WO2019140937A1 - 一种新型酪氨酸激酶抑制剂-安罗替尼在骨肉瘤的应用 - Google Patents

一种新型酪氨酸激酶抑制剂-安罗替尼在骨肉瘤的应用 Download PDF

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WO2019140937A1
WO2019140937A1 PCT/CN2018/106625 CN2018106625W WO2019140937A1 WO 2019140937 A1 WO2019140937 A1 WO 2019140937A1 CN 2018106625 W CN2018106625 W CN 2018106625W WO 2019140937 A1 WO2019140937 A1 WO 2019140937A1
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osteosarcoma
administration
anlotinib
erlotinib
use according
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PCT/CN2018/106625
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French (fr)
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华莹奇
王刚阳
蔡郑东
王卓莹
张涛
汪红胜
孙梦熊
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上海市第一人民医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the invention relates to the field of medicine, in particular to the application of a novel tyrosine kinase inhibitor, rantinib, in osteosarcoma.
  • Anlotinib is a small molecule multi-target tyrosine kinase inhibitor developed by China with independent intellectual property rights. Its compound patent number is WO2008/112407. Clinical approval in 2011, phase I clinical trial Completed in 2013, a variety of cancer clinical trials of erlotinib are underway, including non-small cell lung cancer, soft tissue sarcoma, gastric cancer, colorectal cancer, medullary thyroid carcinoma, differentiated thyroid cancer, esophageal squamous cell carcinoma.
  • a first object of the present invention is to provide a new use of erlotinib for the deficiencies in the prior art.
  • the technical solution adopted by the present invention is: the use of erlotinib and a pharmaceutically acceptable salt thereof for the preparation of a medicament for inhibiting or treating osteosarcoma.
  • erlotinib and its pharmaceutically acceptable salts as a synergist for cisplatin inhibition or treatment of osteosarcoma.
  • the pharmaceutically acceptable salt of erlotinib is ilotinib HCl.
  • the medicament inhibits the growth or metastasis of osteosarcoma.
  • the transfer is migration or invasion.
  • the osteosarcoma is a primary osteosarcoma or a secondary osteosarcoma.
  • the osteosarcoma is an osteogenic osteosarcoma or an osteolytic osteosarcoma.
  • the osteosarcoma is an osteoblast type osteosarcoma, a chondrocyte osteosarcoma or a fibroblast type osteosarcoma.
  • the medicament contains a therapeutically effective amount of ilotinib and a pharmaceutically acceptable carrier.
  • Another object of the present invention is to provide a method of treating osteosarcoma diseases by administering to a subject having osteosarcoma a therapeutically effective amount of ilotinib or a pharmaceutically acceptable salt thereof.
  • the method simultaneously administers a therapeutically effective amount of cisplatin to an individual having an osteosarcoma.
  • the administration route is oral, intravenous, rectal, aerosol, parenteral, ocular administration, pulmonary administration, transdermal administration, vaginal administration, and ear canal administration. Nasal administration or topical administration.
  • the individual is a mammal.
  • the mammal is selected from the group consisting of a human, a mouse, a monkey, a horse, a cow, a pig, a dog, and a cat.
  • the "osteosarcoma” is a primary tumor of a relatively high degree of malignant bone, which is characterized in that the tumor cells directly form bone-like tissue, so it is also called osteosarcoma, but the osteogenesis process of the tumor is not Obviously, osteosarcoma cannot be ruled out.
  • osteosarcoma includes low-grade central osteosarcoma, conventional osteosarcoma, and telangiectal osteosarcoma. , Small cell osteosarcoma, Secondary osteosarcoma, Parosteal osteosarcoma, Periosteal osteosarcoma, High-grade surface osteosarcoma Among them, Conventional osteosarcoma is divided into chondroblastic osteosarcoma, Fibroblastic osteosarcoma and Osteoblastic osteosarcoma.
  • pharmaceutically acceptable salt refers to the presence of a compound that does not cause significant irritation to the administered organism and does not delete the biological activity and properties of the compound.
  • the salt is formed using, but not limited to, the following inorganic or organic acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, ethanol Acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, mandelic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, benzoic acid, Hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid and toluenesulfonic acid.
  • the pharmaceutically acceptable salt can be prepared by a conventional method, for example, by dissolving ilotinib in a water-miscible organic solvent such as acetone, methanol, ethanol and acetonitrile, and adding an excess of an organic or inorganic acid thereto. An aqueous solution is allowed to precipitate the salt from the resulting mixture, from which the solvent and the remaining free acid are removed, and then the precipitated salt is separated.
  • a water-miscible organic solvent such as acetone, methanol, ethanol and acetonitrile
  • inhibiting includes any effect of ameliorating a condition, disease, disorder, etc., such as reducing, reducing, regulating, ameliorating or eliminating, or ameliorating the symptoms thereof.
  • the term "effective amount” means the amount of a drug or agent (ie, erlotinib) that will cause a biological or medical response, such as a tissue, system, animal or human, as sought by a researcher or clinician.
  • the term “effective therapeutic amount” means an amount that results in an improved treatment, cure, prevention or alleviation of a disease, disorder or side effect, or is reduced in comparison to a corresponding subject who does not receive the above amount. The rate at which a disease or condition progresses.
  • pharmaceutically acceptable carrier refers to a medium that is generally accepted in the art for delivery of a biologically active agent to an animal, particularly a mammal, including adjuvants, excipients or vehicles, such as diluents, preservatives.
  • adjuvants such as diluents, preservatives.
  • Agents fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungals, lubricants and dispersing agents, etc.
  • a biologically active agent to an animal, particularly a mammal
  • adjuvants such as diluents, preservatives.
  • Agents fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungals, lubricants and dispersing agents, etc.
  • sweeteners such as a
  • the present invention finds for the first time that rolotinib can inhibit the growth and metastasis of osteosarcoma.
  • Erlotinib can significantly inhibit the cell growth of osteosarcoma cell lines 143B, U2OS, MG63 and SJSA, induce its cycle arrest, and also inhibit osteosarcoma cell migration and invasion.
  • Erlotinib significantly inhibited the increase in tumor volume and weight in the osteosarcoma 143B in situ model, while significantly inhibiting lung metastasis of the tumor.
  • the present invention finds for the first time that erlotinib can enhance the killing effect of chemotherapeutic drug cisplatin on osteosarcoma cells.
  • Human osteosarcoma cell line 143B, U2OS, MG63 and SJSA were purchased from the American Type Culture Collection (ATCC).
  • Anlotinib (a novel tyrosine kinase inhibitor) hydrochloride (Anlotinib Dihydrochloride) is sourced from Zhengda Tianqing Pharmaceutical Group Co., Ltd., which is dissolved in dimethyl sulfoxide. The working mother liquor of 16 mmol/L was placed in a refrigerator at -20 ° C for use, and the DMEM serum culture solution was adjusted to the desired concentration before use.
  • DMEM high glucose medium, fetal bovine serum (American company); cleaved-Caspase3, p-PARP antibody, GAPDH antibody, horseradish peroxidase labeled secondary antibody (Sisi Biotech Co., Ltd.); Dimethyl sulfoxide (Sigma, USA); 0.02% EDTA + 0.25% trypsin (Miltenyi Biotec, Germany); constant temperature incubator (Shanghai Rongyan Instrument Co., Ltd.); flow cytometry (Becton Dickinson, USA); Microplate reader (Molecular Devices, USA).
  • Osteosarcoma cell line 143B, U2OS, MG63 and SJSA were cultured in DMEM complete medium (containing 10% fetal bovine serum, 0.1 g/L streptomycin, 100 U/mL penicillin) at 37 ° C, volume fraction Incubate in a 5% CO 2 incubator. When the cell fusion degree reached about 85%, the cells were digested with 0.02% EDTA + 0.25% trypsin, and then the cells were collected, centrifuged at 1000 r/min for 3 min, and subcultured.
  • DMEM complete medium containing 10% fetal bovine serum, 0.1 g/L streptomycin, 100 U/mL penicillin
  • the 143B, U2OS, MG63 and SJSA cells in the logarithmic growth phase were collected, washed, counted, and divided into PBS.
  • the cells were added to a 96-well plate at a rate of 3000 cells/well. The cells were adhered for 24 hours, and then the concentration of different substances was added.
  • Anlotinib (0, 1, 2, 4, 8, 16, 32, 64 ⁇ M) was cultured for 24 h and then tested. 100 ⁇ L of CCK-8 reagent diluted 10 times with DMEM medium was added to each well, and incubated at 37 ° C for 45 minutes. The absorbance value of each well at 450 nm was measured using a microplate reader.
  • 143B, U2OS, MG63 and SJSA cells were added to a medium containing different concentrations of Anlotinib hydrochloride (0, 1, 2, 4 ⁇ M) for culture. After 24 hours, the cells were collected, centrifuged at 1000 r/min for 1 min, and washed with PBS. The cells were fixed in 70% ethanol, added to the periodic kit PI staining solution, incubated at room temperature for 15 min in the dark, and detected by flow cytometry. Images were analyzed using ModFit software. The experiment was repeated 3 times.
  • 143B, U2OS, MG63 and SJSA cells were added to a culture medium of 2 ⁇ M Anlotinib hydrochloride (with or without 10 ⁇ M cisplatin (DDP)). After 24 hours, the cells were collected, centrifuged at 1000 r/min for 1 min, and washed with PBS. Then, the apoptosis was detected by Annexin-V-FITC/PI apoptosis detection kit. The cells were resuspended in 100 ⁇ L of 1 ⁇ Binding buffer, and 5 ⁇ L of Annexin V-FITC and 2.5 ⁇ L of PI dye were added to protect from light. Shake well and react at room temperature for 15 min, then add 300 ⁇ L of 1 ⁇ Binding buffer, mix, and test by flow cytometry. The test was repeated 3 times.
  • Anlotinib enhances apoptosis of osteosarcoma cells induced by cisplatin (DDP)
  • Anlotinib can significantly inhibit the cell growth of osteosarcoma cell lines 143B, U2OS, MG63 and SJSA, induce its cycle arrest, and also inhibit osteosarcoma cell migration and invasion. Further studies have shown that Anlotinib can also enhance the killing effect of chemotherapeutic drug cisplatin on osteosarcoma cells.
  • Human osteosarcoma cell 143B-luciferase was purchased from the American Type Culture Collection (ATCC).
  • Erlotinib HCl is derived from Zhengda Tianqing Pharmaceutical Group Co., Ltd., and is dissolved in physiological saline to a desired concentration before use.
  • the BALB/c-nu nude mouse was purchased from Shanghai Slack Laboratory Animals Co., Ltd. and was raised at the SPF Laboratory Animal Center of the First People's Hospital affiliated to Shanghai Jiaotong University.
  • 143B-luciferase cells grown in log phase were digested and centrifuged, and 143B-luciferase cells were resuspended in 1.5 mL EP tubes with 4°C pre-cooled PBS at a density of 10 4 / ⁇ L cells.
  • Prepared 143B-luciferase cells were injected into 100 ⁇ L (10 6 cells) of each nude mouse into the medullary cavity of the right tibia of BALB/c-nu nude mice.
  • the nude mice that were successfully modeled were randomly divided into 3 groups of 5 each.
  • the control group was intraperitoneally injected with physiological saline every two days for a dose of 100 ⁇ L; the experimental group was intraperitoneally injected with Anlotinib solution every two days at a dose of 2, 4 mg/kg of Anlotinib hydrochloride every two days for a dose of 100 ⁇ L.
  • the volume of the tumor and the growth state of the nude mice were recorded daily.
  • the lung growth of the tumor growth in nude mice was detected by the IVIS Lumina Imaging System.
  • the tumor volume of the control nude mice was as long as 1500 mm 3 (about 28 days)
  • the nude mice were sacrificed, and the tumor tissues and important organs were weighed and placed in liquid nitrogen and 4% paraformaldehyde (PFA), respectively.
  • PFA paraformaldehyde
  • xylene I room temperature, 5 minutes
  • xylene II room temperature, 5 minutes
  • xylene ethanol (1:1) (room temperature, 5 Minutes), 100% ethanol (room temperature, 3 minutes), 95% ethanol (room temperature, 3 minutes), 85% ethanol (room temperature, 3 minutes), 75% ethanol (room temperature, 3 minutes).
  • paraffin tissue sections were sequentially subjected to double distilled water (room temperature, 10 seconds), hydrochloric acid alcohol solution (room temperature, 10 seconds), and the running tap water was reversed for 20 minutes.
  • Paraffin tissue sections were treated with eosin solution for 1-3 minutes (depending on color change).
  • the lung metastasis of tumor growth in nude mice was detected by the IVIS Lumina Imaging System.
  • the results (a in Figure 9) showed that on the 14th day of the first injection of the drug, 2 nude mice in the control group developed lung metastasis, while the nude mice in the Anlotinib-treated group did not have lung metastasis; on the 28th day of the first injection of the drug In the control group, 4 nude mice showed lung metastasis, while only one of the nude mice in the Anlotinib treatment group (2 mg/kg) showed lung metastasis, and the nude mice in the Anlotinib treatment group (4 mg/kg) showed no lung metastasis.
  • the normalized photon flux statistics showed that the photon flux of the tumor tissue of the nude mice in the Anlotinib treatment group was significantly slower than that of the control group, and on the 28th day of the first injection, compared with the control group. Comparing, the photon flux of tumor tissue in nude mice treated with 2, 4 mg/kg Anlotinib every two days was significantly lower than that of the control nude mice (P ⁇ 0.05; P ⁇ 0.01).
  • Anlotinib significantly inhibits the growth and metastasis of osteosarcoma.

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Abstract

安罗替尼及其药学上可接受的盐在制备抑制或治疗骨肉瘤的药物中的应用。

Description

一种新型酪氨酸激酶抑制剂-安罗替尼在骨肉瘤的应用 技术领域
本发明涉及药物领域，具体地说，是一种新型酪氨酸激酶抑制剂-安罗替尼在骨肉瘤的应用。
背景技术
安罗替尼(Anlotinib)是由我国研发的、具有自主知识产权的小分子多靶点酪氨酸激酶抑制剂，其化合物专利公开号为WO2008/112407。2011年获得临床审批，I期临床试验于2013年完成，安罗替尼多种癌症临床试验正在开展当中，包括非小细胞肺癌、软组织肉瘤、胃癌、结直肠癌、甲状腺髓样癌、分化型甲状腺癌、食管鳞癌等。
国际公布号：WO2008/112407、中国专利号：ZL200880007358.X的专利文献公开了安罗替尼对结肠癌、非小细胞肺癌、肝癌、乳腺癌的抑制活性。但是关于安罗替尼在骨肉瘤中的应用目前还未见报道。
发明内容
本发明第一个目的是，针对现有技术中的不足，提供安罗替尼的新用途。
为实现上述目的，本发明采取的技术方案是：安罗替尼及其药学上可接受的盐在制备抑制或治疗骨肉瘤的药物中的应用。
安罗替尼及其药学上可接受的盐作为顺铂抑制或治疗骨肉瘤的增效剂的应用。
进一步地，安罗替尼药学上可接受的盐为盐酸安罗替尼。
进一步地，所述的药物抑制骨肉瘤生长或转移。
进一步地，所述的转移为迁移或侵袭。
进一步地，所述的骨肉瘤为原发性骨肉瘤或继发性骨肉瘤。
进一步地，所述的骨肉瘤为成骨型骨肉瘤或溶骨型骨肉瘤。
进一步地，所述的骨肉瘤为成骨细胞型骨肉瘤、成软骨细胞型骨肉瘤或成纤维细胞型骨肉瘤。
进一步地，所述的药物中含有有效治疗量的安罗替尼和药学上可接受的载体。
本发明另一目的是，提供一种治疗骨肉瘤疾病的方法，所述的方法为向患有骨肉瘤的个体给予有效治疗量的安罗替尼或其药学上可接受的盐。
作为一个优选例，所述的方法向患有骨肉瘤的个体同时给予有效治疗量的顺铂。
作为另一优选例，给药途径为口服、静脉注射、直肠、气雾剂、非肠道给药、眼部给药、肺部给药、经皮给药、阴道给药、耳道给药、鼻腔给药或局部给药。
作为另一优选例，所述的个体为哺乳动物。
优选地，所述的哺乳动物选自人、鼠类、猴、马、牛、猪、犬和猫。
本发明术语解释如下：
所述“骨肉瘤”(osteosarcoma,OS)是恶性程度较高的骨的原发性肿瘤，其特点是瘤细胞直接形成骨样组织，故也称为成骨肉瘤，但肿瘤的成骨过程不明显者也不能排除骨肉瘤。
根据WHO骨肿瘤分类(2013年，第4版)，骨肉瘤包含低级别中心型骨肉瘤(Low-grade central osteosarcoma)、普通型骨肉瘤(Conventional osteosarcoma)、毛细血管扩张型骨肉瘤(Telangiectatic osteosarcoma)、小细胞骨肉瘤(Small cell osteosarcoma)、继发性骨肉瘤(Secondary osteosarcoma)、骨旁骨肉瘤(Parosteal osteosarcoma)、骨膜骨肉瘤(Periosteal osteosarcoma)、高级别表面骨肉瘤(High-grade surface osteosarcoma)，其中普通型骨肉瘤(Conventional osteosarcoma)又分为成软骨型骨肉瘤(Chondroblastic osteosarcoma)、成纤维型骨肉瘤(Fibroblastic osteosarcoma)和成骨型骨肉瘤(Osteoblastic osteosarcoma)。
术语“药学上可接受的盐”指一种化合物的存在形式，该形式不会引起对给药有机体的重要的刺激，且不会使化合物的生物活性和性质消失。当所述安罗替尼形成药学上可接受的盐的形式时，所述盐使用但不限于以下的无机酸或有机酸而形成：盐酸、氢溴酸、硫酸、磷酸、硝酸、乙酸、乙醇酸、乳酸、丙酮酸、丙二酸、琥珀酸、戊二酸、富马酸、苹果酸、扁桃酸、酒石酸、柠檬酸、抗坏血酸、棕榈酸、马来酸、羟基马来酸、苯甲酸、羟基苯甲酸、苯乙酸、肉桂酸、水杨酸、甲磺酸、苯磺酸和甲苯磺酸。药学上可接受的盐可通过常规方法制备，例如通过将安罗替尼溶解于与水可混溶的有机溶剂(例如丙酮、甲醇、乙醇和乙腈)，向其中添加过量的有机酸或无机酸水溶液，以使得盐从所得混合物中沉淀，从中除去溶剂和剩余的游离酸，然后分离所沉淀的盐。
术语“抑制”或“治疗”包括改善病症、疾病、障碍等的任何效果，例如减轻、减少、调节、改善或消除，或改善其症状。
术语“有效量”意指将会引起例如研究人员或临床医师所寻求的组织、系统、动物或人的生物学或医学响应的药物或药剂(即安罗替尼)的量。此外，术语“有效治疗量”意指这样的量：与未接受上述量的相应受试者相比，所述量引起改善性的治疗、治愈、预防或减轻疾病、病症或副作用，或降低在疾病或病症的进展速度。
术语“药学上可接受的载体”是指本领域中通常接受用于将生物活性剂递送至动物(具体为哺乳动物)的介质，包括佐剂、赋形剂或媒介物，诸如稀释剂、防腐剂、填充剂、流动调控剂、崩解剂、润湿剂、乳化剂、悬浮剂、增甜剂、矫味剂、芳香剂、抗细菌剂、抗真菌剂、润滑剂和分散剂等，这取决于给药途径和剂型的性质。
本发明优点在于：
1、本发明首次发现安罗替尼可抑制骨肉瘤生长和转移。安罗替尼能够明显抑制骨肉瘤细胞系143B、U2OS、MG63和SJSA的细胞生长，诱导其周期阻滞，同时也能够抑制骨肉瘤细胞迁移和侵袭。安罗替尼能够明显抑制骨肉瘤143B原位模型肿瘤体积和重量的增加，同时明显抑制肿瘤的肺转移。
2、本发明首次发现安罗替尼能够增强化疗药物顺铂对骨肉瘤细胞的杀伤作用。
附图说明
附图1：安罗替尼抑制骨肉瘤细胞增殖，*，P<0.05。
附图2：安罗替尼引起骨肉瘤细胞周期阻滞。
附图3：安罗替尼引起骨肉瘤细胞周期阻滞，**，P<0.05。
附图4：安罗替尼增强顺铂(DDP)引起的骨肉瘤细胞凋亡。
附图5：安罗替尼增强顺铂(DDP)引起的骨肉瘤细胞凋亡，*，P<0.05；**，P<0.05。
附图6：安罗替尼抑制骨肉瘤细胞的迁移和侵袭。
附图7：安罗替尼抑制骨肉瘤细胞的迁移和侵袭，*，P<0.05。
附图8：安罗替尼抑制骨肉瘤的生长，*，P<0.05；**，P<0.05。
附图9：安罗替尼抑制骨肉瘤的生长和肺转移，*，P<0.05；**，P<0.05。
附图10：安罗替尼抑制骨肉瘤的肺转移，*，P<0.05；**，P<0.05。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1 细胞实验
1材料和方法
1.1材料
人骨肉瘤细胞株143B，U2OS，MG63和SJSA购自美国菌种保藏中心(ATCC)。Anlotinib(一种新型酪氨酸激酶抑制剂)的盐酸盐(盐酸安罗替尼，Anlotinib Dihydrochloride)来源于正大天晴药业集团股份有限公司，将其溶于二甲基亚砜中，配置成16mmol/L的工作母液，置于-20℃的冰箱中备用，使用前用DMEM血清培养液调至所需浓度。
实验试剂及仪器：DMEM高糖培养基，胎牛血清(美国Thermo公司)；cleaved-Caspase3，p-PARP抗体，GAPDH抗体，辣根过氧化物酶标记二抗(斯信生物科技有限公司)；二甲基亚砜(美国Sigma公司)；0.02％EDTA+0.25％胰蛋白酶(德国Miltenyi Biotec公司)；恒温培养箱(上海茸研仪器公司)；流式细胞仪(美国Becton Dickinson公司)；多功能酶标仪(美国Molecular Devices公司)。
1.2实验方法
1.2.1细胞培养
将骨肉瘤细胞株143B，U2OS，MG63和SJSA培养在DMEM完全培养液中(含有体积分数为10％胎牛血清、0.1g/L链霉素、100U/mL青霉素)，于37℃、体积分数为5％CO 2恒温培养箱中培养。待细胞融合度达到85％左右时，用0.02％EDTA+0.25％胰蛋白酶混合消化液进行消化，然后收集细胞，1000r/min离心3min，传代培养。
1.2.2CCK-8检测细胞增殖情况
收集对数生长期的143B，U2OS，MG63和SJSA细胞，PBS清洗，计数，分管，按3000个/孔细胞数加入96孔板中，培养24h使细胞贴壁，之后加入不同物质的量浓度盐酸Anlotinib(0，1，2，4，8，16，32，64μM)进行培养24h，然后进行检测。每孔加入用DMEM培养基稀释10倍的CCK-8试剂100μL，于37℃孵育45分钟。使用酶标仪检测各孔在450nm处的吸光度值。
1.2.3平板克隆实验
用1μM的盐酸Anlotinib对143B，U2OS，MG63和SJSA细胞进行处理，吸去上层液体，用4％多聚甲醛固定15min，PBS漂洗3次，用0.1％结晶紫染色10min，PBS漂洗2次，风干后拍照。
1.2.4流式细胞仪检测细胞周期
将143B，U2OS，MG63和SJSA细胞加入到含有不同物质的量浓度盐酸Anlotinib(0，1，2，4μM)的培养液中进行培养，24h后收集细胞，1000r/min离心1min，PBS清洗。用70％乙醇固定，加入周期试剂盒PI染液，室温避光孵育15min，用流式细胞仪进行检测。图像使用ModFit软件进行分析。重复实验3次。
1.2.5流式细胞仪检测细胞凋亡
将143B，U2OS，MG63和SJSA细胞加入到2μM的盐酸Anlotinib(含有或者不含有10μM顺铂(DDP))的培养液中进行培养，24h后收集细胞，1000r/min离心1min，PBS清洗。然后使用Annexin-V-FITC/PI细胞凋亡检测试剂盒检测其凋亡情况，将细胞加入到100μL 1×Binding缓冲液中重悬，添加5μL Annexin V-FITC和2.5μL PI染料，进行 避光振荡混匀，室温反应15min，然后再加入300μL 1×Binding缓冲液，混匀，上流式细胞仪进行检测。重复试验3次。
1.2.6Transwell细胞迁移及侵袭实验分析
5×10 4细胞/孔种在Transwell小室的上端，采用1μM盐酸Anlotinib处理143B，U2OS，MG63和SJSA细胞，Transwell小室的上端(侵袭实验需要用10％matrigel预处理4小时)加入2％血清的培养基，下端加入含10％血清的培养基。24小时后，4％多聚甲醛固定，结晶紫染色，显微镜下拍照。
2结果
2.1Anlotinib抑制骨肉瘤细胞增殖
为了评估Anlotinib抗骨肉瘤细胞系143B，U2OS，MG63和SJSA生长的作用，0，1，2，4μM浓度的盐酸Anlotinib处理骨肉瘤细胞系24或48小时，结果(图1)显示，随着Anlotinib浓度的升高，细胞的增殖率下降，相同作用浓度下不同作用时间下Anlotinib对骨肉瘤细胞系的增长也有明显的抑制作用。以上结果表明，Anlotinib对骨肉瘤细胞系的抑制具有明显的抑制作用，并且呈现时间和浓度依赖性。
表1
Figure PCTCN2018106625-appb-000001
2.2Anlotinib引起骨肉瘤细胞周期阻滞
为了研究Anlotinib抑制骨肉瘤增殖的机制，我们以骨肉瘤细胞系143B，U2OS，MG63和SJSA为靶细胞进行细胞周期的检测，结果(图2-3)表明，经过Anlotinib的处理，细胞发生了G2/M周期阻滞。同时伴随着G1期的减少。
2.3Anlotinib增强顺铂(DDP)引起的骨肉瘤细胞凋亡
接下来，我们探究了Anlotinib对骨肉瘤细胞系化疗增敏的作用，将2μM的盐酸Anlotinib，采用经典的Annexin-V-FITC/PI细胞凋亡检测方法，结果(图4-5)表明，Anlotinib能够显著增强顺铂引起的骨肉瘤细胞系的凋亡。
2.4Anlotinib抑制骨肉瘤细胞的迁移和侵袭
为了研究Anlotinib对骨肉瘤细胞系迁移和侵袭的影响，我们采用Transwell分析，结果(图6-7)表明，经过Anlotinib 24小时的处理，Anlotinib大大抑制了骨肉瘤细胞系的迁移和侵袭。
3结论
Anlotinib能够明显抑制骨肉瘤细胞系143B，U2OS，MG63和SJSA的细胞生长，诱导其周期阻滞，同时也能够抑制骨肉瘤细胞迁移和侵袭。进一步研究表明，Anlotinib也能够增强化疗药物顺铂对骨肉瘤细胞的杀伤作用。
实施例2 动物实验
1材料和方法
1.1材料
人骨肉瘤细胞143B-luciferase购自美国菌种保藏中心(ATCC)。盐酸安罗替尼，来源于正大天晴药业集团股份有限公司，使用前将其溶于生理盐水配置成所需浓度。
BALB/c-nu裸鼠购自，购买于上海斯莱克实验动物有限责任公司，饲养于上海交通大学附属第一人民医院标准的SPF级实验动物中心。
实验试剂及仪器：动物活体成像系统IVIS Lumina Imaging System(Xenogen)。
1.2实验方法
1.2.1建立骨肉瘤143B原位模型
取对数期生长的143B-luciferase细胞，消化离心后，将143B-luciferase细胞用4℃预冷过的PBS重悬于1.5mL EP管中，密度为10 4/μL细胞。将准备好的143B-luciferase细胞按照每只裸鼠100μL(10 6个细胞)注射到BALB/c-nu裸鼠的右侧胫骨的骨髓腔内。注射技巧：BALB/c-nu裸鼠异氟烷气体麻醉后，将裸鼠右侧膝关节屈膝90度，沿着裸鼠髌韧带由胫骨结节后方沿着骨髓腔由近端向远端缓慢进针，轻轻推入准备好的细胞悬液，边推打边推针，推入完毕后，停留10秒钟，避免细胞溢出。每天记录观察裸鼠肿瘤变化，待肿瘤体积长至200mm 3时即建模成功。
1.2.2分组及给药
取建模成功的裸鼠，随机分成3组，每组5只。对照组每两天腹腔注射生理盐水，给药体积100μL；实验组按照每两天2、4mg/kg盐酸Anlotinib的剂量，每两天腹腔注射盐酸Anlotinib溶液，给药体积100μL。
每天记录肿瘤的体积和裸鼠的生长状态。肿瘤体积计算公式如下：肿瘤体积＝肿瘤长×肿瘤宽 2/2。
同时在首次注射药物的第0，7，14，21，28天，用动物活体成像系统IVIS Lumina Imaging System分别检测裸鼠肿瘤生长已经肺转移情况。等到对照组裸鼠肿瘤体积长到1500mm 3时候(约28天)，处死裸鼠，取肿瘤组织和重要脏器称重，分别放入液氮和4％多聚甲醛(PFA)中，以备后续实验。
1.2.3H&E染色(苏木素-伊红染色)
(1)染片前先进行脱蜡，将石蜡切片依次经：二甲苯I(室温，5分钟)，二甲苯II(室温，5分钟)，二甲苯:乙醇(1:1)(室温，5分钟)，100％乙醇(室温，3分钟)，95％乙醇(室温，3分钟)，85％乙醇(室温，3分钟)，75％乙醇(室温，3分钟)。
(2)将石蜡组织切片置于苏木精染色(室温，5分钟)。
(3)将石蜡组织切片依次经过双蒸水(室温，10秒)，盐酸酒精溶液(室温，10秒)，流动自来水反蓝20分钟。
(4)反蓝后石蜡组织切片依次经过75％乙醇(室温，3分钟)，85％乙醇(室温，3分钟)，95％乙醇(室温，3分钟)，100％乙醇(室温，3分钟)脱水。
(5)石蜡组织切片经伊红溶液1-3分钟(根据颜色变化决定时间)。
(6)脱水，将石蜡切片依次经：75％乙醇(室温，3分钟)，85％乙醇(室温，3分钟)，95％乙醇(室温，3分钟)，100％乙醇(室温，3分钟)，二甲苯:乙醇(1:1)(室温，5分钟)，二甲苯II(室温，5分钟)，二甲苯I(室温，5分钟)。
(7)脱水后石蜡切片中性树脂封片。
2结果
2.1Anlotinib抑制骨肉瘤的生长
处死裸鼠后，解剖取下裸鼠后肢称重，结果(图8)显示，与对照组裸鼠相比较，每两天给予2、4mg/kg Anlotinib治疗的裸鼠后肢体积显著减小，重量显著降低(P<0.05；P<0.01)，以上结果表明，Anlotinib能明显抑制骨肉瘤的生长。
2.2Anlotinib抑制骨肉瘤的转移
在首次注射药物的第0，7，14，21，28天，用动物活体成像系统IVIS Lumina Imaging System分别检测裸鼠肿瘤生长的肺转移情况。结果(图9中a)显示，在首次注射药物的第14天，对照组有2只裸鼠出现肺转移，而Anlotinib治疗组的裸鼠均未出现肺转移；在首次注射药物的第28天，对照组有4只裸鼠出现肺转移，而Anlotinib治疗组(2mg/kg)的裸鼠只有1只出现肺转移，Anlotinib治疗组(4mg/kg)的裸鼠均未出现肺转移。归一化光子通量统计结果(图9中b)显示，Anlotinib治疗组裸鼠肿瘤组织的光子通量增加要明显慢于对照组，至首次注射药物的第28天，与对照组裸鼠相比较，每两天给予2、4mg/kg Anlotinib治疗的裸鼠肿瘤组织光子通量显著低于对照组裸鼠(P<0.05；P<0.01)。
染色结果(图10)显示，对照组肺转移结节的数量及体积明显大于Anlotinib治疗组(2mg/kg)和Anlotinib治疗组(4mg/kg)。
以上结果表明，Anlotinib能明显抑制骨肉瘤的肺转移。
3结论
Anlotinib明显抑制骨肉瘤的生长和转移。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。

Claims (14)

  1. 安罗替尼及其药学上可接受的盐在制备抑制或治疗骨肉瘤的药物中的应用。
  2. 安罗替尼及其药学上可接受的盐作为顺铂抑制或治疗骨肉瘤的增效剂的应用。
  3. 根据权利要求1或2所述的应用，其特征在于，安罗替尼药学上可接受的盐为盐酸安罗替尼。
  4. 根据权利要求1或2所述的应用，其特征在于，所述的药物抑制骨肉瘤生长或转移。
  5. 根据权利要求4所述的应用，其特征在于，所述的转移为迁移或侵袭。
  6. 根据权利要求1或2所述的应用，其特征在于，所述的骨肉瘤为原发性骨肉瘤或继发性骨肉瘤。
  7. 根据权利要求1或2所述的应用，其特征在于，所述的骨肉瘤为成骨型骨肉瘤或溶骨型骨肉瘤。
  8. 根据权利要求1或2所述的应用，其特征在于，所述的骨肉瘤为成骨细胞型骨肉瘤、成软骨细胞型骨肉瘤或成纤维细胞型骨肉瘤。
  9. 根据权利要求1所述的应用，其特征在于，所述的药物中含有有效治疗量的安罗替尼和药学上可接受的载体。
  10. 一种治疗骨肉瘤疾病的方法，其特征在于，所述的方法为向患有骨肉瘤的个体给予有效治疗量的安罗替尼或其药学上可接受的盐。
  11. 根据权利要求10所述的方法，其特征在于，所述的方法同时向患有骨肉瘤的个体给予有效治疗量的顺铂。
  12. 根据权利要求10所述的方法，其特征在于，给药途径为口服、静脉注射、直肠、气雾剂、非肠道给药、眼部给药、肺部给药、经皮给药、阴道给药、耳道给药、鼻腔给药或局部给药。
  13. 根据权利要求10所述的方法，其特征在于，所述的个体为哺乳动物。
  14. 根据权利要求13所述的方法，其特征在于，所述的哺乳动物选自人、鼠类、猴、马、牛、猪、犬和猫。
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