WO2024092888A1 - 抗血管生成素样蛋白4抗体在制备抑制炎症因子表达的药物中的应用 - Google Patents

抗血管生成素样蛋白4抗体在制备抑制炎症因子表达的药物中的应用 Download PDF

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WO2024092888A1
WO2024092888A1 PCT/CN2022/132616 CN2022132616W WO2024092888A1 WO 2024092888 A1 WO2024092888 A1 WO 2024092888A1 CN 2022132616 W CN2022132616 W CN 2022132616W WO 2024092888 A1 WO2024092888 A1 WO 2024092888A1
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antibody
angiopoietin
protein
inflammatory
expression
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French (fr)
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张鹏
李亮
成文翔
柯丽青
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中国科学院深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

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  • the present application belongs to the field of biomedicine technology and relates to the use of anti-angiopoietin-like protein 4 antibodies in the preparation of drugs for inhibiting the expression of inflammatory factors.
  • Rheumatoid arthritis is a chronic, systemic disease with unknown etiology, characterized by multi-articular, symmetrical, invasive joint inflammation of the small joints of the hands and feet, often accompanied by extra-articular organ involvement and positive serum rheumatoid factor, which can lead to joint deformity and loss of function.
  • the pathology of RA arthritis mainly includes synovial lining cell hyperplasia, massive interstitial inflammatory cell infiltration, microvascular neoplasia, pannus formation, and destruction of cartilage and bone tissue.
  • CN104995210A discloses an anti-GM-CSF antibody for treating rheumatoid arthritis.
  • CN110585428A discloses the use of B cell vaccines in the preparation of drugs for treating rheumatoid arthritis, wherein the B cell vaccines are prepared from Non-B10B cells obtained by sorting spleen cells of collagen-induced arthritis mice; the sorting method is: spleen cells are first surface stained with antibodies CD19-FITC, CD5-APC, and CD1d-PE, and then flow cytometry is performed to collect all B cells except B10 cells (CD19+CD5+CD1dhi) to obtain Non-B10B cells; the preparation method is: Non-B10B cells are stimulated and cultured in the presence of functional antibodies anti-CD40, CpG, type II collagen (CII) and BAFF, and then irradiated with a cobalt source to obtain the vaccine.
  • the B cell vaccines are prepared from Non-B10B cells obtained by sorting spleen cells of collagen-induced arthritis mice; the sorting method is: spleen cells are first surface stained with antibodies CD19-
  • Angiopoietin-like protein 4 contains an N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain, and has three active forms in vivo, namely, full-length angiopoietin-like protein 4 (flANGPTL4), N-terminal angiopoietin-like protein 4 (nANGPTL4) and C-terminal angiopoietin-like protein 4 (cANGPTL4) fragments.
  • Antibodies capable of neutralizing at least one active form of ANGPTL4 are widely used in the treatment of lipid metabolism diseases, such as CN101128485 discloses a monoclonal antibody against ANGPTL4, and also provides the use of the monoclonal antibody in the preparation of a drug for treating a patient's lipid metabolism disease.
  • the present application provides the use of anti-angiopoietin-like protein 4 antibodies in the preparation of drugs for inhibiting the expression of inflammatory factors. It is found that anti-angiopoietin-like protein 4 can exert anti-inflammatory effects by reducing the proinflammatory cytokine interleukin-6 (IL-6) and also has the effect of inhibiting bone destruction, and can be used to prepare drugs for the treatment of bone and joint inflammatory diseases.
  • IL-6 proinflammatory cytokine interleukin-6
  • the present application provides the use of an anti-angiopoietin-like protein 4 antibody in the preparation of a drug for inhibiting the expression of inflammatory factors.
  • the pharmacodynamics of the drug was verified using the collagen-induced arthritis (CIA) animal model, which is a classic animal model in the field of RA research.
  • CIA collagen-induced arthritis
  • the alleviating effect of anti-ANGPTL4 antibodies on joint swelling was evaluated by measuring body weight and paw swelling. Serum testing of the animal model showed that anti-ANGPTL4 antibody treatment can significantly reduce the expression of inflammatory factors.
  • the inflammatory factor comprises interleukin-6 (IL-6).
  • IL-6 interleukin-6
  • the anti-angiopoietin-like protein 4 antibody refers to an antibody that targets and binds to any one of the flANGPTL4 fragments, nANGPTL4 fragments or cANGPTL4 fragments of the full-length angiopoietin-like protein 4.
  • the existing anti-ANGPTL4 antibodies are mainly used as drugs for the treatment of lipid metabolism diseases.
  • anti-ANGPTL4 antibodies disclosed in the art are all suitable for the present application.
  • the anti-ANGPTL4 antibody can be selected from humanized monoclonal antibodies. For specific amino acid sequences, see US10160803B2.
  • the drug further comprises excipients.
  • the excipients include any one or a combination of at least two of a pharmaceutically acceptable carrier, a wetting agent, a disintegrant, an emulsifier, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.
  • the present application provides the use of anti-Angiopoietin-like protein 4 antibodies in the preparation of drugs for treating chronic inflammatory diseases of bones and joints.
  • the present application uses the collagen-induced arthritis (CIA) animal model to verify the pharmacodynamics of anti-ANGPTL4 antibodies.
  • CIA collagen-induced arthritis
  • the chronic bone and joint inflammatory diseases include any one of rheumatoid arthritis, osteoarthritis, bone hyperplasia, scapulohumeral periarthritis, periostitis, bursitis or synovitis, or a combination of at least two thereof.
  • the present application provides the use of an anti-angiopoietin-like protein 4 antibody in the preparation of a drug for treating bone destructive diseases.
  • the bone destruction disease includes any one of femoral head necrosis, refractory fractures or tumor bone metastasis, or a combination of at least two thereof.
  • a mouse animal model of collagen-induced arthritis was constructed, and different doses of anti-ANGPTL4 antibodies were used for therapeutic intervention in the CIA model.
  • the results showed that the anti-ANGPTL4 antibody could alleviate the progression of the disease.
  • the imaging and histological results showed that the bone destruction in the ankle joints of mice treated with the anti-ANGPTL4 antibody was significantly improved, and the drug can be used to develop treatments for femoral head necrosis, refractory fractures, bone destruction caused by tumor bone metastasis and other related diseases.
  • the present application provides a use of an anti-angiopoietin-like protein 4 antibody for non-therapeutic purposes in the preparation of an inhibitor of inflammatory factor expression.
  • the inhibitor refers to an agent that can inhibit the expression of inflammatory factors.
  • anti-angiopoietin-like protein 4 antibody can regulate the expression level of the pro-inflammatory cytokine interleukin-6 (IL-6) in vivo, and it can be used as an inhibitor in the basic research of interleukin-6 (IL-6) related behavior.
  • IL-6 interleukin-6
  • the present application provides a method for inhibiting the expression of inflammatory factors for the purpose of non-disease diagnosis and/or treatment, the method comprising:
  • Anti-angiopoietin-like protein 4 antibody is used to prepare the method for inhibiting the expression of inflammatory factors, wherein the inflammatory factors include interleukin-6.
  • anti-angiopoietin-like protein 4 antibodies can be used as inhibitors in basic research on interleukin-6 (IL-6) related behaviors, such as constructing an animal model with low interleukin-6 (IL-6) expression.
  • IL-6 interleukin-6
  • anti-ANGPTL4 antibodies can inhibit the expression of the inflammatory factor interleukin-6 (IL-6), relieve local synovial hyperplasia and infiltration of inflammatory cells in joints, and inhibit bone destruction. They can be used to prepare drugs for the treatment of bone and joint inflammatory diseases, make up for the shortcoming that most RA drugs currently used in clinical practice have a single effect, provide new ideas and new means for the treatment of rheumatoid arthritis, and are of great significance to the field of rheumatoid arthritis treatment.
  • IL-6 interleukin-6
  • Figure 1 is a graph showing changes in mouse body weight.
  • FIG. 2 is a graph showing the clinical scoring results of mouse joint swelling.
  • FIG3 is a graph showing the expression level of ANGPTL4 in mouse serum.
  • FIG4 is a graph showing the expression of IL-6 in mouse serum.
  • FIG5 is a three-dimensional image of the mouse ankle joint by Micro-CT.
  • FIG. 6 is a histological staining image of pathological sections of mouse ankle joints.
  • This application specifically validates the pharmacodynamics of the drug using the classic CIA animal model in the field of RA research.
  • the anti-ANGPTL4 antibody was evaluated for its effect on alleviating joint swelling and its toxic effects at the therapeutic dose by measuring body weight and paw swelling, and the expression of cytokine IL-6 was analyzed by serological tests collected from the animal model; the effect of anti-ANGPTL4 antibody treatment on bone destruction in the ankle joint of RA was analyzed by Micro-CT scanning data and histological staining results.
  • This example constructs a model and conducts anti-ANGPTL4 antibody treatment intervention.
  • mice Thirty-two SPF-grade 8-week-old male DBA/1 immune normal mice were purchased and raised in an SPF-grade animal room. The mice were randomly divided into groups according to their average weight, with 8 mice in each group. The mice were divided into a blank control group (CON), a model group (CIA), a low-dose treatment group (50 mg/kg), and a high-dose treatment group (200 mg/kg).
  • An immune program inducer containing 2 mg/mL bovine type II collagen and 4 mg/mL complete Freund's adjuvant was prepared. After full emulsification, 100 ⁇ L of immune program inducer was subcutaneously injected into the tail of the mice in all groups except the blank control group to construct the CIA model.
  • anti-ANGPTL4 antibody (amino acid sequence see US10160803B2) was intraperitoneally injected at the above dose, twice a week, and the model group was injected with PBS as a control until the end point.
  • the entire experiment lasted for 7 weeks, and body weight and paw swelling were measured every week.
  • the body weight measurement results are shown in Figure 1.
  • the weight of mice decreased to varying degrees; in the third week after modeling, intraperitoneal injection treatment was started, and the mice in each group recovered to normal weight gain.
  • the weight growth rate of mice in the 200 mg/kg treatment group was higher than that in the CIA group, indicating that the antibody treatment had no obvious drug toxicity.
  • Clinical scoring of joint swelling (specific method reference: N.
  • mice were taken 7 weeks after modeling in Example 1, and after anesthetizing the mice, blood was collected from the heart, and the serum was obtained by static separation; the ELISA detection kit was used to detect the changes in the expression of ANGPTL4 in the serum and the changes in the content of the cytokine IL-6.
  • the results of the changes in the expression of ANGPTL4 are shown in Figure 3.
  • the expression of ANGPTL4 in the serum of the mice in the low-dose treatment group (50 mg/kg) and the high-dose treatment group (200 mg/kg) was significantly reduced, indicating that ANGPTL4 in the serum can be effectively neutralized.
  • the results of the changes in the content of the cytokine IL-6 are shown in Figure 4.
  • the results show that compared with the CIA model group, after treatment with 200 mg/kg of anti-ANGPTL4 antibodies, the expression level of the inflammatory factor IL-6 in the serum of the mice decreased.
  • mice were taken 7 weeks after modeling in Example 1, and the mice were euthanized.
  • the ankle tissue was dissected and separated, and the anterior and posterior tibial muscles were taken out and cut off from the tibia.
  • the intact paws and ankle joints of the mice were retained and fixed with 10% formalin.
  • Micro-CT (SKYSCAN 1176, Bruker, Belgium) was used to scan the ankle joints of the mice. After the scan data was reconstructed, the degree of joint damage in each group was observed. The results are shown in Figure 5.
  • the joint surface of the CON group was smooth and intact; the bone surface of the CIA group was rough and the bone destruction was serious.
  • the bone destruction of the finger joints was improved, indicating that the anti-ANGPTL4 antibody has a therapeutic effect of inhibiting inflammatory bone destruction, and the therapeutic effect is dose-dependent.
  • This example conducts local histological staining analysis of joints.
  • the bone tissues that had completed CT scanning in Example 3 were sequentially decalcified, dehydrated, and routinely paraffin-embedded, and then HE staining was performed after making tissue sections.
  • the results are shown in Figure 6.
  • the surface of the ankle cartilage of the animals in the CON group was smooth, the joint cavity was intact, and the joint space was obvious; a large number of inflammatory cells were infiltrated in the ankle joints of the animals in the CIA modeling group, the joint space was narrowed, the cartilage surface was destroyed, and the synovial tissue was hyperplastic; while in the anti-ANGPTL4 antibody treatment group, the degree of joint cavity destruction and inflammatory cell infiltration was alleviated, and the degree of bone destruction was reduced.
  • the present application constructs a mouse animal model of collagen-induced arthritis (CIA) to simulate the disease state of RA, applies different doses of anti-ANGPTL4 antibodies for therapeutic intervention in the CIA model, and evaluates the alleviating effect of anti-ANGPTL4 antibodies on joint swelling and the toxic effects at the therapeutic dose by measuring body weight and paw swelling.
  • CIA collagen-induced arthritis
  • Serum detection of the animal model shows that anti-ANGPTL4 antibody treatment can significantly reduce the expression of cytokine IL-6; Micro-CT scanning data and histological staining results show that anti-ANGPTL4 antibody treatment can effectively improve bone destruction of the ankle joint of RA, and can alleviate local synovial hyperplasia and infiltration of inflammatory cells in the joint, proving that anti-ANGPTL4 antibodies can be used to prepare drugs for the treatment of bone and joint inflammatory diseases.

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Abstract

抗血管生成素样蛋白4 抗体在制备抑制炎症因子表达的药物中的应用。通过构建胶原诱导性关节炎(CIA)的小鼠动物模型，模拟RA的疾病状态，应用不同剂量的抗 ANGPTL4 抗体对 CIA 模型进行治疗干预，发现抗 ANGPTL4 抗体治疗可以明显降低细胞因子IL-6的表达，可以有效改善RA对踝关节的骨破坏，并可以缓解关节局部的滑膜增生和炎症细胞的浸润，证明了抗 ANGPTL4抗体可应用于制备治疗骨及关节炎症疾病的药物。

Description

抗血管生成素样蛋白4抗体在制备抑制炎症因子表达的药物中的应用 技术领域
本申请属于生物医药技术领域，涉及抗血管生成素样蛋白4抗体在制备抑制炎症因子表达的药物中的应用。
背景技术
类风湿关节炎(RA)是一种病因未明的慢性、以炎性滑膜炎为主的系统性疾病，其特征是手、足小关节的多关节、对称性、侵袭性关节炎症，经常伴有关节外器官受累及血清类风湿因子阳性，可以导致关节畸形及功能丧失。RA关节炎的病理主要有滑膜衬里细胞增生、间质大量炎性细胞浸润，以及微血管的新生、血管翳的形成及软骨和骨组织的破坏等。
目前，已将靶向肿瘤坏死因子α(TNF-α)、B细胞或T细胞的生物化合物(诸如抗体)用于治疗RA，但仍有许多患者未能对这些疗法响应。如CN104995210A公开一种用于治疗类风湿关节炎的抗GM-CSF抗体。CN110585428A公开B细胞疫苗在制备治疗类风湿关节炎药物中的应用，所述B细胞疫苗由胶原诱导性关节炎小鼠的脾脏细胞经分选后获得的Non-B10B细胞制备而成；所述分选方法是：脾脏细胞先经抗体CD19-FITC、CD5-APC、CD1d-PE进行表面染色，然后进行流式细胞分选，收集除B10细胞(CD19+CD5+CD1dhi)之外的所有B细胞，获得Non-B10B细胞；所述制备方法是：将Non-B10B细胞在有功能抗体anti-CD40、CpG、二型胶原(CII)和BAFF存在的条件下刺激培养，然后再经钴源照射，获得疫苗。
血管生成素样蛋白4(ANGPTL4)含有一个N末端卷曲螺旋结构域和一个C末端纤维蛋白原样结构域，在体内有3种活性形式，分别为全长血管生成素样蛋白4(flANGPTL4)，N端血管生成素样蛋白4(nANGPTL4)和C端血管生成素样蛋白4(cANGPTL4)片段。能够中和ANGPTL4的至少一种活性形式的抗体被广泛应用于治疗脂质代谢疾病，如CN101128485公开一种针对ANGPTL4的单克隆抗体，还提供了单克隆抗体在制备治疗患者的脂质代谢疾病的药物中的应用。
综上所述，开发新型的类风湿关节炎治疗药物，为治疗类风湿关节炎提供 新思路、新手段，对于类风湿关节炎治疗领域具有重要意义。
发明内容
本申请针对目前临床上多数类风湿关节炎药物作用效果单一的问题，以及对新型的类风湿关节炎治疗药物的需求，提供了抗血管生成素样蛋白4抗体在制备抑制炎症因子表达的药物中的应用，发现抗血管生成素样蛋白4可以通过降低促炎细胞因子白介素-6(IL-6)发挥抗炎作用，并兼具抑制骨破坏的作用，可应用于制备治疗骨及关节炎症疾病的药物。
第一方面，本申请提供抗血管生成素样蛋白4抗体在制备抑制炎症因子表达的药物中的应用。
本申请中，经过RA研究领域经典的胶原诱导性关节炎(CIA)动物模型进行了药物的药效学验证，在实验过程中，通过测量体重和足爪肿胀评价了抗ANGPTL4抗体对关节肿胀的缓解作用，通过对动物模型的血清检测表明抗ANGPTL4抗体治疗可以明显降低炎症因子的表达。
优选地，所述炎症因子包括白介素-6(IL-6)。
本申请中，所述抗血管生成素样蛋白4抗体指靶向结合全长血管生成素样蛋白4的flANGPTL4片段、nANGPTL4片段或cANGPTL4片段中任意一种的抗体，现有的抗ANGPTL4抗体临床应用范围主要是治疗脂质代谢疾病的药物。
可以理解，本领域内公开的抗ANGPTL4抗体均适用于本申请，具体地，所述抗ANGPTL4抗体可选自人源化单克隆抗体，具体氨基酸序列参见US10160803B2。
优选地，所述药物还包括辅料。
优选地，所述辅料包括药学上可接受的载体、润湿剂、崩解剂、乳化剂、增溶剂、渗透压调节剂、表面活性剂、包衣材料、着色剂、pH调节剂、抗氧化剂、抑菌剂或缓冲剂中的任意一种或至少两种的组合。
第二方面，本申请提供抗血管生成素样蛋白4抗体在制备治疗慢性骨及关节炎症疾病的药物中的应用。
本申请利用胶原诱导性关节炎(CIA)动物模型对抗ANGPTL4抗体进行药物的药效学验证，Micro-CT扫描数据以及组织学染色结果显示，抗ANGPTL4抗体可以有效改善RA的踝关节的骨破坏，并可以缓解关节局部的滑膜增生和炎 症细胞的浸润。
优选地，所述慢性骨及关节炎症疾病包括类风湿关节炎、骨关节炎、骨质增生、肩周炎、骨膜炎、滑囊炎或滑膜炎中任意一种或至少两种的组合。
第三方面，本申请提供抗血管生成素样蛋白4抗体在制备治疗骨破坏疾病的药物中的应用。
优选地，所述骨破坏疾病包括股骨头坏死、难愈性骨折或肿瘤骨转移中的任意一种或至少两种的组合。
本申请中，通过构建胶原诱导性关节炎(CIA)的小鼠动物模型，应用不同剂量的抗ANGPTL4抗体对CIA模型进行治疗干预，结果显示抗ANGPTL4抗体可并可缓解疾病的进展，影像学及组织学结果显示抗ANGPTL4抗体治疗后的小鼠踝关节明显改善骨破坏情况，可用于开发治疗股骨头坏死、难愈性骨折、肿瘤骨转移引发的骨破坏等相关性疾病。
第四方面，本申请提供一种以非治疗为目的的抗血管生成素样蛋白4抗体在制备炎症因子表达的抑制剂中的应用。
本申请中，所述抑制剂指能够抑制炎症因子表达的试剂。
本申请中，发现抗血管生成素样蛋白4抗体能够调控体内促炎细胞因子白介素-6(IL-6)表达水平，其可作为抑制剂，应用于白介素-6(IL-6)相关行为的基础研究中。
第五方面，本申请提供一种以非疾病诊断和/或治疗为目的的抑制炎症因子表达的方法，所述方法包括：
利用抗血管生成素样蛋白4抗体制备抑制炎症因子表达，所述炎症因子包括白介素-6。
本申请中，可利用抗血管生成素样蛋白4抗体作为抑制剂应用于白介素-6(IL-6)相关行为的基础研究中，如构建白介素-6(IL-6)低表达动物模型。
与现有技术相比，本申请具有以下有益效果：
本申请首次发现抗ANGPTL4抗体可抑制炎症因子白介素-6(IL-6)的表达，能够缓解关节局部的滑膜增生和炎症细胞的浸润，兼具抑制骨破坏的作用，可应用于制备治疗骨及关节炎症疾病的药物，弥补目前临床上多数RA药物作用效果单一的缺点，为治疗类风湿关节炎提供新思路、新手段，对于类风湿关节炎治疗领域具有重要意义。
附图说明
图1为小鼠体重变化图。
图2为小鼠关节肿胀临床评分结果图。
图3为小鼠血清中ANGPTL4表达量图。
图4为小鼠血清中IL-6表达量图。
图5为小鼠踝骨关节Micro-CT的三维图。
图6为小鼠踝骨关节病理切片组织学染色图。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
本申请具体通过RA研究领域经典的CIA动物模型进行了药物的药效学验证。在实验过程中，通过测量体重和足爪肿胀评价了抗ANGPTL4抗体对关节肿胀的缓解作用以及该治疗剂量下的毒性作用，通过对动物模型所收集的血清学检测分析细胞因子IL-6的表达；通过Micro-CT扫描数据以及组织学染色结果分析抗ANGPTL4抗体治疗对RA的踝关节的骨破坏的影响。
实施例1
本实施例进行模型构建以及抗ANGPTL4抗体治疗干预。
购买SPF级8周龄的雄性DBA/1免疫正常小鼠32只，饲养于SPF级动物房，小鼠按平均体重进行随机分组，每组8只；将小鼠分为空白对照组(CON)，模型组(CIA)，低剂量治疗组(50mg/kg)、高剂量治疗组(200mg/kg)，配制含有2mg/mL的牛II型胶原和4mg/mL的完全氟氏佐剂的免疫程序诱导剂，充分乳化后，除空白对照组外，其余组在小鼠尾部进行皮下注射100μL免疫程序诱导剂，进行CIA模型的构建；造模后第3周开始，按照前述剂量经腹腔注射予以抗ANGPTL4抗体(氨基酸序列参见US10160803B2)治疗，每周给药2次，模型组注射PBS作为对照，直至终点取材。整个实验维持7周，每周进行体重 以及足爪肿胀测量，体重测量结果如图1所示。在造模后第1周，小鼠体重均有不同程度下降；造模后第3周开始腹腔注射给药治疗，各组小鼠恢复正常增重的生理状态，200mg/kg治疗组小鼠体重增长率高于CIA组，表明该抗体治疗无明显的药物毒性。关节肿胀临床评分(具体方法参考文献：N.Choudhary,L.K.Bhatt,and K.S.Prabhavalkar,Experimental animal models for rheumatoid arthritis.Immunopharmacology and Immunotoxicology 40(2018)193-200.)，结果如图2所示，表明抗ANGPTL4抗体治疗可缓解CIA动物模型中足爪肿胀的临床症状。
实施例2
本实施例进行ANGPTL4表达量及炎症因子表达变化的检测。
取实施例1造模7周后小鼠，麻醉小鼠后，进行心脏取血，静置分离获取血清；使用ELISA检测试剂盒，检测血清中ANGPTL4表达量变化以及细胞因子IL-6的含量变化。ANGPTL4表达量变化结果如图3所示，与CIA组相比，低剂量治疗组(50mg/kg)、高剂量治疗组(200mg/kg)的小鼠血清ANGPTL4表达量显著降低，表明可有效中和血清中的ANGPTL4。细胞因子IL-6的含量变化结果如图4所示，结果表明，与CIA模型组相比，经过200mg/kg的抗ANGPTL4抗体治疗后，小鼠血清中炎症因子IL-6的表达水平下降。
实施例3
本实施例进行Micro-CT分析。
取实施例1造模7周后小鼠，对小鼠进行安乐死，解剖分离脚踝组织，取出胫骨前后肌肉，从胫骨中断截断，保留小鼠完整的足爪及踝关节，用10％福尔马林固定。采用Micro-CT(SKYSCAN 1176，Bruker，Belgium)扫描小鼠踝关节，对扫描数据重建后观察各组关节破损程度，结果如图5所示，CON组关节表面光滑且完整；CIA组骨表面粗糙且骨破坏现象严重，50mg/kg和200mg/kg抗ANGPTL4抗体治疗后，指端关节骨破坏情况改善，表明抗ANGPTL4抗体有抑制炎性骨破坏的治疗效果，且该治疗效果呈现剂量依赖性。
实施例4
本实施例进行关节局部的组织学染色分析。
将实施例3已完成CT扫描的骨组织依次进行脱钙、脱水及常规的石蜡包埋后，制作组织切片后进行HE染色。结果如图6所示，CON组动物踝关节软骨表面光滑，关节腔完整，关节间隙明显；CIA造模组动物组踝关节处可见大量 炎性细胞浸润，关节间隙变窄，软骨表面破坏，滑膜组织增生；而抗ANGPTL4抗体治疗组，关节腔破坏程度和炎性细胞浸润程度缓解，骨破坏程度减轻。
综上所述，本申请通过构建胶原诱导性关节炎(CIA)的小鼠动物模型，模拟RA的疾病状态，应用不同剂量的抗ANGPTL4抗体对CIA模型进行治疗干预，通过测量体重和足爪肿胀评价了抗ANGPTL4抗体对关节肿胀的缓解作用以及该治疗剂量下的毒性作用，通过对动物模型的血清检测表明，抗ANGPTL4抗体治疗可以明显降低细胞因子IL-6的表达；Micro-CT扫描数据以及组织学染色结果显示，抗ANGPTL4抗体治疗可以有效改善RA的踝关节的骨破坏，并可以缓解关节局部的滑膜增生和炎症细胞的浸润，证明了抗ANGPTL4抗体可应用于制备治疗骨及关节炎症疾病的药物。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (10)

  1. 抗血管生成素样蛋白4抗体在制备抑制炎症因子表达的药物中的应用。
  2. 根据权利要求1所述的应用，其中，所述炎症因子包括白介素-6。
  3. 根据权利要求1所述的应用，其中，所述抗血管生成素样蛋白4抗体靶向结合血管生成素样蛋白4的flANGPTL4片段、nANGPTL4片段或cANGPTL4片段中任意一种。
  4. 根据权利要求1所述的应用，其中，所述药物还包括辅料。
  5. 根据权利要求4所述的应用，其中，所述辅料包括药学上可接受的载体、润湿剂、崩解剂、乳化剂、增溶剂、渗透压调节剂、表面活性剂、包衣材料、着色剂、pH调节剂、抗氧化剂、抑菌剂或缓冲剂中的任意一种或至少两种的组合。
  6. 抗血管生成素样蛋白4抗体在制备治疗慢性骨及关节炎症疾病的药物中的应用。
  7. 根据权利要求6所述的应用，其中，所述慢性骨及关节炎症疾病包括类风湿关节炎、骨关节炎、骨质增生、肩周炎、骨膜炎、滑囊炎或滑膜炎中任意一种或至少两种的组合。
  8. 抗血管生成素样蛋白4抗体在制备治疗骨破坏疾病的药物中的应用。
  9. 根据权利要求8所述的应用，其中，所述骨破坏疾病包括股骨头坏死、难愈性骨折或肿瘤骨转移中的任意一种或至少两种的组合。
  10. 一种以非疾病诊断和/或治疗为目的的抑制炎症因子表达的方法，其包括：
    利用抗血管生成素样蛋白4抗体抑制炎症因子表达；
    其中，所述炎症因子包括白介素-6。
PCT/CN2022/132616 2022-11-03 2022-11-17 抗血管生成素样蛋白4抗体在制备抑制炎症因子表达的药物中的应用 WO2024092888A1 (zh)

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