WO2021155764A1 - Bi853520与化疗药物的联用 - Google Patents

Bi853520与化疗药物的联用 Download PDF

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WO2021155764A1
WO2021155764A1 PCT/CN2021/074371 CN2021074371W WO2021155764A1 WO 2021155764 A1 WO2021155764 A1 WO 2021155764A1 CN 2021074371 W CN2021074371 W CN 2021074371W WO 2021155764 A1 WO2021155764 A1 WO 2021155764A1
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cancer
tumor
group
drug
pld
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PCT/CN2021/074371
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English (en)
French (fr)
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王在琪
张江伟
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应世生物科技(南京)有限公司
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Priority to CN202180012434.1A priority Critical patent/CN115052603B/zh
Application filed by 应世生物科技(南京)有限公司 filed Critical 应世生物科技(南京)有限公司
Priority to CA3165980A priority patent/CA3165980A1/en
Priority to JP2022547735A priority patent/JP2023515765A/ja
Priority to CN202311560963.2A priority patent/CN117599060A/zh
Priority to US17/797,867 priority patent/US20230079055A1/en
Priority to KR1020227028705A priority patent/KR20220137666A/ko
Priority to AU2021215747A priority patent/AU2021215747A1/en
Priority to BR112022015150A priority patent/BR112022015150A2/pt
Priority to MX2022009612A priority patent/MX2022009612A/es
Priority to EP21750093.3A priority patent/EP4101453A4/en
Publication of WO2021155764A1 publication Critical patent/WO2021155764A1/zh
Priority to US18/347,324 priority patent/US12097200B2/en
Priority to US18/633,164 priority patent/US20240261285A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the invention belongs to the field of medicinal chemistry. Specifically, the present invention relates to the use of BI853520 or a pharmaceutically acceptable salt thereof in the preparation of drugs for the treatment of tumors in combination with chemotherapeutics.
  • Cancer is one of the most serious diseases that threaten human life and health in modern times. Worldwide, the number of cancer deaths reaches 7 million every year. At present, the treatment of cancer mainly adopts four methods: surgical treatment, drug treatment, radiotherapy and immunotherapy. In addition, there are adjuvant therapies. Among them, the drug treatment of cancer includes targeted drug therapy and chemotherapy. Among the current various treatment methods, targeted drug therapy and chemotherapy still occupy an important position and are important standard therapies for cancer. For example, carboplatin/paclitaxel combined chemotherapy is the first-line treatment for ovarian cancer, and chemotherapy drugs such as paclitaxel are also indispensable in the treatment of gastric cancer. However, the biggest problem facing targeted drug therapy and chemotherapeutic drug monotherapy is drug resistance, including spontaneous drug resistance and adaptive drug resistance, resulting in a low overall chemotherapy response rate and limited duration of response.
  • the present invention provides a use of BI853520 or a pharmaceutically acceptable salt thereof in the preparation of a drug for the treatment of tumors in combination with a chemotherapeutic drug, wherein the BI853520 is 2-fluoro-5-methoxy-4 -[(4-(2-Methyl-3-oxo-2,3-dihydro-1H-isoindole-4-oxy)-5-trifluoromethyl-pyrimidin-2-yl)amino] -N-(1-methyl-piperidin-4-yl)benzamide (see WO2010058032), the structure of the compound is as follows:
  • the chemotherapeutic drug is PLD, taxanes or cisplatin.
  • the tumor does not include NRAS mutated tumors.
  • the tumor is lymphocytic acute leukemia, granulocytic acute leukemia, malignant lymphoma, breast cancer, lung cancer, ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, nerve Bladder cancer, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, testicular cancer, stomach cancer, liver cancer, pancreatic cancer or melanoma.
  • the tumor is prostate cancer, esophageal cancer, ovarian cancer, gastric cancer or lung cancer.
  • the lung cancer is preferably squamous cell lung cancer.
  • the chemotherapeutic drug is PLD.
  • the tumor is ovarian cancer, especially platinum-resistant ovarian cancer.
  • the chemotherapeutic drugs are taxanes, especially docetaxel and paclitaxel.
  • the tumor is prostate cancer, esophageal cancer or gastric cancer.
  • the pharmaceutically acceptable salt is BI853520 tartrate.
  • the present invention provides the use of BI853520 or a pharmaceutically acceptable salt thereof and a chemotherapeutic drug in the preparation of a drug for the treatment of tumors, and the structure of the compound is:
  • the chemotherapeutic drug is PLD, taxanes or cisplatin.
  • the tumor does not include NRAS mutated tumors.
  • the tumor is lymphocytic acute leukemia, granulocytic acute leukemia, malignant lymphoma, breast cancer, lung cancer, ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, nerve Bladder cancer, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, testicular cancer, stomach cancer, liver cancer, pancreatic cancer or melanoma.
  • the tumor is prostate cancer, esophageal cancer, ovarian cancer, gastric cancer or lung cancer.
  • the lung cancer is preferably squamous cell lung cancer.
  • the chemotherapy drug is PLD.
  • the tumor is ovarian cancer, especially platinum-resistant ovarian cancer.
  • the chemotherapeutic drugs are taxanes, especially docetaxel and paclitaxel.
  • the tumor is prostate cancer, esophageal cancer or gastric cancer.
  • the pharmaceutically acceptable salt is BI853520 tartrate.
  • the present invention provides a drug combination, characterized in that the drug combination includes BI853520 or a pharmaceutically acceptable salt thereof, and a chemotherapeutic drug;
  • the structural formula of BI853520 is as follows:
  • the chemotherapeutic drug is PLD, taxanes or cisplatin
  • the chemotherapy drug is PLD.
  • the chemotherapeutic drugs are taxanes, especially docetaxel and paclitaxel.
  • the pharmaceutically acceptable salt is BI853520 tartrate.
  • the present invention provides a method for treating tumors, which is characterized in that it comprises administering BI853520 or a pharmaceutically acceptable salt thereof and a chemotherapeutic drug to a subject; the structure of the compound is as follows:
  • the method includes administering BI853520 or a pharmaceutically acceptable salt thereof and an effective amount of a chemotherapeutic drug to the subject.
  • the chemotherapeutic drug is PLD, taxanes or cisplatin.
  • an effective amount of BI853520 and an effective amount of PLD, taxanes or cisplatin especially an effective amount of BI853520 and an effective amount of PLD, taxanes or cisplatin.
  • the tumor does not include NRAS mutated tumors.
  • the tumor is lymphocytic acute leukemia, granulocytic acute leukemia, malignant lymphoma, breast cancer, lung cancer, ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, nerve Bladder cancer, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, testicular cancer, stomach cancer, liver cancer, pancreatic cancer or melanoma.
  • the tumor is prostate cancer, esophageal cancer, ovarian cancer, gastric cancer or lung cancer.
  • the lung cancer is preferably squamous cell lung cancer.
  • the chemotherapy drug is PLD.
  • the tumor is ovarian cancer, especially platinum-resistant ovarian cancer.
  • the chemotherapeutic drugs are taxanes, especially docetaxel and paclitaxel.
  • the tumor is prostate cancer, esophageal cancer or gastric cancer.
  • the BI853520 or a pharmaceutically acceptable salt thereof and the chemotherapeutic drug are administered simultaneously, alternately or sequentially.
  • the pharmaceutically acceptable salt is BI853520 tartrate.
  • Figure 1a depicts the median tumor volume of BI 853520 and PLD as a single agent and combined use in a mouse model of human ovarian cancer (TOV-21G cell line), which shows the anti-tumor activity of different test groups.
  • the test groups are group A (blank control group); group B (administration once a day, the BI 853520 group with a dose of 12.5 mg/kg); group C (administration once a week, the PLD group with a dose of 1 mg/kg) ; Group D (combined medication group of BI 853520 and PLD, BI 853520 is administered once a day at a dose of 12.5 mg/kg, and PLD is administered once a week at a dose of 1 mg/kg).
  • Figure 1b depicts the median tumor volume of BI 853520 and PLD as a single agent and in combination in a mouse model of human ovarian cancer (SKOV-3 cell line), which shows the anti-tumor activity of different test groups.
  • the test groups were group A (blank control group); group B (BI 853520 group with a dose of 50 mg/kg once a day); group C (PLD group with a dose of 5 mg/kg once a week); Group D (combined medication group of BI 853520 and PLD, BI 853520 was administered once a day at a dose of 50 mg/kg, and PLD was administered once a week at a dose of 5 mg/kg).
  • Figure 1c depicts the median tumor volume of BI 853520 and PLD as a single agent and combined use in a mouse model of human ovarian cancer (A2780 cell line), which shows the anti-tumor activity of different test groups.
  • the test groups are group A (blank control group); group B (administration once a day, the BI 853520 group with a dose of 12.5 mg/kg); group C (administration once a day, the BI 853520 group with a dose of 25 mg/kg) ; D group (once a day, dose of 50mg/kg BI 853520 group); E group (once a week, dose of 5mg/kg PLD group); F group (BI 853520 and PLD combined drug group , BI 853520 is administered once a day at a dose of 50 mg/kg, and PLD is administered once a week at a dose of 5 mg/kg).
  • Figure 2 depicts the tumor growth kinetics of the CTG-1166 model, which shows the anti-tumor activity of different test groups.
  • the experimental groups were blank control groups; BI 853520 group administered twice a day at a dose of 50 mg/kg; PLD group administered once a week at a dose of 3 mg/kg; BI 853520 and PLD combination group, BI 853520 It is administered twice a day at a dose of 50 mg/kg, and PLD is administered once a week at a dose of 3 mg/kg; the carboplatin and paclitaxel combination group, carboplatin, is administered once a week for three weeks at a dose of 25 mg/ kg, paclitaxel, administered once a week for three weeks at a dose of 20 mg/kg.
  • Figure 3 depicts the tumor volume of BI853520 combined with docetaxel in the PDX model, which shows the anti-tumor activity of different test groups.
  • the test groups were blank control group 1/blank control group 2; BI 853520 group with a dose of 50 mg/kg once a day; Docetaxel group with a dose of 10 mg/kg once a week; BI 853520 and In the combination group of docetaxel, BI 853520 is administered once a day at a dose of 50 mg/kg, and docetaxel is administered once a week at a dose of 10 mg/kg.
  • Figure 4 depicts the tumor volume (mm 3 ) of a nude mouse xenograft model of human lung squamous cell carcinoma (cell line NCI-H520), which shows the anti-tumor effects of different test groups, which are blank control groups; BI 853520 group with a dose of 50 mg/kg once a day; a cisplatin group with a dose of 5 mg/kg once a week; a combination group of BI 853520 and cisplatin with a dose of BI 853520 once a day. It is 50 mg/kg, and cisplatin is administered once a week at a dose of 5 mg/kg.
  • Figure 5 depicts the tumor volume (mm 3 ) of a nude mouse xenograft model of human esophageal cancer (cell line KYSE-270), which shows the anti-tumor effects of different test groups.
  • the test groups are blank control groups; once a day Administration, the BI 853520 group at a dose of 50 mg/kg; once a week, the paclitaxel group at a dose of 10 mg/kg; the combination group of BI 853520 and paclitaxel, BI 853520 is administered once a day at a dose of 50 mg/kg, Paclitaxel is administered once a week at a dose of 10 mg/kg.
  • Figure 6 depicts the tumor volume (mm 3 ) of a nude mouse xenograft model of human esophageal cancer (cell line KYSE-70), which shows the anti-tumor effects of different test groups, which are blank control groups; once a day Administration, the BI 853520 group at a dose of 50 mg/kg; once a week, the docetaxel group at a dose of 10 mg/kg; the combination group of BI 853520 and docetaxel, BI 853520 is administered once a day, The dose is 50 mg/kg, docetaxel is administered once a week, and the dose is 10 mg/kg.
  • FIG. 7 depicts tumor volume in nude mouse xenograft model of human prostate cancer cells (cell line PC-3) in (mm 3), which shows the antitumor effect of the different test groups, the test group were control group; once daily Administration: BI 853520 group at a dose of 25 mg/kg; docetaxel group at a dose of 7.5 mg/kg once a week; BI 853520 combined with docetaxel group, BI 853520 administered once a day , The dose is 25mg/kg, docetaxel is administered once a week, the dose is 7.5mg/kg.
  • Figure 8 depicts the tumor volume (mm 3 ) of a nude mouse xenograft model of human gastric cancer (cell line HS 746T), which shows the anti-tumor effects of different test groups, the test groups are blank control groups; once a day BI 853520 group with a dose of 50mg/kg; Docetaxel group with a dose of 10mg/kg once a week; BI 853520 and Docetaxel group with a dose of BI 853520 once a day with a dose of 50mg/kg, docetaxel is administered once a week at a dose of 10mg/kg.
  • the present invention relates to the use of BI853520 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating tumors in combination with a chemotherapeutic drug.
  • the invention also relates to the use of BI853520 or a pharmaceutically acceptable salt thereof and a chemotherapeutic drug in the preparation of a drug for treating tumors.
  • the present invention also relates to a method for treating tumors, which is characterized in that it comprises administering to a subject an effective amount of BI853520 or a pharmaceutically acceptable salt thereof and a chemotherapeutic drug.
  • chemotherapy refers to a systemic treatment of malignant tumors, which can be administered orally, intravenously, or in the body cavity.
  • Chemotherapy drugs will circulate throughout most organs and tissues throughout the body. Chemotherapeutic drugs can act on different links of tumor cell growth and reproduction, inhibiting or killing tumor cells. It is currently one of the most effective methods for the treatment of malignant tumors.
  • the chemotherapeutic drug is PLD, docetaxel, paclitaxel or cisplatin.
  • the tumor does not include NRAS mutant tumors.
  • the tumor is selected from lymphocytic acute leukemia, granulocytic acute leukemia, malignant lymphoma, breast cancer, undifferentiated small cell bronchial lung cancer, non-small cell bronchial lung cancer, non-small cell lung cancer, Squamous cell lung cancer, ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, esophagus Cancer, testicular cancer, stomach cancer, liver cancer, pancreatic cancer and melanoma.
  • the tumor is ovarian cancer, gastric cancer, or squamous cell lung cancer.
  • chemotherapeutic drug is PLD.
  • PLD polyethylene glycol liposome adriamycin, also known as doxorubicin liposome.
  • ком ⁇ онент refers to the use of two or more drugs for the treatment of one disease.
  • the BI853520 or a pharmaceutically acceptable salt thereof is used in combination with a chemotherapeutic drug, which may further include other drugs.
  • the BI853520 or a pharmaceutically acceptable salt thereof and the chemotherapeutic drug can be administered simultaneously, alternately or sequentially.
  • NRAS refers to an oncogene, a member of the RAS oncogene family, which also includes two other genes: KRAS and HRAS. These genes play an important role in cell division, cell differentiation and apoptosis.
  • NRAS mutation means that when a pathogenic mutation occurs in the NRAS gene, the N-Ras protein encoded by it will be in a state of continuous activation, leading to uncontrolled cell proliferation and the formation of tumors.
  • pharmaceutically acceptable refers to non-toxic, biologically tolerable, and suitable for administration to a subject.
  • pharmaceutically acceptable salt refers to a non-toxic, biologically tolerable salt suitable for administration to a subject
  • a pharmaceutically acceptable salt of the compound refers to a non-toxic , Biologically tolerable acid addition salts suitable for administration to subjects, including but not limited to: acid addition salts formed by the compound and inorganic acids, such as hydrochloride, hydrobromide, and carbonic acid Salts, bicarbonates, phosphates, sulfates, sulfites, nitrates, etc.; and acid addition salts formed by the compounds and organic acids, such as formate, acetate, malate, and maleic acid Salt, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, benzoate, salicylate , Stearates and salts formed with alkane dicarboxylic acids of the formula HO
  • subject refers to mammals and non-mammals.
  • Mammal refers to any member of the mammalian class, which includes but is not limited to: humans; non-human primates, such as chimpanzees and other apes and monkey species; farm animals, such as cows, horses, sheep, goats, and pigs; Domestic animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs; and so on. Examples of non-mammals include but are not limited to birds and the like.
  • the term "subject” does not limit a specific age or gender. In some embodiments, the subject is a human.
  • treatment refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect can be therapeutic, including partial or substantially obtaining one or more of the following results: partial or full reduction of the degree of disease, disease or syndrome; improvement of clinical symptoms or indicators related to the disease; or delay, inhibition or reduction The likelihood of the disease, condition, or syndrome progressing.
  • BI853520 or a pharmaceutically acceptable salt thereof
  • a chemotherapeutic drug especially PLD, docetaxel or cisplatin
  • BI853520 or a pharmaceutically acceptable salt thereof
  • chemotherapeutic drugs especially PLD, docetaxel or cisplatin
  • determining the effective amount also depends on the degree, severity and type of cell proliferation. The skilled person will be able to determine the appropriate dosage based on these and other factors.
  • the "effective amount" of any other therapeutic agent will depend on the type of drug used.
  • the appropriate dosage is known for approved therapeutic agents, and can be adjusted by the skilled person according to the condition of the subject, the type of condition to be treated, and the amount of the compound or its pharmaceutically acceptable salt. In the absence of a clear indication of the quantity, some quantity should be assumed.
  • the effective dose of BI853520 (or a pharmaceutically acceptable salt thereof) may be 10 ⁇ g-2000 mg. This example is non-limiting.
  • the effective amount of chemotherapeutic drugs especially PLD, docetaxel or cisplatin is known to those skilled in the art.
  • BI853520 (or a pharmaceutically acceptable salt thereof) can be administered by any suitable method of administration. Suitable methods include oral, intravenous, intramuscular or subcutaneous administration to the subject.
  • the BI853520 (or a pharmaceutically acceptable salt thereof) can be orally administered together with a pharmaceutically acceptable carrier such as an inert diluent or an absorbable edible carrier. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be mixed directly with the patient's food.
  • a pharmaceutically acceptable carrier such as an inert diluent or an absorbable edible carrier.
  • the compound or a pharmaceutically acceptable salt thereof may be combined with one or more excipients and used as ingestible tablets, buccal tablets, lozenges, capsules, elixirs, Use in the form of suspension, syrup or glutinous rice paper capsules. These preparations contain an effective amount of the BI853520 (or a pharmaceutically acceptable salt thereof).
  • Tablets, lozenges, pills, capsules, etc. may further include: binders, such as tragacanth, acacia, corn starch or gelatin; excipients, such as dicalcium phosphate; disintegrating agents, such as corn starch, potato starch , Alginic acid, etc.; lubricants, such as magnesium stearate; or sweeteners, such as sucrose, fructose, lactose or aspartame; or flavoring agents.
  • binders such as tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • disintegrating agents such as corn starch, potato starch , Alginic acid, etc.
  • lubricants such as magnesium stearate
  • sweeteners such as sucrose, fructose, lactose or aspartame; or flavoring agents.
  • the BI853520 (or a pharmaceutically acceptable salt thereof) can also be administered intravenously or intraperitoneally by infusion or injection.
  • the solution of BI853520 (or a pharmaceutically acceptable salt thereof) and a chemotherapeutic drug (especially PLD, docetaxel or cisplatin) can be prepared in water, optionally mixed with a non-toxic surfactant.
  • Exemplary pharmaceutical dosage forms for injection or infusion include: sterile aqueous solutions, dispersions, or sterile powders containing active ingredients, which are suitable for extemporaneous preparation of sterile injection or infusion solutions or dispersions.
  • the final dosage form should be sterile, fluid and stable under the conditions of production and storage.
  • a sterile injectable solution can be prepared by mixing the required amount of BI853520 (or a pharmaceutically acceptable salt thereof) and various other ingredients mentioned above into an appropriate solvent, and then filtering and sterilizing.
  • the preferred preparation methods may be vacuum drying and freeze-drying techniques, which can produce a powder of the active ingredient plus any other required ingredients that exist after the previous sterile filtration.
  • the amount of BI853520 (or a pharmaceutically acceptable salt thereof) or chemotherapeutics (especially PLD, docetaxel or cisplatin) required for treatment can not only vary with the specific salt selected, but can also vary with the administration
  • the route of medicine, the nature of the disease to be treated, and the age and condition of the patient vary, and ultimately can be determined by the attending physician or clinician.
  • the dosage may be in the range of about 0.1 to about 50 mg/kg body weight per day.
  • the required dose may conveniently be presented as a single dose or in divided doses administered at appropriate intervals.
  • Athymic female BomTac:NMRI-Foxn1nu mice were purchased from Taconic, Denmark. After arriving in the animal room, the mice adapt to the new environment for at least 3 days before being used in the experiment. These animals were kept under standard conditions (temperature 21.5 ⁇ 1.5°C and 55 ⁇ 10% humidity), standardized diet and freely provided autoclaved tap water.
  • the transponders Datamars T-IS 8010 FDX-B and LabMax II fixed subcutaneously implanted in the neck area are used to identify each mouse.
  • the cage card shows the study number, animal identification number, compound and dose level, route of administration, and the animal's dosing schedule throughout the experiment.
  • mice To establish subcutaneous tumors, human cancer cells were collected by trypsinization, centrifuged, washed, and suspended in a suitable medium. Then 100 ⁇ l of a cell suspension containing 5 ⁇ 10 6 -1 ⁇ 10 7 was injected subcutaneously into the right flanks of nude mice (1 site per mouse). When the tumor grew to an appropriate size, the mice were randomly assigned to the treatment group and the blank control group.
  • BI853520 was synthesized according to the method in patent WO2010058032. Suspend the dry powder in an equimolar volume of 1M HCl and dilute it in an appropriate volume of 0.5% Natrosol to reach the required concentration for each experiment.
  • the mice were checked daily for abnormalities, and the body weight was measured three times a week (Monday, Wednesday, and Friday). Animals were sacrificed at the end of the study (approximately three weeks after the start of treatment); during the study period, for ethical reasons, animals with tumor necrosis or tumors larger than 2000 mm 3 would be sacrificed early.
  • the tumor volume and body weight parameters were statistically evaluated.
  • the absolute value of the tumor volume and the percentage change in body weight (refer to the initial weight on day 1) were used.
  • a non-parametric method was used to calculate the number of observations, median, minimum and maximum.
  • the median tumor volume of each treatment group T and the median tumor volume of the control group C are used to calculate the TGI from day 1 to day d:
  • the tumor volume and body weight parameters were statistically evaluated.
  • the absolute value is used.
  • a non-parametric method was used to calculate the number of observations, median, minimum and maximum.
  • the median tumor volume of each treatment group T and the median tumor volume of the control group C are used to calculate the TGI from day 1 to day d:
  • C 1 , T 1 the median tumor volume of the control group and the treatment group on the first day of the experiment
  • Example 1 Study on the anti-tumor activity of BI 853520 in combination with PLD in a mouse model of human ovarian cancer.
  • TOV-21G cells carrying KRAS and PIK3CA gene mutations were obtained from ATCC (CRL-11730).
  • MCB and WCB were established according to BI RCV GmbH&Co KG standards.
  • the cells were cultured in T175 tissue culture flasks, and the medium used was RPMI-1640 supplemented with L-glutamine, sodium pyruvate, non-essential amino acids and 10% heat-inactivated fetal bovine serum.
  • the cells were cultured at 37°C and 5% CO 2 .
  • A2780 cells carrying PTEN gene mutation were obtained from ECACC (93112519).
  • MCB and WCB were established according to BI RCV GmbH&Co KG standards.
  • the cells were cultured in T175 tissue culture flasks, and the medium used was: RPMI-1640+Glutamax supplemented with 10% heat-inactivated fetal bovine serum.
  • the cells were cultured at 37°C and 5% CO 2 .
  • SKOV-3 cells carrying CDKN2, MLH1, PIK3CA and TP53 gene mutations were obtained from ATCC (HTB-77).
  • MCB and WCB were established according to BI RCV GmbH&Co KG standards.
  • Cells were cultured in T175 tissue culture flasks, and the medium used was IMDM+L-Glutamax and supplemented with 10% heat-inactivated fetal bovine serum for non-essential amino acids. The cells were cultured at 37°C and 5% CO 2 .
  • the A2780 cells were suspended in ice-cold PBS+5% FCS and matrigel (1:1) with reduced growth factors, and the cell inoculation amount was 5 ⁇ 10 6 cells/ml.
  • the TOV-21G cells were suspended in ice-cold PBS+5% FCS, and the cell inoculation amount was 5 ⁇ 10 6 cells/ml.
  • the mice were randomly assigned to the treatment group and the blank control group.
  • the pH of the BI853520 formulation is between 3 and 4.
  • PLD PLD (Doxil) was purchased from Johnson & Johnson and dissolved in a sterile 5% glucose solution.
  • B BI 853520: 12.5mg/kg (once a day, administered by gavage needle)
  • B BI 853520: 50mg/kg (once a day, administered by gavage needle)
  • B BI 853520: 12.5mg/kg (once a day, administered by gavage needle)
  • the unilateral declining wilcoxon test was used to compare each dose of the test compound with the control group.
  • the reduction in tumor volume was used as a therapeutic effect, and the weight loss was used as a side effect.
  • the P value of tumor volume (efficacy parameter) has been compared and adjusted many times, while the P value of body weight (tolerability parameter) has not been adjusted to avoid ignoring possible side effects.
  • SKOV-3 cell line is less sensitive to BI 853520 and PLD, and the required drug dose is higher (50mg/kg, 5mg/kg).
  • the combination therapy is significantly more effective than the single agent, and the tolerability is not bad.
  • the TGI of group E was 99% (p ⁇ 0.0001), and 4 out of 10 animals had tumor regression (Table 1c; Table 4).
  • BI 853520 can be observed at medium and high dose levels (25mg/kg, 50mg/kg), but no tumor regression is found.
  • High-dose PLD has high efficacy and can cause 40% of tumors to regress.
  • the combined use of BI 853520 and PLD can produce higher efficacy, and 60% of the regression, and the tolerability is still very good.
  • BI 853520 and PLD showed a synergistic effect in the ovarian cancer models of the three cell lines, and the tolerance was not affected.
  • Example 2 Combination of BI853520 and PLD in graduates Research on anti-tumor activity in PDX ovarian cancer model.
  • mice approximately 7 weeks old (at the start of dosing) were purchased from Taconic, Denmark. Body weight (at the time of initial administration) is approximately 20 grams. After arriving in the animal room, the mice adapt to the new environment for at least 3 days.
  • the immune-compromised female mice are housed in a HEPA ventilated cage ( IVC, Innovive USA), at 68-74°F (20-23°C) and 30-70% humidity, after 12 hours of light in a light-dark cycle.
  • the animals were fed water (reverse osmosis, 2ppm Cl2) and a standard diet (Teklad 2919; 19% protein, 9% fat and 4% fiber) ad libitum. Compared with day 0, animals with a weight loss of ⁇ 10% can get DietAel 76A ( Portland, ME). 3 in each group.
  • mice Implantation in mice from graduates Tumor cells in an ovarian model, which was established from patients previously treated with platinum therapy. After the tumors reached 1000-1500 mm 3 , they were collected and the tumor fragments were implanted SC into the left flanks of female pre-study mice. Each study animal was implanted in a specific passage batch. The tumor volume was measured twice a week with a caliper to monitor tumor growth, and the tumor volume (TV) was calculated using the formula (0.52 ⁇ [length ⁇ width 2]). When the TV reached about 250-350mm 3 (average 300mm 3 ), the animals were matched to the tumor size and assigned to the control group or treatment group (research mice), and the administration was started on day 0.
  • the animals were weighed with a digital scale every day, and the TV was measured twice a week.
  • the average tumor volume of the control group reached about 1500 mm 3 or until the 60th day, the study ended.
  • the study is extended to after the 60th day, and the dosing is also extended to the end of the study.
  • the tumor volume reaches approximately 1500 mm 3 , individual animals can be removed from the study.
  • the design of the anti-tumor effect study is shown in Table 3.
  • Blank control group 1 0.5% Natrosol
  • Blank control group 2 5% sterile glucose.
  • BI853520 was synthesized according to the method in patent WO2010058032 and stored at room temperature and protected from light.
  • the blank control group for oral administration is 0.5% Natrosol.
  • the blank control group for intravenous administration was 5% sterile glucose.
  • Carboplatin is a pre-prepared 10mg/mL stock solution.
  • PLD is a pre-prepared 2mg/mL stock solution.
  • Paclitaxel is a pre-prepared 6mg/mL stock solution.
  • the animals were observed every day from day 0 and weighed twice a week. Record the data of each group, including the weight and average weight of each animal, record the average weight change percentage (%vD 0 ) of each group relative to day 0, and plot %vD 0 when the study is completed. Animal deaths were recorded every day, and were determined as drug-related (D), technology (T), tumor-related (B) or unknown (U) based on weight loss and visual observation.
  • the single-agent or combination therapy group with an average %vD 0 >20% and/or >10% mortality was considered to be higher than the MTD of the treatment in the evaluated regimen.
  • the maximum mean %vD 0 (lowest point of body weight) for each treatment group was reported at the end of the study.
  • the inhibitory effect on tumor growth was determined by calculating TGI (100% x [1-(final MTV of the treatment group-initial MTV)/(final MTV of the control group-initial MTV)]). Start treatment on day 0. The tumor volume of the treatment group was compared with the control group on the day the study was completed.
  • PR and TFS are considered not to include CR.
  • TFS CR that continues to exist until the study is completed
  • the selected endpoint includes as many groups as possible and as many animals as possible in each group.
  • the tumor volume of animals taken out early due to tumor volume ⁇ 1500mm 3 is carried forward for analysis, but it shall not exceed 4 consecutive measurement time points. Compare the tumor volume of the BI 853520+PLD combination treatment group, the BI 853520 group and the PLD group, and the total tumor volume of the BI 853520 group and the PLD group.
  • Example 3 Study on the anti-tumor activity of BI853520 and docetaxel in combination with PDX gastric cancer model
  • mice Female BomTac:NMRI-Foxn1nu mice were purchased from Taconic, Denmark. The mice are housed in separate ventilated cages (TECNIPLAST Sealsafe TM -IVC-System, TECNIPLAST, Hohenpeissenberg, Germany), and type II or III cages are selected according to the number of experimental animals. Under 25 ⁇ 1°C and 40-70% humidity, after 14L: 10D light and dark cycle, light and air exchange (AC), the speed in the cage is 60-65 AC/hour. The animals were fed water (filtered and acidified (pH 2.5) sterile tap water) and animal diet (Teklad Global 19% protein squeeze diet (T.2019S.12)) ad libitum. The water and diet are changed twice a week, and all materials are autoclaved before use.
  • TECNIPLAST Sealsafe TM -IVC-System TECNIPLAST Sealsafe TM -IVC-System
  • TECNIPLAST TECNIPLAST, Hohenpeissenberg,
  • the GXA 3039 gastric cancer PDX model was used in this study.
  • Tumor cells from the GXA 3039 gastric cancer PDX model were implanted in immunodeficient mice. After the initial implantation, tumor cells were established and characterized (1st generation). After the tumor cells were passaged until a stable growth pattern was established, they were removed from the mice, and the tumors were cut into pieces (with a margin of 3-4 mm in length) and placed in PBS containing 10% penicillin/streptomycin. The tumor fragments were implanted subcutaneously on the side of female pre-study mice. Measure the tumor volume with calipers every day to monitor tumor growth.
  • the tumor volume is greater than 1500mm 3 , the weight loss is greater than 18%, or severe conditions such as numbness and pain occur, the animal will be put to death in advance. If the number of surviving mice is less than 70% of the initial number (ie less than 6 out of 8), the entire group is terminated. In order to be able to monitor the reproliferation of the tumor after the end of the administration, le Kaplan-Meier statistics were performed (this rule does not apply to the group where the tumor has been remission). Tolerance was assessed using the largest group of median weight loss (BWL) for each group.
  • BWL median weight loss
  • TGI tumor growth
  • a represents the length and b represents the vertical tumor diameter
  • T 0 and C 0 are the median ATV of the test group and the blank control group on day 0;
  • T x and C x are the corresponding median ATV on day X; day X is the second day after the last dosing of BI 853520 (QD dosing), one week after the last docetaxel dosing (every Weekly administration) or the last day of at least 70% of the animals, whichever occurs earlier.
  • the TGI value has the following meanings (assuming C x >C 0 ):
  • the growth rate of the tumor is reduced: 100%>TGI>0%
  • endpoint indicators used to assess efficacy include: time to tumor volume doubling/quadruple and tumor growth delay/tumor progression time/tumor reproliferation.
  • Tumor volume doubling/quadruple time the time for the test group and the control group to double/quadruple the tumor volume (T d /T q ) is defined as the time interval required for each group to reach 200%/400% of the median RTV ( In days).
  • Tumor growth delay/tumor progression time/tumor repopulation To evaluate the time difference of tumor progression/tumor repopulation (tumor growth delay, TGD) after tumor regression during the no-dose observation period, Kaplan-Meier survival analysis was used. A relative tumor volume of 400% was defined as the endpoint.
  • Blank control group 1 0.5% Natrosol
  • Blank control group 2 0.9% saline.
  • BI853520 was synthesized according to the method in patent WO2010058032 and stored at room temperature and protected from light. BI 853520 was dissolved in 0.5% Natrosol to prepare a solution with a concentration of 5 mg/ml and administered at a dose of 50 mg/kg. Divide the solution into two equal parts, store at room temperature, protect from light, and use within one week.
  • Docetaxel Purchase Sanofi Avents and store at 4°C. On the day of administration, the docetaxel solution was diluted with 0.9% saline to prepare a concentration of 1 mg/ml, and the solution was administered at a dose of 10 mg/kg.
  • the blank control group for oral administration is 0.5% Natrosol.
  • the blank control group administered intravenously was 0.9% saline.
  • the dosage is 10ml/kg.
  • BI 853520 was administered first, and then docetaxel was administered immediately.
  • the body weight determined at the end of the dosing period (that is, on the day when the TGI value was counted) was used, and a two-tailed Mann-Whitney-Wilcoxon U test was performed without parameters.
  • the Bonferroni-Holm method was not used to correct the p-value for adjusting body weight, and if the p-value was in the range of 0.10 ⁇ p-value ⁇ 0.05, the increase in weight loss was considered indicative.
  • p-value ⁇ 0.05 means that tumor suppression or weight loss is significant.
  • the GraphPad Prism biological analysis software version 6.01 for Windows, GraphPad software, San Diego, California, USA, www.graphpad.com was used for statistical calculations.
  • TGI of the BI 853520 group was 0%, which did not show any anti-tumor efficacy (Table 8, Figure 3).
  • the TGI of the docetaxel group was 113%, which was statistically significant compared with the control group (Table 8, Figure 3).
  • the TGI of the BI 853520 and docetaxel combined group was 124%, which was statistically significant compared with the control group (Table 8, Figure 3).
  • T d was extended from about 7-8 days to 138 days, and T q was not reached until the end of the experiment (Table 8, Figure 3).
  • the P value has been compared to the control group, s represents a significant difference, and ns represents a non-significant difference
  • nr means not reached (for example, the median RTV of the group is always less than 200%/400%)
  • nr means not achieved, no weight loss (for example, the median RBW of the group is always greater than 100%)
  • BI 853520 and docetaxel showed a synergistic effect in the GXA 3039 gastric cancer PDX model, and it has a good inhibitory effect on tumor reproliferation after stopping the drug. The tolerance of the combination is not affected.
  • Example 4 The efficacy of BI 853520 in a mouse model of human lung squamous cell carcinoma (cell line NCI-H520)
  • NCI-H520 cells were obtained from ATCC (HTB-182). MCB and WCB were established according to BI RCV GmbH&Co KG standards. The cells were cultured in T175 tissue culture flasks. The medium used was GlutaMAX+F175K medium supplemented with 10% heat-inactivated fetal bovine serum and 1.5g/l sodium bicarbonate. The cells were cultured at 37°C and 5% CO 2 . Maintain the cell concentration of the culture between 8 ⁇ 10 6 to 12 ⁇ 10 7 /tissue culture flask.
  • mice Suspend NCI-H520 cells in ice-cold PBS+5% FCS, and then inject 100 ⁇ l of cell suspension containing 5 ⁇ 10 6 cells subcutaneously into the right flanks of nude mice (1 site per mouse) . When the tumor formed and reached a median volume of 50 mm 3 (14 days after cell injection), the mice were randomly assigned to a treatment group and a blank control group.
  • the pH of the BI853520 formulation is 3.
  • B BI 853520: 50mg/kg (once a day, administered by gavage needle)
  • Cisplatin 5mg/kg (injection into the intraperitoneal cavity once a week)
  • the tumor volume was statistically evaluated at the end of the experiment on the 22nd day.
  • Statistical evaluation uses the student's t-test function in Microsoft Excel, using two-tailed distribution and two-sample variance types.
  • the median animal weight increased by 9.1% (Table 10).
  • the treated animals showed the smallest change in body weight (-0.1%) (Table 10).
  • BI 853520 shows statistically significant tumor suppressor activity at 50mg/kg (p ⁇ 0.05), and it is well tolerated.
  • BI 853520 is used in combination with cisplatin (5mg/kg, IP, q7d), better tumor suppressor activity is observed, and there is no significant weight loss, and it is well tolerated.
  • BI 853520 is effective in the NCI-H520 human lung squamous cell carcinoma xenograft model, and its combined use with cisplatin shows a better anti-tumor effect than any single agent.
  • Example 5 Study on the anti-tumor activity of BI 853520 combined with paclitaxel in a mouse model (cell line KYSE-270).
  • KYSE-270 is a cell line of esophageal cancer (Public Health England, catalog number 94072021). The cells were cultured in T175 tissue culture flasks, and the medium used was: RPMI-1640+HAMF2 (1:1) supplemented with 2% calf serum and 2nM glutamine. The cells were cultured at 37°C and 5% CO 2 .
  • KYSE-270 cells were suspended in PBS+5% FCS, and the cell inoculation amount was 5 ⁇ 10 6 cells/ml.
  • the mice were randomly assigned to the treatment group and the blank control group.
  • B BI 853520: 50mg/kg (once a day, administered by gavage needle)
  • the tumors in group A reached a median volume of 1032 mm 3 on the 13th day of treatment (Figure 5; Table 11).
  • Example 6 Study on the anti-tumor activity of BI 853520 combined with docetaxel in a mouse model (cell lines KYSE-70, PC-3 and HS 746T).
  • KYSE-70 approximately 8-10 weeks old mice. 7-10 per group.
  • PC-3 approximately 6-week-old mice. 7-10 per group.
  • HS 746T approximately 6-week-old mice. 7-10 per group
  • KYSE-70 is a cell line of esophageal cancer (HPA strain collection, catalog number 94072012). Both PC-3 and HS 746T were purchased from ATCC. Cells were cultured in T175 tissue culture flasks, and the medium used was: RPMI-1640 supplemented with 10% calf serum. The cells were cultured at 37°C and 5% CO 2 .
  • KYSE-70 cells were suspended in PBS+5% FCS, and the cell inoculation amount was 5 ⁇ 10 6 cells/(50uL culture medium+50uL Matrigel).
  • the tumor formed and reached 67-93mm 3 11 days after cell injection
  • the mice were randomly assigned to the treatment group and the blank control group.
  • the PC-3 cells were suspended in PBS+5% FCS, and the cell inoculation amount was 5 ⁇ 10 6 cells/(50uL culture medium+50uL Matrigel).
  • the tumor formed and reached about 100 mm 3 (11 days after cell injection) the mice were randomly assigned to the treatment group and the blank control group.
  • the HS 746T cells were suspended in PBS+5% FCS, and the cell inoculation amount was 1 ⁇ 10 7 cells/(50uL culture medium+50uL Matrigel).
  • the mice were randomly assigned to the treatment group and the blank control group.
  • B BI 853520: 50mg/kg (once a day, administered by gavage needle)
  • B BI 853520: 25mg/kg (once a day, administered by gavage needle)
  • a tumor on day 42 the median tumor growth to a volume of 798mm 3 76mm 3 in volume position (FIG. 6; Table 12).
  • the body weight of the animals increased by 7.0% (Table 12).
  • a tumor at day 35 the median tumor growth to a volume 70mm 3 in volume of 809mm 3 in position (FIG. 7; Table 12).
  • the body weight of the animals was reduced by 3.2% (Table 12).
  • the single agent 25mg/kg BI 853520 and the single agent 7.5mg/kg docetaxel have a certain inhibitory effect on tumor growth.
  • the combined use of the two drugs has a significant synergistic effect.
  • the tumors in 6 out of 7 animals regressed, and the tumor growth was still inhibited after the drug was stopped. Very well tolerated.

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Abstract

本发明涉及BI853520或其药学上可接受的盐在制备用于与化疗药物联用治疗肿瘤的药物中的用途。

Description

BI853520与化疗药物的联用
本申请要求于2020年2月5日递交的中国专利申请第202010080757.1号的优先权，在此全文引用上述中国专利申请公开的内容以作为本申请的一部分。
技术领域
本发明属于药物化学领域。具体地，本发明涉及BI853520或其药学上可接受的盐在制备用于与化疗药物联用治疗肿瘤的药物中的用途。
背景技术
癌症是当代最严重的威胁人类生命和健康的疾病之一，在世界范围内，每年癌症死亡人数达七百万。目前，癌症的治疗主要采用四种方法：手术治疗、药物治疗、放射治疗及免疫治疗，此外，还有辅助疗法。其中癌症的药物治疗包括靶向药物疗法和化疗。在当前的各种治疗方法中，靶向药物疗法和化疗仍然占重要地位，是癌症的重要的标准疗法。例如，卡铂/紫杉醇联用的化疗方案是卵巢癌的一线治疗方案，化疗药物如紫杉醇类药物也是胃癌治疗方案中不可或缺的。但是，靶向药物疗法和化疗药物单药治疗中面临的最大问题是耐药性问题，包括自发性耐药和适应性耐药，造成化疗总体缓解率低且缓解持续时间有限。
因此，找到一种方法去提高化疗中单药的疗效，并进一步克服耐药问题是目前癌症治疗迫切需要解决的技术难题。
发明内容
一方面，本发明提供了一种BI853520或其药学上可接受的盐在制备用于与化疗药物联用治疗肿瘤的药物中的用途，所述BI853520为2-氟-5-甲氧基-4-[(4-(2-甲基-3-氧代-2,3-二氢-1H-异吲哚-4-氧基)-5-三氟甲基-嘧啶-2-基)氨基]-N-(1-甲基-哌啶-4-基)苯甲酰胺(参见WO2010058032)，所述化合物的结构如下：
Figure PCTCN2021074371-appb-000001
可选的，所述化疗药物为PLD、紫杉烷类或顺铂。
可选的，所述肿瘤不包括NRAS突变的肿瘤。
可选的，所述肿瘤为淋巴细胞性急性白血病、粒细胞性急性白血病、恶性淋巴瘤、乳腺癌、肺癌、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、尤文肉瘤、母细胞瘤、神经母细胞瘤、膀胱癌、甲状腺癌、前列腺癌、头颈部鳞癌、鼻咽癌、食道癌、睾丸癌、胃癌、肝癌、胰腺癌或黑色素瘤。
可选的，所述肿瘤为前列腺癌、食道癌、卵巢癌、胃癌或肺癌。所述肺癌优选为鳞状细胞肺癌。
可选的，所述所述化疗药物为PLD。
可选的，所述肿瘤为卵巢癌，尤其是铂耐药的卵巢癌。
可选的，所述化疗药物为紫衫烷类，尤其是多西他赛和紫杉醇。
可选的，所述肿瘤为前列腺癌、食道癌或胃癌。
可选的，所述药学上可接受的盐为BI853520酒石酸盐。
另一方面，本发明提供了BI853520或其药学上可接受的盐与化疗药物在制备用于治疗肿瘤的药物中的用途，所述化合物的结构为：
Figure PCTCN2021074371-appb-000002
可选的，所述化疗药物为PLD、紫杉烷类或顺铂。
可选的，所述肿瘤不包括NRAS突变的肿瘤。
可选的，所述肿瘤为淋巴细胞性急性白血病、粒细胞性急性白血病、恶性淋巴瘤、乳腺癌、肺癌、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、尤文肉瘤、母细胞瘤、神经 母细胞瘤、膀胱癌、甲状腺癌、前列腺癌、头颈部鳞癌、鼻咽癌、食道癌、睾丸癌、胃癌、肝癌、胰腺癌或黑色素瘤。
可选的，所述肿瘤为前列腺癌、食道癌、卵巢癌、胃癌或肺癌。所述肺癌优选为鳞状细胞肺癌。
可选的，所述化疗药物为PLD。
可选的，所述肿瘤为卵巢癌，尤其是铂耐药的卵巢癌。
可选的，所述化疗药物为紫衫烷类，尤其是多西他赛和紫杉醇。
可选的，所述肿瘤为前列腺癌、食道癌或胃癌。
可选的，所述药学上可接受的盐为BI853520酒石酸盐。
另一方面，本发明提供了一种药物组合，其特征在于，所述药物组合包括BI853520或其药学上可接受的盐，和化疗药物；所述BI853520的结构式如下：
Figure PCTCN2021074371-appb-000003
可选的，所述化疗药物为PLD、紫杉烷类或顺铂
可选的，所述化疗药物为PLD。
可选的，所述化疗药物为紫衫烷类，尤其是多西他赛和紫杉醇。可选的，所述药学上可接受的盐为BI853520酒石酸盐。
另一方面，本发明提供了一种治疗肿瘤的方法，其特征在于，其包括向受试者施用BI853520或其药学上可接受的盐和化疗药物；所述化合物的结构如下：
Figure PCTCN2021074371-appb-000004
可选的，所述方法包括向受试者施用BI853520或其药学上可接受的盐和有效量的化疗药物。
可选的，所述化疗药物为PLD、紫杉烷类或顺铂。
可选的，尤其是有效量的BI853520和有效量的PLD、紫杉烷类或顺铂。
可选的，所述肿瘤不包括NRAS突变的肿瘤。
可选的，所述肿瘤为淋巴细胞性急性白血病、粒细胞性急性白血病、恶性淋巴瘤、乳腺癌、肺癌、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、尤文肉瘤、母细胞瘤、神经母细胞瘤、膀胱癌、甲状腺癌、前列腺癌、头颈部鳞癌、鼻咽癌、食道癌、睾丸癌、胃癌、肝癌、胰腺癌或黑色素瘤。
可选的，所述肿瘤为前列腺癌、食道癌、卵巢癌、胃癌或肺癌。所述肺癌优选为鳞状细胞肺癌。
可选的，所述化疗药物为PLD。
可选的，所述肿瘤为卵巢癌，尤其是铂耐药的卵巢癌。
可选的，所述化疗药物为紫衫烷类，尤其是多西他赛和紫杉醇。
可选的，所述肿瘤为前列腺癌、食道癌或胃癌。
可选的，所述BI853520或其药学上可接受的盐，和化疗药物同步、交替或序贯给药。
可选的，其特征在于，所述药学上可接受的盐为BI853520酒石酸盐。
附图说明
图1a描述了BI 853520和PLD单药和联用在人卵巢癌小鼠模型中(TOV-21G细胞系)的肿瘤中位体积，其显示了不同试验组的抗肿瘤活性作用。试验组分别为A组(空白对照组)；B组(每天一次给药，剂量为12.5mg/kg的BI 853520组)；C组(每周给药一次，剂量为1mg/kg的PLD组)；D组(BI 853520和PLD的联合用药组，BI 853520每天给药一次，剂量为12.5mg/kg，PLD每周给药一次，剂量为1mg/kg)。
图1b描述了BI 853520和PLD单药和联用在人卵巢癌小鼠模型中(SKOV-3细胞系)的肿瘤中位体积，其显示了不同试验组的抗肿瘤活性作用。试验组分别为A组(空白对照组)；B组(每天一次给药，剂量为50mg/kg的BI 853520组)；C组(每周给药一次， 剂量为5mg/kg的PLD组)；D组(BI 853520和PLD的联合用药组，BI 853520每天给药一次，剂量为50mg/kg，PLD每周给药一次，剂量为5mg/kg)。
图1c描述了BI 853520和PLD单药和联用在人卵巢癌小鼠模型中(A2780细胞系)的肿瘤中位体积，其显示了不同试验组的抗肿瘤活性作用。试验组分别为A组(空白对照组)；B组(每天一次给药，剂量为12.5mg/kg的BI 853520组)；C组(每天一次给药，剂量为25mg/kg的BI 853520组)；D组(每天一次给药，剂量为50mg/kg的BI 853520组)；E组(每周给药一次，剂量为5mg/kg的PLD组)；F组(BI 853520和PLD的联合用药组，BI 853520每天给药一次，剂量为50mg/kg，PLD每周给药一次，剂量为5mg/kg)。
图2描述了CTG-1166模型的肿瘤生长动力学，其显示了不同试验组的抗肿瘤活性作用。试验组分别为空白对照组；每天给药两次，剂量为50mg/kg的BI 853520组；每周给药一次，剂量为3mg/kg的PLD组；BI 853520和PLD的联合用药组，BI 853520每天给药两次，剂量为50mg/kg，PLD每周给药一次，剂量为3mg/kg；卡铂和紫杉醇联合用药组，卡铂，每周给药一次，持续三周，剂量为25mg/kg，紫杉醇，每周给药一次，持续三周，剂量为20mg/kg。
图3描绘了BI853520和多西他赛联用在PDX模型中的肿瘤体积，其显示了不同试验组的抗肿瘤活性作用。试验组分别为空白对照组1/空白对照组2；每天一次给药，剂量为50mg/kg的BI 853520组；每周给药一次，剂量为10mg/kg的多西他赛组；BI 853520和多西他赛的联合用药组，BI 853520每天给药一次，剂量为50mg/kg，多西他赛每周给药一次，剂量为10mg/kg。
图4描绘了人肺鳞状细胞癌(细胞系NCI-H520)的裸鼠异种移植模型的肿瘤体积(mm 3)，其显示了不同试验组的抗肿瘤作用，试验组分别为空白对照组；每日一次给药，剂量为50mg/kg的BI 853520组；每周给药一次，剂量为5mg/kg的顺铂组；BI 853520和顺铂的联合用药组，BI 853520每天给药一次，剂量为50mg/kg，顺铂每周给药一次，剂量为5mg/kg。
图5描绘了人食道癌(细胞系KYSE-270)的裸鼠异种移植模型的肿瘤体积(mm 3)，其显示了不同试验组的抗肿瘤作用，试验组分别为空白对照组；每日一次给药，剂量为50mg/kg的BI 853520组；每周给药一次，剂量为10mg/kg的紫杉醇组；BI 853520和紫杉醇的联合用药组，BI 853520每天给药一次，剂量为50mg/kg，紫杉醇每周给药一次，剂量为10mg/kg。
图6描绘了人食道癌(细胞系KYSE-70)的裸鼠异种移植模型的肿瘤体积(mm 3)，其显示了不同试验组的抗肿瘤作用，试验组分别为空白对照组；每日一次给药，剂量为50mg/kg的BI 853520组；每周给药一次，剂量为10mg/kg的多西他赛组；BI 853520和多西他赛的联合用药组，BI 853520每天给药一次，剂量为50mg/kg，多西他赛每周给药一次，剂量为10mg/kg。
图7描绘了人前列腺癌(细胞系PC-3)的裸鼠异种移植模型的肿瘤体积(mm 3)，其显示了不同试验组的抗肿瘤作用，试验组分别为空白对照组；每日一次给药，剂量为25mg/kg的BI 853520组；每周给药一次，剂量为7.5mg/kg的多西他赛组；BI 853520和多西他赛的联合用药组，BI 853520每天给药一次，剂量为25mg/kg，多西他赛每周给药一次，剂量为7.5mg/kg。
图8描绘了人胃癌(细胞系HS 746T)的裸鼠异种移植模型的肿瘤体积(mm 3)，其显示了不同试验组的抗肿瘤作用，试验组分别为空白对照组；每日一次给药，剂量为50mg/kg的BI 853520组；每周给药一次，剂量为10mg/kg的多西他赛组；BI 853520和多西他赛的联合用药组，BI 853520每天给药一次，剂量为50mg/kg，多西他赛每周给药一次，剂量为10mg/kg。
实施例
提供下面的实施例以进一步阐述本发明。应理解，这些实施例仅用于举例说明本发明，而不用于限制本发明的范围。
下列实施例中未注明具体条件的实验方法均可以按照这类反应的常规条件进行或者按照制造厂商所建议的条件进行。
以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
本发明涉及一种BI853520或其药学上可接受的盐在制备用于与化疗药物联用治疗肿瘤的药物中的用途。本发明还涉及一种BI853520或其药学上可接受的盐与化疗药物在制备用于治疗肿瘤的药物中的用途。本发明还涉及了一种治疗肿瘤的方法，其特征在于，其包括向受试者施用有效量的BI853520或其药学上可接受的盐和化疗药物。
本文所用术语“化疗”是指一种全身治疗恶性肿瘤手段，可采用口服、静脉和体腔给药等方式，化疗药物会随着血液循环遍布全身的绝大部分器官和组织。化疗药物能作用在肿瘤细胞生长繁殖的不同环节上，抑制或杀死肿瘤细胞。是目前治疗恶性肿瘤最有效的手段之一。在一些实施方案中，所述化疗药物为PLD、多西他赛、紫杉醇或顺铂。
在一些实施方案中，所述肿瘤不包括NRAS突变的肿瘤。在一些实施例中，所述肿瘤选自淋巴细胞性急性白血病、粒细胞性急性白血病、恶性淋巴瘤、乳腺癌、未分化小细胞性支气管肺癌、非小细胞性支气管肺癌、非小细胞肺癌、鳞状细胞肺癌、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、尤文肉瘤、母细胞瘤、神经母细胞瘤、膀胱癌、甲状腺癌、前列腺癌、头颈部鳞癌、鼻咽癌、食道癌、睾丸癌、胃癌、肝癌、胰腺癌和黑色素瘤。在一些实施例中，所述肿瘤为卵巢癌、胃癌或鳞状细胞肺癌。
本发明的另一个实施方案是所述化疗药物为PLD。
本文所用术语“PLD”是指聚乙二醇脂质体阿霉素，也称多柔比星脂质体。
本文所述的术语“联用”，“联合治疗”和“联合用药”等是指两种或两种以上药物用于一种疾病的治疗。在一些实施例中，所述BI853520或其药学上可接受的盐与化疗药物联用，其可以进一步包括其它药物。在一些实施例中，所述BI853520或其药学上可接受的盐与化疗药物可以同步、交替或序贯给药。
本文所述的术语“NRAS”是指是一种致癌基因，是RAS致癌基因家族的一员，其中还包括另外两个基因：KRAS和HRAS。这些基因在细胞分裂、细胞分化和细胞凋亡中有着重要的作用。
本文所述的术语“NRAS突变”是指当NRAS基因发生致病性突变，其所编码的N-Ras蛋白将处于持续激活的状态，导致细胞增殖失控，进而形成肿瘤。
本文所用的术语“药学上可接受的”指的是无毒的、生物学上可耐受的，适合给受试者施用的。
本文所用的术语“药学上可接受的盐”指的是无毒的、生物学上可耐受的适合给受试者施用的盐，所述化合物药学上可接受的盐指的是无毒的、生物学上可耐受的适合给受试者施用的酸加成盐，包括但不限于：所述化合物与无机酸形成的酸加成盐，例如盐酸盐、氢溴酸盐、碳酸盐、碳酸氢盐、磷酸盐、硫酸盐、亚硫酸盐、硝酸盐等；以及所述化合物与有机酸形成的酸加成盐，例如甲酸盐、乙酸盐、苹果酸盐、马来酸盐、富马酸盐、酒石酸盐、琥珀酸盐、柠檬酸盐、乳酸盐、甲磺酸盐、对甲苯磺酸盐、2-羟基乙磺酸盐、苯甲酸盐、水杨酸盐、硬脂酸盐和与式HOOC-(CH 2) n-COOH(其中n是0-4)的链烷二羧酸形成的盐等。药学上可接受的盐可以通过本领域众所周知的常规方法获得，例如通过使足够碱性的化合物例如胺与提供生理上可接受的阴离子的合适的酸反应来获得。在一些实施方案中，所述盐为酒石酸盐。
本文所用的术语“受试者”是指哺乳动物和非哺乳动物。哺乳动物是指哺乳类的任何 成员，其包括但不限于：人；非人灵长类动物，如黑猩猩及其它猿类和猴类物种；农场动物，如牛、马、绵羊、山羊和猪；家畜，如兔、狗和猫；实验室动物，包括啮齿类动物，如大鼠、小鼠和豚鼠；等等。非哺乳动物的例子包括但不限于鸟等。术语“受试者”并不限定特定的年龄或性别。在一些实施方案中，受试者是人。
如本文所用术语“治疗”是指获得期望的药理和/或生理作用。该作用可以是治疗性的，包括部分或基本获得以下一项或多项结果：部分或全部减轻疾病、病症或综合症的程度；改善与疾病相关的临床症状或指标；或延迟、抑制或降低疾病，病症或综合症进展的可能性。
如本文所用术语“有效量”是指BI853520(或其药学上可接受的盐)或化疗药物(尤其是PLD、多西他赛或者顺铂)，其足以降低或改善疾病或病症的严重性，持续时间，进展或发作，延迟或阻止疾病或病症的进展，引起疾病或病症的消退或延迟症状的复发或进展，或增强或改善另一种疗法的治疗效果。给予受试者的BI853520(或其药学上可接受的盐)和化疗药物(尤其是PLD、多西他赛或者顺铂)的精确量将取决于各种因素，例如给定的药物或化合物，药物制剂，给药途径，疾病类型，病症，所治疗的受试者或宿主的身份等，但是仍然可以由本领域技术人员常规确定。例如，确定有效量还取决于细胞增殖的程度，严重性和类型。技术人员将能够根据这些和其他因素确定合适的剂量。当与其他治疗剂共同给药时，例如，当与抗癌剂共同给药时，任何其他治疗剂的“有效量”将取决于所用药物的类型。合适的剂量对于批准的治疗剂是已知的，并且可以由技术人员根据受试者的病情，所治疗的病情的类型以及所述化合物或其药学上可接受的盐的量进行调整。在没有明确注明量的情况下，应假定有一些量。所述BI853520(或其药学上可接受的盐)的有效剂量可以是10μg-2000mg。此实施例是非限制性的。化疗药物(尤其是PLD、多西他赛或者顺铂)的有效量是本领域技术人员已知的。
可以通过任何合适的给药方法来给予BI853520(或其药学上可接受的盐)。合适的方法包括对受试者口服，静脉，肌肉或皮下施用。
因此，所述BI853520(或其药学上可接受的盐)可以与药学上可接受的载体例如惰性稀释剂或可吸收的食用载体一起口服给药。它们可以封装在硬壳或软壳明胶胶囊中，可以压制成片剂，或者可以直接与患者的食物混合。对于口服治疗性给药，所述化合物或其药学上可接受的盐可以与一种或多种赋形剂组合，并以可摄取的片剂，口腔片剂，锭剂，胶囊，酏剂，悬浮液，糖浆或糯米纸囊剂等形式使用。这些制剂中包含有效量的所述BI853520(或其药学上可接受的盐)。
片剂，锭剂，丸剂，胶囊剂等可进一步包括：粘合剂，例如黄芪胶，阿拉伯胶，玉米淀粉或明胶；赋形剂，如磷酸二钙；崩解剂，例如玉米淀粉，马铃薯淀粉，海藻酸等；润滑剂，例如硬脂酸镁；或甜味剂，例如蔗糖，果糖，乳糖或阿斯巴甜；或调味剂。
所述BI853520(或其药学上可接受的盐)也可以通过输注或注射静脉内或腹膜内给药。所述BI853520(或其药学上可接受的盐)和化疗药物(尤其是PLD、多西他赛或者顺铂)的溶液可以在水中制备，任选地与无毒的表面活性剂混合。
用于注射或输注的示例性药物剂型包括：无菌水溶液，分散液，或包含活性成分的无菌粉末，该无菌粉末适合于临时制备无菌注射或输注溶液或分散液。无论如何，最终剂型在生产和储存条件下均应无菌，流动且稳定。
无菌注射溶液可以通过将所需量的所述BI853520(或其药学上可接受的盐)与所需的上述各种其他成分掺入适当的溶剂中，然后过滤灭菌来制备。对于用于制备无菌注射溶液的无菌粉末，优选的制备方法可以是真空干燥和冷冻干燥技术，其可以产生活性成分加上先前无菌过滤后存在的任何其他所需成分的粉末。
用于治疗所需的BI853520(或其药学上可接受的盐)、或化疗药物(尤其是PLD、多西他赛或者顺铂)的量不仅可以随所选择的特定盐而变化，还可以随给药途径，所治疗疾病的性质以及患者的年龄和状况而变化，并且最终可以由主治医师或临床医生自行决定。然而，一般而言，剂量可以在每天约0.1至约50mg/kg体重的范围内。
所需剂量可以方便地以单剂量或以适当间隔施用的分剂量存在。
所用的缩写含义如下：
ATCC            美国典型培养物保藏中心
ATVs            绝对肿瘤体积
BID             每天给药两次
BWL             体重减轻
CV              变异系数
CO 2             二氧化碳
CR              完整回应
d               天
ECACC           欧洲细胞培养物保藏中心
EDTA            乙二胺四乙酸
FCS             小牛血清
H               小时
kg              千克
IV              静脉给药
IP              腹腔给药
0.5％Natrosol   0.5％羟乙基纤维素
mg              毫克
ml              毫升
mm 3              立方毫米
MCB             主细胞库
Mean            平均值
MTD             最大耐受量
MTV             肿瘤体积平均值
N/n             样品量
PBS             磷酸盐缓冲盐
PDX             患者来源的异种移植瘤模型
PLD             聚乙二醇脂质体阿霉素
RPMI 1640       RPMI 1640培养基
p.o.            经口
PR              部分响应
qd/QD           每天
SEM             均数标准误
TFS             无肿瘤的幸存者
TGI             肿瘤生长抑制
TV              肿瘤体积
WCB             工作细胞库
通用的异种移植模型的抗肿瘤研究方法：
无胸腺雌性BomTac：NMRI-Foxn1nu小鼠购自丹麦Taconic。在到达动物房后，老鼠在新的环境下适应至少3天，然后再用于实验。这些动物饲养在标准条件下(温度21.5± 1.5℃和55±10％湿度)，标准化饮食和随意提供高压灭菌的自来水。皮下植入颈部区域的应答器Datamars T-IS 8010 FDX-B和LabMax II固定式读取器，用来识别每只小鼠。笼卡上显示研究编号，动物识别号，化合物和剂量水平，给药途径以及在整个实验过程中动物的给药时间表都保留在动物身上。
为建立皮下肿瘤，用胰酶消化法采集人类癌细胞，离心、洗涤并悬浮在合适的培养基中。然后将100μl含有5×10 6-1×10 7的细胞悬液皮下注射到裸鼠的右侧胁腹(每只小鼠1个位点)。当肿瘤生长到合适大小时，将小鼠随机分配到治疗组和空白对照组。
BI853520按照专利WO2010058032中的方法合成。将干粉悬浮在等摩尔体积的1M HCl中，并在适当体积的0.5％Natrosol中稀释，以达到每个实验所需的浓度。
每周用卡尺测量肿瘤直径三次(周一、周三和周五)。根据公式计算每个肿瘤的体积[以mm 3为单位]，“肿瘤体积＝长度×直径 2×π/6”。为了监测治疗的副作用，每日检查小鼠的异常情况，并每周三次(周一、周三和周五)测定体重。动物在研究结束时处死(大约在治疗开始后三周)；在研究期间，出于伦理原因，肿瘤坏死或肿瘤大小超过2000mm 3的动物会提前被处死。
在实验结束时，对肿瘤体积和体重参数进行统计评估。肿瘤体积绝对值和体重的变化百分比(参考第1天的初始重量)被使用。采用了非参数方法，计算了观察次数、中位数、最小值和最大值。为了快速了解可能的治疗效果，每个治疗组T的肿瘤体积中位数和对照组C的肿瘤体积中位数被用于计算从第1天到第d天的TGI：
在实验结束时，对肿瘤体积和体重参数进行统计评估。对于肿瘤体积，使用绝对值。采用了非参数方法，计算了观察次数，中位数，最小值和最大值。为了快速了解可能的治疗效果，每个治疗组T的肿瘤体积中位数和对照组C的肿瘤体积中位数被用于计算从第1天到第d天的TGI：
从第1天到第d天的TGI：
Figure PCTCN2021074371-appb-000005
其中，C 1，T 1＝在实验开始第一天对照组和治疗组的肿瘤体积中位数
C d，T d＝第d天实验结束时对照组和治疗组肿瘤体积的中位数
采用合适的统计学方法进行评估。显著性水平固定在α＝5％。p值(调整后)小于0.05的被认为显示出组间的统计学显著性差异，当0.05≤p值<0.10时，差异被视为指示性差异。
实施例1：BI 853520与PLD联用在人卵巢癌小鼠模型中的抗肿瘤活性研究。
使用通用的异种移植模型的抗肿瘤研究方法。大约6周龄小鼠。每组7-10只。
从ATCC(CRL-11730)获得了携带KRAS和PIK3CA基因突变的TOV-21G细胞。根据BI RCV GmbH&Co KG标准建立了MCB和WCB。细胞在T175组织培养瓶中培养，所用培养基为：补充有L-谷氨酰胺，丙酮酸钠，非必需氨基酸和10％热灭活的胎牛血清的RPMI-1640。细胞在于37℃和5％CO 2下培养。
从ECACC(93112519)获得了携带PTEN基因突变的A2780细胞。根据BI RCV GmbH&Co KG标准建立了MCB和WCB。细胞在T175组织培养瓶中培养，所用培养基为：补充了10％热灭活胎牛血清的RPMI-1640+Glutamax。细胞在37℃和5％CO 2下培养。
从ATCC(HTB-77)获得了携带CDKN2，MLH1，PIK3CA和TP53基因突变的SKOV-3细胞。根据BI RCV GmbH&Co KG标准建立了MCB和WCB。细胞在T175组织培养瓶中培养，所用培养基为IMDM+L-Glutamax和补充有10％热灭活胎牛血清的非必须氨基酸。细胞在37℃和5％CO 2下培养。
将A2780细胞悬浮在冰冷的PBS+5％FCS和生长因子降低的基质胶(1:1)中，细胞接种量为5×10 6细胞/ml。将TOV-21G细胞悬浮于冰冷的PBS+5％FCS中，细胞接种量5×10 6细胞/ml。当肿瘤形成并达到5-8mm直径时，将小鼠随机分配到治疗组和空白对照组。
BI853520配方的pH值在3到4之间。
PLD(Doxil)购自强生公司，并溶于无菌的5％葡萄糖溶液中。
当肿瘤中位体积在70至110mm 3之间时开始治疗。
TOV-21G：
空白对照组中10只动物，治疗组中每组7只动物
A：空白对照组：0.5％Natrosol
B：BI 853520：12.5mg/kg(每日一次，通过灌胃针给药)
C：PLD：1mg/kg(每周一次静脉推注)
D：B+C联用
SKOV-3：
空白对照组中10只动物，治疗组中每组7只动物
A：空白对照组：0.5％Natrosol
B：BI 853520：50mg/kg(每日一次，通过灌胃针给药)
C：PLD：5mg/kg(每周一次静脉推注)
D：B+C联用
A2780：
空白对照组和治疗组各10只动物
A：空白对照组：0.5％Natrosol
B：BI 853520：12.5mg/kg(每日一次，通过灌胃针给药)
C：BI 853520：25mg/kg(每日一次，通过灌胃针给药)
D：BI 853520：50mg/kg(每日一次，通过灌胃针给药)
E：PLD：5mg/kg(每周一次静脉推注)
F：D+E联用
这三个实验使用一样的方法，但分别进行了评估。统计评估实验结束时(第15天或第22天)的肿瘤体积和体重参数。
在细胞系A2780中，动物2、6、7和8的值(参考值：0.5％Natrosol；0.9％NaCl)，动物12和17(12.5mg/kg BI 853520)和动物36(50mg/kgBI 853520)，都是因为肿瘤达到了临界体积，出于伦理原因而处死了。
采用单侧递减wilcoxon试验，将每个剂量的试验化合物与对照组进行比较，肿瘤体积减少作为治疗效果，体重减少作为副作用。根据Bonferroni-Holm对肿瘤体积(疗效参数)的P值进行了多次比较调整，而体重(耐受性参数)的P值未进行调整，以免忽略可能的副作用。
TOV-21G：
A组动物体重减轻了1.5％(图3a；表5)，其肿瘤在治疗的第22天达到了654mm 3的中位体积(图1a；表1)。
B组肿瘤没有消退，TGI为67％(p＝0.0023)(图1a；表1)。动物体重增加了3.1％(与A组相比，p＝0.9977)(表2)。
C组TGI为83％(p＝0.002)，肿瘤无消退(图1a；表1)。动物体重增加了1.6％(与A组相比，p＝0.9649)(表2)。
D组TGI为106％(相对于A组，p＝0.0002；相对于B组，p＝0.0006；C组，p＝0.0006)，并且7只动物中有6只的肿瘤消退(图1a；表1)。动物体重增加了1.4％(相对于A组，p＝0.9335；相对于B组，p＝0.1914；相对于C组，p＝0.5000)(表2)。
研究表明，TOV-21G细胞系对BI 853520和PLD都非常敏感，因此，两种药物均以低剂量(12.5mg/kg；1mg/kg)，单药的情况下显示出很好的疗效，但并未诱导肿瘤消退。相比之下，两药联合使用，7只动物中有6只动物的肿瘤消退，且耐受性很好。
SKOV-3:
A组体重增加了0.3％(表5)，其肿瘤在治疗的第22天达到了608mm 3的中位体积(图1b；表4)。
B组TGI为57％(p＝0.0136)，无肿瘤消退(图1b；表1)。动物体重增加了5.5％(与A组相比，p＝0.9977)(表2)。
C组TGI为66％(p＝0.0136)，无肿瘤消退(图1b；表1)。动物体重增加了1.5％(相对于A组，p＝0.6302)(表2)。
D组TGI为90％(与A组相比，p＝0.0003；与B组和C组相比p＝0.0070)，7只动物中有1只肿瘤消退(图1b；表1)。动物体重减少了1.4％(相对于A组p＝0.1349；相对于B组，p＝0.0006；相对于C组，p＝0.1588)(表2)。
研究表明，SKOV-3细胞系对BI 853520和PLD的敏感性均较低，且所需药物剂量较高(50mg/kg，5mg/kg)。与单药相比联合疗法明显更有效，且耐受性也不差。
A2780：
A组体重增加了12.5％(图1c；表2)，其治疗第15天的肿瘤中位体积为1170mm 3(图1c；表1)。
B组TGI为19％(p＝0.1965)，无肿瘤消退(图1c；表1)。动物体重增加了10.6％(相对于A组，p＝0.5251)(表2)。
C组TGI为83％(p＝0.0134)，无肿瘤消退(图1c；表1)。动物体重增加了12.6％(相对于A组，p＝0.4789)(表2)。
D组TGI为85％(p＝0.0185)，无肿瘤消退(图1c；表1)。动物体重增加了10.1％(相 对于A组，p＝0.2280)(表2)。
E组TGI为99％(p<0.0001)，10只动物中4只动物肿瘤消退(图1c；表4)。动物体重增加了2.7％(相对于A组，p＝0.0156)(表2)。
F组TGI为100％(与A组相比，p<0.0001；与B组相比，p<0.0001；与C组相比，p＝0.0376)。10只动物中有6只肿瘤消退(图1c；表1)。动物体重增加了3.8％(与A组相比，p＝0.0080；与B组相比，p＝0.0021；与C组相比，p＝0.5147)(表2)。
研究表明，在A2780细胞系中，BI 853520在中、高剂量水平(25mg/kg，50mg/kg)下能观察到疗效，但未发现肿瘤消退。高剂量的PLD具有很高的功效，可导致40％的肿瘤消退。BI 853520和PLD中联用可产生更高的疗效，并有60％的消退，且耐受性依然很好。
BI 853520与PLD的联用在三个细胞系的卵巢癌模型中均显示出协同效应，且耐受性不受影响。
表1：肿瘤中位体积
Figure PCTCN2021074371-appb-000006
表2：试验结束时体重中位数改变
Figure PCTCN2021074371-appb-000007
Figure PCTCN2021074371-appb-000008
实施例2：BI853520和PLD联用在Champions
Figure PCTCN2021074371-appb-000009
PDX卵巢癌模型中的抗肿瘤活性研究。
通用实验方法如下：
雌性BomTac：大约7周龄(开始给药时)的NMRI-Foxn1nu小鼠购自丹麦Taconic。体重(开始给药时)大约20克。在到达动物房后，小鼠在新的环境下适应至少3天。将免疫功能低下的雌性小鼠圈养在HEPA通风笼中(
Figure PCTCN2021074371-appb-000010
IVC，Innovive USA)，在68-74°F(20-23℃)和30-70％湿度下，经过12小时明暗循环中的光照。随意给动物喂水(反渗透，2ppm Cl2)和标准饮食(Teklad 2919；19％蛋白质，9％脂肪和4％纤维)。与第0天相比，体重减轻≥10％的动物可免费获得DietAel 76A(
Figure PCTCN2021074371-appb-000011
Portland，ME)。每组3个。
在小鼠中植入来自Champions
Figure PCTCN2021074371-appb-000012
卵巢模型的肿瘤细胞，该模型是从先前接受铂类疗法治疗的患者中建立的。肿瘤达到1000-1500mm 3后，将它们收集起来，并将肿瘤片段SC植入雌性预研究小鼠的左侧胁腹。每只研究动物均植入特定的传代批次。每周用卡尺测量肿瘤体积两次以监测肿瘤生长，并使用公式(0.52×[长度×宽度 2])计算肿瘤体积(TV)。当TV达到约250-350mm 3(平均300mm 3)时，将动物与肿瘤大小匹配，并分配在对照组或治疗组(研究小鼠)，并在第0天开始给药。开始给药后，每天使用数字秤对动物称重，每周测量两次TV。当对照组的平均肿瘤体积达到约1500mm 3或直至第60天时，研究结束。在某些模型中，研究扩展至第60天后，并给药也扩展至研究结束。当肿瘤体积达到约1500mm 3时，可以将个体动物从研究中移除。抗肿瘤作用研究的设计如表3。
表3：人卵巢癌ChampionsTumor
Figure PCTCN2021074371-appb-000013
PDX模型的抗肿瘤作用研究设计
Figure PCTCN2021074371-appb-000014
空白对照组1：0.5％Natrosol；空白对照2：5％无菌葡萄糖。
BI853520按照专利WO2010058032中的方法合成，并在室温下避光保存。
口服给药的空白对照组是0.5％Natrosol。静脉给药的空白对照组是5％无菌葡萄糖。
卡铂为预先配制的10mg/mL的储备液。PLD为预先配制的2mg/mL的储备液。紫杉醇为预先配制的6mg/mL的储备液。这些标准化疗药物均由Champions提供。
为了监测治疗的副作用，从第0天开始，每天观察动物，并每周称重两次。记录每组的数据，包括每只的体重和平均体重，记录各组相对于第0天的平均体重变化百分比(％vD 0)，并在研究完成时绘制％vD 0。每天记录动物死亡，并根据体重减轻和肉眼观察将其确定为药物相关(D)，技术(T)，肿瘤相关(B)或未知(U)。平均％vD 0>20％和/或>10％死亡率的单药或联合治疗组被认为高于所评估方案中治疗的MTD。研究结束时报告了每个治疗组的最大平均％vD 0(体重最低点)。
通过计算TGI(100％x[1-(治疗组的最终MTV–初始MTV)/(对照组的最终MTV-初始MTV)])确定对肿瘤生长的抑制作用。在第0天开始治疗。将研究完成当天治疗组的肿瘤体积与对照组进行比较。
用于评估疗效的其他终点指标包括：CR，PR和TFS的数量。PR和TFS被认为不包括CR。
PR：连续2次测量，TV≤第0天TV的30％
CR：连续2次测量，检测不到TV(<4×4mm 2)
TFS：持续存在直到研究完成的CR
研究结束时使用数据进行统计分析。使用单向方差分析对肿瘤体积进行统计学比较，随后进行Newman-Keuls多重比较试验以比较所有组之间的差异。选择的终点包括尽可能多的组和每组尽可能多的动物。将由于肿瘤体积≥1500mm 3而提早取出的动物的肿瘤体积结转进行分析，但不得超过4个连续的测量时间点。比较BI 853520+PLD联合治疗组，BI 853520组和PLD组的肿瘤体积，以及BI 853520组和PLD组的肿瘤体积总和。
以模型CTG-1166为例：
空白对照组在第17天达到终点。使用PLD，和卡铂/紫杉醇进行治疗没有明显的抗肿瘤活性(图2，表4)。继续进行实验，显示BI 853520组，BI 853520和PLD联合治疗组对肿瘤生长有明显的抑制作用(图2，表4)。且BI 853520和PLD联合治疗组有2个PR(图2，表4)。在卡铂和紫杉醇耐药的情况下，联合治疗组显示很好的抑制作用。且所有组耐受性均较好，没有动物死亡(表5)。
表4：CTG-1166模型中的抗肿瘤活性
Figure PCTCN2021074371-appb-000015
表5：CTG-1166模型中的动物体重
Figure PCTCN2021074371-appb-000016
Figure PCTCN2021074371-appb-000017
按照上述通用的实验方法，在CTG-0252，CTG-0257，CTG-0791，CTG-0868，CTG-0956，CTG-0958，CTG-0964，CTG-0992，CTG-1086，CTG-1180，CTG-1301,CTG-1395，CTG-1427，CTG-1433，CTG-1498，CTG-1602，CTG-1624，CTG-1627，CTG-1649，CTG-1677，CTG-1678和CTG-1809中也分别对肿瘤抑制活性做了测试。结果显示在上述模型中，BI 853520和PLD的联合用药组，均有较好的协同作用，且具有好的抗肿瘤活性(TGI>70％)；对于卡铂和紫杉醇耐药的模型(CTG-0791，CTG-0956，CTG-0964，CTG-0992，CTG-1180，CTG-1301，CTG-1433，CTG-1498和CTG-1809)中，BI 853520和PLD的联合用药组仍具有好的抗肿瘤活性(TGI>70％)(表6)。
表6：不同卵巢癌PDX模型的抗肿瘤活性
Figure PCTCN2021074371-appb-000018
Figure PCTCN2021074371-appb-000019
天：实验结束天数
TGI>70％(有响应)；30％<TGI<70％(中等的)；TGI<30％(无响应)
研究表明，在不同的PDX模型中，BI 853520和PLD中联用可产生更高的疗效，且耐受性依然很好。尤其对于卡铂和紫杉醇耐药的模型，仍表现出较好的抗肿瘤活性。
实施例3：BI853520和多西他赛联用在PDX胃癌模型中的抗肿瘤活性研究
雌性BomTac：NMRI-Foxn1nu小鼠购自丹麦Taconic。将小鼠圈养在单独的通风笼中(TECNIPLAST Sealsafe TM-IVC-System，TECNIPLAST，Hohenpeissenberg，Germany)，根据实验动物的多少选择II型或III型笼子。在25±1℃和40-70％湿度下，经过14L：10D明暗循环中的光照，换气(AC)，笼中的速度为60-65 AC/小时。随意给动物喂水(过滤和酸化(pH 2.5)的无菌自来水)和动物饮食(Teklad Global 19％蛋白质挤压饮食(T.2019S.12))。水和饮食每周更换两次，在使用之前，所有材料均高压灭菌。
本研究中使用GXA 3039胃癌PDX模型。在免疫缺陷小鼠中植入来自GXA 3039胃癌PDX模型的肿瘤细胞，在初次植入后，建立并表征了肿瘤细胞(第1代)。当肿瘤细胞传代直至建立稳定的生长模式后，从小鼠中取出后，将肿瘤切成片段(边缘长度为3-4mm)，并置于含有10％青霉素/链霉素的PBS中。将肿瘤片段植入雌性预研究小鼠的侧面皮下。每天用卡尺测量肿瘤体积以监测肿瘤生长，当TV达到50-250mm 3(最好80-200mm 3)时，将他们随机到对照组或治疗组(使得各组的肿瘤中位体积和平均肿瘤体积约为100-150mm 3)。对未随机分组的动物实施安乐死。将随机分组的日期指定为实验的第0天，并在第1天开始给药。开始给药后，每周两次观察及测量动物称重(如果记录到体重减轻超过15％时，每天测量一次)及绝对肿瘤体积(ATV)。当肿瘤溃疡，穿透皮肤，肿瘤体积大于1500mm 3，体重减轻大于18％，或出现麻木疼痛等严重状况时，动物会提前被处死。如果存活的小鼠少于最初数量的70％(即少于8只中的6只)，则终止整个组。为了能够在给药结束后监测肿瘤的再增殖，进行le Kaplan-Meier统计(该规则不适用于肿瘤已缓解的组)。使用各组体重减轻(BWL)中位数的最大组评估耐受性。
通过计算TGI确定对肿瘤生长的抑制作用。使用ATV中位数绝对肿瘤体积计算TGI，计算公式如下：
ATV＝a×b 2×0.5
其中，a代表长度，b代表垂直肿瘤直径；
Figure PCTCN2021074371-appb-000020
其中，T 0和C 0是测试组和空白对照组第0天的ATV中位数；
T x和C x是相应的第X天的ATV中位数；第X天是BI 853520最后一次给药(QD给药)的第二天，多西他赛最后一次给药后的一周(每周给药)或至少70％的动物的最后一天，以较早发生者为准。
TGI值具有以下含义(假设C x>C 0)：
试验组肿瘤总体缓解：TGI>100％
测试肿瘤的体积不变(停滞)：TGI＝100％
与对照组相比，肿瘤的生长速度降低：100％>TGI>0％
与对照组相比肿瘤生长率相同：TGI＝0
与对照相比，肿瘤生长刺激：TGI<0
用于评估疗效的其他终点指标包括：肿瘤体积加倍/四倍的时间和肿瘤生长延迟/肿瘤进展时间/肿瘤再增殖。
肿瘤体积加倍/四倍的时间：测试组和对照组的肿瘤体积加倍/四倍的时间(T d/T q)定义为各组达到中位RTV的200％/400％所需的时间间隔(以天为单位)。通过将第X天(T x)肿瘤的绝对体积除以第0天(T 0)肿瘤的绝对体积，再乘以100，得出第X天的单个肿瘤的相对体积(RTV x[％])，公式如下：
Figure PCTCN2021074371-appb-000021
肿瘤生长延迟/肿瘤进展时间/肿瘤再增殖：为了评估无剂量观察期内肿瘤消退后肿瘤进展/肿瘤再增殖的时间差异(肿瘤生长延迟，TGD)，使用Kaplan-Meier生存分析。相对肿瘤体积为400％定义为终点。
抗肿瘤作用研究的设计如表7。
表7：抗肿瘤作用研究设计
Figure PCTCN2021074371-appb-000022
Figure PCTCN2021074371-appb-000023
空白对照组1：0.5％Natrosol；空白对照2：0.9％盐水。
BI853520按照专利WO2010058032中的方法合成，并在室温下避光保存。BI 853520溶解在0.5％Natrosol中来制备浓度为5mg/ml的溶液，以50mg/kg的剂量给药。将溶液分成两等份，在室温下保存，避光并在一周内使用。
多西他赛：购买子Sanofi Avents,在4℃下保存。在给药日通过用0.9％盐水稀释多西他赛溶液来制备浓度为1mg/ml的浓度，以10mg/kg的剂量给药溶液。
口服给药的空白对照组是0.5％Natrosol。静脉给药的空白对照组是0.9％盐水。剂量均为10ml/kg。对于联合治疗组，BI 853520先给药，然后立即再给药多西他赛。
当在实验中记录到明显的体重减轻时，将采取以下措施：
对于体重减轻超过15％的动物，不予给药；
对于体重减轻>15％的动物，每日测量体重；
对于体重减轻超过15％的动物可方便获取饲料和水；
当个别动物的相对体重至少达到85％时恢复用药。
研究结束时使用数据进行统计分析。使用单尾非参数Mann-Whitney-Wilcoxon U检验，显著性水平α设置为0.05。从U检验获得的p值使用Bonferroni-Holm校正调整。
为了评估使用RTV的400％作为试验终点对肿瘤进展/肿瘤生长延迟(TGD)的时间差异的统计显著性，Kaplan-Meier生存方式统计量与对数秩Mantel-Cox检验相结合使用成对比较。
另外，为了评估治疗的耐受性，使用了在给药阶段结束时确定的体重(即在TGI值被统计的当天)，进行了两尾无参数Mann-Whitney-Wilcoxon U检测。为了不忽略治疗的可能不利影响，未使用Bonferroni-Holm法校正调整体重的p值，并且如果p值在0.10≥p值≥0.05范围内，则认为体重减轻增加具有指示性。
按照惯例，p值≤0.05代表抑制肿瘤或体重减轻具有显著性。使用GraphPad Prism生物分析软件(适用于Windows的6.01版，GraphPad软件，美国圣地亚哥加利福尼亚，www.graphpad.com)进行统计计算。
研究显示：BI 853520组TGI为0％，其未显示任何抗肿瘤功效(表8，图3)。多西他赛组TGI为113％，与对照组相比，数据具有统计学显著性(表8，图3)。BI 853520和多西他赛联 用组TGI为124％，与对照组相比，数据具有统计学显著性(表8，图3)。联合用药组除在肿瘤抑制方面表现出比单药组更好的效果外，在给药结束后对于肿瘤再生长的延迟方面表现出更突出的优势。将T d从约7-8天延长到138天，且一直到实验结束也未达到T q(表8，图3)。
在所有组中均未观察到或只有很少的BWL中位数最大值达到2.9％。BI 853520组均未记录到BWL中位数(表9)。耐受性均较好。
表8：抗肿瘤(胃癌)活性
Figure PCTCN2021074371-appb-000024
P值均已相对于对照组，s代表显著性差异，ns代表不显著
nr代表没达到(例如组的RTV中位数总是小于200％/400％)
表9：体重减轻和存活率
Figure PCTCN2021074371-appb-000025
nr代表没达到，没有体重减轻(例如组的RBW中位数总是大于100％)
BI 853520与多西他赛的联用在GXA 3039胃癌PDX模型中显示出协同效应，且停药后对肿瘤的再增殖有很好的抑制作用。联用耐受性不受影响。
实施例4：BI 853520在人肺鳞状细胞癌的小鼠模型中的功效(细胞系NCI-H520)
使用通用的异种移植模型的抗肿瘤研究方法。大约6周龄小鼠。每组5只。
从ATCC(HTB-182)获得NCI-H520细胞。根据BI RCV GmbH&Co KG标准建立了MCB和WCB。细胞在T175组织培养瓶中培养，所用培养基为：添加10％热灭活胎牛血清和1.5g/l碳酸氢钠的GlutaMAX+F175K培养基，细胞在37℃和5％CO 2下培养。将培养物的细胞浓度维持在8×10 6到12×10 7/组织培养瓶之间。
将NCI-H520细胞悬浮在冰冷的PBS+5％FCS中，然后将100μl含有5×10 6个细胞的细胞悬液皮下注射到裸鼠的右侧胁腹(每只小鼠1个位点)。当肿瘤形成并达到50mm 3的体积中位数(细胞注射后14天)时，将小鼠随机分配到治疗组和空白对照组。
BI853520配方的pH值为3。
顺铂溶于0.9％盐水中
空白对照组中10只动物，治疗组中每组7只动物
A：空白对照组：0.5％Natrosol/0.9％盐水
B：BI 853520：50mg/kg(每日一次，通过灌胃针给药)
C：顺铂：5mg/kg(每周一次腹腔注射)
D：B+C联用
在第22天实验结束时对肿瘤体积进行统计学评估。统计评估是使用Microsoft Excel中的学生t检验功能，利用两尾分布和两样本等方差类型。
A组动物体重增加了5.8％(表10)，并且在治疗的第22天，他们的肿瘤的中位体积为721mm 3(表10)。
B组TGI为54％(p＝0.007)(图4，表10)。动物体重中位数增加了9.1％(表10)。
C组可显着抑制肿瘤生长，TGI为74％(p＝0.0004，表10，图4)。治疗的耐受性良好，体重中位数增加了9.6％(表10)。
D组TGI为90％(p＝0.00003)，且和2/7肿瘤消退(表10，图4)。治疗的动物显示出最小的体重变化(-0.1％)(表10)。
研究表明：BI 853520在50mg/kg下显示出统计学上显着的肿瘤抑制活性(p<0.05)，且耐受性较好。此外，当BI 853520与顺铂(5mg/kg，IP，q7d)联合使用时，观察到更好的肿瘤抑制活性，且没有明显的体重减轻，有良好的耐受性。
BI 853520在NCI-H520人肺鳞状细胞癌异种移植模型中有效，与顺铂联合使用显示出比任一单药更好的抗肿瘤作用。
表10：肿瘤中位体积
治疗 肿瘤中位体积[mm 3] TGI中位数[％] 肿瘤消退 体重中位数改变[％]
A 721   0/10 +5.8
B 352 54 0/7 +9.1
C 228 74 0/7 +9.6
D 116 90 2/7 -0.1
实施例5：BI 853520与紫衫醇联用在小鼠模型中的抗肿瘤活性研究(细胞系KYSE-270)。
使用通用的异种移植模型的抗肿瘤研究方法。大约8-10周龄小鼠。每组7-10只。
KYSE-270是食道癌的细胞系(英格兰公共卫生，目录号94072021)。细胞在T175组织培养瓶中培养，所用培养基为：补充有2％小牛血清和2nM谷氨酰胺的RPMI-1640+HAMF2(1:1)。细胞在于37℃和5％CO 2下培养。
将KYSE-270细胞悬浮在PBS+5％FCS中，细胞接种量为5×10 6细胞/ml。当肿瘤形成并达到94-252mm 3(细胞注射后13天)时，将小鼠随机分配到治疗组和空白对照组。
空白对照组中10只动物，治疗组中每组7只动物
A：空白对照组：0.5％Natrosol/0.9NaCl
B：BI 853520：50mg/kg(每日一次，通过灌胃针给药)
C：紫杉醇：10mg/kg(每周一次静脉推注)
D：B+C联用
试验结束时，通过精确的Wilcoxon检验进行比较。
A组肿瘤在治疗的第13天达到了1032mm 3的中位体积(图5；表11)。动物体重减轻了9.1％(表11)，且有一只动物由于第9天体重严重下降，不得不提前实施安乐死。
B组与对照组相比，显著延缓肿瘤生长，TGI为106％(p＝0.0003)(图5；表11)。且7只动物中有6只肿瘤发生消退，且全部存活。动物体重增加了2.8％(与A组相比，p＝0.9999)(表11)。
C组与对照组相比，对肿瘤生长没有影响，TGI为2％(p＝0.3788)，肿瘤无消退(图5；表11)。动物体重下降了9.7％(与A组相比，p＝0.7320)(表11)。
D组与对照组相比，显著延迟肿瘤生长，TGI为110％(相对于A组，p＝0.0003)，并且7只动物中有7只的肿瘤消退(图5；表11)。动物体重增加了3.5％(相对于A组，p＝1.0000)(表11)。
研究表明，在皮下人KYSE-270食道癌模型中，单药50mg/kg BI 853520对肿瘤生长有抑制作用，但是单药10mg/kg紫杉醇没有作用。相比之下，两药联合使用，抑制效果更好，且7只动物中有7只动物的肿瘤消退，耐受性很好。尤其在停药后，肿瘤大小无明显增长。
表11：肿瘤中位体积
Figure PCTCN2021074371-appb-000026
实施例6：BI 853520与多西他赛联用在小鼠模型中的抗肿瘤活性研究(细胞系KYSE-70，PC-3和HS 746T)。
使用通用的异种移植模型的抗肿瘤研究方法。
KYSE-70：大约8-10周龄小鼠。每组7-10只。
PC-3：大约6周龄小鼠。每组7-10只。
HS 746T：大约6周龄小鼠。每组7-10只
KYSE-70是食道癌的细胞系(HPA菌种保藏，目录号94072012)。PC-3和HS 746T均购买自ATCC.细胞在T175组织培养瓶中培养，所用培养基为：补充有10％小牛血清的RPMI-1640。细胞在于37℃和5％CO 2下培养。
将KYSE-70细胞悬浮在PBS+5％FCS中，细胞接种量为5×10 6细胞/(50uL培养液+50uL Matrigel)。当肿瘤形成并达到67-93mm 3(细胞注射后11天)时，将小鼠随机分配到治疗组和空白对照组。
将PC-3细胞悬浮在PBS+5％FCS中，细胞接种量为5×10 6细胞/(50uL培养液+50uL Matrigel)。当肿瘤形成并达到100mm 3左右(细胞注射后11天)时，将小鼠随机分配到治 疗组和空白对照组。
将HS 746T细胞悬浮在PBS+5％FCS中，细胞接种量为1×10 7细胞/(50uL培养液+50uL Matrigel)。当肿瘤形成并达到117mm 3(细胞注射后10天)时，将小鼠随机分配到治疗组和空白对照组。
KYSE-70及HS 746T：
空白对照组中10只动物，治疗组中每组7只动物
A：空白对照组：0.5％Natrosol/5葡萄糖
B：BI 853520：50mg/kg(每日一次，通过灌胃针给药)
C：多西他赛：10mg/kg(每周一次静脉推注)
D：B+C联用
PC-3：
空白对照组中10只动物，治疗组中每组7只动物
A：空白对照组：0.5％Natrosol/5葡萄糖
B：BI 853520：25mg/kg(每日一次，通过灌胃针给药)
C：多西他赛：7.5mg/kg(每周一次静脉推注)
D：B+C联用
实验结束时，通过精确的Wilcoxon检验进行比较。
KYSE-70：
A组肿瘤在第42天，肿瘤从76mm 3的中位体积增长到798mm 3的中位体积(图6；表12)。动物体重增加了7.0％(表12)。
B组与对照组相比，未显著延缓肿瘤生长，TGI为42％(p＝0.1574)(图6；表12)。且7只动物中有1只肿瘤发生消退。动物体重增加了8.8％(与A组相比，p＝0.7189)(表12)。
C组对照组相比，显著延缓肿瘤生长，TGI为100％(p＝0.0002)，且在第28天，7只动物中有4只肿瘤发生消退(图6；表12)。动物体重增加了5.6％(与A组相比，p＝0.2681)(表12)。
D组与对照组相比，显著延迟肿瘤生长，TGI为111％(相对于A组，p＝0.0002)，并且7只动物中有7只的肿瘤消退(图6；表11)。动物体重增加了2.5％(相对于A组，p＝0.0093)(表12)。
研究表明，在皮下人KYSE-70食道癌模型中，单药50mg/kg BI 853520对肿瘤生长无抑制作用，但是单药10mg/kg多西他赛有抑制作用。相比之下，两药联合使用，抑制效果更好，7只动物中有6只动物的肿瘤消退，且中位肿瘤体积达到了0。耐受性很好。
PC-3：(28天停药)
A组肿瘤在第35天，肿瘤从70mm 3的中位体积增长到809mm 3的中位体积(图7；表12)。动物体重降低了3.2％(表12)。
B组与对照组相比，显著延缓肿瘤生长，TGI为66％(图7；表12)。动物体重增加了2.4％(表12)。
C组对照组相比，显著延缓肿瘤生长，TGI为92％，且在第28天，7只动物中有3只肿瘤发生消退(图7；表12)。动物体重增加了10％(表12)。
D组与对照组相比，显著延迟肿瘤生长，TGI为108％，并且7只动物中有6只的肿瘤消退(图7；表12)。动物体重增加了7.8％(表12)。
研究表明，在皮下人PC-3前列腺癌模型中，单药25mg/kg BI 853520和单药7.5mg/kg多西他赛对肿瘤生长有一定的抑制作用。两药联合使用，有明显的协同效应，7只动物中有6只动物的肿瘤消退，且停药后肿瘤生长仍受到抑制。耐受性很好。
HS 746T：(28天停药)
A组肿瘤在第24天，肿瘤从117mm 3的中位体积增长1684mm 3的中位体积(图8；表12)。动物体重增加了8.4％(表12)。
B组与对照组相比，显著延缓肿瘤生长，TGI为93％(图8；表12)。且8只动物中有1只肿瘤发生消退。动物体重增加了7.3％(表12)。
C组对照组相比，显著延缓肿瘤生长，TGI为108％，且在第28天，8只动物中全部发生肿瘤消退(图8；表12)。动物体重增加了6.7％(表12)。
D组与对照组相比，显著延迟肿瘤生长，TGI为108％，并且8只动物中全部发生肿瘤消退(图8；表12)。动物体重增加了9.3％(表12)。
研究表明，在皮下人HS 746T胃癌模型中，单药50mg/kg BI 853520或单药10mg/kg多西他赛有抑制作用。相比之下，两药联合使用，抑制效果更好，8只动物中全部动物出现肿瘤消退，且停药后肿瘤生长仍受到抑制，尤其在联合用药组，有更持久的抗肿瘤效果。耐受性很好。
表12：肿瘤中位体积
Figure PCTCN2021074371-appb-000027
在本申请全文中引用的所有参考文献(包括文献参考文献，已发布的专利，已公开的专利申请和同时待审的专利申请)的内容在此明确地全文引入作为参考。除非另有定义，否则本文所用的所有技术和科学术语均与本领域普通技术人员通常已知的含义一致。
本说明书中公开的所有特征可以以任何组合进行组合。本说明书中公开的每个特征可以由具有相同，等同或相似目的的替代特征代替。因此，除非另有明确说明，否则所公开的每个特征仅是一系列等同或相似特征的示例。
根据以上描述，本领域技术人员可以容易地确定本发明的基本特征，并且在不脱离本发明的精神和范围的情况下，可以对本发明进行各种改变和修改以使其适应各种用途和条件。因此，其他实施例也在所附权利要求的范围内。

Claims (32)

  1. BI853520或其药学上可接受的盐在制备用于与化疗药物联用治疗肿瘤的药物中的用途，所述BI853520的结构式如下：
    Figure PCTCN2021074371-appb-100001
  2. 如权利要求1所述的用途，其特征在于，所述化疗药物为PLD、紫杉烷类或顺铂。
  3. 如权利要求1-2中任一项所述的用途，其特征在于，所述肿瘤为淋巴细胞性急性白血病、粒细胞性急性白血病、恶性淋巴瘤、乳腺癌、肺癌、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、尤文肉瘤、母细胞瘤、神经母细胞瘤、膀胱癌、甲状腺癌、前列腺癌、头颈部鳞癌、鼻咽癌、食道癌、睾丸癌、胃癌、肝癌、胰腺癌或黑色素瘤。
  4. 如权利要求1-3中任一项所述的用途，其特征在于，所述肿瘤为前列腺癌、食道癌、卵巢癌、胃癌或肺癌。
  5. 如权利要求1-4中任一项所述的用途，其特征在于，所述化疗药物为PLD。
  6. 如权利要求1-5中任一项所述的用途，其特征在于，所述肿瘤为卵巢癌，尤其是铂耐药的卵巢癌。
  7. 如权利要求1-4中任一项所述的用途，其特征在于，所述化疗药物为紫衫烷类，尤其是多西他赛和紫杉醇。
  8. 如权利要求7所述的用途，其特征在于，所述肿瘤为前列腺癌、食道癌或胃癌。
  9. 如权利要求1-8中任一项所述的用途，其特征在于，所述药学上可接受的盐为BI853520酒石酸盐。
  10. BI853520或其药学上可接受的盐与化疗药物在制备用于治疗肿瘤的药物中的用途，所述化合物的结构为：
    Figure PCTCN2021074371-appb-100002
  11. 如权利要求10所述的用途，其特征在于，所述化疗药物为PLD、紫杉烷类或顺铂。
  12. 如权利要求10-11中任一项所述的用途，其特征在于，所述肿瘤如权利要求3或权利要求4所定义。
  13. 如权利要求10-12中任一项所述的用途，其特征在于，所述化疗药物为PLD。
  14. 如权利要求10-13中任一项所述的用途，其特征在于，所述肿瘤为卵巢癌，尤其是铂耐药的卵巢癌。
  15. 如权利要求10-12中任一项所述的用途，其特征在于，所述化疗药物为紫衫烷类，尤其是多西他赛和紫杉醇。
  16. 如权利要求15所述的用途，其特征在于，所述肿瘤为前列腺癌、食道癌或胃癌。
  17. 如权利要求10-16中任一项所述的用途，其特征在于，所述药学上可接受的盐为BI853520酒石酸盐。
  18. 药物组合，其特征在于，所述药物组合包括BI853520或其药学上可接受的盐，和化疗药物；所述BI853520的结构式如下：
    Figure PCTCN2021074371-appb-100003
  19. 如权利要求18所述的药物组合，其特征在于，所述化疗药物为PLD、紫杉烷类或顺铂。
  20. 如权利要求19所述的药物组合，其特征在于，化疗药物为PLD。
  21. 如权利要求19所述的药物组合，其特征在于，化疗药物为紫衫烷类，尤其是多西他赛和紫杉醇。
  22. 如权利要求18-21中任一项所述的药物组合，其特征在于，所述药学上可接受的盐为BI853520酒石酸盐。
  23. 一种治疗肿瘤的方法，其特征在于，其包括向受试者施用BI853520或其药学上可接受的盐和化疗药物；所述BI853520的结构式如下：
    Figure PCTCN2021074371-appb-100004
    尤其是有效量的BI853520或其药学上可接受的盐和有效量的化疗药物。
  24. 如权利要求23所述的方法，其特征在于，所述化疗药物为PLD、紫杉烷类或顺铂。
  25. 如权利要求23-24中任一项所述的方法，其特征在于，所述肿瘤为淋巴细胞性急性白血病、粒细胞性急性白血病、恶性淋巴瘤、乳腺癌、肺癌、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、尤文肉瘤、母细胞瘤、神经母细胞瘤、膀胱癌、甲状腺癌、前列腺癌、头颈部鳞癌、鼻咽癌、食道癌、睾丸癌、胃癌、肝癌、胰腺癌或黑色素瘤。
  26. 如权利要求23-25中任一项所述的方法，其特征在于，所述肿瘤为卵巢癌、胃癌、前列腺癌、食道癌或肺癌。
  27. 如权利要求23-26中任一项所述的方法，其特征在于，所述化疗药物为PLD。
  28. 如权利要求23-26中任一项所述的方法，其特征在于，所述肿瘤为卵巢癌，尤其是铂耐药的卵巢癌。
  29. 如权利要求23-26中任一项所述的方法，其特征在于，所述化疗药物为紫衫烷类，尤其是多西他赛和紫杉醇。
  30. 如权利要求29所述的方法，其特征在于，所述肿瘤为前列腺癌、食道癌或胃癌。
  31. 如权利要求23-30中任一项所述的方法，其特征在于，所述BI853520或其药学上可接受的盐，和化疗药物同步、交替或序贯给药。
  32. 如权利要求23-31中任一项所述的方法，其特征在于，所述药学上可接受的盐为BI853520酒石酸盐。
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