TW202313026A

TW202313026A - Pharmaceutical composition comprising protein kinase inhibitor and medical use thereof

Info

Publication number: TW202313026A
Application number: TW111119725A
Authority: TW
Inventors: 周游; 山松; 張星; 先平魯; 李靈杰; 張鈺; 李丹丹; 師丹丹; 黃琳; 黃寧
Original assignee: 大陸商深圳微芯生物科技股份有限公司; 大陸商成都微芯藥業有限公司
Priority date: 2021-05-26
Filing date: 2022-05-26
Publication date: 2023-04-01
Also published as: CN117377492A; WO2022247844A1

Abstract

The present invention provides a pharmaceutical composition comprising a protein kinase inhibitor and medical use thereof, belonging to the technical field of medicine. The present invention proves through experiments that protein kinase inhibitors have a good therapeutic effect for treating sarcoma, in particular, N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthamide has a good therapeutic effect on three diseases subtypes of osteosarcoma, synovial sarcoma, and Kaposi's sarcoma.

Description

包含蛋白激酶抑制劑的藥物組合物及其醫藥用途Pharmaceutical compositions comprising protein kinase inhibitors and their medical use

本發明涉及藥物技術領域，具體涉及包含蛋白激酶抑制劑的藥物組合物及其醫藥用途。The invention relates to the field of pharmaceutical technology, in particular to a pharmaceutical composition containing a protein kinase inhibitor and its medical application.

肉瘤（sarcoma）是一種常見的腫瘤疾病，是由間葉組織（包括纖維結締組織、脂肪、肌肉、脈管、骨、軟骨組織等）發生的惡性腫瘤，這些腫瘤表現出向某種間葉組織分化的特點。其命名方式是在組織來源名稱之後加「肉瘤」，如纖維肉瘤（fibrosarcoma）、橫紋肌肉瘤（rhabdomyosarcoma）、骨肉瘤（osteosarcoma）、滑膜肉瘤（synovial sarcoma）、血管肉瘤（angiosarcoma），如卡波西肉瘤（Kaposi’s sarcoma）等。骨肉瘤是由腫瘤性成骨細胞、骨樣組織所組成，為起源於成骨組織的惡性腫瘤。骨肉瘤發病率在原發性惡性腫瘤中占據首位。該瘤惡性程度甚高，癒後極差，可於數月內出現肺部轉移，截肢後3–5年存活率僅為5–20%。骨肉瘤可發生在任何年齡，但大多在10–25歲，男性較多，腫瘤多處於骨端，偶發生於骨幹或骨骺。滑膜肉瘤是一種惡性程度很高的軟組織肉瘤，起源於具有滑膜分化的間葉細胞，但罕見於關節內。滑膜肉瘤是第四常見的軟組織肉瘤，好發於15–40歲，它很少從關節滑膜發生，而是從關節附近的軟組織內發生，有時甚至遠離關節，好發於四肢，約70%發生於下肢，特別在膝關節附近，其次為足及踝部，上肢以肘部為多。另外與關節無關的部位如頭頸部、腹壁、後腹膜也可發生。卡波西肉瘤又稱Kaposi肉瘤（KS）、卡波濟肉瘤，是1872年匈牙利皮膚科專家Kaposi首先報道的一種罕見的腫瘤，在非洲多見，並可形成地方性流行，且多發於60歲以上的老人。近10年來，在歐美男性同性戀者中出現了爆發性流行，同時，卡波濟肉瘤與愛滋病有一定的關係，它被認為是愛滋病的一種常見腫瘤。卡波西肉瘤是一種由8型疱疹病毒引起的多灶性血管腫瘤，具有局部侵襲性，典型病變表現為皮膚多發性斑點狀、斑塊狀或結節狀病損，也可累及黏膜、淋巴結和內臟器官。根據臨床和流行病學特點，KS有4種不同類型：經典惰性型、非洲地方性、醫源性、獲得性免疫缺陷症候群相關性。經典型KS特徵是出現紫色、紅藍色或深棕色斑丘疹、斑塊和結節，也可形成潰瘍。尤其常見於肢體末端，可伴有淋巴水腫。此病一般為惰性，淋巴結和內臟不常受累。可伴有造血細胞性惡性病變。地方性KS可有皮膚病變，病程較長。淋巴腺病型是地方性KS的一個亞型，見於非洲兒童，進展快速，具有高度致命性。醫源性KS相對常見，發生在實性器官移植或免疫抑制治療之後數月或數年。AIDS相關性KS為侵襲性最強的KS，皮膚病損最常見於面部、生殖器和下肢。常見口腔黏膜、淋巴結、胃腸道和肺受累。肉瘤目前主要採用以手術為主，化療和放療為輔的治療手法，但均預後較差。目前仍需進一步開發肉瘤的治療方案。Sarcoma is a common tumor disease, which is a malignant tumor of mesenchymal tissue (including fibrous connective tissue, fat, muscle, blood vessel, bone, cartilage tissue, etc.), and these tumors show differentiation into a certain mesenchymal tissue specialty. Its naming method is to add "sarcoma" after the name of tissue source, such as fibrosarcoma (fibrosarcoma), rhabdomyosarcoma (rhabdomyosarcoma), osteosarcoma (osteosarcoma), synovial sarcoma (synovial sarcoma), angiosarcoma (angiosarcoma), such as Kabo Western sarcoma (Kaposi's sarcoma) and so on. Osteosarcoma is composed of neoplastic osteoblasts and bone-like tissues, and is a malignant tumor originating from bone-forming tissues. The incidence of osteosarcoma occupies the first place among primary malignant tumors. The tumor has a very high degree of malignancy, and the prognosis is extremely poor. Lung metastases can occur within a few months, and the 3-5-year survival rate after amputation is only 5-20%. Osteosarcoma can occur at any age, but most of them are between 10 and 25 years old. There are more men, and the tumors are mostly located at the end of the bone, and occasionally occur in the diaphysis or epiphysis. Synovial sarcoma is a highly malignant soft tissue sarcoma that arises from mesenchymal cells with synovial differentiation but is rare in joints. Synovial sarcoma is the fourth most common soft tissue sarcoma. It occurs frequently in 15–40 years old. It rarely occurs from the synovial membrane of the joint, but from the soft tissue near the joint, sometimes even far away from the joint. It usually occurs in the extremities, about 70% occurred in the lower extremities, especially near the knee joint, followed by the feet and ankles, and the elbows in the upper extremities. In addition, non-joint parts such as the head and neck, abdominal wall, and retroperitoneum can also occur. Kaposi's sarcoma, also known as Kaposi's sarcoma (KS), Kaposi's sarcoma, is a rare tumor first reported by the Hungarian dermatologist Kaposi in 1872. It is more common in Africa and can form endemic epidemics, and it mostly occurs at the age of 60 above the elderly. In the past 10 years, there has been an explosive epidemic among gay men in Europe and the United States. At the same time, Kaposi's sarcoma has a certain relationship with AIDS, and it is considered to be a common tumor of AIDS. Kaposi's sarcoma is a multifocal vascular tumor caused by herpes virus type 8. It is locally aggressive. The typical lesion is multiple macular, plaque or nodular lesions on the skin. It can also involve mucosa, lymph nodes and internal organs. According to clinical and epidemiological characteristics, there are 4 different types of KS: classic indolent type, African endemic, iatrogenic, and acquired immunodeficiency syndrome-related. Classic KS is characterized by purple, reddish-blue or dark brown maculopapules, plaques and nodules, and can also form ulcers. It is especially common in the extremities and may be accompanied by lymphedema. The disease is generally indolent, with infrequent lymph node and visceral involvement. May be accompanied by hematopoietic malignant lesions. Endemic KS may have skin lesions with a longer course. Lymphadenopathy is a subtype of endemic KS that occurs in African children and is rapidly progressive and highly lethal. Iatrogenic KS is relatively common, occurring months or years after solid organ transplantation or immunosuppressive therapy. AIDS-associated KS was the most aggressive type of KS, with skin lesions most commonly on the face, genitals, and lower extremities. Oral mucosa, lymph nodes, gastrointestinal tract, and lung involvement are common. Sarcomas are currently mainly treated with surgery, supplemented by chemotherapy and radiotherapy, but the prognosis is poor. Treatment options for sarcomas still need to be further developed.

WO 2010/139180 A1公開了一種蛋白激酶和組蛋白去乙醯化酶雙靶點抑制劑的通式化合物結構，並具體公開了N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(6,7-二甲氧基喹唑啉-4-氧)-1-萘醯胺；N-(2-胺基-4-氟苯基)-6-(6,7-二甲氧基喹唑啉-4-氧)-1-萘醯胺；N-(2-胺基-4-氟苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(6,7-二甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基-4-氟苯基)-6-(6,7-二甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(8-(三氯甲基)喹啉-4-氧)-1-萘醯胺的化合物結構、合成工藝及其體外PDGF和VEGF配體依賴性細胞增殖試驗的實驗數據，並進一步公開了這些化合物的體外細胞增殖抑制試驗數據，包括對A-498、A549、Bel-7402、HCT-8、MCF-7等多種腫瘤細胞增殖的抑制試驗數據，及用於肺癌、腸癌、肝癌的動物模型實驗數據。目前還未發現有蛋白激酶抑制劑可用於治療肉瘤，如滑膜肉瘤、骨肉瘤、卡波西肉瘤的相關研究和報道。WO 2010/139180 A1 discloses a general formula compound structure of a protein kinase and histone deacetylase dual-target inhibitor, and specifically discloses N-(2-aminophenyl)-6-(7-methyl Oxyquinoline-4-oxo)-1-naphthamide; N-(2-aminophenyl)-6-(6,7-dimethoxyquinazoline-4-oxo)-1-naphthalene Amide; N-(2-amino-4-fluorophenyl)-6-(6,7-dimethoxyquinazoline-4-oxy)-1-naphthamide; N-(2-amine N-(2-aminophenyl)-6-(6,7-di Methoxyquinoline-4-oxo)-1-naphthylamide; N-(2-amino-4-fluorophenyl)-6-(6,7-dimethoxyquinoline-4-oxo) -1-naphthylamide; N-(2-aminophenyl)-6-(quinoline-4-oxygen)-1-naphthylamide; N-(2-aminophenyl)-6-(8 -(trichloromethyl)quinoline-4-oxygen)-1-naphthylamide compound structure, synthesis process and experimental data of in vitro PDGF and VEGF ligand-dependent cell proliferation test, and further disclose the details of these compounds In vitro cell proliferation inhibition test data, including the inhibition test data of A-498, A549, Bel-7402, HCT-8, MCF-7 and other tumor cell proliferation, and the data of animal model experiments for lung cancer, intestinal cancer, and liver cancer . So far, no related studies and reports have been found that protein kinase inhibitors can be used to treat sarcomas, such as synovial sarcoma, osteosarcoma, and Kaposi's sarcoma.

有鑒於此，本發明的目的在於提供一種蛋白抑制劑的在肉瘤治療領域的新醫藥用途。In view of this, the object of the present invention is to provide a new medical application of a protein inhibitor in the field of sarcoma treatment.

為實現上述發明目的，本發明提供如下技術方案：In order to realize the foregoing invention object, the present invention provides following technical scheme:

第一方面，本發明提供蛋白激酶抑制劑在製備預防和／或治療肉瘤的藥物中的應用；優選地，所述肉瘤為骨肉瘤、軟組織肉瘤、血管肉瘤中的一種或多種；優選地，所述軟組織肉瘤為滑膜肉瘤；優選地，所述血管肉瘤為卡波西肉瘤。In a first aspect, the present invention provides the use of protein kinase inhibitors in the preparation of drugs for the prevention and/or treatment of sarcoma; preferably, the sarcoma is one or more of osteosarcoma, soft tissue sarcoma, and angiosarcoma; preferably, the The soft tissue sarcoma is synovial sarcoma; preferably, the angiosarcoma is Kaposi's sarcoma.

第二方面，本發明提供蛋白激酶抑制劑在預防和／或治療肉瘤疾病中的應用；優選地，所述肉瘤為骨肉瘤、軟組織肉瘤、血管肉瘤中的一種或多種；優選地，所述軟組織肉瘤為滑膜肉瘤；優選地，所述血管肉瘤為卡波西肉瘤。In a second aspect, the present invention provides the application of protein kinase inhibitors in the prevention and/or treatment of sarcoma; preferably, the sarcoma is one or more of osteosarcoma, soft tissue sarcoma, and angiosarcoma; preferably, the soft tissue The sarcoma is synovial sarcoma; preferably, said angiosarcoma is Kaposi's sarcoma.

第三方面，本發明提供一種預防和／或治療肉瘤的藥物組合物，所述藥物組合物以蛋白激酶抑制劑為主要活性成分，並包含藥學上可接受的輔料；優選地，所述肉瘤為骨肉瘤、軟組織肉瘤、血管瘤中的一種或多種；優選地，所述軟組織肉瘤為滑膜肉瘤；優選地，所述血管肉瘤為卡波西肉瘤。In a third aspect, the present invention provides a pharmaceutical composition for the prevention and/or treatment of sarcoma, the pharmaceutical composition uses protein kinase inhibitors as the main active ingredient, and contains pharmaceutically acceptable excipients; preferably, the sarcoma is One or more of osteosarcoma, soft tissue sarcoma, and hemangioma; preferably, the soft tissue sarcoma is synovial sarcoma; preferably, the angiosarcoma is Kaposi's sarcoma.

在一些實施方案中，所述藥學上可接受的輔料選自潤滑劑、黏合劑、填充劑、防腐劑、表面活性劑、著色劑、矯味劑、乳化劑、助懸劑、稀釋劑、膠凝劑、崩解劑、pH調節劑、增溶劑的一種或兩種及以上。In some embodiments, the pharmaceutically acceptable adjuvant is selected from lubricants, binders, fillers, preservatives, surfactants, colorants, flavoring agents, emulsifiers, suspending agents, diluents, gelling agents One or two or more of agents, disintegrants, pH regulators, and solubilizers.

在一些實施方案中，所述藥物組合物是片劑、膠囊、粉劑、液體製劑、懸浮劑、針劑、緩釋製劑等常規劑型，優選口服片劑、膠囊或口服液體。In some embodiments, the pharmaceutical composition is in conventional dosage forms such as tablets, capsules, powders, liquid preparations, suspensions, injections, sustained-release preparations, preferably oral tablets, capsules or oral liquids.

第四方面，本發明提供一種預防和／或治療肉瘤的方法，包括向有需要的個體施用預防和／或治療有效劑量的如上所述的藥物組合物；優選地，所述肉瘤為骨肉瘤、軟組織肉瘤、血管瘤中的一種或多種；優選地，所述軟組織肉瘤為滑膜肉瘤；優選地，所述血管肉瘤為卡波西肉瘤。In a fourth aspect, the present invention provides a method for preventing and/or treating sarcoma, comprising administering a preventive and/or therapeutically effective dose of the above-mentioned pharmaceutical composition to an individual in need; preferably, the sarcoma is osteosarcoma, One or more of soft tissue sarcoma and hemangioma; preferably, the soft tissue sarcoma is synovial sarcoma; preferably, the hemangiosarcoma is Kaposi's sarcoma.

在一些實施方案中，本發明中，所述蛋白激酶抑制劑選自Aurora B抑制劑，和／或，VEGFR抑制劑，和／或，PDGFR抑制劑，和／或 c-Kit／CSF1R抑制劑。In some embodiments, in the present invention, the protein kinase inhibitor is selected from Aurora B inhibitors, and/or, VEGFR inhibitors, and/or, PDGFR inhibitors, and/or c-Kit/CSF1R inhibitors.

在一些實施方案中，本發明中，所述蛋白激酶抑制劑選自以下化合物或其藥學上可接受的鹽、晶型、異構體、前藥或代謝產物：N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(6,7-二甲氧基喹唑啉-4-氧)-1-萘醯胺；N-(2-胺基-4-氟苯基)-6-(6,7-二甲氧基喹唑啉-4-氧)-1-萘醯胺；N-(2-胺基-4-氟苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(6,7-二甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基-4-氟苯基)-6-(6,7-二甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(8-(三氯甲基)喹啉-4-氧)-1-萘醯胺，或選自下述Aurora B抑制劑／VEGFR抑制劑／PDGFR抑制劑／c-Kit抑制劑／CSF1R抑制劑中的一種或多種：塞尼舍替（cenisertib）、橙皮苷（hesperidin）、安羅替尼（anlotinib）、海那替尼（henatinib）、阿帕替尼（Apatinib）、索拉非尼（sorafenib）、瑞格非尼（regorafenib）、卡博替尼（Cometriq）、侖伐替尼（Lenvatinib）、凡德他尼（vandetanib）、舒尼替尼（Sunitinib）、伊馬替尼（Imatinib）、克萊拉尼（Crenolanib）、阿伐替尼（Avapritinib）、馬賽替尼（Masitinib）、尼洛替尼（Nilotinib）、艾克利單抗（Axatilimab）、培西替尼（Pexidartinib）。In some embodiments, in the present invention, the protein kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, crystal forms, isomers, prodrugs or metabolites: N-(2-aminophenyl Base)-6-(7-methoxyquinoline-4-oxygen)-1-naphthylamide; N-(2-aminophenyl)-6-(6,7-dimethoxyquinazoline -4-oxo)-1-naphthylamide; N-(2-amino-4-fluorophenyl)-6-(6,7-dimethoxyquinazoline-4-oxy)-1-naphthalene Amide; N-(2-amino-4-fluorophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthylamide; N-(2-aminophenyl) -6-(6,7-dimethoxyquinoline-4-oxo)-1-naphthamide; N-(2-amino-4-fluorophenyl)-6-(6,7-dimethyl Oxyquinoline-4-oxo)-1-naphthamide; N-(2-aminophenyl)-6-(quinoline-4-oxy)-1-naphthamide; N-(2-amine phenyl)-6-(8-(trichloromethyl)quinoline-4-oxo)-1-naphthamide, or selected from the following Aurora B inhibitors/VEGFR inhibitors/PDGFR inhibitors/c- One or more of Kit inhibitors/CSF1R inhibitors: cenisertib, hesperidin, anlotinib, henatinib, apatinib ), sorafenib (sorafenib), regorafenib (regorafenib), cabozantinib (Cometriq), lenvatinib (Lenvatinib), vandetanib (vandetanib), sunitinib (Sunitinib), Imatinib, Crenolanib, Avapritinib, Masitinib, Nilotinib, Axatilimab, Pecitinib (Pexidartinib).

在一些實施方案中，本發明中，所述蛋白激酶抑制劑選自N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺或其藥學上可接受的鹽、晶型、異構體、前藥或代謝產物。優選地，本發明中，所述蛋白激酶抑制劑選自N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺及其晶型。In some embodiments, in the present invention, the protein kinase inhibitor is selected from N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxygen)-1-naphthamide or a pharmaceutically acceptable salt, crystal form, isomer, prodrug or metabolite thereof. Preferably, in the present invention, the protein kinase inhibitor is selected from N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxygen)-1-naphthamide and its crystals type.

在一些實施方案中，所述晶型為非溶劑化晶型。In some embodiments, the crystalline form is an ansolvated crystalline form.

在一些實施方案中，所述非溶劑化晶型選自N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺晶型A、晶型B、晶型C。In some embodiments, the non-solvated crystal form is selected from N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxygen)-1-naphthamide crystal form A , Form B, Form C.

N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺非溶劑化晶體、晶型A、晶型B、晶型C的公開資訊見WO 2018/059429 A1的專利公開文本，WO 2018/059429 A1的全文通過引用的方式並入本申請。Disclosure of N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthamide non-solvated crystal, crystal form A, crystal form B, and crystal form C See the patent publication of WO 2018/059429 A1 for information, and the entirety of WO 2018/059429 A1 is incorporated into this application by reference.

本發明中，所述晶型包括非溶劑化晶型、溶劑化晶型和無定型結構。In the present invention, the crystal forms include non-solvated crystal forms, solvated crystal forms and amorphous structures.

本發明中，所述異構體包括構象異構體，對映異構體和非對應異構體。In the present invention, the isomers include conformational isomers, enantiomers and diastereoisomers.

本發明所述的「可藥用鹽」或「藥學上可接受的鹽」是指N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺與藥學上可接受的酸進行反應製得的酸加成鹽，或者其中具有酸性基團的化合物和藥學上可接受的鹼反應生成的鹽。其中，所述的藥學上可接受的酸較佳的選自無機酸（如鹽酸、硫酸、磷酸或氫溴酸等），和有機酸（如草酸、馬來酸、富馬酸、蘋果酸、酒石酸、檸檬酸或苯甲酸等）；所述藥學上可接受的鹼較佳的選自氫氧化鈉、氫氧化鉀、氫氧化鈣、碳酸鈉、碳酸氫鉀、氨水或碳酸氫銨等。The "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" of the present invention refers to N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxygen)-1 - an acid addition salt prepared by reacting a naphthamide with a pharmaceutically acceptable acid, or a salt formed by reacting a compound with an acidic group with a pharmaceutically acceptable base. Wherein, the pharmaceutically acceptable acid is preferably selected from inorganic acids (such as hydrochloric acid, sulfuric acid, phosphoric acid or hydrobromic acid, etc.), and organic acids (such as oxalic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid or benzoic acid, etc.); the pharmaceutically acceptable base is preferably selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium bicarbonate, ammonia water or ammonium bicarbonate, etc.

本發明中的前藥，也稱前體藥物、藥物前體、前驅藥物等，是指藥物經過化學結構修飾後得到的在體外無活性或活性較小、在體內經酶或非酶的轉化釋放出活性藥物而發揮藥效的化合物。Prodrugs in the present invention, also known as prodrugs, drug precursors, prodrugs, etc., refer to drugs that are inactive or less active in vitro and released through enzymatic or non-enzymatic conversion in vivo after chemical structure modification. A compound that exerts a medicinal effect by producing an active drug.

本發明中的代謝產物，是指化合物在動物個體體內經新陳代謝後得到的中間代謝產物和最終代謝產物。Metabolites in the present invention refer to the intermediate metabolites and final metabolites obtained after the compound is metabolized in the body of an individual animal.

本發明中，預防和／或治療有效劑量中的「有效劑量」是指用藥介於最小有限量和極量之間，並能對機體產生明顯效應而不引起毒性反應的劑量。In the present invention, the "effective dose" in the preventive and/or therapeutic effective dose refers to the dose between the minimum limited amount and the maximum dose, which can produce obvious effects on the body without causing toxic reactions.

本發明中，有需要的個體中的「個體」是指脊椎動物。在某些實施方案中，脊椎動物指哺乳動物。哺乳動物包括，但不限於，牲畜（諸如牛）、寵物（諸如貓、犬、和馬）、靈長類動物、小鼠和大鼠。在某些實施方案中，哺乳動物指人。In the present invention, the "individual" in the individual in need refers to a vertebrate. In certain embodiments, a vertebrate is a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, a mammal is a human.

本發明的有益效果：本發明提供了蛋白激酶抑制劑的一種新的醫藥用途，通過動物實驗證明，蛋白激酶抑制劑用於治療肉瘤具有良好的治療效果，尤其是N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺針對骨肉瘤、滑膜肉瘤和卡波西肉瘤具有良好的治療效果。Beneficial effects of the present invention: the present invention provides a new medical application of protein kinase inhibitors. It has been proved by animal experiments that protein kinase inhibitors have a good therapeutic effect for the treatment of sarcoma, especially N-(2-aminophenyl base)-6-(7-methoxyquinoline-4-oxygen)-1-naphthamide has good therapeutic effects on osteosarcoma, synovial sarcoma and Kaposi's sarcoma.

本發明公開了蛋白激酶抑制劑用於肉瘤的藥用組合物和新醫藥用途，本領域技術人員可以借鑒本文內容，適當進行優化或等效變換。特別需要指出的是，所有類似的替換和改動對本領域技術人員來說是顯而易見的，它們都被視為包括在本發明。本發明所述應用和藥用組合物已經通過較佳實施例進行了描述，相關人員明顯能在不脫離本發明內容、精神和範圍內對本文所述應用和藥用組合物進行改動或適當變更與組合，來實現和應用本發明技術。The invention discloses a pharmaceutical composition and a new medical application of a protein kinase inhibitor for sarcoma. Those skilled in the art can refer to the contents of this article for appropriate optimization or equivalent transformation. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The application and pharmaceutical composition of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes to the application and pharmaceutical composition described herein without departing from the content, spirit and scope of the present invention and combination, to realize and apply the technology of the present invention.

以下就本發明所提供的蛋白激酶抑制劑用於治療肉瘤的藥用組合物及醫藥用途做進一步說明。The pharmaceutical composition and medical application of the protein kinase inhibitor provided by the present invention for treating sarcoma will be further described below.

試驗材料和儀器Test Materials and Instruments

（1）材料及來源(1) Materials and sources

人骨肉瘤細胞系MG63、U2OS、143B，人滑膜肉瘤細胞系SW982和人卡波西（Kaposi）肉瘤細胞系SK-UT-1均購自美國菌種保藏中心（American type culture collection, ATCC），用含1 %雙抗（青鏈黴素，Hyclone）和10%胎牛血清（Gibco）的DMEM培養液（Hyclone）培養。Human osteosarcoma cell lines MG63, U2OS, 143B, human synovial sarcoma cell line SW982 and human Kaposi sarcoma cell line SK-UT-1 were purchased from American type culture collection (ATCC), Cultured in DMEM medium (Hyclone) containing 1% double antibody (penicillin, streptomycin, Hyclone) and 10% fetal bovine serum (Gibco).

N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺（蛋白激酶抑制劑，以下稱為「供試品」）：來源於深圳微芯生物科技股份有限公司，將其溶於二甲基亞碸（Dimethyl sulfoxide, DMSO；上海生工，DN3039A）中，配置成20 mM的工作母液，置於−20 ℃的冰箱中備用，使用時根據各項實驗需要配置相應終濃度的工作溶液。N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthamide (protein kinase inhibitor, hereinafter referred to as "test article"): derived from Shenzhen Microchip Biotechnology Co., Ltd., dissolved it in dimethyl sulfoxide (DMSO; Shanghai Sangong, DN3039A) to make a 20 mM working mother solution, and put it in a −20 °C refrigerator for later use. When using, configure the working solution with the corresponding final concentration according to the needs of each experiment.

CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS)試劑盒（Promega, G5421）CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) Kit (Promega, G5421)

結晶紫染色液（上海碧雲天生物技術有限公司，C0121）Crystal violet staining solution (Shanghai Biyuntian Biotechnology Co., Ltd., C0121)

碘化丙啶（Propidium iodide, PI；上海生工，A601112）Propidium iodide (PI; Shanghai Sangong, A601112)

核糖核酸酶A（RNase A；上海生工，B500474）Ribonuclease A (RNase A; Shanghai Sangong, B500474)

Triton X-100（上海生工，A110694）Triton X-100 (Shanghai Sangong, A110694)

抗體：Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb (Cell Signaling, #5625), β-Actin Antibody (AC-15)(Santa Cruz, sc-69879), Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling, #7074), HRP-conjugated Goat Anti-Mouse IgG（上海生工，D110087）。Antibodies: Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb (Cell Signaling, #5625), β-Actin Antibody (AC-15)(Santa Cruz, sc-69879), Anti-rabbit IgG, HRP-linked Antibody ( Cell Signaling, #7074), HRP-conjugated Goat Anti-Mouse IgG (Shanghai Sangong, D110087).

BALB/C (nu/nu)雌性裸鼠（SPF級），5周齡，體重14–18g，購自廣東省實驗動物中心，共60隻。在IVC籠具中按SPF條件飼餵，注射瘤株前飼養約1周，以使裸鼠適應新環境。BALB/C (nu/nu) female nude mice (SPF grade), 5 weeks old, weighing 14–18 g, were purchased from Guangdong Experimental Animal Center, 60 in total. The nude mice were fed under SPF conditions in IVC cages, and fed for about 1 week before injection of the tumor strain, so that the nude mice could adapt to the new environment.

0.2% CMC-Na助懸劑（用於製備供試品懸液並作為對照溶劑）：在200 mL去離子水中，分多次加入共0.4 g CMC-Na，邊加邊攪拌，然後在沸水浴上加熱攪拌直至溶液澄清。冷却後離心取上清，於高溫高壓滅菌後加入0.2 g吐溫-20混勻，室溫備用。0.2% CMC-Na suspending agent (for the preparation of the test product suspension and as a reference solvent): in 200 mL deionized water, add a total of 0.4 g CMC-Na several times, stir while adding, and then place in a boiling water bath Stir over heat until the solution is clear. After cooling, centrifuge to take the supernatant, add 0.2 g Tween-20 after high temperature and high pressure sterilization, mix well, and set aside at room temperature.

（2）實驗試劑及儀器：(2) Experimental reagents and instruments:

超淨工作臺（蘇淨安泰）Ultra-clean workbench (Sujing Antai)

CO ₂細胞培養箱（RS Biotech, Galaxy S） _CO2 cell incubator (RS Biotech, Galaxy S)

倒置螢光顯微鏡（廣州市明美科技有限公司）Inverted fluorescence microscope (Guangzhou Mingmei Technology Co., Ltd.)

細胞計數板（上海求精生化試劑儀器有限公司）Cell counting plate (Shanghai Qiujing Biochemical Reagent Instrument Co., Ltd.)

Tecan infinite F50光吸收酶標儀Tecan infinite F50 absorbance microplate reader

冷凍高速離心機（Eppendorf）Refrigerated high-speed centrifuge (Eppendorf)

BD FACSCelestaTM流式細胞儀BD FACSCelestaTM flow cytometer

蛋白電泳及轉膜裝置（Bio-Rad）Protein electrophoresis and membrane transfer device (Bio-Rad)

實施例1　蛋白激酶抑制劑抑制肉瘤細胞的增殖試驗研究Example 1 Protein Kinase Inhibitors Inhibit the Proliferation of Sarcoma Cells

接種細胞：將消化收集的MG63、U2OS、SW982、143B和SK-UT-1細胞按每孔1000個/190 μL接種到96孔細胞培養板中，並留出BLANK孔不接種細胞，只加入相同體積的培養液；5 % CO ₂、37 ℃培養。 Inoculate cells: Inoculate MG63, U2OS, SW982, 143B and SK-UT-1 cells collected by digestion into 96-well cell culture plates at 1000 cells/190 μL per well, and leave the BLANK wells without inoculating cells, only add the same Volume of culture medium; 5 % CO ₂ , 37 ℃ culture.

給藥：接種培養24小時後給藥。首先用DMSO將供試品稀釋成每孔終濃度的1000倍（即使每孔最終都加入了1/1000體積的DMSO），再將供試品的DMSO稀釋液按1 : 50稀釋到培養液中。最終每孔按終濃度0、1、3、6、9 μM供試品加入10 μL相應的上述溶液。Administration: administration 24 hours after inoculation. First, dilute the test product with DMSO to 1000 times the final concentration of each well (even if 1/1000 volume of DMSO is finally added to each well), and then dilute the DMSO dilution of the test product into the culture medium by 1:50 . Finally, add 10 μL of the corresponding above-mentioned solution to each well according to the final concentration of 0, 1, 3, 6 and 9 μM of the test substance.

檢測：藥物作用120小時後檢測，將96孔培養板中的培養液吸出，每孔加入50 μL新鮮的無酚紅1640培養液及10 μL CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay（MTS）試劑盒檢測液，共60 μL。37 ℃孵育1–2小時，通過光吸收酶標儀讀取每孔490 nm波長處的吸光度值。Detection: After 120 hours of drug action, the culture solution in the 96-well culture plate was aspirated, and 50 μL of fresh phenol red-free 1640 culture solution and 10 μL of CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) reagent were added to each well Box detection solution, a total of 60 μL. Incubate at 37°C for 1–2 hours, and read the absorbance value of each well at a wavelength of 490 nm with an optical absorbance microplate reader.

數據處理與分析：根據BLANK孔的讀數求平均值OD490-BLK-A（背景值），各孔讀數减去OD490-BLK-A後獲得扣除背景值的各給藥孔的OD490-T及陰性對照孔的OD490-T0。求得陰性對照孔OD490-T0的平均值OD490-T0-A，各給藥孔相對細胞存活率根據公式進行計算：相對細胞存活率(%) = (OD490-T ÷ OD490-T0-A)×100%。Data processing and analysis: Calculate the average OD490-BLK-A (background value) based on the readings of the BLANK wells, and subtract the OD490-BLK-A from the readings of each well to obtain the OD490-T and negative control of each administration well with the background value deducted OD490-T0 of the wells. Obtain the average value OD490-T0-A of negative control well OD490-T0, and the relative cell survival rate of each administration well is calculated according to the formula: relative cell survival rate (%)=(OD490-T ÷ OD490-T0-A)× 100%.

試驗結果：通過MTS檢測發現，與溶劑對照相比，供試品對各細胞系的增殖均有不同程度的抑制作用且具有明顯的劑量依賴性，即供試品終濃度越高，各細胞系的相對細胞存活率越低。尤其在U2OS、SW982、143B和SK-UT-1中，3 μM供試品的抑制率即已接近或超過50%。（圖1）Test results: Through MTS detection, it was found that compared with the solvent control, the test product had different degrees of inhibitory effects on the proliferation of each cell line and had a significant dose-dependent effect, that is, the higher the final concentration of the test product, the greater the effect of each cell line. The relative cell viability is lower. Especially in U2OS, SW982, 143B and SK-UT-1, the inhibition rate of 3 μM test substance is close to or exceeds 50%. (figure 1)

實施例2　蛋白激酶抑制劑抑制肉瘤細胞的克隆形成能力試驗研究Example 2 Experimental study on the ability of protein kinase inhibitors to inhibit the colony formation of sarcoma cells

接種細胞：將消化收集的MG63、U2OS、143B、SK-UT-1細胞按每孔1000個/2 mL接種到6孔細胞培養板中，培養過夜待細胞貼壁後給藥。Inoculation of cells: Inoculate MG63, U2OS, 143B, and SK-UT-1 cells collected by digestion into 6-well cell culture plates at 1000 cells/2 mL per well, culture overnight and administer after the cells adhere to the wall.

給藥：每孔分別按終濃度0、1、3、6、9 μM加入供試品，繼續培養，並2–3天更換培養液及相應藥物一次，給藥周期 7 天。Administration: Add the test substance to each well at the final concentration of 0, 1, 3, 6, and 9 μM, continue to cultivate, and replace the culture medium and corresponding drugs once every 2-3 days, and the administration cycle is 7 days.

染色觀察：棄去培養液，按2 mL/孔加入PBS溶液小心洗滌一次，棄去PBS後每孔加入0.5 mL 95 %乙醇溶液固定細胞5分鐘。棄去乙醇溶液，每孔加入0.5 mL結晶紫染色液，室溫放置20分鐘。棄去染色液，用自來水洗滌各孔，將培養板倒置於吸水紙上吸乾水分，自然乾燥後觀察各孔染色情况，每個染色點記為1個克隆（即由單個細胞***增殖形成的細胞團）。Staining observation: Discard the culture medium, add PBS solution at 2 mL/well to wash carefully once, discard PBS, add 0.5 mL 95% ethanol solution to each well to fix the cells for 5 minutes. Discard the ethanol solution, add 0.5 mL of crystal violet staining solution to each well, and place at room temperature for 20 minutes. Discard the staining solution, wash each well with tap water, place the culture plate upside down on absorbent paper to absorb the water, and observe the staining of each well after natural drying. Each staining point is recorded as a clone (that is, a cell formed by a single cell division and proliferation). group).

試驗結果：結晶紫染色結果表明，供試品對肉瘤細胞的克隆形成能力具有抑制作用，且供試品對各細胞系的克隆形成能力均呈劑量依賴性。在MG63中6 μM供試品對克隆形成能力的抑制超過50%，在143B和SK-UT-1中3 μM供試品的抑制強度達到或超過80%，在U2OS中1 μM供試品的抑制強度已接近50%。（圖2）Test results: The results of crystal violet staining showed that the test substance had an inhibitory effect on the colony formation ability of sarcoma cells, and the test substance had a dose-dependent effect on the clone formation ability of each cell line. In MG63, 6 μM of the test substance inhibited the colony formation ability by more than 50%, in 143B and SK-UT-1, the inhibitory intensity of 3 μM of the test substance reached or exceeded 80%, in U2OS, 1 μM of the test substance inhibited The inhibition strength is close to 50%. (figure 2)

實施例3　蛋白激酶抑制劑抑制肉瘤的作用機制探索研究Example 3 Exploration and research on the mechanism of action of protein kinase inhibitors in inhibiting sarcoma

試驗方法：通過流式細胞術觀察不同劑量供試品對143B、SW982、U2OS和SK-UT-1細胞周期狀態的影響。通過流式細胞術檢測細胞的PI染色情况可以顯示每個細胞的DNA含量，從而分辨細胞處於G0/G1期（DNA含量 = 2N）、S期（2N ＜ DNA含量＜ 4N）或G2/M期（DNA含量 = 4N），此外，當DNA含量＞ 4N時，表明這可能是一個多倍體細胞；DNA含量＜ 2N時，該細胞可能處於凋亡晚期。Test method: Observe the effect of different doses of the test substance on the cell cycle state of 143B, SW982, U2OS and SK-UT-1 by flow cytometry. PI staining of cells can be detected by flow cytometry to display the DNA content of each cell, so as to distinguish whether the cells are in G0/G1 phase (DNA content = 2N), S phase (2N < DNA content < 4N) or G2/M phase (DNA content = 4N). In addition, when the DNA content is > 4N, it may be a polyploid cell; when the DNA content is < 2N, the cell may be in the late stage of apoptosis.

試驗過程：Experimental procedure:

細胞的培養及給藥：將對數生長期的143B、SW982、U2OS和SK-UT-1細胞消化收集，按每孔10 ⁶個/2 mL接種到六孔板內，各接種12孔，正常培養過夜。上述四種細胞各分為4組，每組3孔，分別給予終濃度0、1、3、6 μM的供試品，培養48小時後收集細胞。 Cell culture and administration: Digest and collect 143B, SW982, U2OS and SK-UT-1 cells in the logarithmic growth phase, inoculate 10 ⁶ cells/2 mL per well into six-well plates, each inoculate 12 wells, and culture normally overnight. The above-mentioned four kinds of cells were divided into 4 groups, each group had 3 wells, and the final concentrations of 0, 1, 3, and 6 μM of the test substance were given respectively, and the cells were collected after 48 hours of culture.

樣品收集：供試品作用完成後，將培養液及懸浮狀態細胞轉移至15 mL離心管中，用PBS輕柔清洗孔中貼壁狀態的細胞1次，清洗液轉入同一支離心管；向培養板中剩下的貼壁細胞中加入300 μL胰酶消化液，於37 ℃孵育5分鐘，待細胞充分離散後，分別加入含10% FBS的培養液終止消化反應，並反覆吸打重懸細胞後合並至對應的15 mL離心管，室溫、800 rpm離心10分鐘，去上清，以PBS洗1次後重懸於300 μL PBS中待用。按樣品數量在1.5 mL離心管中以700 μL每管加入無水乙醇，置冰上預冷備用。將上述收集到的細胞樣品懸液逐管、逐滴滴入預冷的700 μL無水乙醇，然後輕柔顛倒混勻數次，至此細胞已收集並固定，樣品於4 ℃靜置至少12小時。Sample collection: After the effect of the test product is completed, transfer the culture medium and cells in suspension to a 15 mL centrifuge tube, gently wash the adherent cells in the well once with PBS, and transfer the cleaning solution to the same centrifuge tube; Add 300 μL trypsin digestion solution to the remaining adherent cells in the plate and incubate at 37 °C for 5 minutes. After the cells are fully dispersed, add culture medium containing 10% FBS to terminate the digestion reaction, and resuspend the cells by pipetting repeatedly After that, they were combined into corresponding 15 mL centrifuge tubes, centrifuged at room temperature and 800 rpm for 10 minutes, removed the supernatant, washed once with PBS, and resuspended in 300 μL PBS for use. According to the number of samples, 700 μL of absolute ethanol was added to 1.5 mL centrifuge tubes, and pre-cooled on ice for later use. Add the cell sample suspension collected above into 700 μL of pre-cooled absolute ethanol drop by tube, and then gently invert and mix several times. By now, the cells have been collected and fixed, and the samples are left to stand at 4 °C for at least 12 hours.

染色及上機檢測：將PBS與20 mg/mL的PI儲備液及10 mg/mL的RNase A溶液及10% Triton X-100按1000 : 2.5 : 2 : 10的比例混勻成為工作染液。將固定好的細胞樣品於4 ℃、1000 rpm離心10分鐘，吸淨上清並用PBS洗滌兩次，按每管500 μL重懸於上述工作染液中。37 ℃避光孵育30分鐘後，將染色好的細胞樣品於4 ℃、1000 rpm離心10分鐘，吸淨上清並用200 μL PBS洗滌重懸，用200目不鏽鋼網過濾細胞，濾液上機進行流式細胞周期分析（在激發波長488 nm波長處檢測紅色螢光，同時檢測光散射情况；每個樣本計數10000個細胞）。Staining and detection on the machine: mix PBS with 20 mg/mL PI stock solution, 10 mg/mL RNase A solution and 10% Triton X-100 at a ratio of 1000: 2.5: 2: 10 to become a working dye solution. The fixed cell samples were centrifuged at 1000 rpm at 4 °C for 10 minutes, the supernatant was aspirated and washed twice with PBS, and resuspended in the above working dye solution in 500 μL per tube. After incubating at 37°C in the dark for 30 minutes, centrifuge the stained cell samples at 4°C and 1000 rpm for 10 minutes, aspirate the supernatant and wash and resuspend with 200 μL PBS, filter the cells with a 200-mesh stainless steel mesh, and flow the filtrate onto the machine. Cell cycle analysis (detection of red fluorescence at an excitation wavelength of 488 nm and detection of light scattering; 10,000 cells per sample).

數據處理及統計分析：採用FlowJo軟體進行細胞DNA含量分析和光散射分析，獲得各樣品中的周期分布數據，通過t檢驗比較各組間差異（P ＜ 0.01為差異顯著）。試驗結果顯示：143B在3 μM供試品作用下發生了明顯的G2/M期和多倍體細胞比例的增加，當劑量達到6 μM時，143B中G2/M期和多倍體細胞比例的增加更為顯著；SW982則是在6 μM供試品作用下發生明顯的G2/M期和多倍體細胞比例增加；U2OS在3或6 μM供試品作用下最為明顯的變化是S期細胞比例的减少，同時凋亡細胞的比例有所增加；SK-UT-1的DNA含量在1 μM供試品作用下即已發生明顯變化（S期細胞减少、凋亡細胞增加），3 μM的供試品使G2/M期和多倍體細胞顯著增加，而當供試品劑量達到6 μM時，G2/M期、多倍體及凋亡細胞的總比例已達到90%左右。（圖3）Data processing and statistical analysis: FlowJo software was used for cell DNA content analysis and light scattering analysis to obtain cycle distribution data in each sample, and the differences among groups were compared by t test (P < 0.01 means significant difference). The test results showed that: 143B had a significant increase in the proportion of G2/M phase and polyploid cells under the action of 3 μM of the test substance. When the dose reached 6 μM, the proportion of G2/M phase and polyploid cells in 143B increased significantly. The increase was more significant; SW982 had a significant increase in the proportion of G2/M phase and polyploid cells under the action of 6 μM test substance; the most obvious change of U2OS was S phase cells under the action of 3 or 6 μM test substance At the same time, the proportion of apoptotic cells increased; the DNA content of SK-UT-1 had changed significantly under the action of 1 μM of the test substance (decreased cells in S phase and increased apoptotic cells), and the DNA content of 3 μM The test substance can significantly increase the G2/M phase and polyploid cells, and when the dose of the test substance reaches 6 μM, the total proportion of G2/M phase, polyploid and apoptotic cells has reached about 90%. (image 3)

實施例4　蛋白激酶抑制劑引起部分肉瘤細胞凋亡的實驗研究Example 4 Experimental research on apoptosis of some sarcoma cells induced by protein kinase inhibitors

接種細胞及給藥：將消化收集的143B和SK-UT-1細胞按每孔10 ⁶個/2 mL接種到六孔細胞培養板中，培養過夜待細胞貼壁後給藥，每孔分別按終濃度0、1、3、6 μM加入供試品，繼續培養48小時。 Inoculation of cells and administration: inoculate 143B and SK-UT-1 cells collected by digestion into six-well cell culture plates at 10 ⁶ cells/2 mL per well, culture overnight and administer after the cells adhered to the wall. The final concentration of 0, 1, 3, 6 μM was added to the test product, and the culture was continued for 48 hours.

樣品收集與處理：供試品作用48小時後，用細胞刮刀輕輕將細胞刮下來，轉移至15 mL離心管中，再每孔加入1 mL PBS洗滌一遍，清洗液轉入同一支離心管，4 ℃離心機1200 rpm離心5分鐘，棄去上清，細胞沉澱加入500 μL PBS重懸，轉移至1.5 mL離心管，4 ℃離心機1200 rpm離心5分鐘，棄上清。細胞沉澱加入110 μL 1× 蛋白上樣緩衝液，劇烈振蕩混勻後置於金屬浴，100 ℃煮沸10分鐘，取出樣品管冰浴冷却後待用（可於−80 ℃冰箱保存）。Sample collection and processing: After 48 hours of exposure to the test product, gently scrape the cells with a cell scraper, transfer them to a 15 mL centrifuge tube, add 1 mL PBS to each well to wash once, transfer the cleaning solution to the same centrifuge tube, Centrifuge at 1200 rpm at 4°C for 5 minutes, discard the supernatant, resuspend the cell pellet in 500 μL PBS, transfer to a 1.5 mL centrifuge tube, centrifuge at 1200 rpm at 4°C for 5 minutes, and discard the supernatant. Add 110 μL of 1× protein loading buffer to the cell pellet, vortex vigorously, place in a metal bath, boil at 100 °C for 10 minutes, take out the sample tube and cool it in an ice bath for later use (it can be stored in a −80 °C refrigerator).

SDS-PAGE電泳及western blot檢測：將上述蛋白樣品離心後取上清進行SDS-PAGE電泳，每孔上樣體積40 μL，電泳條件為恒壓140 V，50分鐘；電泳結束後，在Bio-Rad turbo半乾轉印儀的電極板上鋪上一層與膠大小相同並經轉膜緩衝液浸透的濾紙，再鋪上提前用甲醇活化好的PVDF膜，將PAGE膠鋪在膜上，趕走氣泡後再鋪一層與膠大小相同並經轉膜緩衝液浸透的濾紙，啟動裝置開始轉印，條件為25 V，7分鐘；完成轉印後，將膜放在雜交袋中，加入封閉液，封口，室溫下搖床搖動1小時。封閉結束取出膜按照Marker指示大小將膜剪開，放入新的雜交袋，分別加入按比例配置的一抗溶液（Cleaved PARP抗體按照1 : 1000稀釋, β-Actin抗體按照1:2000稀釋，抗體稀釋液為PBS + 1‰ Tween 20 + 5% BSA），趕氣泡後封口，室溫下孵育1小時，用PBST洗滌4次，每次5 分鐘；洗滌後將膜分別放入對應二抗中（Anti-rabbit二抗和Anti-Mouse二抗均按照1 : 2000比例用抗體稀釋液稀釋）室溫搖床搖動孵育1小時，孵育結束之後用PBST洗滌3次，每次5分鐘，最後用PBS洗滌一次，5分鐘。將洗好的膜放到顯影儀中，每條膜滴加200 μL預混的ECL發光液，操作儀器進行影像記錄。SDS-PAGE electrophoresis and western blot detection: Centrifuge the above protein samples and take the supernatant for SDS-PAGE electrophoresis. The loading volume of each well is 40 μL. The electrode plate of the Rad turbo semi-dry transfer machine is covered with a layer of filter paper that is the same size as the gel and soaked in the transfer buffer, and then covered with a PVDF membrane that has been activated with methanol in advance, and the PAGE gel is spread on the membrane to remove After the air bubbles, spread a layer of filter paper with the same size as the gel and soaked in the transfer buffer, start the device to start the transfer, the condition is 25 V, 7 minutes; after the transfer is completed, put the membrane in the hybridization bag, add the blocking solution, Seal and shake on a shaker at room temperature for 1 hour. After sealing, take out the membrane and cut the membrane according to the size indicated by the Marker, put it into a new hybridization bag, and add the primary antibody solution configured in proportion (Cleaved PARP antibody is diluted at 1:1000, β-Actin antibody is diluted at 1:2000, antibody Diluent is PBS + 1‰ Tween 20 + 5% BSA), seal the seal after removing air bubbles, incubate at room temperature for 1 hour, wash 4 times with PBST, 5 minutes each time; after washing, put the membrane into the corresponding secondary antibody ( Both Anti-rabbit secondary antibody and Anti-Mouse secondary antibody were diluted with antibody diluent at a ratio of 1:2000) and incubated on a shaker at room temperature for 1 hour. After the incubation, washed 3 times with PBST for 5 minutes each time, and finally washed with PBS Once, 5 minutes. Put the washed membranes into a developing device, add 200 μL of premixed ECL luminescent solution dropwise to each membrane, and operate the instrument for image recording.

試驗結果：通過western blot實驗，在143B和SK-UT-1細胞系中驗證了蛋白激酶抑制劑對肉瘤的促凋亡效應。在不同劑量供試品的作用後，143B細胞中沒有檢出PARP蛋白剪切體，而SK-UT-1細胞中則檢測到了PARP蛋白剪切體且呈劑量依賴性增加趨勢。結果表明，供試品在143B細胞中可能不是通過誘導細胞凋亡的機制抑制腫瘤，而在SK-UT-1中則可以誘導腫瘤細胞的凋亡——與流式細胞術實驗所檢測到的情况一致。（圖4）Test results: Through western blot experiments, the pro-apoptotic effect of protein kinase inhibitors on sarcoma was verified in 143B and SK-UT-1 cell lines. After different doses of the test substance, no PARP protein splicing body was detected in 143B cells, but PARP protein splicing body was detected in SK-UT-1 cells and showed a dose-dependent increase trend. The results show that the test substance may not inhibit tumors through the mechanism of inducing apoptosis in 143B cells, but it can induce apoptosis of tumor cells in SK-UT-1 - which is consistent with that detected by flow cytometry experiments The situation is consistent. (Figure 4)

實施例5　蛋白激酶抑制劑在裸鼠荷瘤模型中觀察供試品的體內抑瘤藥效試驗研究Example 5 Protein Kinase Inhibitor Observation of In vivo Tumor Inhibition Drug Effect Test Research of Test Article in Nude Mouse Tumor-bearing Model

裸鼠移植瘤模型的構建：大量擴增培養143B細胞並使細胞保持在對數生長狀態。待細胞數量達到所需後消化收集細胞，並用大量PBS充分清洗2次以去除胰酶和血清成分，室溫、800 rpm離心10分鐘，棄上清。用不含FBS的DMEM培養液重懸細胞，調整細胞濃度至5×10 ⁶個 / 200 μL。按每針200 μL將細胞懸液注射至裸鼠腋窩中部外側皮下，每隻裸鼠注射一針。接種細胞後，每天觀察傷口癒合情况及成瘤情况，適時用游標卡尺測量腫瘤最長徑（length）及與之垂直的最寬徑（width），通過公式Tumor Volume = length × (width) ²/2計算腫瘤體積。當腫瘤體積（Tumor Volume）達到約100 mm ³時視為成瘤，即可進行給藥實驗。 Construction of xenograft tumor model in nude mice: a large number of 143B cells were expanded and cultured, and the cells were kept in a logarithmic growth state. After the number of cells reached the required level, the cells were digested and collected, washed twice with a large amount of PBS to remove trypsin and serum components, centrifuged at room temperature at 800 rpm for 10 minutes, and the supernatant was discarded. Resuspend the cells in DMEM medium without FBS, and adjust the cell concentration to 5×10 ⁶ cells/200 μL. The cell suspension was injected subcutaneously into the middle and outer side of the axilla of nude mice at 200 μL per injection, and each nude mouse was injected with one injection. After inoculating the cells, observe the wound healing and tumor formation every day, measure the longest diameter (length) and the widest diameter (width) perpendicular to it with a vernier caliper in due course, and calculate by the formula Tumor Volume = length × (width) ² /2 tumor volume. When the tumor volume (Tumor Volume) reaches about 100 ^mm3 , it is considered as a tumor, and the drug administration experiment can be carried out.

給藥及數據採集：將成瘤裸鼠隨機分成3組（即溶劑對照組、供試品 5 mg/kg組和供試品 10 mg/kg組）、每組8隻，標記後分籠飼養並開始給藥。每隻裸鼠給藥前測量體重，按每千克體重計算給藥劑量，即溶劑對照組按每克體重10 μL的CMC-Na溶液、供試品 5或10 mg/kg組按每克體重10 μL的0.5或1 mg/mL 供試品-CMC-Na懸濁液進行灌胃給藥。每隻裸鼠每天定時灌胃給藥1次，觀察到有個體所荷腫瘤體積達到2000 mm ³時視為實驗終點。給藥周期：24天；給藥方式：口服灌胃。 Dosing and data collection: The tumor-forming nude mice were randomly divided into 3 groups (the solvent control group, the test product 5 mg/kg group, and the test product 10 mg/kg group), 8 in each group, and raised in separate cages after marking and start dosing. Measure the body weight of each nude mouse before administration, and calculate the dosage per kilogram of body weight, that is, the solvent control group uses 10 μL of CMC-Na solution per gram of body weight, and the test product 5 or 10 mg/kg group uses 10 μL of CMC-Na solution per gram of body weight. μL of 0.5 or 1 mg/mL test article-CMC-Na suspension was administered by intragastric administration. Each nude mouse was intragastrically administered once a day at regular intervals, and when the tumor volume of an individual reached 2000 mm ³ was observed, it was regarded as the end point of the experiment. Administration cycle: 24 days; administration method: oral gavage.

試驗結果：通過對各組裸鼠荷瘤體積的記錄分析發現，相對於溶劑對照組，供試品5 mg/kg組的荷瘤體積得到了一定抑制，而供試品10 mg/kg組的荷瘤體積則得到了更為明顯的抑制。同時，相關給藥並沒有引起裸鼠體重的顯著變化。（圖5）Test results: Through the record analysis of the tumor-bearing volume of nude mice in each group, it was found that compared with the solvent control group, the tumor-bearing volume of the 5 mg/kg group of the test product was inhibited to a certain extent, while the tumor-bearing volume of the 10 mg/kg group of the test product The tumor-bearing volume was more significantly suppressed. At the same time, the relevant administration did not cause significant changes in the body weight of nude mice. (Figure 5)

綜合上述實驗結果可以看出：Based on the above experimental results, it can be seen that:

供試品可顯著抑制人骨肉瘤細胞系MG63、U2OS、143B，人滑膜肉瘤細胞系SW982和人卡波西（Kaposi）肉瘤細胞系SK-UT-1等的生長和克隆形成，在不同的肉瘤細胞模型中以誘導G2/M期阻滯、細胞多倍體化和／或細胞凋亡等機制抑制腫瘤。實驗動物的體內藥效結果也表明，供試品用於肉瘤的治療是安全、有效的。The test product can significantly inhibit the growth and clone formation of human osteosarcoma cell lines MG63, U2OS, 143B, human synovial sarcoma cell line SW982 and human Kaposi sarcoma cell line SK-UT-1, etc. In cell models, tumors are inhibited by mechanisms such as induction of G2/M phase arrest, cell polyploidization and/or apoptosis. The in vivo pharmacodynamic results of experimental animals also show that the test product is safe and effective for the treatment of sarcoma.

無。none.

圖1繪示N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺抑制肉瘤細胞的增殖。圖2繪示N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺抑制肉瘤細胞的克隆形成能力。圖3繪示N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺引起肉瘤細胞周期阻滯並誘導多倍體。圖4繪示N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺引起部分肉瘤細胞凋亡。圖5繪示N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺在裸鼠荷瘤模型體內抑制肉瘤生長。 Figure 1 shows that N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthylamide inhibits the proliferation of sarcoma cells. Figure 2 shows that N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthylamide inhibits the clonogenicity of sarcoma cells. Figure 3 shows that N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthamide causes sarcoma cell cycle arrest and induces polyploidy. FIG. 4 shows that N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthamide induces apoptosis in some sarcoma cells. FIG. 5 shows that N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthylamide inhibits the growth of sarcoma in a tumor-bearing nude mouse model.

Claims

一種蛋白激酶抑制劑在製備預防和／或治療肉瘤的藥物中的應用。Application of a protein kinase inhibitor in the preparation of drugs for preventing and/or treating sarcoma.

如請求項1所述的應用，其中，該肉瘤為骨肉瘤、軟組織肉瘤、血管肉瘤中的一種或多種；優選地，該軟組織肉瘤為滑膜肉瘤；優選地，該血管肉瘤為卡波西肉瘤。The application as claimed in claim 1, wherein the sarcoma is one or more of osteosarcoma, soft tissue sarcoma, and angiosarcoma; preferably, the soft tissue sarcoma is synovial sarcoma; preferably, the angiosarcoma is Kaposi's sarcoma .

如請求項1所述的應用，其中，該蛋白激酶抑制劑選自Aurora B抑制劑，和／或，VEGFR抑制劑，和／或，PDGFR抑制劑，和／或 c-Kit／CSF1R抑制劑。The application according to claim 1, wherein the protein kinase inhibitor is selected from Aurora B inhibitors, and/or, VEGFR inhibitors, and/or, PDGFR inhibitors, and/or c-Kit/CSF1R inhibitors.

如請求項1至3任一所述的應用，其中，該蛋白激酶抑制劑選自以下化合物或其藥學上可接受的鹽、晶型、異構體、前藥或代謝產物：N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(6,7-二甲氧基喹唑啉-4-氧)-1-萘醯胺；N-(2-胺基-4-氟苯基)-6-(6,7-二甲氧基喹唑啉-4-氧)-1-萘醯胺；N-(2-胺基-4-氟苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(6,7-二甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基-4-氟苯基)-6-(6,7-二甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(8-(三氯甲基)喹啉-4-氧)-1-萘醯胺，或選自下述Aurora B抑制劑／VEGFR抑制劑／PDGFR抑制劑／c-Kit抑制劑／CSF1R抑制劑中的一種或多種：塞尼舍替（cenisertib）、橙皮苷（hesperidin）、安羅替尼（anlotinib）、海那替尼（henatinib）、阿帕替尼（Apatinib）、索拉非尼（sorafenib）、瑞格非尼（regorafenib）、卡博替尼（Cometriq）、侖伐替尼（Lenvatinib）、凡德他尼（vandetanib）、舒尼替尼（Sunitinib）伊馬替尼（Imatinib）、克萊拉尼（Crenolanib）、阿伐替尼（Avapritinib）、馬賽替尼（Masitinib）、尼洛替尼（Nilotinib）、艾克利單抗（Axatilimab）、培西替尼（Pexidartinib）。The application as described in any one of claims 1 to 3, wherein the protein kinase inhibitor is selected from the following compounds or their pharmaceutically acceptable salts, crystal forms, isomers, prodrugs or metabolites: N-(2 -Aminophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthylamide; N-(2-aminophenyl)-6-(6,7-dimethoxy N-(2-amino-4-fluorophenyl)-6-(6,7-dimethoxyquinazoline-4-oxy) -1-naphthylamide; N-(2-amino-4-fluorophenyl)-6-(7-methoxyquinoline-4-oxygen)-1-naphthylamide; N-(2-amine N-(2-amino-4-fluorophenyl)-6-(6, 7-dimethoxyquinoline-4-oxo)-1-naphthamide; N-(2-aminophenyl)-6-(quinoline-4-oxo)-1-naphthamide; N- (2-Aminophenyl)-6-(8-(trichloromethyl)quinoline-4-oxo)-1-naphthamide, or selected from the following Aurora B inhibitors/VEGFR inhibitors/PDGFR inhibitors One or more of the following agents/c-Kit inhibitors/CSF1R inhibitors: cenisertib, hesperidin, anlotinib, henatinib, apa Apatinib, sorafenib, regorafenib, cabozantinib (Cometriq), lenvatinib, vandetanib, sunitinib (Sunitinib) Imatinib, Crenolanib, Avapritinib, Masitinib, Nilotinib, Axatilimab, Pexidartinib.

如請求項4所述的應用，其中，該蛋白激酶抑制劑選自N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺及其晶型；優選地，該晶型為非溶劑化晶型；優選地，該非溶劑化晶型選自N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺晶型A、晶型B、晶型C。Application as described in claim item 4, wherein, the protein kinase inhibitor is selected from N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxygen)-1-naphthamide and crystal forms thereof; preferably, the crystal form is a non-solvated crystal form; preferably, the non-solvated crystal form is selected from N-(2-aminophenyl)-6-(7-methoxyquinoline- 4-Oxo)-1-naphthylamide crystal form A, crystal form B, and crystal form C.

一種預防和／或治療肉瘤的藥物組合物，以蛋白激酶抑制劑為主要活性成分，並包含藥學上可接受的輔料；優選地，該肉瘤為骨肉瘤、軟組織肉瘤、血管瘤中的一種或多種；優選地，該軟組織肉瘤為滑膜肉瘤；優選地，該血管肉瘤為卡波西肉瘤。A pharmaceutical composition for preventing and/or treating sarcoma, with a protein kinase inhibitor as the main active ingredient and containing pharmaceutically acceptable auxiliary materials; preferably, the sarcoma is one or more of osteosarcoma, soft tissue sarcoma, and hemangioma ; Preferably, the soft tissue sarcoma is synovial sarcoma; Preferably, the angiosarcoma is Kaposi's sarcoma.

如請求項6所述的藥物組合物，其中，該蛋白激酶抑制劑選自Aurora B抑制劑，和／或，VEGFR抑制劑，和／或，PDGFR抑制劑，和／或 c-Kit／CSF1R抑制劑。The pharmaceutical composition according to claim 6, wherein the protein kinase inhibitor is selected from Aurora B inhibitors, and/or, VEGFR inhibitors, and/or, PDGFR inhibitors, and/or c-Kit/CSF1R inhibitors agent.

如請求項6或7所述的藥物組合物，其中，該蛋白激酶抑制劑選自以下化合物，或其藥學上可接受的鹽、晶型、異構體、前藥或代謝產物：N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(6,7-二甲氧基喹唑啉-4-氧)-1-萘醯胺；N-(2-胺基-4-氟苯基)-6-(6,7-二甲氧基喹唑啉-4-氧)-1-萘醯胺；N-(2-胺基-4-氟苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(6,7-二甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基-4-氟苯基)-6-(6,7-二甲氧基喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(喹啉-4-氧)-1-萘醯胺；N-(2-胺基苯基)-6-(8-(三氯甲基)喹啉-4-氧)-1-萘醯胺，或選自下述Aurora B抑制劑／VEGFR抑制劑／PDGFR抑制劑／c-Kit抑制劑／CSF1R抑制劑中的一種或多種：塞尼舍替（cenisertib）、橙皮苷（hesperidin）、安羅替尼（anlotinib）、海那替尼（henatinib）、阿帕替尼（Apatinib）、索拉非尼（sorafenib）、瑞格非尼（regorafenib）、卡博替尼（Cometriq）、侖伐替尼（Lenvatinib）、凡德他尼（vandetanib）、舒尼替尼（Sunitinib）、伊馬替尼（Imatinib）、克萊拉尼（Crenolanib）、阿伐替尼（Avapritinib）、馬賽替尼（Masitinib）、尼洛替尼（Nilotinib）、艾克利單抗（Axatilimab）、培西替尼（Pexidartinib）。The pharmaceutical composition as claimed in item 6 or 7, wherein the protein kinase inhibitor is selected from the following compounds, or pharmaceutically acceptable salts, crystal forms, isomers, prodrugs or metabolites thereof: N-( 2-aminophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthamide; N-(2-aminophenyl)-6-(6,7-dimethyl Oxyquinazoline-4-oxo)-1-naphthamide; N-(2-amino-4-fluorophenyl)-6-(6,7-dimethoxyquinazoline-4-oxo )-1-naphthamide; N-(2-amino-4-fluorophenyl)-6-(7-methoxyquinoline-4-oxygen)-1-naphthamide; N-(2- Aminophenyl)-6-(6,7-dimethoxyquinoline-4-oxo)-1-naphthamide; N-(2-amino-4-fluorophenyl)-6-(6 ,7-dimethoxyquinoline-4-oxo)-1-naphthamide; N-(2-aminophenyl)-6-(quinoline-4-oxo)-1-naphthamide; N -(2-Aminophenyl)-6-(8-(trichloromethyl)quinoline-4-oxo)-1-naphthamide, or selected from the following Aurora B inhibitors/VEGFR inhibitors/PDGFR One or more of inhibitors/c-Kit inhibitors/CSF1R inhibitors: cenisertib, hesperidin, anlotinib, henatinib, a Apatinib, sorafenib, regorafenib, cabozantinib (Cometriq), lenvatinib, vandetanib, sunitinib Sunitinib, Imatinib, Crenolanib, Avapritinib, Masitinib, Nilotinib, Axatilimab , Pexidartinib.

如請求項8所述的藥物組合物，其中，該蛋白激酶抑制劑選自N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺及其晶型，優選地，該晶型為非溶劑化晶型，優選地，該非溶劑化晶型包括N-(2-胺基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘醯胺的晶型A、晶型B和晶型C。The pharmaceutical composition as claimed in item 8, wherein the protein kinase inhibitor is selected from N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxygen)-1-naphthalene Amide and its crystal form, preferably, the crystal form is a non-solvated crystal form, preferably, the non-solvated crystal form includes N-(2-aminophenyl)-6-(7-methoxyquinoline - crystalline form A, crystalline form B and crystalline form C of 4-oxo)-1-naphthamide.

如請求項6至9任一項所述的藥物組合物，其中，該藥學上可接受的輔料選自潤滑劑、黏合劑、填充劑、防腐劑、表面活性劑、著色劑、矯味劑、乳化劑、助懸劑、稀釋劑、膠凝劑、崩解劑、pH調節劑、增溶劑的一種或兩種及以上；優選地，該藥物組合物是片劑、膠囊、粉劑、液體製劑、懸浮劑、針劑或緩釋製劑。The pharmaceutical composition as described in any one of claims 6 to 9, wherein the pharmaceutically acceptable adjuvant is selected from lubricants, adhesives, fillers, preservatives, surfactants, coloring agents, flavoring agents, emulsifying One or two or more of agent, suspending agent, diluent, gelling agent, disintegrating agent, pH regulator, solubilizer; preferably, the pharmaceutical composition is tablet, capsule, powder, liquid preparation, suspension doses, injections or sustained-release preparations.