model group were significant. Higher than the normal group, and the content of the anti-inflammatory factor IL-10 was not different from the normal group.
  • the administration groups of Hydrostatin-SN10 and PEG-SN10 significantly reduced the content of collagen-specific antibody IgG and pro-inflammatory factors IL-17, TNF- ⁇ , IL-6 and IFN- ⁇ in serum. Increase the content of anti-inflammatory factor IL-10.
  • the mouse trabecular Micro-CT results showed that the normal group of trabecular bone was intact, while the CIA model group had severe bone destruction and significant voids compared with the normal group; compared with the model group, Hydrostatin- The degree of bone destruction of SN10, PEG-SN10 and the positive drug infliximab was significantly reduced.
  • Figure 10 shows the results of trabecular bone measurement by analyzing bone mineral density (BMD), bone volume/tissue volume (BV/TV), bone area/tissue volume (BS/TV), and number of trabeculae (Th. N), trabecular thickness (Tb.Th), bone surface area/bone volume (BS/BV), model factor of trabecular bone (Tb.pf), trabecular separation (Tb.sp) and other bone parameters, analysis Hydrostatin-SN10, PEG-SN10 relief of bone destruction.
  • the results showed that the model group had bone mineral density (BMD), bone volume/tissue volume (BV/TV), bone area/tissue volume (BS/TV), number of trabeculae (Th. N), trabecular bone compared with the normal group.
  • Tb.Th thickness
  • BS/BV bone surface area/bone volume
  • Tb.pf trabecular model factor
  • Tb.sp trabecular separation
  • BMD Bone mineral density
  • BV/TV bone volume/tissue volume
  • BS/TV bone area/tissue volume
  • Th.N number of trabeculae
  • the HE staining results of the mouse joint showed that the synovial tissue of the joint capsule of the normal group was intact, the fibrous membrane was clearly visible, no exudation was observed in the cystic cavity, and no obvious inflammatory cells were observed in the synovial membrane and subserosal tissue.
  • CIA model group showed severe joint structure damage, synovial hyperplasia, severe cartilage damage, severe inflammatory cell infiltration; while the administration group Hydrostatin-SN10 showed complete joint structure, severe synovial hyperplasia and more serious inflammatory cell infiltration .
  • PEG-SN10 and infliximab showed complete joint structure and weak inflammatory cell infiltration, and joint damage was significantly weakened.
  • Osteoclasts are a type of bone tissue that performs the function of bone resorption. Osteoclasts and osteoblasts function functionally. Tartrate-resistant phosphatase, TRAP, is a specific marker enzyme for osteoclasts, and its expression and secretion are closely related to the differentiation and function of osteoclasts. As shown in Figure 13, the anti-tartaric acid phosphatase was significantly increased in the CIA model group compared with the normal group; Hydrostatin-SN10 and PEG-SN10 reduced the resistance to tartrate phosphatase and reduced osteoclasts compared with the model group. The amount of this, thereby reducing the erosion of bone tissue.
  • Treg cells are a special type of immunoregulatory cells with two major functional features: immune non-reactivity and immunosuppression. It can inhibit the activity and function of immune cells such as B cells and T cells by direct contact with cells and secretion of inhibitory cytokines, etc., and plays an extremely important role in preventing autoimmune diseases.
  • Treg cell abnormalities are closely related to the development of RA.
  • Foxp3 is specifically expressed in Treg cells and plays an important role in the proliferation and function of Treg, so Foxp3 can be used as a biomarker of Treg.
  • the expression levels of Fxop3 in Hydrostatin-SN10, PEG-SN10 and infliximab were significantly increased, that is, the number of Treg cells was increased, and the immunosuppressive effect was significantly enhanced. Thereby inhibiting the development of rheumatoid arthritis.
  • Th1 mainly secretes IFN- ⁇ , mainly mediates cellular immune response, induces the production of inflammatory factors, macrophage activation, etc., mainly induces the production of autoimmune diseases
  • Th2 mainly secretes IL-4, mainly mediator fluid immunity, Inhibits monocytes and promotes the release of inflammatory cytokines.
  • Th1/Th2 ratio imbalance plays an important role in the pathogenesis of RA.
  • Th17 cells secrete high levels of IL-17, which stimulates FLS secretion of matrix metalloproteinase-1, IL-1 ⁇ , TNF- ⁇ , etc., and induces NF- ⁇ B receptor activator ligands to participate in the bone destruction and tissue of RA. Inflammatory damage.
  • the effect was observed by measuring the expression of helper T cells in splenocytes.
  • the expression levels of IFN- ⁇ and IL-17 in Hydrostatin-SN10, PEG-SN10 and infliximab were significantly down-regulated, and the expression of IL-4 was up-regulated. That is, Hydrostatin-SN10 and PEG-SN10 may inhibit the expression and release of inflammatory factors and reduce the damage to bone by regulating the changes of Th1, Th2 and Th17.

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Abstract

提供青环海蛇蛇毒来源的选择性TNFR1拮抗肽Hydrostatin-SN10在制备预防和治疗类风湿性关节炎的药物中的应用。

Description

[根据细则37.2由ISA制定的发明名称]　选择性TNFR1拮抗肽在制备预防和治疗类风湿性关节炎的药物中的应用 技术领域
本发明涉及生物医药技术领域，具体地说，是一种青环海蛇蛇毒来源的选择性TNFR1拮抗肽Hydrostatin-SN10及其在类风湿性关节炎中的应用。
背景技术
类风湿性关节炎(Rheumatoid arthritis，RA)是一种以关节滑膜炎为特征的的慢性自身免疫性疾病。主要临床表现为小关节滑膜所致的关节肿痛，滑膜炎症和增生导致软骨和骨的侵蚀，关节间隙变窄，晚期因严重骨质破坏、吸收导致关节僵直、畸形、功能障碍。在美国，RA被认为是影响人类健康的五大疾病(心血管疾病，阿尔兹海默症，癌症，艾滋病，类风湿性关节炎)之一。RA在任何年龄均可发病，以20-50岁最多。全球大约有0.24％的人在遭受RA的折磨，有极高的致残率，预后不良(Cross,M.,Smith,E.,Hoy,D.,Carmona,L.,Wolfe,F.,Vos,T.,Williams,B.,Gabriel,S.,Lassere,M.,Johns,N.,Buchbinder,R.,Woolf,A.,March,L.,2014.The global burden of rheumatoid arthritis:estimates from the global burden of disease 2010 study.Ann.Rheum.Dis.73,1316–132.)。到目前为止，RA的发病机制并不明确，但遗传因素，环境因素都与疾病的发生发展密切相关。研究表明类风湿性关节炎的严重程度受炎性介质的影响较大，主要的炎性介质有肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-17(IL-17)以及白细胞介素-1(IL-1)等，其中TNF-α在RA的发病机制中起着非常重要的作用。当前，在类风湿性关节炎临床治疗中，针对TNF-α的药物如阿达木单抗，英夫利昔单抗等取得了很好的临床效果，不仅能够改善症状，还能够抑制关节的破坏。然而，这类抗TNF-α单抗药物因完全封闭了TNF-α的生物学功能，导致机体的免疫自稳、免疫监视功能受到影响，给患者带来了易发生结核病感染、产生新的自身免疫疾病甚至诱发肿瘤的风险。
TNF-α在机体内的过量表达及TNF-α/TNFRs信号通路的异常激活与类风湿关节炎等自身免疫性疾病的发生发展有着密切关系。随着对RA发病机制的研究深入，当前更多的研究是把治疗的靶点转向TNFRs。然而，相对于TNF-α单抗等生物制剂，TNFRs的小分子拮抗剂的基础和应用研究进展缓慢。总体 上从抗炎角度来说，TNFR1主要传递促炎、凋亡信号，通过选择性地阻断TNFR1传递的信号通路来封闭TNF-α的生物学功能，已成为该类药物研发的热点。目前尚无一种具有高度选择性的TNFR1拮抗药物上市应用于临床。本课题组在前期工作基础中，生物淘选青环海蛇蛇毒毒腺噬菌体展示文库，获得了一种抗炎活性肽Hydrostatin-SN1(中国专利文献CN103030687A)。通过结构优化，获得候选肽Hydrostatin-SN10，采用BIAcore(基于表面等离子共振技术的生物大分子相互作用分析技术)和MST(微量热泳动)技术分析Hydrostatin-SN10与TNF-α、TNFR1、TNFR2之间的相互作用，证明Hydrostatin-SN10可直接与TNFR1结合，并且与TNF-α和TNFR2不结合，靶点特异，是一种选择性TNFR1拮抗肽。在专利文献CN103030687A中，公开了Hydrostatin-SN1能够治疗与TNF-α相关的疾病，并公开了TNF-α相关的疾病是类风湿性关节炎，根据在先申请公开的机理，并不能显而易见的推导出本发明Hydrostatin-SN10能够具有治疗类风湿性关节炎的用途。
然而，单纯的小分子多肽，其相对分子质量较小，在体内极易被肾小球快速滤过以及被相关蛋白酶降解，稳定性差，血浆半衰期很短，无法正常发挥其生物功能，难以达到有效治疗效果，这在一定程度上制约了它的发展和应用。为了解决这一问题，对多肽药物进行改造修饰或与其他材料融合来提高其稳定性已经成为生物医药领域的研究热点。目前用作多肽和蛋白质的化学修饰剂很多，而单甲氧基聚乙二醇(mPEG)及其衍生物是应用最广泛的修饰剂之一。采用PEG修饰的优势主要有：1、改善药代动力学特性，延长血浆半衰期；2、增加溶解度和稳定性(热稳定性、抗酸碱能力、抗变性剂能力及抗蛋白酶水解能力)；3、降低免疫原性和毒性；4、提高体内的生物活性。目前已有多种PEG化的蛋白质和多肽类药物被批准进入临床应用，如PEG-TNF-α抗体Fab片段、PEG-促红细胞生成多肽等。因此，我们在前期工作的基础上，对Hydrostatin-SN10进行结构修饰，获得本发明产物PEG-SN10，本发明着重研究PEG-SN10在类风湿性关节炎中的作用。
发明内容
本发明的目的在于提供一种青环海蛇蛇毒来源的选择性TNFR1拮抗肽Hydrostatin-SN10及其在类风湿性关节炎中的应用，本发明的另一目的在于提供一种基于mPEG2000(单甲氧基聚乙二醇2000)修饰的选择性TNFR1拮抗 肽Hydrostatin-SN10，即PEG-SN10在类风湿性关节炎中的应用。
本发明人所在课题组(江海龙，中国人民解放军第二军医大学2015年硕士学位论文，海蛇蛇毒抗炎活性肽Hydrostatin-SN1的结构优化和抗炎机制研究)通过将Hydrostatin-SN1(22AA)截短，得到Hydrostatin-SN10(10AA)，利用表面等离子共振技术(SPR)发现其能与TNFR1结合，结合能力约为K D＝2.8μM，高于Hydrostatin-SN1与TNFR1的结合能力(K D＝32μM)，然而，是否选择性拮抗TNFR1不清楚；动物模型结果表明其具有一定的抗炎活性。
本发明的主要技术方案是：通过建立牛Ⅱ型胶原诱导的类风湿性关节炎(CIA)动物模型，证明Hydrostatin-SN10及PEG-SN10具有治疗类风湿性关节炎的用途。
本发明的第一方面，提供一种选择性TNFR1拮抗肽Hydrostatin-SN10，所述的选择性TNFR1拮抗肽Hydrostatin-SN10的氨基酸序列如SEQ ID NO:2所示。
所述的选择性TNFR1拮抗肽Hydrostatin-SN10，其合成方法为：利用固相合成技术合成Hydrostatin-SN10，并以HPLC及MS分析其纯度和分子量，分子量为1250.29道尔顿，等电点为4.39。
本发明的第二方面，提供一种选择性TNFR1拮抗肽Hydrostatin-SN10的编码基因，其核苷酸序列如SEQ ID NO:1所示。
本发明的第三方面，提供上述的选择性TNFR1拮抗肽Hydrostatin-SN10在制备预防和治疗类风湿性关节炎的药物中的应用。
优选的，所述的预防或治疗类风湿性关节炎的药物为：以选择性TNFR1拮抗肽Hydrostatin-SN10为唯一的活性成分，或包含选择性TNFR1拮抗肽Hydrostatin-SN10的药物组合物。
本发明的第四方面，提供一种基于mPEG2000(单甲氧基聚乙二醇2000)修饰的选择性TNFR1拮抗肽Hydrostatin-SN10，即PEG-SN10，所述的mPEG2000的羧基共价连接至Hydrostatin-SN10肽链N-端天冬氨酸的游离氨基上。用平均分子量约2000道尔顿的mPEG(单甲氧基聚乙二醇)修饰Hydrostatin-SN10能增加Hydrostatin-SN10的半衰期和稳定性。所述的mPEG2000的结构简式可表示为：CH 3O-(CH 2CH 2O) n-COOH，其中所述n为聚合度，n＝35～45，其平均分子量为2000道尔顿。
本发明的第五方面，提供上述的基于mPEG2000修饰的选择性TNFR1拮抗肽PEG-SN10在制备预防和治疗类风湿性关节炎的药物中的应用。
优选的，所述的预防和治疗类风湿性关节炎的药物为：以PEG-SN10为唯一的活性成分，或包含PEG-SN10的药物组合物。
优选的，所述的药物组合物和药剂学上的常规药用辅料制成药物制剂。
优选的，所述的药物制剂是片剂、颗粒剂、分散剂、胶囊剂、软胶囊剂、滴丸、注射剂、粉针剂或气雾剂等。
优选的，上述的预防和治疗类风湿性关节炎的药物选择性拮抗TNFR1。
本发明采用BIAcore(基于表面等离子共振技术的生物大分子相互作用分析技术)和MST(微量热泳动)技术分析Hydrostatin-SN10与TNF-α、TNFR1、TNFR2之间的相互作用，证明Hydrostatin-SN10可直接与TNFR1结合，并且与TNF-α和TNFR2不结合。本发明采用牛Ⅱ型胶原诱导的类风湿性关节炎动物模型，观察本发明提供的多肽的治疗作用。结果显示Hydrostatin-SN10及PEG-SN10腹腔注射给药后，Hydrostatin-SN10及PEG-SN10能够有效降低CIA小鼠的关节炎指数；显著降低CIA小鼠血清中胶原特异性抗体IgG及促炎因子IL-17、TNF-α、IL-6、IFN-γ的水平且显著提高抑炎因子IL-10的水平，通过炎症因子的作用发挥抗炎作用；通过micro-CT扫描，可显著改善小鼠骨小梁骨质破坏程度，从骨质破坏角度观察Hydrostatin-SN10及PEG-SN10具有显著的抗炎效果；可显著改善CIA小鼠的关节损伤，滑膜增生和炎性细胞浸润且能够显著减少关节中TNF-α的表达量，显著减少破骨细胞的数量，从关节病理角度观察具有显著的抗炎效果；Hydrostatin-SN10及PEG-SN10能够调节Th1/Th2及Th17/Treg比例失衡，即Hydrostatin-SN10及PEG-SN10可以通过免疫调节发挥抗类风湿性关节炎的作用。
上述研究结果表明，选择性TNFR1拮抗肽Hydrostatin-SN10及PEG-SN10均具有良好的抗类风湿性关节炎的作用。本发明寻找到了有效防治类风湿性关节炎的药物。
附图说明
图1 Hydrostatin-SN10的HPLC分析结果。
图2 Hydrostatin-SN10的MS分析结果。
图3采用BIAcore(SPR技术)分析Hydrostatin-SN10与TNFR1的结合 能力；其中，A、SN10与TNFR1的相互作用，结合解离常数K D值约为2.8μM；B、SN10与TNF-α的相互作用，不结合；C、SN10对TNF-α-TNFR1结合的竞争性抑制作用(TNFR1在芯片上)；D、SN10对TNF-α-TNFR1结合的竞争性抑制作用(TNF-α在芯片上)；E、SN10对TNF-α-TNFR2结合的竞争性抑制作用。
图4是采用MST技术分析Hydrostatin-SN10与TNFR1的结合能力；其中，A、SN10与TNFR1的相互作用，结合解离常数KD值约为2.8μM；B、SN10与TNF-α的相互作用，不结合；C、SN10与TNFR2的相互作用，不结合；D、SN10对TNFR1-TNF-α结合的竞争性抑制作用，(TNFR1荧光标记)；E、SN10对TNFR1-TNF-α结合的竞争性抑制作用，(TNF-α荧光标记)；F、SN10对TNFR2-TNF-α结合的竞争性抑制作用，(TNFR2荧光标记)。
图5.Hydrostatin-SN10和PEG-SN10对CIA小鼠脚掌肿胀的改善情况。
图6.Hydrostatin-SN10和PEG-SN10对CIA小鼠临床评分的改善情况。
图7.Hydrostatin-SN10和PEG-SN10对CIA小鼠胶原特异性抗体IgG及血清中炎症因子的影响(n＝6)。
图8.Hydrostatin-SN10和PEG-SN10对CIA小鼠后肢关节的影响(n＝12)。
图9.Hydrostatin-SN10和PEG-SN10对CIA小鼠骨小梁的影响(n＝12)。
图10.Hydrostatin-SN10和PEG-SN10对CIA小鼠骨小梁参数的影响(n＝12)。
图11.Hydrostatin-SN10和PEG-SN10对CIA小鼠踝关节病变的影响，组织切片HE染色光镜图，其中A为正常组，B为模型组，C为Hydrostatin-SN10，D为PEG-SN10，E为英夫利昔单抗组。
图12.Hydrostatin-SN10和PEG-SN10对CIA小鼠TNF-α表达量的影响，其中A为正常组，B为模型组，C为Hydrostatin-SN10，D为PEG-SN10，E为英夫利昔单抗组。
图13.Hydrostatin-SN10和PEG-SN10对CIA小鼠TRAP表达量的影响，其中A为正常组，B为模型组，C为Hydrostatin-SN10，D PEG-SN10，E为英夫利昔单抗组。
图14.Hydrostatin-SN10和PEG-SN10对CIA小鼠Treg细胞中Foxp3表达量的影响(n＝6)。
图15.Hydrostatin-SN10和PEG-SN10对CIA小鼠Treg细胞中Foxp3表达量的影响(n＝6)。
图16.Hydrostatin-SN10和PEG-SN10对CIA小鼠T细胞中Th1，Th2，Th17的百分比的影响(n＝6)。
图17.Hydrostatin-SN10和PEG-SN10对CIA小鼠T细胞中Th1，Th2，Th17的百分比的影响(n＝6)。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
用实施例1制得的Hydrostatin-SN10进行实施例2-5的实验。以下实施例中所用PEG-SN10，由强耀生物科技有限公司合成，经HPLC检测纯度≥98％。
实施例1：青环海蛇选择性TNFR1拮抗肽Hydrostatin-SN10的合成
以固相多肽合成技术合成Hydrostatin-SN10，并以HPLC(图1)和MS(图2)分析其纯度及分子量，结果可知其纯度>97％，分子量为1250.29g/mol。
实施例2：BIAcore分析Hydrostatin-SN10与TNFR1的结合能力。
1、running buffer以10μl/min的流速流过CM-5传感芯片中设定的通道，直至达到基线水平。
2、用仪器推荐的buffer活化芯片各通道的表面反应基团。
3、用EP buffer溶解TNFR1和TNFR2冻干粉，按一定的浓度进样，使之包被于芯片表面，再用1mol/L乙醇胺封闭芯片。测定动力学曲线前要对再生条件进行测试，以选择合适的再生条件。
4、当running buffer跑至基线稳定后，将系列浓度的多肽进样，中间浓度的多肽重复进样一次，记录每个浓度的响应值。
如图3所示，Hydrostatin-SN10能与TNFR1直接相互作用，与TNFR1的结合能力约为2.8μM；Hydrostatin-SN10与TNF-α不结合，并能够竞争性抑制TNFR1与TNF-α的相互作用。
实施例3：MST分析Hydrostatin-SN10与TNFR1的结合能力。
1、Hydrostatin-SN10与TNF-α、TNFR1、TNFR2的相互作用：
以1:1稀释比例配制系列梯度浓度的Hydrostatin-SN10，将等体积的荧光标记TNF-α/TNFR1/TNFR2 200nM与Hydrostatin-SN10均匀混合后避光孵育30min，用毛细吸管吸取适量的样品上机检测，观察相对荧光值的时间轨迹和热泳动Thermophoresis的剂量-响应曲线，并通过软件NTAffinityAnalysis v2.0.2拟合计算亲和力K D值，判断是否有特异性结合趋势。
2、Hydrostatin-SN10对TNF-α与TNFR1/TNFR2结合的竞争性抑制作用：
以1:1稀释比例配制系列梯度浓度的TNF-α，将等体积的荧光标记TNFR1/TNFR2 200nM与TNF-α均匀混合后避光孵育30min，用毛细吸管吸取适量的样品上机检测，软件拟合求出阳性对照TNFR1/TNFR2与TNF-α的K D值；将400nM TNFR1和400μM Hydrostatin-SN10等体积混合后，再与系列浓度的TNF-α等体积混合孵育30min，用毛细吸管吸取适量的样品上机检测，软件拟合求出K D值。比较加入Hydrostatin-SN10前后TNF-α饱和浓度、响应值amplitude及亲和力常数K D值的变化。
3、Hydrostatin-SN10对TNF-α与TNFR1/TNFR2结合的竞争性抑制作用(方法同2)。
如图4所示，MST实验结果表明，Hydrostatin-SN10靶点特异，能与TNFR1直接相互作用，与TNFR1的结合能力约为2.8μM；且只与TNFR1结合，具有选择性，而不与TNF-α、TNFR2结合，能竞争性抑制TNFR1和TNF-α的相互作用。
实施例4：Hydrostatin-SN10和PEG-SN10在SD大鼠体内的血浆半衰期检测。
采用购自Genzyme公司的ELISA试剂盒，根据产品说明书进行测定。血清样品用肝素化的50μL毛细管从后眶内采集，在处理后的1min、2min、3min、5min、10min、15min、20min、30min、45min、1h、2h、4h、6h、8h收集血液样品。静置2h后，将样品离心，把得到的上清液贮存在-20℃以备检测。
表1
Figure PCTCN2018077862-appb-000001
如表1所示，结果表明，PEG修饰后PEG-SN10比Hydrostatin-SN10的血浆半衰期延长。
实施例5：Hydrostatin-SN10及PEG-SN10对类风湿性关节炎的治疗作用
一、实验动物
DBA/1小鼠，雄性，8～10周，体重18～20g，30只。
二、主要试剂
牛Ⅱ型胶原(Bovine TypeⅡCollagen，Chondrex)、完全弗氏佐剂(Complete Freund's Adjuvant，Chondrex)、不完全弗氏佐剂(Incomplete Freund's Adjuvant，Chondrex)、辣根过氧化物酶标记的IgG抗体，(Ebioscience)、小鼠IL-17ELISA检测试剂盒(Ebioscience)、小鼠LEGENDplex TM多因子(TNF-α，IL-6，IL-10，IFN-γ)流式检测试剂盒(Biolegend，美国)、True-Nuclear TMMouse Treg Flow TMKit(FOXP3Alexa
Figure PCTCN2018077862-appb-000002
488/CD4APC/CD25PE)(Biolegend，美国)、FITC anti-mouse CD3(Biolegend，美国)、PE/Cy7anti-mouse CD4(Biolegend，美国)、PerCP/Cy5.5anti-mouse IFN-γ(Biolegend，美国)、PE/Dazzle TM594anti-mouse IL-4(Biolegend，美国)、Alexa
Figure PCTCN2018077862-appb-000003
647anti-mouse IL-17A(Biolegend，美国)、FOXP3Fix/Perm Buffer(4x)(Biolegend，美国)。
三、实验方法
DBA/1小鼠适应3天后，编号称重，开始造模。随机取6只作为正常组，其余为实验组。
第0天：取牛Ⅱ型胶原(4mg/ml)与等体积CFA(4mg/ml)涡旋混合，至滴于水上不分散为较好，小鼠尾根部皮内注射100ul/只。
第21天：取牛Ⅱ型胶原(4mg/ml)与等体积IFA(4mg/ml)涡旋混合至滴于水上不分散为较好，小鼠尾根部皮内注射100ul/只。第30天观察造模成功，模型组(PBS)，给药组-SN10(1600ug/kg)，给药组PEG-SN10(1600ug/kg)，英夫利昔单抗(4mg/kg)当天开始腹腔注射给药，每日1次，连续给药18天。
四、试验结果
采用Graphpad Prism 5对数据进行One-Way ANOVA统计学分析P<0.05时认为差异有统计学意义。“*”表示P<0.05，“**”表示P<0.01，“***”表示P<0.001。
1.Hydrostatin-SN10、PEG-SN10对小鼠关节炎指数评分的影响。
如图5，图6所示，初次免疫29天，各组小鼠关节炎指数逐渐增高即炎症逐渐加重，随着时间的延长，小鼠后肢肿胀程度逐渐消退，但关节出现强直或畸形，达到最高评分。与模型组相比，给药组Hydrostatin-SN10及PEG-SN10关节炎指数均有所减轻，同时病情发展减缓。
2.Hydrostatin-SN10、PEG-SN10对CIA小鼠血清中胶原特异性抗体及炎症因子的影响
用ELISA试剂盒及多因子试剂盒测定各组小鼠血清中的胶原特异性抗体IgG及促炎因子IL-17，TNF-α，IL-6，IFN-γ及抑炎因子IL-10的含量，各组含量如图7所示：与正常对照组相比，模型组小鼠血清中胶原特异性抗体IgG及促炎因子IL-17，TNF-α，IL-6，IFN-γ的含量显著高于正常组，而抑炎因子IL-10的含量与正常组无差异。与模型组相比，给药组Hydrostatin-SN10、PEG-SN10均能显著降低血清中胶原特异性抗体IgG及促炎因子IL-17，TNF-α，IL-6，IFN-γ的含量且显著升高抑炎因子IL-10的含量。
3.Micro-CT检测Hydrostatin-SN10、PEG-SN10对CIA小鼠骨质破坏的影响
如图8所示：小鼠后肢Micro-CT检查结果表明，正常组关节面光滑，关节结构清晰可见；CIA模型组关节面粗糙，骨质破坏严重；而给药组Hydrostatin-SN10、PEG-SN10及阳性药英夫利昔单抗骨质破坏程度显著减轻。
如图9所示：小鼠骨小梁Micro-CT结果显示正常组骨小梁结构完整，而CIA模型组与正常组相比骨质破坏严重，有明显空洞；与模型组相比，Hydrostatin-SN10、PEG-SN10及阳性药英夫利昔单抗骨质破坏程度明显减轻。
图10所示，骨小梁的测定结果显示：通过分析骨密度(BMD)、骨体积/组织体积(BV/TV)、骨面积/组织体积(BS/TV)、小梁的数量(Th.N)、小梁厚度(Tb.Th)、骨表面积/骨体积(BS/BV)、骨小梁的模式因子(Tb.pf)、小梁的分离度(Tb.sp)等骨参数，分析Hydrostatin-SN10、PEG-SN10对骨质破坏的缓解情况。结果表明与正常组相比，模型组骨密度(BMD)、骨体积/组织体积(BV/TV)、骨面积/组织体积(BS/TV)、小梁的数量(Th.N)、小梁厚度(Tb.Th)等参数显著下调，骨表面积/骨体积(BS/BV)、骨小梁的模式因子(Tb.pf)、小梁的分离度(Tb.sp)等参数显著上调；与模型组相比， Hydrostatin-SN10及PEG-SN10组骨密度(BMD)、骨体积/组织体积(BV/TV)、骨面积/组织体积(BS/TV)、小梁的数量(Th.N)、小梁厚度(Tb.Th)等参数显著上调，骨表面积/骨体积(BS/BV)、骨小梁的模式因子(Tb.pf)、小梁的分离度(Tb.sp)等参数显著下调。
4.HE染色观察Hydrostatin-SN10、PEG-SN10对CIA小鼠关节破坏的影响
如图11所示，小鼠关节HE染色结果表明，正常组小鼠关节囊滑膜组织完整，纤维膜清晰可见，囊腔内未见渗出，滑膜、滑膜下组织未见明显炎症细胞浸润；CIA模型组可见关节结构破坏严重，滑膜增生，软骨损伤严重，严重的炎性细胞浸润；而给药组Hydrostatin-SN10可见关节结构完整，滑膜增生严重且较严重的炎性细胞浸润。而PEG-SN10及英夫利昔单抗可见关节结构完整及微弱的炎性细胞浸润，关节损伤显著减弱。
5.免疫组化观察Hydrostatin-SN10、PEG-SN10对CIA小鼠TNF-α表达量的影响
前期结果显示，Hydrostatin-SN10靶点特异，能够与TNFR1特异性结合，而不与TNF-α、TNFR2结合，且Hydrostatin-SN10能够竞争性抑制TNF-α与TNFR1的结合。从而减少TNF-α的表达量，如图12所示，与正常组相比，模型组TNF-α的表达量增加；与模型组相比，Hydrostatin-SN10、PEG-SN10关节中TNF-α的表达量减少。
6.TRAP染色观察Hydrostatin-SN10、PEG-SN10的对关节中破骨细胞的影响
破骨细胞是骨组织成分的一种，行使骨吸收的功能。破骨细胞与成骨细胞在功能上相对应。抗酒石酸磷酸酶即TRAP为破骨细胞的特异性标志酶，其表达和分泌与破骨细胞的分化及功能具有重要的关系。如图13所示，与正常组相比，CIA模型组小鼠抗酒石酸磷酸酶明显增多；与模型组相比，Hydrostatin-SN10、PEG-SN10能够减少抗酒石酸磷酸酶的含量，减少破骨细胞的数量，从而减少对骨组织的侵蚀。
7.Hydrostatin-SN10、PEG-SN10对CIA小鼠Treg细胞中Foxp3表达量的影响
Treg细胞是一种特殊的免疫调节细胞，具有免疫无反应性和免疫抑制性 两大功能特征。可通过细胞间直接接触和分泌抑制性细胞因子等抑制B细胞、T细胞等免疫细胞的活性和功能，对于防止自身免疫性疾病具有极其重要的作用。
证据表明，RA患者体内存在Treg细胞数量的改变，提示Treg细胞的异常与RA的发生发展密切相关。Foxp3在Treg细胞中特异性表达，对Treg的增殖和功能起重要作用，故Foxp3可以作为Treg的生物标志物。如图14和图15所示：与模型组相比，Hydrostatin-SN10、PEG-SN10及英夫利昔单抗组Fxop3的表达量均显著提高，即Treg细胞的数量提高，免疫抑制作用明显加强，从而抑制类风湿性关节炎的发生发展。
8.Hydrostatin-SN10、PEG-SN10对CIA小鼠脾脏中Th1，Th2，Th17的表达量的影响
Th1：主要分泌IFN-γ，主要介导细胞免疫应答，诱导炎性因子的产生，巨噬细胞活化等，主要诱导自身免疫疾病的产生；Th2：主要分泌IL-4，主要介导体液免疫，抑制单核细胞，促进炎性细胞因子的释放。且Th1/Th2比例失衡在RA发病机制中起着重要的作用。
研究表明：Th17细胞高分泌IL-17，通过刺激FLS分泌基质金属蛋白酶-1，IL-1β，TNF-α等协调诱导NF-κB受体激活因子配体等参与RA的骨质破坏和组织的炎性损伤。通过测定脾细胞中辅助性T细胞的表达，观察其作用。如图16和图17所示，与模型组相比，Hydrostatin-SN10、PEG-SN10及英夫利昔单抗组IFN-γ，IL-17的表达量显著下调，IL-4的表达量上调，即Hydrostatin-SN10、PEG-SN10可能是通过调节Th1，Th2，Th17的变化，抑制炎症因子的表达和释放，减少对骨质的破坏。
以上结果表明，Hydrostatin-SN10和PEG-SN10能够有效治疗Ⅱ型胶原诱导的小鼠类风湿性关节炎动物模型。
以上已对本发明创造的实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
序列表
Figure PCTCN2018077862-appb-000004

Claims (9)

  1. 一种选择性TNFR1拮抗肽Hydrostatin-SN10在制备预防和治疗类风湿性关节炎的药物中的应用，所述的选择性TNFR1拮抗肽Hydrostatin-SN10的编码基因的核苷酸序列如SEQ ID NO:1所示；所述的选择性TNFR1拮抗肽Hydrostatin-SN10的氨基酸序列如SEQ ID NO:2所示。
  2. 根据权利要求1所述的应用，其特征在于，所述的选择性TNFR1拮抗肽Hydrostatin-SN10的分子量为1250.29道尔顿。
  3. 根据权利要求1所述的应用，其特征在于，所述的预防和治疗类风湿性关节炎的药物选择性拮抗TNFR1。
  4. 根据权利要求1所述的应用，其特征在于，所述的预防或治疗类风湿性关节炎的药物为：以选择性TNFR1拮抗肽Hydrostatin-SN10为唯一的活性成分，或包含选择性TNFR1拮抗肽Hydrostatin-SN10的药物组合物。
  5. 一种基于mPEG2000修饰的选择性TNFR1拮抗肽PEG-SN10在制备预防或治疗类风湿性关节炎的药物中的应用，其特征在于，所述的mPEG2000的羧基共价连接至Hydrostatin-SN10肽链N-端天冬氨酸的游离氨基上，所述的Hydrostatin-SN10的氨基酸序列如SEQ ID NO:2所示。
  6. 根据权利要求5所述的应用，其特征在于，所述的mPEG2000的平均分子量为2000道尔顿。
  7. 根据权利要求5所述的应用，其特征在于，所述的预防和治疗类风湿性关节炎的药物为：以PEG-SN10为唯一的活性成分，或包含PEG-SN10的药物组合物。
  8. 根据权利要求4或7所述的应用，其特征在于，所述的药物组合物和药剂学上的常规药用辅料制成药物制剂。
  9. 根据权利要求8所述的应用，其特征在于，所述的药物制剂是片剂、颗粒剂、分散剂、胶囊剂、软胶囊剂、滴丸、注射剂、粉针剂或气雾剂。
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