WO2022247844A1 - 包含蛋白激酶抑制剂的药物组合物及其医药用途 - Google Patents

包含蛋白激酶抑制剂的药物组合物及其医药用途 Download PDF

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WO2022247844A1
WO2022247844A1 PCT/CN2022/094828 CN2022094828W WO2022247844A1 WO 2022247844 A1 WO2022247844 A1 WO 2022247844A1 CN 2022094828 W CN2022094828 W CN 2022094828W WO 2022247844 A1 WO2022247844 A1 WO 2022247844A1
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sarcoma
inhibitors
naphthylamide
oxo
aminophenyl
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PCT/CN2022/094828
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English (en)
French (fr)
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周游
山松
张星
鲁先平
李灵杰
张钰
李丹丹
师丹丹
黄琳
黄宁
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深圳微芯生物科技股份有限公司
成都微芯药业有限公司
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Priority to CN202280032836.2A priority Critical patent/CN117377492A/zh
Publication of WO2022247844A1 publication Critical patent/WO2022247844A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the field of pharmaceutical technology, in particular to a pharmaceutical composition containing a protein kinase inhibitor and its medical application.
  • 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.) differentiated features.
  • the naming method is to add "sarcoma" after the name of the tissue source, such as fibrosarcoma (fibrosarcoma), rhabdomyosarcoma (rhabdomyosarcoma), osteosarcoma (osteosarcoma), synovial sarcoma (synovial sarcoma), angiosarcoma (angiosarcoma), such as Kaposi Sarcoma (Kaposi's sarcoma) and so on.
  • fibrosarcoma fibrosarcoma
  • rhabdomyosarcoma rhabdomyosarcoma
  • osteosarcoma osteosarcoma
  • synovial sarcoma synovial sarcoma
  • 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. It can metastasize to the lungs within a few months, and the survival rate within 3 to 5 years after amputation is only 5 to 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 backbone 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 in the 15 to 40 years old. It rarely occurs from the synovium of the joint, but from the soft tissue near the joint, sometimes even far away from the joint. It occurs in the limbs, about 70 % occurred in the lower limbs, especially near the knee joint, followed by the feet and ankles, and more in the elbows of the upper limbs.
  • 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) or Kaposi's sarcoma
  • Kaposi's sarcoma is a rare tumor first reported by the Hungarian dermatologist Kaposi in 1872. older people. 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 and typically manifests as multiple macular, plaque, or nodular lesions on the skin. It can also involve mucous membranes, Lymph nodes and internal organs.
  • KS classic indolent, African endemic, iatrogenic, and acquired immunodeficiency syndrome-associated.
  • 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.
  • WO2010139180A1 discloses a general compound structure of a protein kinase and histone deacetylase dual-target inhibitor, and specifically discloses N-(2-aminophenyl)-6-(7-methoxyquinoline- 4-oxo)-1-naphthylamide; N-(2-aminophenyl)-6-(6,7-dimethoxyquinazoline-4-oxygen)-1-naphthylamide; N-(2- Amino-4-fluorophenyl)-6-(6,7-dimethoxyquinazoline-4-oxo)-1-naphthylamide; N-(2-amino-4-fluorophenyl)-6- (7-methoxyquinoline-4-oxygen)-1-naphthylamide; N-(2-aminophenyl)-6-(6,7-dimethoxyquinoline-4-oxygen)-1- Naphthylamide; N-(2-amino-4-
  • the object of the present invention is to provide a new medical application of a protein inhibitor in the field of sarcoma treatment.
  • 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 soft tissue sarcoma is synovial sarcoma; preferably, the angiosarcoma is Kaposi's sarcoma.
  • the unit dose of the protein kinase inhibitor is 1-20 mg.
  • the unit dose of the protein kinase inhibitor is 5-10 mg.
  • the unit dose of the protein kinase inhibitor is 5 mg.
  • 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 The soft tissue sarcoma is synovial sarcoma; preferably, the angiosarcoma is Kaposi's sarcoma.
  • the present invention provides a pharmaceutical composition for preventing and/or treating sarcoma
  • the pharmaceutical composition uses protein kinase inhibitors as the main active ingredient and includes pharmaceutically acceptable adjuvants; preferably, the sarcoma It 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.
  • the unit dose of the protein kinase inhibitor is 1-20 mg.
  • the unit dose of the protein kinase inhibitor is 5-10 mg.
  • the unit dose of the protein kinase inhibitor is 5 mg.
  • the pharmaceutically acceptable adjuvant is selected from lubricants, binders, fillers, preservatives, surfactants, colorants, flavoring agents, emulsifiers, suspending agents, diluents, gelling agents , disintegrants, pH regulators, and solubilizers, or two or more of them.
  • 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.
  • 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 angiosarcoma is Kaposi's sarcoma.
  • the protein kinase inhibitor is selected from Aurora B inhibitors, and/or, VEGFR inhibitors, and/or, PDGFR inhibitors, and/or c-Kit/CSF1R inhibitors.
  • 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-oxygen)-1-naphthylamide; N-(2-aminophenyl)-6-(6,7-dimethoxyquinazoline-4- Oxygen)-1-naphthylamide; N-(2-amino-4-fluorophenyl)-6-(6,7-dimethoxyquinazoline-4-oxygen)-1-naphthylamide; N-( 2-amino-4-fluorophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthylamide; N-(2-aminophenyl)-6-(6,7-di Methoxyquinoline-4-oxo)-1-naphthyl
  • the protein kinase inhibitor is selected from N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxygen)-1-naphthylamide or Pharmaceutically acceptable salts, crystal forms, isomers, prodrugs or metabolites.
  • the protein kinase inhibitor is selected from N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthylamide and its crystal forms.
  • the crystalline form is an ansolvated crystalline form.
  • the non-solvated crystal form is selected from N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxygen)-1-naphthylamide crystal form A, crystal Form B, crystal form C.
  • N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxygen)-1-naphthylamide or a pharmaceutically acceptable salt, crystal type, isomer in the composition or the prepared medicine, its unit dosage is 1-20mg (with active ingredient N-(2-aminophenyl)-6-(7-methoxyquinoline-4 -oxy)-1-naphthylamide).
  • N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxygen)-1-naphthylamide or a pharmaceutically acceptable salt, crystal Type, isomer in the composition or the prepared medicine, its unit dose is 5-10mg (with active ingredient N-(2-aminophenyl)-6-(7-methoxyquinoline-4 -oxy)-1-naphthylamide).
  • N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxygen)-1-naphthylamide or a pharmaceutically acceptable salt, crystal type, isomer in the composition or the prepared medicine, its unit dosage is 5 mg (with the active ingredient N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxo )-1-naphthylamide).
  • the crystal forms include non-solvated crystal forms, solvated crystal forms and amorphous structures.
  • the isomers include conformational isomers, enantiomers and diastereoisomers.
  • 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 naphthylamide with a pharmaceutically acceptable acid, or a salt formed by reacting a compound with an acidic group with a pharmaceutically acceptable base.
  • 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.
  • 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.
  • 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.
  • 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.
  • the "individual" in the individual in need refers to a vertebrate.
  • 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.
  • a mammal is a human.
  • 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-aminobenzene base)-6-(7-methoxyquinoline-4-oxy)-1-naphthylamide has good therapeutic effects on osteosarcoma, synovial sarcoma and Kaposi's sarcoma.
  • N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthylamide inhibits the growth of sarcoma in a tumor-bearing nude mouse model.
  • Figure 6 N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxygen)-1-naphthylamide tumor volume in each group of mice in SK-ES-1 human sarcoma model growth curve.
  • the invention discloses a pharmaceutical composition and a new medical application of a protein kinase inhibitor for sarcoma.
  • a protein kinase inhibitor for sarcoma for sarcoma.
  • Those skilled in the art can refer to the contents of this article for appropriate optimization or equivalent transformation.
  • 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.
  • 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), Culture in DMEM medium (Hyclone) containing 1% double antibody (penicillin, Hyclone) and 10% fetal bovine serum (Gibco).
  • test article N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthylamide (protein kinase inhibitor, hereinafter referred to as "test article"): sourced from Shenzhen Microelectronics Core Biotechnology Co., Ltd., dissolved it in dimethyl sulfoxide (Dimethyl sulfoxide, DMSO; Shanghai Sangong, DN3039A), configured it into a 20mM working mother solution, and placed it in a refrigerator at -20°C for use. Each experiment needs to prepare the working solution with the corresponding final concentration.
  • DMSO dimethyl sulfoxide
  • MTS Non-Radioactive Cell Proliferation Assay
  • Ribonuclease A (RNase A; Shanghai Sangong, B500474)
  • Triton X-100 (Shanghai Sangong, A110694)
  • Antibody Cleaved PARP(Asp214)(D64E10) Rabbit mAb (Cell Signaling, #5625), ⁇ -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) 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.
  • CMC-Na suspending agent for preparing need testing product suspension and as contrast solvent: in 200mL deionized water, add altogether 0.4g CMC-Na in several times, stir while adding, then in boiling water bath Heat and stir until the solution is clear. After cooling, centrifuge to take the supernatant, add 0.2g Tween-20 after high temperature and high pressure sterilization, mix well, and set aside at room temperature.
  • 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 ⁇ L per well, and leave the BLANK wells without inoculating cells, only add the same volume culture medium; 5% CO 2 , 37°C culture.
  • 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 ⁇ L of the corresponding above-mentioned solution to each well according to the final concentration of 0, 1, 3, 6, and 9 ⁇ M of the test substance.
  • Detection After 120 hours of drug action, the culture solution in the 96-well culture plate was sucked out, and 50 ⁇ L of fresh phenol red-free 1640 culture solution and 10 ⁇ L CellTiter were added to each well. AQueous Non-Radioactive Cell Proliferation Assay (MTS) kit detection solution, 60 ⁇ L in total. Incubate at 37°C for 1-2 hours, and read the absorbance value of each well at a wavelength of 490nm with a light absorption microplate reader.
  • MTS Non-Radioactive Cell Proliferation Assay
  • 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 ⁇ M test substance is close to or exceeds 50%. (figure 1)
  • Inoculation of cells Inoculate MG63, U2OS, 143B, and SK-UT-1 cells collected by digestion into 6-well cell culture plates at 1000/2mL per well, culture overnight and administer after the cells adhere to the wall.
  • 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.
  • 6 ⁇ M of the test substance inhibited the ability of colony formation by more than 50%
  • 143B and SK-UT-1 the inhibitory intensity of 3 ⁇ M of the test substance reached or exceeded 80%
  • U2OS the inhibitory intensity of 1 ⁇ M of the test substance was already Closer to 50%.
  • 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.
  • Cell culture and administration Digest and collect 143B, SW982, U2OS and SK-UT-1 cells in the logarithmic growth phase, inoculate 10 6 cells/2mL into six-well plates, each inoculate 12 wells, and culture normally overnight. The above four kinds of cells were divided into 4 groups, each group had 3 wells, and the test substances were administered with final concentrations of 0, 1, 3, and 6 ⁇ M, respectively, and the cells were collected after 48 hours of culture.
  • Sample collection After the effect of the test product is completed, transfer the culture medium and cells in suspension to a 15mL centrifuge tube, gently wash the adherent cells in the well once with PBS, and transfer the cleaning solution to the same centrifuge tube; transfer to the culture plate Add 300 ⁇ L of trypsin digestion solution to the remaining adherent cells in the medium, 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 and merge Transfer to the corresponding 15mL centrifuge tube, centrifuge at room temperature and 800rpm for 10 minutes, remove the supernatant, wash once with PBS and resuspend in 300 ⁇ L PBS for use.
  • Staining and on-machine detection Mix PBS with 20mg/mL PI stock solution, 10mg/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-mentioned working dye solution in 500 ⁇ L per tube.
  • 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 ⁇ M of the test product. When the dose reached 6 ⁇ M, the increase in the proportion of G2/M phase and polyploid cells in 143B was even greater.
  • Cell inoculation and administration inoculate the 143B and SK-UT-1 cells collected by digestion into six-well cell culture plates at 10 6 cells/2mL per well, cultivate overnight and administer after the cells adhere to the wall. Concentrations of 0, 1, 3, 6 ⁇ M were added to the test substance, and the culture was continued for 48 hours.
  • Sample collection and processing After 48 hours of exposure to the test product, gently scrape the cells with a cell scraper, transfer to a 15mL centrifuge tube, then add 1ml PBS to each well to wash once, transfer the cleaning solution to the same centrifuge tube, and keep at 4°C Centrifuge at 1200 rpm for 5 minutes, discard the supernatant, resuspend the cell pellet in 500 ⁇ L PBS, transfer to a 1.5 mL centrifuge tube, centrifuge at 1200 rpm for 5 minutes at 4°C, and discard the supernatant.
  • SDS-PAGE electrophoresis and western blot detection Centrifuge the above protein samples and take the supernatant for SDS-PAGE electrophoresis. Cover the electrode plate of the semi-dry transfer machine with a layer of filter paper that is the same size as the gel and soaked in the transfer buffer, and then cover the PVDF membrane that has been activated with methanol in advance, and spread the PAGE gel on the membrane to drive away the air bubbles. Then spread a layer of filter paper with the same size as the gel and soaked in the transfer buffer, start the device to start transfer, the condition is 25V, 7 minutes; after the transfer is completed, put the membrane in the hybridization bag, add blocking solution, seal , shake on a shaker at room temperature for 1 hour.
  • 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)
  • 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 800 rpm for 10 minutes at room temperature, and the supernatant was discarded. Resuspend the cells in DMEM medium without FBS, and adjust the cell concentration to 5 ⁇ 10 6 cells/200 ⁇ L. The cell suspension was injected subcutaneously into the middle and outer side of the armpits of nude mice at 200 ⁇ L per injection, and each nude mouse was injected with one injection.
  • Tumor Volume length ⁇ (width) 2 /2 Calculate the tumor volume.
  • Tumor Volume reaches about 100 mm 3 , it is considered as a tumor, and the drug administration experiment can be carried out.
  • the tumor-forming nude mice were randomly divided into 3 groups (i.e. the solvent control group, the test product 5mg/kg group and the test product 10mg/kg group), 8 in each group, raised in separate cages after marking, and began to give medicine.
  • the body weight of each nude mouse is measured before administration, and the dosage is calculated per kilogram of body weight, that is, the solvent control group uses 10 ⁇ L of CMC-Na solution per gram of body weight, and the test product 5 or 10 mg/kg group uses 0.5 Or 1mg/mL test article-CMC-Na suspension is carried out 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.
  • 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, it inhibits tumors by inducing 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.
  • SK-ES-1 cells purchased from American type culture collection (ATCC), cultured with McCoy's 5A containing 1% double antibody (penicillin, streptomycin, Hyclone) and 15% fetal bovine serum (ExcellBio) liquid (Gibco) culture.
  • Fetal bovine serum Supplier: ExcellBio; Lot number: 11H305;
  • McCoy's 5A Supplier: GIBCO; Lot Number: 2318827;
  • Matrigel Supplier: CORNING; Lot No.: 1084001;
  • BALB/c nude mice male, 7-9 weeks old, purchased from Changzhou Branch of Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • test article N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxo)-1-naphthylamide (protein kinase inhibitor, hereinafter referred to as "test article"): sourced from Shenzhen Microelectronics Core Biotechnology Co., Ltd., dissolved it in dimethyl sulfoxide (Dimethyl sulfoxide, DMSO; Shanghai Sangong, DN3039A), configured it into a 20mM working mother solution, and placed it in a refrigerator at -20°C for use. Each experiment needs to prepare the working solution with the corresponding final concentration.
  • DMSO dimethyl sulfoxide
  • Preparation of the test product and the reference substance accurately weigh an appropriate amount of the test product, dissolve it in a solvent (sterile aqueous solution containing 0.2% CMC-Na and 0.1% Tween-80), and configure the final concentration to be 0.5 mg/ ml, 1mg/ml, 2mg/ml of the test solution, stored at 2-8°C.
  • a solvent sterile aqueous solution containing 0.2% CMC-Na and 0.1% Tween-80
  • SK-ES-1 cells were cultured in McCoy's 5A medium containing 15% fetal calf serum. SK-ES-1 cells in exponential growth phase were collected, resuspended in PBS and Matrigel (1:1) to a suitable concentration for subcutaneous inoculation of mice.
  • tumor volume (mm 3 ) 1/2 ⁇ (a ⁇ b 2 ), where a represents the long diameter and b represents the short diameter), they were randomly divided into groups according to tumor size.
  • model mice were randomly divided into 4 groups (vehicle control group, test product 5mg/kg group, test product 10mg/kg group and test product 20mg/kg group), 8 in each group, labeled Afterwards, they were reared in cages and started to administer drugs.
  • the day of grouping was defined as day 0, and the administration began on the day of grouping, with oral or gavage administration once a day for a total of three weeks.
  • the body weight of each nude mouse was measured before administration, and the dosage was calculated per kilogram of body weight, that is, the administration groups of 5mg/kg, 10mg/kg and 20mg/kg were given 5mg/kg, 10mg/kg and 20mg/kg every day respectively.
  • the volume of each administration is 10mL/kg; the vehicle control group is given an equal volume of vehicle. From the day of administration, the body weight of the mice was recorded every day; the tumor volume of the mice was measured and recorded on days 0, 4, 7, 11, 14, 18, and 21.
  • T/C Relative tumor proliferation rate
  • mice in the vehicle control group after three weeks of administration were 1203.75 mm 3 .
  • the average tumor volume of the test product 5 mg/kg administration group was 792.28 mm 3 after three weeks of administration, and the tumor inhibition rate TGI (%) was 34.18%.
  • the average tumor volumes of the 10mg/kg and 20mg/kg administration groups of the test product were 279.03mm 3 and 375.59mm 3 after three weeks of administration, and the tumor inhibition rates TGI (%) were 76.82% and 68.80% respectively, compared with the vehicle Statistically significant differences were shown in the control group (p ⁇ 0.05). See Figure 6 for the growth of tumors in each administration group and control group.
  • the experimental result of embodiment 6 shows: need testing product has the effect of inhibiting tumor growth to SK-ES-1 human source sarcoma tumor model under 5mg/kg dosage; SK-ES-1 human sarcoma tumor model can significantly inhibit tumor growth; tumor-bearing mice tolerate the test product well at the test dose.
  • the test product is safe and effective for the in vivo treatment of sarcoma.

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Abstract

包含蛋白激酶抑制剂的药物组合物及其医药用途。蛋白激酶抑制剂用于治疗肉瘤具有良好的治疗效果，尤其是N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺针对骨肉瘤、滑膜肉瘤和卡波西肉瘤三种亚型病症具有良好的治疗效果。

Description

包含蛋白激酶抑制剂的药物组合物及其医药用途 技术领域
本发明涉及药物技术领域，具体涉及包含蛋白激酶抑制剂的药物组合物及其医药用途。
背景技术
肉瘤(sarcoma)是一种常见的肿瘤疾病，是由间叶组织(包括纤维***、脂肪、肌肉、脉管、骨、软骨组织等)发生的恶性肿瘤，这些肿瘤表现出向某种间叶组织分化的特点。其命名方式是在组织来源名称之后加“肉瘤″，如纤维肉瘤(fibrosarcoma)、横纹肌肉瘤(rhabdomyosarcoma)、骨肉瘤(osteosarcoma)、滑膜肉瘤(synovialsarcoma)、血管肉瘤(angiosarcoma)，如卡波西肉瘤(Kaposi′s sarcoma)等。骨肉瘤是由肿瘤性成骨细胞、骨样组织所组成，为起源于成骨组织的恶性肿瘤。骨肉瘤发病率在原发性恶性肿瘤中占据首位。该瘤恶性程度甚高，愈后极差，可于数月内出现肺部转移，截肢后3～5年存活率仅为5～20％。骨肉瘤可发生在任何年龄，但大多在10～25岁，男性较多，肿瘤多处于骨端，偶发生于骨干或骨骺。滑膜肉瘤是一种恶性程度很高的软组织肉瘤，起源于具有滑膜分化的间叶细胞，但罕见于关节内。滑膜肉瘤是第四常见的软组织肉瘤，好发于15～40岁，它很少从关节滑膜发生，而是从关节附近的软组织内发生，有时甚至远离关节，好发于四肢，约70％发生于下肢，特别在膝关节附近，其次为足及踝部，上肢以肘部为多。另外与关节无关的部位如头颈部、腹壁、后腹膜也可发生。卡波西肉瘤又称Kaposi肉瘤(KS)、卡波济肉瘤，是1872年匈牙利皮肤科专家Kaposi首先报道的一种罕见的肿瘤，在非洲多见，并可形成地方性流行，且多发于60岁以上的老人。近10年来，在欧美男性同性恋者中出现了暴发性流行，同时，卡波济肉瘤与艾滋病有一定的关系，它被认为是艾滋病的一种常见肿瘤。卡波西肉瘤是一种由8型疱疹病毒引起的多灶性血管肿瘤，具有局部侵袭性，典型病变表现为皮肤多发性斑点状、斑块状或结节状病损，也可累及黏膜、***和内脏器官。根据临床和流行病学特点，KS有4种不同类型：经典惰性型、非洲地方性、医源性、获得性免疫缺陷综合征相关性。经典型KS特征是出现紫色、红蓝色或深棕色斑丘疹、斑块和结节，也可形成溃疡。尤其常见于肢体末端，可伴有淋巴水肿。此病一般为惰性，***和内脏不常受累。可伴有造血细胞性恶性病变。地方性KS可有皮肤病变，病程较长。淋巴腺病型是地方性KS的一个亚型，见于非洲儿童，进展快速，具有高度致命性。医源性KS相对常见，发生在实性器官移植或免疫抑制治疗之后数月或数年。AIDS相关性KS为侵袭性最强的KS，皮肤病损最常见于面部、生殖器和下肢。常见口腔黏膜、***、胃肠道和肺受累。肉瘤目前主要采用以手术为主，化疗和放疗为辅的治疗手法，但均预后较差。目前仍需进一步开发肉瘤的治疗方案。
WO2010139180A1公开了一种蛋白激酶和组蛋白去乙酰化酶双靶点抑制剂的通式化合物结构， 并具体公开了N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(6，7-二甲氧基喹唑啉-4-氧)-1-萘酰胺；N-(2-氨基-4-氟苯基)-6-(6，7-二甲氧基喹唑啉-4-氧)-1-萘酰胺；N-(2-氨基-4-氟苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(6，7-二甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基-4-氟苯基)-6-(6，7-二甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(8-(三氯甲基)喹啉-4-氧)-1-萘酰胺的化合物结构、合成工艺及其体外PDGF和VEGF配体依赖性细胞增殖试验的实验数据，并进一步公开了这些化合物的体外细胞增殖抑制试验数据，包括对A-498、A549、Bel-7402、HCT-8、MCF-7等多种肿瘤细胞增殖的抑制试验数据，及用于肺癌、肠癌、肝癌的动物模型实验数据。目前还未发现有蛋白激酶抑制剂可用于治疗肉瘤，如滑膜肉瘤、骨肉瘤、卡波西肉瘤的相关研究和报道。
发明内容
有鉴于此，本发明的目的在于提供一种蛋白抑制剂的在肉瘤治疗领域的新医药用途。
为实现上述发明目的，本发明提供如下技术方案：
第一方面，本发明提供蛋白激酶抑制剂在制备预防和/或治疗肉瘤的药物中的应用；优选的，所述肉瘤为骨肉瘤、软组织肉瘤、血管肉瘤中的一种或多种；优选的，所述软组织肉瘤为滑膜肉瘤；优选的，所述血管肉瘤为卡波西肉瘤。
在一些实施方案中，上述制备预防和/或治疗肉瘤的药物的应用，所述蛋白激酶抑制剂的单位剂量为1-20mg。
在一些实施方案中，上述制备预防和/或治疗肉瘤的药物的应用，所述蛋白激酶抑制剂的单位剂量为5-10mg。
在一些实施方案中，上述制备预防和/或治疗肉瘤的药物的应用，所述蛋白激酶抑制剂的单位剂量为5mg。
第二方面，本发明提供蛋白激酶抑制剂在预防和/或治疗肉瘤疾病中的应用；优选的，所述肉瘤为骨肉瘤、软组织肉瘤、血管肉瘤中的一种或多种；优选的，所述软组织肉瘤为滑膜肉瘤；优选的，所述血管肉瘤为卡波西肉瘤。
第三方面，本发明提供一种预防和/或治疗肉瘤的药物组合物，所述药物组合物以蛋白激酶抑制剂为主要活性成分，并包含药学上可接受的辅料；优选的，所述肉瘤为骨肉瘤、软组织肉瘤、血管瘤中的一种或多种；优选的，所述软组织肉瘤为滑膜肉瘤；优选的，所述血管肉瘤为卡波西肉瘤。
在一些实施方案中，上述预防和/或治疗肉瘤的药物组合物，所述蛋白激酶抑制剂的单位剂量为1-20mg。
在一些实施方案中，上述预防和/或治疗肉瘤的药物组合物，所述蛋白激酶抑制剂的单位剂量为5-10mg。
在一些实施方案中，上述预防和/或治疗肉瘤的药物组合物，所述蛋白激酶抑制剂的单位剂量为5mg。
优选的，所述药学上可接受的辅料选自润滑剂、粘合剂、填充剂、防腐剂、表面活性剂、着色剂、矫味剂、乳化剂、助悬剂、稀释剂、胶凝剂、崩解剂、pH调节剂、增溶剂的一种或两种及以上。
在一些实施方案中，所述药物组合物是片剂、胶囊、粉剂、液体制剂、悬浮剂、针剂、缓释制剂等常规剂型，优选口服片剂、胶囊或口服液体。
第四方面，本发明提供一种预防和/或治疗肉瘤的方法，包括向有需要的个体施用预防和/或治疗有效剂量的如上所述的药物组合物；优选的，所述肉瘤为骨肉瘤、软组织肉瘤、血管瘤中的一种或多种；优选的，所述软组织肉瘤为滑膜肉瘤；优选的，所述血管肉瘤为卡波西肉瘤。
在一些实施方案中，本发明中，所述蛋白激酶抑制剂选自Aurora B抑制剂，和/或，VEGFR抑制剂，和/或，PDGFR抑制剂，和/或c-Kit/CSF1R抑制剂。
在一些实施方案中，本发明中，所述蛋白激酶抑制剂选自以下化合物或其药学上可接受的盐、晶型、异构体、前药或代谢产物：N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(6，7-二甲氧基喹唑啉-4-氧)-1-萘酰胺；N-(2-氨基-4-氟苯基)-6-(6，7-二甲氧基喹唑啉-4-氧)-1-萘酰胺；N-(2-氨基-4-氟苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(6，7-二甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基-4-氟苯基)-6-(6，7-二甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(8-(三氯甲基)喹啉-4-氧)-1-萘酰胺，或选自下述Aurora B抑制剂/VEGFR抑制剂/PDGFR抑制剂/c-Kit抑制剂/CSF1R抑制剂中的一种或多种：塞尼舍替(cenisertib)、橙皮苷(hesperidin)、安罗替尼(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)、仑伐替尼(Lenvatinib)、凡德他尼(vandetanib)、舒尼替尼(Sunitinib)、伊马替尼(Imatinib)、克莱拉尼(Crenolanib)、阿伐替尼(Avapritinib)，马赛替尼(Masitinib)、尼洛替尼(Nilotinib)、艾克利单抗(Axatilimab)、培西替尼(Pexidartinib)。
在一些实施方案中，本发明中，所述蛋白激酶抑制剂选自N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺或其药学上可接受的盐、晶型、异构体、前药或代谢产物。优选的，本发明中，所述蛋白激酶抑制剂选自N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺及其晶型。
在一些实施方案中，所述晶型为非溶剂化晶型。
在一些实施方案中，所述非溶剂化晶型选自N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺晶型A、晶型B、晶型C。
在一些实施方案中，本申请中，N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺或其药学上可接受的盐、晶型、异构体，在所述组合物或制备的药物中，其单位剂量为1-20mg(以活性成分N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺计)。
在一些实施方案中，本申请中，N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺或其药学上可接受的盐、晶型、异构体，在所述组合物或制备的药物中，其单位剂量为5-10mg(以活性成分N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺计)。
在一些实施方案中，本申请中，N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺或其药学上可接受的盐、晶型、异构体，在所述组合物或制备的药物中，其单位剂量为5mg(以活性成分N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺计)。
N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺非溶剂化晶体、晶型A、晶型B、晶型C的***息见WO2018059429A1的专利公开文本，WO2018059429A1的全文通过引用的方式并入本申请。
本发明中，所述晶型包括非溶剂化晶型、溶剂化晶型和无定型结构。
本发明中，所述异构体包括构象异构体，对映异构体和非对应异构体。
本发明所述的“可药用盐″或“药学上可接受的盐″是指N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺与药学上可接受的酸进行反应制得的酸加成盐，或者其中具有酸性基团的化合物和药学上可接受的碱反应生成的盐。其中，所述的药学上可接受的酸较佳的选自无机酸(如盐酸、硫酸、磷酸或氢溴酸等)，和有机酸(如草酸、马来酸、富马酸、苹果酸、酒石酸、柠檬酸或苯甲酸等)；所述药学上可接受的碱较佳的选自氢氧化钠、氢氧化钾、氢氧化钙、碳酸钠、碳酸氢钾、氨水或碳酸氢铵等。
本发明中的前药，也称前体药物、药物前体、前驱药物等，是指药物经过化学结构修饰后得到的在体外无活性或活性较小、在体内经酶或非酶的转化释放出活性药物而发挥药效的化合物。
本发明中的代谢产物，是指化合物在动物个体体内经新陈代谢后得到的中间代谢产物和最终代谢产物。
本发明中，预防和/或治疗有效剂量中的“有效剂量″是指用药介于最小有限量和极量之间，并能对机体产生明显效应而不引起毒性反应的剂量。
本发明中，有需要的个体中的“个体″是指脊椎动物。在某些实施方案中，脊椎动物指哺乳动物。哺乳动物包括，但不限于，牲畜(诸如牛)、宠物(诸如猫、犬、和马)、灵长类动物、小鼠和大鼠。在某些实施方案中，哺乳动物指人。
本发明的有益效果：本发明提供了蛋白激酶抑制剂的一种新的医药用途，通过动物实验证明，蛋白激酶抑制剂用于治疗肉瘤具有良好的治疗效果，尤其是N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺针对骨肉瘤、滑膜肉瘤和卡波西肉瘤具有良好的治疗效果。
附图说明
图1：N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺抑制肉瘤细胞的增殖。
图2：N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺抑制肉瘤细胞的克隆形成能力。
图3：N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺引起肉瘤细胞周期阻滞并诱导多倍体。
图4：N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺引起部分肉瘤细胞凋亡。
图5：N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺在裸鼠荷瘤模型体内抑制肉瘤生长。
图6：N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺在SK-ES-1人源肉瘤模型中各组小鼠肿瘤体积的生长曲线。
具体实施方式
本发明公开了蛋白激酶抑制剂用于肉瘤的药用组合物和新医药用途，本领域技术人员可以借鉴本文内容，适当进行优化或等效变换。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明所述应用和药用组合物已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述应用和药用组合物进行改动或适当变更与组合，来实现和应用本发明技术。
以下就本发明所提供的蛋白激酶抑制剂用于治疗肉瘤的药用组合物及医药用途做进一步说明。
试验材料和仪器
(1)材料及来源
人骨肉瘤细胞系MG63、U2OS、143B，人滑膜肉瘤细胞系SW982和人卡波西(Kaposi)肉瘤细胞系SK-UT-1均购自美国菌种保藏中心(American type culture collection，ATCC)，用含1％双抗(青链霉素，Hyclone)和10％胎牛血清(Gibco)的DMEM培养液(Hyclone)培养。
N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺(蛋白激酶抑制剂，以下称为“供试品″)：来源于深圳微芯生物科技股份有限公司，将其溶于二甲基亚砜(Dimethyl sulfoxide，DMSO；上海生工，DN3039A)中，配置成20mM的工作母液，置于-20℃的冰箱中备用，使用时根据各项实验需要配置相应终浓度的工作溶液。
CellTiter
Figure PCTCN2022094828-appb-000001
AQueous Non-Radioactive Cell Proliferation Assay(MTS)试剂盒(Promega，G5421)
结晶紫染色液(上海碧云天生物技术有限公司，C0121)
碘化丙啶(Propidium iodide，PI；上海生工，A601112)
核糖核酸酶A(RNase A；上海生工，B500474)
Triton X-100(上海生工，A110694)
抗体：Cleaved PARP(Asp214)(D64E10)
Figure PCTCN2022094828-appb-000002
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)。
BALB/C(nu/nu)雌性裸鼠(SPF级)，5周龄，体重14～18g，购自广东省实验动物中心，共60只。在IVC笼具中按SPF条件饲喂，注射瘤株前饲养约1周，以使裸鼠适应新环境。
0.2％CMC-Na助悬剂(用于制备供试品悬液并作为对照溶剂)：在200mL去离子水中，分多次加入共0.4g CMC-Na，边加边搅拌，然后在沸水浴上加热搅拌直至溶液澄清。冷却后离心取上清，于高温高压灭菌后加入0.2g吐温-20混匀，室温备用。
(2)实验试剂及仪器：
超净工作台(苏净安泰)
CO 2细胞培养箱(RS Biotech，Galaxy S)
倒置荧光显微镜(广州市明美科技有限公司)
细胞计数板(上海求精生化试剂仪器有限公司)
Tecan infinite F50光吸收酶标仪
冷冻高速离心机(Eppendorf)
BD FACSCelestaTM流式细胞仪
蛋白电泳及转膜装置(Bio-Rad)
实施例1蛋白激酶抑制剂抑制肉瘤细胞的增殖试验研究
接种细胞：将消化收集的MG63、U2OS、SW982、143B和SK-UT-1细胞按每孔1000个/190μL接种到96孔细胞培养板中，并留出BLANK孔不接种细胞，只加入相同体积的培养液；5％CO 2、37℃培养。
给药：接种培养24小时后给药。首先用DMSO将供试品稀释成每孔终浓度的1000倍(即使每孔最终都加入了1/1000体积的DMSO)，再将供试品的DMSO稀释液按1:50稀释到培养液中。最终每孔按终浓度0、1、3、6、9μM供试品加入10μL相应的上述溶液。
检测：药物作用120小时后检测，将96孔培养板中的培养液吸出，每孔加入50μL新鲜的无酚红1640培养液及10μL CellTiter
Figure PCTCN2022094828-appb-000003
AQueous Non-Radioactive Cell Proliferation Assay(MTS)试剂盒检测液，共60μL。37℃孵育1～2小时，通过光吸收酶标仪读取每孔490nm波长处的吸光度值。
数据处理与分析：根据BLANK孔的读数求平均值OD490-BLK-A(背景值)，各孔读数减去OD490-BLK-A后获得扣除背景值的各给药孔的OD490-T及阴性对照孔的OD490-T0。求得阴性对照孔OD490-T0的平均值OD490-T0-A，各给药孔相对细胞存活率根据公式进行计算：相对细胞存活率(％)＝(OD490-T÷OD490-T0-A)×100％。
试验结果：通过MTS检测发现，与溶剂对照相比，供试品对各细胞系的增殖均有不同程度的抑制作用且具有明显的剂量依赖性，即供试品终浓度越高，各细胞系的相对细胞存活率越低。尤其在U2OS、SW982、143B和SK-UT-1中，3μM供试品的抑制率即已接近或超过50％。(图1)
实施例2蛋白激酶抑制剂抑制肉瘤细胞的克隆形成能力试验研究
接种细胞：将消化收集的MG63、U2OS、143B、SK-UT-1细胞按每孔1000个/2mL接种到6孔细胞培养板中，培养过夜待细胞贴壁后给药。
给药：每孔分别按终浓度0、1、3、6、9μM加入供试品，继续培养，并2～3天更换培养液及相应药物一次，给药周期7天。
染色观察：弃去培养液，按2mL/孔加入PBS溶液小心洗涤一次，弃去PBS后每孔加入0.5mL 95％乙醇溶液固定细胞5分钟。弃去乙醇溶液，每孔加入0.5mL结晶紫染色液，室温放置20分钟。弃去染色液，用自来水洗涤各孔，将培养板倒置于吸水纸上吸干水分，自然干燥后观察各孔染色情况，每个染色点记为1个克隆(即由单个细胞***增殖形成的细胞团)。
试验结果：结晶紫染色结果表明，供试品对肉瘤细胞的克隆形成能力具有抑制作用，且供试品对各细胞系的克隆形成能力均呈剂量依赖性。在MG63中6μM供试品对克隆形成能力的抑制超过50％，在143B和SK-UT-1中3μM供试品的抑制强度达到或超过80％，在U2OS中1μM供试品的抑制强度已接近50％。(图2)
实施例3蛋白激酶抑制剂抑制肉瘤的作用机制探索研究
试验方法：通过流式细胞术观察不同剂量供试品对143B、SW982、U2OS和SK-UT-1细胞周期状态的影响。通过流式细胞术检测细胞的PI染色情况可以显示每个细胞的DNA含量，从而分辨细胞处于G0/G1期(DNA含量＝2N)、S期(2N＜DNA含量＜4N)或G2/M期(DNA含量＝4N)，此外，当DNA含量＞4N时，表明这可能是一个多倍体细胞；DNA含量＜2N时，该细胞可能处于凋亡晚期。
试验过程：
细胞的培养及给药：将对数生长期的143B、SW982、U2OS和SK-UT-1细胞消化收集，按每孔10 6个/2mL接种到六孔板内，各接种12孔，正常培养过夜。上述四种细胞各分为4组，每组3孔，分别给予终浓度0、1、3、6μM的供试品，培养48小时后收集细胞。
样品收集：供试品作用完成后，将培养液及悬浮状态细胞转移至15mL离心管中，用PBS轻柔清洗孔中贴壁状态的细胞1次，清洗液转入同一支离心管；向培养板中剩下的贴壁细胞中加入300μL胰酶消化液，于37℃孵育5分钟，待细胞充分离散后，分别加入含10％FBS的培养液终止消化反应，并反复吸打重悬细胞后合并至对应的15mL离心管，室温、800rpm离心10分钟，去上清，以PBS洗1次后重悬于300μL PBS中待用。按样品数量在1.5mL离心管中以700μL每管加入无水乙醇，置冰上预冷备用。将上述收集到的细胞样品悬液逐管、逐滴滴入预冷的700μL无水乙醇，然后轻柔颠倒混匀数次，至此细胞已收集并固定，样品于4℃静置至少12小时。
染色及上机检测：将PBS与20mg/mL的PI储备液及10mg/mL的RNase A溶液及10％Triton X-100按1000:2.5:2:10的比例混匀成为工作染液。将固定好的细胞样品于4℃、1000rpm离心10分钟，吸净上清并用PBS洗涤两次，按毎管500μL重悬于上述工作染液中。37℃避光孵育30分钟后，将染色好的细胞样品于4℃、1000rpm离心10分钟，吸净上清并用200μL PBS洗涤重悬，用200目不锈钢网过滤细胞，滤液上机进行流式细胞周期分析(在激发波长488nm波长处检测红色荧光，同时检测光散射情况；每个样本计数10000个细胞)。
数据处理及统计分析：采用FlowJo软件进行细胞DNA含量分析和光散射分析，获得各样品中的周期分布数据，通过t检验比较各组间差异(P＜0.01为差异显著)。试验结果显示：143B在3μM供试品作用下发生了明显的G2/M期和多倍体细胞比例的增加，当剂量达到6μM时，143B中G2/M期和多倍体细胞比例的增加更为显著；SW982则是在6μM供试品作用下发生明显的G2/M期和多倍体细胞比例增加；U2OS在3或6μM供试品作用下最为明显的变化是S期细胞比例的减少，同时凋亡细胞的比例有所增加；SK-UT-1的DNA含量在1μM供试品作用下即已发生明显变化(S期细胞减少、凋亡细胞增加)，3μM的供试品使G2/M期和多倍体细胞显著增加，而当供试品剂量达到6μM时，G2/M期、多倍体及凋亡细胞的总比例已达到90％左右。(图3)
实施例4蛋白激酶抑制剂引起部分肉瘤细胞凋亡的实验研究
接种细胞及给药：将消化收集的143B和SK-UT-1细胞按每孔10 6个/2mL接种到六孔细胞培养板中，培养过夜待细胞贴壁后给药，每孔分别按终浓度0、1、3、6μM加入供试品，继续培养48小时。
样品收集与处理：供试品作用48小时后，用细胞刮刀轻轻将细胞刮下来，转移至15mL离心管中，再每孔加入1ml PBS洗涤一遍，清洗液转入同一支离心管，4℃离心机1200rpm离心5分钟，弃去上清，细胞沉淀加入500μL PBS重悬，转移至1.5mL离心管，4℃离心机1200rpm离心5分钟，弃上清。细胞沉淀加入110μL 1×蛋白上样缓冲液，剧烈振荡混匀后置于金属浴，100℃煮沸10分钟，取出样品管冰浴冷却后待用(可于-80℃冰箱保存)。
SDS-PAGE电泳及western blot检测：将上述蛋白样品离心后取上清进行SDS-PAGE电泳，每孔上样体积40μL，电泳条件为恒压140V，50分钟；电泳结束后，在Bio-Rad turbo半干转印仪的电极板上铺上一层与胶大小相同并经转膜缓冲液浸透的滤纸，再铺上提前用甲醇活化好的PVDF膜，将PAGE胶铺在膜上，赶走气泡后再铺一层与胶大小相同并经转膜缓冲液浸透的滤纸，启动装置开始转印，条件为25V，7分钟；完成转印后，将膜放在杂交袋中，加入封闭液，封口，室温下摇床摇动1小时。封闭结束取出膜按照Marker指示大小将膜剪开，放入新的杂交袋，分别加入按比例配置的一抗溶液(Cleaved PARP抗体按照1:1000稀释，β-Actin抗体按照1:2000稀释，抗体稀释液为PBS+1‰Tween 20+5％BSA)，赶气泡后封口，室温下孵育1小时，用PBST洗涤4次，每次5分钟；洗涤后将膜分别放入对应二抗中(Anti-rabbit二抗和Anti-Mouse二抗均按照1:2000比例用抗体稀释液稀释)室温摇床摇动孵育1小时，孵育结束之后用PBST洗涤3次，每次5分钟，最后用PBS洗涤一次，5分钟。将洗好的膜放到显影仪中，每条膜滴加200μL预混的ECL发光液，操作仪器进行影像记录。
试验结果：通过western blot实验，在143B和SK-UT-1细胞系中验证了蛋白激酶抑制剂对肉瘤的促凋亡效应。在不同剂量供试品的作用后，143B细胞中没有检出PARP蛋白剪切体，而SK-UT-1细胞中则检测到了PARP蛋白剪切体且呈剂量依赖性增加趋势。结果表明，供试品在143B细胞中可能不是通过诱导细胞凋亡的机制抑制肿瘤，而在SK-UT-1中则可以诱导肿瘤细胞的凋亡——与流式细胞术实验所检测到的情况一致。(图4)
实施例5蛋白激酶抑制剂在裸鼠荷瘤模型中观察供试品的体内抑瘤药效试验研究
裸鼠移植瘤模型的构建：大量扩增培养143B细胞并使细胞保持在对数生长状态。待细胞数量达到所需后消化收集细胞，并用大量PBS充分清洗2次以去除胰酶和血清成分，室温、800rpm离心10分钟，弃上清。用不含FBS的DMEM培养液重悬细胞，调整细胞浓度至5×10 6个/200μL。按每针200μL将细胞悬液注射至裸鼠腋窝中部外侧皮下，每只裸鼠注射一针。接种细胞后，每天观察伤口愈合情况及成瘤情况，适时用游标卡尺测量肿瘤最长径(length)及与之垂直的最宽径(width)，通过公式Tumor Volume＝length×(width) 2/2计算肿瘤体积。当肿瘤体积(Tumor Volume)达到约100mm 3时视为成瘤，即可进行给药实验。
给药及数据采集：将成瘤裸鼠随机分成3组(即溶剂对照组、供试品5mg/kg组和供试品10mg/kg组)、每组8只，标记后分笼饲养并开始给药。每只裸鼠给药前测量体重，按每千克体重计算给药剂量，即溶剂对照组按每克体重10μL的CMC-Na溶液、供试品5或10mg/kg组按每克体重10μL的0.5或1mg/mL供试品-CMC-Na悬浊液进行灌胃给药。每只裸鼠每天定时灌胃给药1次，观察到有个体所荷肿瘤体积达到2000mm 3时视为实验终点。给药周期：24天；给药方式：口服灌胃。
试验结果：通过对各组裸鼠荷瘤体积的记录分析发现，相对于溶剂对照组，供试品5mg/kg组的荷瘤体积得到了一定抑制，而供试品10mg/kg组的荷瘤体积则得到了更为明显的抑制。同时，相关给药并没有引起裸鼠体重的显著变化。(图5)
综合上述实验结果可以看出：
供试品可显著抑制人骨肉瘤细胞系MG63、U2OS、143B，人滑膜肉瘤细胞系SW982和人卡波西(Kaposi)肉瘤细胞系SK-UT-1等的生长和克隆形成，在不同的肉瘤细胞模型中以诱导G2/M期阻滞、细胞多倍体化和/或细胞凋亡等机制抑制肿瘤。实验动物的体内药效结果也表明，供试品用于肉瘤的治疗是安全、有效的。
实施例6蛋白激酶抑制剂在人源肉瘤SK-ES-1细胞株皮下异种移植雌性BALB/c Nude小鼠模型中的药效学评价
主要试剂、材料及来源：
SK-ES-1细胞：购自美国菌种保藏中心(American type culture collection，ATCC)，用含1％双抗(青链霉素，Hyclone)和15％胎牛血清(ExcellBio)的McCoy's 5A培养液(Gibco)培养。
胎牛血清：供应商：ExcellBio；批号：11H305；
McCoy's 5A：供应商：GIBCO；批号：2318827；
Matrigel：供应商：CORNING；批号：1084001；
BALB/c nude小鼠：购于江苏集萃药康生物科技股份有限公司常州分公司，7-9周龄，雄性。
N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺(蛋白激酶抑制剂，以下称为“供试品″)：来源于深圳微芯生物科技股份有限公司，将其溶于二甲基亚砜(Dimethyl sulfoxide，DMSO；上海生工，DN3039A)中，配置成20mM的工作母液，置于-20℃的冰箱中备用，使用时根据各项实验需要配置相应终浓度的工作溶液。
试验准备：
供试品和对照品的配制：准确称量供试品适量，溶于溶媒(含有0.2％CMC-Na和0.1％吐温-80的灭菌水溶液)中，配置成终浓度分别为0.5mg/ml、1mg/ml、2mg/ml的供试品溶液，2-8℃保存。
试验过程：
细胞培养：SK-ES-1细胞培养在含15％胎牛血清的McCoy's 5A培养液中。收集指数生长期的SK-ES-1细胞，PBS和Matrigel(1:1)重悬至适合浓度用于小鼠皮下接种。
人源肉瘤SK-ES-1细胞株皮下异种移植雌性BALB/c Nude小鼠模型构建：50只雌性小鼠右侧皮下接种1×10 7SK-ES-1细胞。待肿瘤平均体积为90mm 3时(肿瘤体积计算公式：肿瘤体积(mm 3)＝1/2×(a×b 2)，其中a表示长径，b表示短径)，根据肿瘤大小随机分组。
给药及数据采集：模型小鼠随机分成4组(即溶媒对照组、供试品5mg/kg组、供试品10mg/kg组和供试品20mg/kg组)，每组8只，标记后分笼饲养并开始给药。分组当天定义为第0天，给药开始于分组当天，口服灌胃给药，每天给药一次，共给药三周。每只裸鼠给药前测量体重，按每千克体重计算给药剂量，即5mg/kg、10mg/kg和20mg/kg的给药组分别每天每次给予5mg/kg、10mg/kg和20mg/kg的供试品，每次给药体积10mL/kg；溶媒对照组给予等体积的溶媒。自给药当天开始，每天记录小鼠的体重；第0、4、7、11、14、18、21天测量并记录小鼠的肿瘤体积。
疗效评价标准：
相对肿瘤增殖率，T/C(％)，即在某一时间点，治疗组和对照组肿瘤体积或瘤重的百分比值。计算公式为：T/C％＝T TV/C TV×100％(T TV：治疗组平均肿瘤体积；C TV：对照组平均肿瘤体积)。
肿瘤抑制率，TGI(％)，计算公式为：TGI％＝(1-T/C)×100％(T和C分别为治疗组和对照组在某一特定时间点的肿瘤体积(TV))。
试验结果：
疗效：溶媒对照组小鼠给药三周后的平均肿瘤体积为1203.75mm 3。供试品5mg/kg给药组在给药三周后的平均肿瘤体积为792.28mm 3，肿瘤抑制率TGI(％)为34.18％。供试品10mg/kg和20mg/kg给药组在给药三周后的平均肿瘤体积分别为279.03mm 3和375.59mm 3，肿瘤抑制率TGI(％)分别为76.82％和68.80％，相对溶媒对照组统计学上均体现出显著性差异(p＜0.05)。各给药组和对照组肿瘤生长情况见图6。
安全性：给药组5mg/kg、10mg/kg和20mg/kg各治疗组均无动物死亡，没有表现明显的药物毒性，治疗期间耐受良好。治疗组和对照组给药后小鼠体重变化见表1。
表1.在SK-ES-1人源肉瘤模型中各组体重变化情况
Figure PCTCN2022094828-appb-000004
Figure PCTCN2022094828-appb-000005
注：数据以“平均值±标准误差”表示。
实施例6的实验结果表明：供试品在5mg/kg剂量下对SK-ES-1人源肉瘤肿瘤模型具有一定抑制肿瘤生长的作用；供试品在10mg/kg和20mg/kg剂量下对SK-ES-1人源肉瘤肿瘤模型具有显著抑制肿瘤生长的作用；荷瘤小鼠对供试品在测试剂量下耐受良好。综上，供试品对肉瘤的体内治疗安全、有效。

Claims (11)

  1. 蛋白激酶抑制剂在制备预防和/或治疗肉瘤的药物中的应用；优选的，所述肉瘤为骨肉瘤、软组织肉瘤、血管肉瘤中的一种或多种；优选的，所述软组织肉瘤为滑膜肉瘤；优选的，所述血管肉瘤为卡波西肉瘤；优选的，所述蛋白激酶抑制剂选自Aurora B抑制剂，和/或，VEGFR抑制剂，和/或，PDGFR抑制剂，和/或c-Kit/CSF1R抑制剂。
  2. 蛋白激酶抑制剂在预防和/或治疗肉瘤疾病中的应用；优选的，所述肉瘤为骨肉瘤、软组织肉瘤、血管肉瘤中的一种或多种；优选的，所述软组织肉瘤为滑膜肉瘤；优选的，所述血管肉瘤为卡波西肉瘤；优选的，所述蛋白激酶抑制剂选自Aurora B抑制剂，和/或，VEGFR抑制剂，和/或，PDGFR抑制剂，和/或c-Kit/CSF1R抑制剂。
  3. 如权利要求1或2所述的应用，其特征在于，所述蛋白激酶抑制剂选自以下化合物或其药学上可接受的盐、晶型、异构体、前药或代谢产物：N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(6，7-二甲氧基喹唑啉-4-氧)-1-萘酰胺；N-(2-氨基-4-氟苯基)-6-(6，7-二甲氧基喹唑啉-4-氧)-1-萘酰胺；N-(2-氨基-4-氟苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(6，7-二甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基-4-氟苯基)-6-(6，7-二甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(8-(三氯甲基)喹啉-4-氧)-1-萘酰胺，或选自下述Aurora B抑制剂/VEGFR抑制剂/PDGFR抑制剂/c-Kit抑制剂/CSF1R抑制剂中的一种或多种：塞尼舍替(cenisertib)、橙皮苷(hesperidin)、安罗替尼(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)、仑伐替尼(Lenvatinib)、凡德他尼(vandetanib)、舒尼替尼(Sunitinib)、舒尼替尼(Sunitinib)、伊马替尼(Imatinib)、克莱拉尼(Crenolanib)、阿伐替尼(Avapritinib)、马赛替尼(Masitinib)、尼洛替尼(Nilotinib)、艾克利单抗(Axatilimab)、培西替尼(Pexidartinib)。
  4. 如权利要求3所述的应用，其特征在于：所述蛋白激酶抑制剂选自N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺及其晶型；优选的，所述晶型为非溶剂化晶型；优选的，所述非溶剂化晶型选自N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺晶型A、晶型B、晶型C。
  5. 如权利要求4所述的应用，其特征在于：所述蛋白激酶抑制剂的单位剂量为1-20mg，优选为5-10mg，最优选为5mg。
  6. 一种预防和/或治疗肉瘤的药物组合物，其特征在于：所述药物组合物以蛋白激酶抑制剂为主要活性成分，并包含药学上可接受的辅料；优选的，所述肉瘤为骨肉瘤、软组织肉瘤、血管瘤中的一种或多种；优选的，所述软组织肉瘤为滑膜肉瘤；优选的，所述血管肉瘤为卡波西肉瘤；优选的，所述蛋白激酶抑制剂选自Aurora B抑制剂，和/或，VEGFR抑制剂，和/或，PDGFR抑制剂，和/或c-Kit/CSF1R抑制剂。
  7. 如权利要求6所述的药物组合物，其特征在于，所述蛋白激酶抑制剂选自以下化合物，或其药学上可接受的盐、晶型、异构体、前药或代谢产物：N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(6，7-二甲氧基喹唑啉-4-氧)-1-萘酰胺；N-(2-氨基-4-氟苯基)-6-(6，7-二甲氧基喹唑啉-4-氧)-1-萘酰胺；N-(2-氨基-4-氟苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(6，7-二甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基-4-氟苯基)-6-(6，7-二甲氧基喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(喹啉-4-氧)-1-萘酰胺；N-(2-氨基苯基)-6-(8-(三氯甲基)喹啉-4-氧)-1-萘酰胺，或选自下述Aurora B抑制剂/VEGFR抑制剂/PDGFR抑制剂/c-Kit抑制剂/CSF1R抑制剂中的一种或多种：塞尼舍替(cenisertib)，橙皮苷(hesperidin)，安罗替尼(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)，仑伐替尼(Lenvatinib)，凡德他尼(vandetanib)、舒尼替尼(Sunitinib)，舒尼替尼(Sunitinib)，伊马替尼(Imatinib)、克莱拉尼(Crenolanib)、阿伐替尼(Avapritinib)，马赛替尼(Masitinib)、尼洛替尼(Nilotinib)，艾克利单抗(Axatilimab)、培西替尼(Pexidartinib)。
  8. 如权利要求7所述的药物组合物，其特征在于：所述蛋白激酶抑制剂选自N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺及其晶型，优选的，所述晶型为非溶剂化晶型，优选的，所述非溶剂化晶型包括N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺的晶型A、晶型B和晶型C。
  9. 如权利要求6-8任一项所述的药物组合物，其特征在于，所述药学上可接受的辅料选自润滑剂、粘合剂、填充剂、防腐剂、表面活性剂、着色剂、矫味剂、乳化剂、助悬剂、稀释剂、胶凝剂、崩解剂、pH调节剂、增溶剂的一种或两种及以上；优选的，所述药物组合物是片剂、胶囊、粉剂、液体制剂、悬浮剂、针剂或缓释制剂。
  10. 如权利要求6-9任一项所述的药物组合物，其特征在于：所述蛋白激酶抑制剂的单位剂量为1-20mg，优选为5-10mg，最优选5mg。
  11. 一种预防和/或治疗肉瘤的方法，其特征在于：向有需要的个体施用预防和/或治疗有效剂量的如权利要求6-10任一项所述的药物组合物；优选的，所述肉瘤为骨肉瘤、软组织肉瘤、血管瘤中的一种或多种；优选的，所述软组织肉瘤为滑膜肉瘤；优选的，所述血管肉瘤为卡波西肉瘤。
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