WO2013037127A1 - Antitumour pharmaceutical composition and use thereof - Google Patents

Antitumour pharmaceutical composition and use thereof Download PDF

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Publication number
WO2013037127A1
WO2013037127A1 PCT/CN2011/079757 CN2011079757W WO2013037127A1 WO 2013037127 A1 WO2013037127 A1 WO 2013037127A1 CN 2011079757 W CN2011079757 W CN 2011079757W WO 2013037127 A1 WO2013037127 A1 WO 2013037127A1
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tumor
dexamethasone
dipyridamole
pharmaceutical composition
weight ratio
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PCT/CN2011/079757
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French (fr)
Chinese (zh)
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刘秀均
甄永苏
郑艳波
李毅
吴淑英
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中国医学科学院医药生物技术研究所
浙江普洛康裕制药有限公司
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Priority to PCT/CN2011/079757 priority Critical patent/WO2013037127A1/en
Publication of WO2013037127A1 publication Critical patent/WO2013037127A1/en

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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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • 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 present invention is in the field of medicinal chemistry, and in particular, relates to an antitumor drug composition and use thereof. Background technique
  • the strategy for developing new anti-tumor drugs includes two aspects: finding drugs for tumor cells and drugs for regulating tumor microenvironment.
  • drugs acting on tumor cells mainly induce tumor cell proliferation, induce tumor cell apoptosis, and induce tumor cells. Differentiation works.
  • the drugs that regulate the tumor microenvironment mainly play the role of inhibiting tumor angiogenesis or interfering with tumor vascular network; regulating immune cells and related cytokines in tumor infiltration; regulating growth factor secretion and growth factor receptor expression; Inhibition of specific enzyme secretion and regulation of corresponding inhibitors; Interfering with the transport, uptake and efflux of intracellular and extracellular substances in tumor cells.
  • Dipyr idamole is a non-tumor therapeutic drug synthesized in the 1980s. Its pharmaceutically acceptable derivatives or analogues such as mopidamole, BIBW22BS, RA25 or its pharmacy
  • An acceptable salt is a non-nitrate coronary artery dilator that has the effect of expanding the coronary vessels and promoting the formation of collateral circulation. Dipyridamole also has the effect of inhibiting platelet aggregation and preventing thrombosis.
  • the drug is included as a cardiovascular dilating agent in the Pharmacopoeia of the People's Republic of China (2000 edition) and the United States Pharmacopoeia (XXI-XXI II), but because of its so-called "coronary theft” phenomenon, ie heparin, coumarins and fibrin When dissolved drugs are combined, they can cause bleeding tendency, so they have become clinically unnecessary.
  • Dipyridamole is a potent nucleoside transport inhibitor in the mechanism of drug action by blocking the balanced nucleoside transporter hENTl (Nat Med, 1997, 3: 89-93) and hENT2 (Biochem. J, 1997, 328: 739-43) inhibits nucleoside transport.
  • Ubenimex (Bes tat in, BEN) and pharmaceutically acceptable derivatives or analogs thereof include AHPA-VaK Bestat in Hydrochloride and the like.
  • Ubumex is a small molecule dipeptide compound isolated from the fermentation broth of Streptomyces ol ivoret icul i by Japanese scholar Umezawa in 1976. It can competitively inhibit various aminopeptidase activities. It exerts a dual anti-tumor effect by promoting the immune function of the body and directly acting on tumor tissues. In 1987, it was marketed as a new anticancer drug with immunomodulatory function in Japan for the adjuvant treatment of malignant tumors.
  • Ubumex exhibits a multi-faceted immune activity that not only enhances lymphocyte function, but also activates monocyte macrophages to enhance the killing activity of NK cells.
  • Molecular mechanism studies have shown that its target is leucine aminopeptidase (Leu-AP) and aminopeptidase B (AP-B) located on the surface of immune cells, which can catalyze the cleavage of the amino terminus of the substrate to make the antigen The molecule is inactivated.
  • Ubumex inhibits the activity of aminopeptidase by chelation with zinc ions in the active center of the enzyme. Due to its immune-enhancing function, Ubumex is often used in the treatment of tumor chemotherapy, radiotherapy and postoperative surgery. It can be combined with chemotherapy, radiotherapy and combined therapy for leukemia, multiple myeloma and solid tumors such as lung cancer and breast cancer.
  • Ubumex can also exert anti-tumor activity by directly acting on tumor tissues.
  • Aminopeptidase N (CD13) is highly expressed in tumor neovascular endothelial cells, which promotes tumor vascular formation by degrading extracellular matrix, promoting endothelial cell invasion, and regulating the activity of certain growth factors and cytokines (Int J Cancer, 1993) , 54: 137-43).
  • Certain tumor cells such as human fibrosarcoma HT-1080 cell aminopeptidase N, also exhibit high expression and are closely related to invasion and metastasis of tumor cells (Int J Cancer, 1993, 54: 137-43).
  • Ubumex can inhibit the activity of aminopeptidase N, thereby effectively inhibiting the formation of tumor blood vessels and preventing the metastasis of tumor cells.
  • Ubumex can also directly induce tumor cell apoptosis (Biomed Pharmacother. 1996; 50: 283-9), thereby exerting an anti-tumor effect.
  • Glucocorticoids such as dexamethasone (Dexamethason, DEX) and pharmaceutically acceptable derivatives or analogs thereof, such as dexamethasone acetate, dexamethasone sodium silicate, dexamethasone palmitate, hydrocortisone , cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone and other pharmacological effects such as anti-inflammatory, immunosuppressive, anti-toxin, anti-shock.
  • DEX dexamethasone
  • pharmaceutically acceptable derivatives or analogs thereof such as dexamethasone acetate, dexamethasone sodium silicate, dexamethasone palmitate, hydrocortisone , cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone and other pharmacological effects such as anti-inflammatory, immunosuppressive,
  • glucocorticoids include the C3 carbonyl group, the ⁇ 4 and 17 ⁇ ketol side chains of the adrenocortical hormone, and the 17 ⁇ - 0 ⁇ and 11 ⁇ - ⁇ unique to glucocorticoids.
  • glucocorticoids not only include endogenous substances with the above characteristics and activities, but also many synthetic drugs with similar structure and activity optimized by structure.
  • glucocorticoid drugs are a kind of clinical application. drug.
  • GR Glucocort icoid Receptor
  • glucocorticoid-receptor complex is located at the apex of the inflammatory regulatory network and is capable of inhibiting multiple inflammatory pathways.
  • glucocorticoids induce and activate Annexin I, induce MKP-1 and inhibit C0X-2 transcription, inhibit prostaglandin synthesis, and control inflammatory responses.
  • glucocorticoid-receptor complexes can also rapidly regulate inflammation through non-transcriptional pathways (Nat Med, 2002 8: 473-9). Tumors are closely related to inflammation, and the occurrence of inflammation promotes the development of tumors. Dexamethasone controls the inflammatory response through multiple pathways and inhibits tumor development.
  • Dexamethasone also inhibits tumor angiogenesis, and Yano A uses prostate cancer. Studies conducted have shown that dexamethasone inhibits tumor blood vessels (Cl in Cancer Res, 2006, 12: 3003-9) and lymphangiogenesis (Cl in Cancer Res, 2006, 12: 6012-7), Wilson C et al. Studies have shown that dexamethasone enhances the anti-tumor angiogenesis activity of docetaxel (Br J Cancer, 2008, 99: 2054-64).
  • dexamethasone can reduce some side effects of chemotherapy in cancer patients, and reduce the symptoms of nausea and vomiting after chemotherapy.
  • Dexamethasone is also commonly used in the treatment of some hematological malignancies.
  • thal/dex thal/dex
  • thal/dex thal/dex
  • dexamethasone with cyclophosphamide, vincristine and doxorubicin Hyper-CVAD
  • Hyper-CVAD doxorubicin
  • dexamethasone is commonly used to inhibit the permeability of tumor-associated blood vessels to reduce the development of edema.
  • dipyridamole and dexamethasone are used as non-antitumor drugs for cardiovascular and anti-inflammatory drugs.
  • Dexamethasone is only used as an adjunct to hematologic malignancies, and Ubmen is only The adjuvant anti-tumor drugs are not used as first-line anti-tumor drugs. Summary of the invention
  • the present inventors hope to find a novel pharmaceutical composition which is low in toxicity, high in efficiency, and capable of comprehensively regulating the tumor microenvironment. After intensive research and creative labor, a novel pharmaceutical composition is obtained, and it is surprisingly found that the pharmaceutical composition The substance has a good anti-tumor effect (significantly superior to the effect of any one of the components alone), and the toxic side effect is small.
  • the following invention is thus provided:
  • One aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising two or three of the following three components (active ingredients) of A, B and C:
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or adjuvant.
  • the pharmaceutical composition is an antitumor pharmaceutical composition. More specifically, the tumor is selected from one or more of liver cancer, squamous cell carcinoma, lung cancer, and pancreatic cancer.
  • composition according to any one of the present invention, wherein the pharmaceutically acceptable derivative of dipyridamole is one or more selected from the group consisting of mopidamole, BIBW BS and RA25.
  • the pharmaceutically acceptable derivative of dipyridamole also includes a pharmaceutically acceptable salt of dipyridamole.
  • the pharmaceutical composition according to any one of the present invention wherein the pharmaceutically acceptable derivative of umbrel is AHPA-Val and/or Bes tat in Hydrochloride.
  • the pharmaceutically acceptable derivative of ursinide also includes a pharmaceutically acceptable salt of umbrel.
  • the pharmaceutical composition according to any one of the present invention wherein the pharmaceutically acceptable derivative of dexamethasone is selected from the group consisting of dexamethasone acetate, dexamethasone sodium phosphate, dexamethasone sodium sulphate, and hydrogenated One or more of pine, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, and betamethasone.
  • the pharmaceutically acceptable derivative of dexamethasone also includes a pharmaceutically acceptable salt of dexamethasone.
  • a pharmaceutical composition according to any one of the present invention which is characterized by any one or more of the following (1) to (13):
  • the pharmaceutical composition of the present invention can be produced by a conventional method in the pharmaceutical field.
  • a conventional physical mixing method may be carried out by mixing two or three of the A, B or C.
  • the invention adopts the clone formation method to detect the in vitro cytotoxicity of the drug, and uses the mouse tumor model and the human cancer tumor mouse transplantation model to observe the in vivo experimental effect of the drug.
  • cytology and molecular biology methods were used to study the mechanism of action of effective drugs, and histotoxicity was studied by histopathological methods. Animal Tests It has been confirmed that the antitumor drug of the present invention has remarkable antitumor activity in animals and has low systemic toxicity, and is expected to be a new drug having antitumor activity.
  • Another aspect of the invention relates to the use of a pharmaceutical composition according to any of the inventions in the manufacture of an antitumor drug or a medicament or agent for inhibiting tumor cells in vivo or in vitro.
  • the tumor is selected from one or more of liver cancer, squamous cell carcinoma, lung cancer, and pancreatic cancer.
  • a further aspect of the invention relates to a method of anti-tumor comprising the step of administering to a subject an effective amount of a pharmaceutical composition according to any one of the inventions.
  • the tumor is selected from the group consisting of liver cancer, squamous cell carcinoma, lung cancer, and pancreatic cancer. One or more.
  • the anti-tumor pharmaceutical composition of the present invention can be orally administered to a patient in need of treatment in the form of a composition, and the dosage is generally from 100 to 500 mg/person/day, depending on the age, weight and condition of the patient. Etc., determined by the physician.
  • a further aspect of the invention relates to a method of inhibiting tumor cells in vivo or in vitro, comprising the step of using an effective amount of a pharmaceutical composition according to any of the inventions.
  • the tumor is selected from one or more of liver cancer, squamous cell carcinoma, lung cancer, and pancreatic cancer.
  • pharmaceutically acceptable carrier or adjuvant means a conventional pharmaceutical carrier or adjuvant in the pharmaceutical field, such as a diluent, an excipient such as water, a filler, such as starch, sucrose, etc., a binder such as a cellulose derivative. Gelatin, polyvinylpyrrolidone, etc., lubricants, such as talc.
  • an effective amount refers to a dose that can achieve treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.
  • subject can refer to a patient or other animal that receives the composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog, a monkey, a cow, Horse and so on.
  • the present invention provides a novel antitumor pharmaceutical composition which is effective for the treatment of tumors mainly by acting on the tumor microenvironment.
  • the pharmaceutical composition of the present invention is characterized by the regulation of tumor microenvironment drugs which act on multiple targets and multiple pathways; the side effects caused by cytotoxic drugs are expected to be alleviated.
  • the invention combines dipyridamole, umbrel and dexamethasone for the treatment of tumors.
  • Dipyridamole, umbrel and dexamethasone are not cytotoxic drugs, so when used alone, there is only moderate antitumor effect at tolerable doses. About 20-60%.
  • the inventors combined the three and two, and found that the tumor inhibition rate can be increased to 60-70%.
  • the present inventors have surprisingly discovered that the combination of dipyridamole, umbrel and dexamethasone has significant anti-tumor activity in animals, and the tumor inhibition rate can reach more than 90%, for human liver cancer, human lung cancer.
  • the treatment effect of human pancreatic cancer is much better than the corresponding clinical first-line drugs 5-fluorouracil, gemcitabine, capecitabine, etc., for human epidermal cancer A431 with high expression of epidermal growth factor receptor (EGFR), its therapeutic effect and clinical
  • the first-line drug gefitinib is equally effective, and the system toxicity is extremely low, and it is expected to be a pharmaceutical composition with high anti-tumor activity.
  • the combination of dipyridamole, umbrel and dexamethasone 22, and the combination of the three for tumor treatment are the first, and no relevant reports have been reported at home and abroad.
  • Fig 1 dipyridamole, ubenimex combination of dexamethasone inhibition of tumor growth in mice liver cancer H 22.
  • Fig. 2 Tumor growth inhibition effect of dipyridamole, umbrel and dexamethasone on transplanted human liver cancer BEL-7402.
  • Fig. 3 Inhibition of tumor growth by transplanting human hepatoma HepG2 into mice by dipyridamole, umbrel and dexamethasone.
  • Fig. 4 Inhibition of tumor growth by transplanting human squamous cell carcinoma A431 with dipyridamole, umbrel and dexamethasone.
  • Fig. 5 Inhibition of tumor growth by transplanting human lung cancer PG with dipyridamole, umbrel and dexamethasone.
  • Fig. 6 Tumor growth inhibition effect of dipyridamole, umbrel and dexamethasone on transplanted human lung cancer A549.
  • Fig. 7 Inhibition of tumor growth by transplanting human pancreatic MPAC with dipyridamole, umbrel and dexamethasone.
  • Fig. 8 Inhibition of tumor growth by transplanting human pancreas SW1990 with dipyridamole, umbrel and dexamethasone.
  • Fig. 9 Pathological section of pathological examination of various organ tissues of HepG2 mice bearing dipyridamole, umbrel and dexamethasone.
  • the tissue and sample administration used in Fig. 9A - Fig. 9P are as follows:
  • Fig. 10 Wes tern blot to detect the effect of drugs on the expression of tissue protein in transplanted tumor of mouse liver cancer H 22 mice.
  • Fig. 11 Effect of two-dimensional electrophoresis on the expression of tissue protein in human liver cancer Be-Bad 7402 mice.
  • Fig. 11A is the control group
  • Fig. 11 B is the DPM+BEN+DEX group. detailed description
  • the abbreviations of the respective drugs are: dipyridamole (DPM), umbrel (BEN), and dexamethasone (DEX).
  • Example 1 Preparation of a sample of a composition comprising dipyridamole and umbrelzine: Dipyridamole, 'J is 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 900 mg or 1600 mg, Uzbek Division 1
  • Another J is 1mg, 10mg, 20mg, 40mg, 60mg, 80mg, 100mg, 300mg, 900mg, 1600mg or 3200mg, dipyridamole and umbrel can be mixed and mixed. As shown in Table 1, the intersection in the table is the recipe.
  • Table 1 Formulations of compositions containing dipyridamole and umbrel
  • Example 2 Preparation of a sample of a composition comprising dipyridamole and dexamethasone: Dipyridamole, 'J is 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 900 mg or 1600 mg, dexamethasone Further, 'J is 0.1 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 20 mg, 40 mg, 80 mg or 160 mg. Mix dipyridamole and dexamethasone, and mix. As shown in Table 2, the intersection in the table is For the recipe.
  • Table 2 Formulations of compositions containing dipyridamole and dexamethasone
  • Example 3 Preparation of a sample of a composition comprising umbrel and dexamethasone:
  • Ubimetrex is 1 mg, 10 mg, 20 mg, 40 mg, 60 mg, 80 mg, 100 mg, 300 mg, 900 mg, 1600 mg or 3200 mg, respectively.
  • Dexamethasone is divided into '1 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 20 mg, 40 mg, 80 mg or 160 mg.
  • Formulation 1 The formulation of Example 1 is mixed with 0.1 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 20 mg, 40 mg, 80 mg or 160 mg of dexamethasone, respectively.
  • Formulation 2 The formulation of Example 2 was mixed with 1 mg, 10 mg, 20 mg, 40 mg, 60 mg, 80 mg, 100 mg, 300 mg, 900 mg, 1600 mg or 3200 mg of urinium, respectively.
  • Example 3 The formulation of Example 3 was mixed with 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 900 mg or 1600 mg of dipyridamole, respectively.
  • Example 5 In vitro cytotoxicity test of drugs
  • Drugs and Reagents Dipyridamole: Standard, purchased from China National Institute for the Control of Pharmaceutical and Biological Products; Ubumex: API, Zhejiang Plo Kangyu Pharmaceutical Co., Ltd.; Dexamethasone: Standard, purchased from China Pharmaceutical and Biological Products Laboratory; 5-fluorouracil injection for Shanghai Xudong Haipu Pharmaceutical Co., Ltd.;
  • liver cancer BEL-7402 cells were purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.
  • Test drug A sample of the pharmaceutical composition selected from the preparation of Examples 1 - 4.
  • Example 6 In vivo antitumor activity of drugs The inhibition of tumor growth in a pair of mice
  • the test drug is selected from the samples of the pharmaceutical compositions prepared in Examples 1-4. Dipyridamole: Standard, purchased from China National Institute for the Control of Pharmaceutical and Biological Products; Ubumex: API, Zhejiang Plo Kangyu Pharmaceutical Co., Ltd.; Dexamethasone: Standard, purchased from China National Institute for the Control of Pharmaceutical and Biological Products; Pyramidazole tablets: Shanxi Yabao Pharmaceutical Group Co., Ltd. production; dexamethasone acetate tablets: Tianjin Lisheng Pharmaceutical Co., Ltd. production; Ubimex (Bai Shixin) plastic bottles: Zhejiang Plo Kangyu Pharmaceutical Co., Ltd.
  • Kunming mice (clean grade II) are female, 6-8 weeks old, weighing 18-22 g, provided by the Experimental Animal Center of the Academy of Military Medical Sciences, license number SCXK (Army) 2007-004.
  • Table 7 Inhibition of tumor growth of mouse liver cancer H 22 by dipyridamole combined with different doses of dexamethasone
  • Table 8 Inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of mouse liver cancer H 22
  • Table 9 Growth inhibition of mouse liver cancer H 22 by different doses of dipyridamole combined with umbrel and dexamethasone
  • Table 10 Inhibition of growth of mouse liver cancer H 22 by different doses of umbrel with dipyridamole and dexamethasone Dosage number of animals, body weight, tumor weight (g)
  • Table 11 Growth inhibitory effects of different doses of dexamethasone combined with dipyridamole and umbrel on liver cancer H 22 in mice
  • Fig. 1 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of mouse liver cancer H 22 .
  • Table 13 Effect of dipyridamole, umbrel and dexamethasone on mean survival time of mice bearing liver cancer H 22 Dose median survival time
  • Example 7 In vivo antitumor activity of drugs The growth inhibition of human tumors transplanted in a pair of mice
  • the test drug is selected from the sample of the pharmaceutical composition prepared in Example 4.
  • Dipyridamole Standard, purchased from China National Institute for the Control of Pharmaceutical and Biological Products;
  • Ubumex API, Zhejiang Plo Kangyu Pharmaceutical Co., Ltd.;
  • Dexamethasone Standard, purchased from China National Institute for the Control of Pharmaceutical and Biological Products;
  • Pyramidazole tablets Shanxi Yabao Pharmaceutical Group Co., Ltd. production; dexamethasone acetate tablets: Tianjin Lisheng Pharmaceutical Co., Ltd.
  • mice NIHnu/nu mice were female, 6-8 weeks old, weighing 18-22 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., license number SCXK (Beijing) 2006-0009.
  • mice Human tumor cells cultured in vitro were inoculated into the armpits of one side of NIHnu/nu mice. After 2-3 passages, the tumors were subcultured under the armpits and cut into small pieces of 1.5 legs and 3 pieces. The mice were subcutaneously on one side of the armpits. After the tumors were grown to 100 to 300 legs 3, they were randomly grouped according to tumor size, and oral administration was started. The control group was given physiological saline, and the other groups were administered with the drugs in the respective examples.
  • the inoculation was administered for 7 days, once a day for 10 times and sacrificed for 17 days. Compared with the control group *P ⁇ 0.01.
  • Table 15 Growth inhibition of dipyridamole, umbrel and dexamethasone on transplanted human liver cancer BEL-7402 (17d)
  • Fig. 2 Dipyridamole, Ubumex and Dexamethasone for transplantation of human liver Tumor growth inhibition of cancer BEL-7402.
  • Table 16 Growth inhibition of dipyridamole, umbrel and dexamethasone on human hepatoma HepG2 transplanted (28d)
  • the cells were administered for 7 days, and were administered 5 times a week for 3 weeks and 28 days, and no animals died in each group. Compared with the control group *P ⁇ 0.01.
  • Fig. 3 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human hepatic carcinoma HepG2.
  • Fig. 4 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human squamous cell carcinoma A431.
  • Table 18 Inhibition of tumor growth by transplanting human lung cancer PG with dipyridamole, umbrel and dexamethasone (17d)
  • Fig. 5 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human lung cancer PG.
  • Table 19 Growth inhibition of lung cancer A549 in mice transplanted with dipyridamole, umbrel and dexamethasone (17 d )
  • the drug was administered for 7 days, once a day for 10 times, and gemcitabine was administered 3 times on days 7, 10, and 13, respectively. He died in 17 days. Compared with the control group * ⁇ 0. 01.
  • Table 20 Inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human lung cancer A549 (28 d)
  • Fig. 6 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human lung cancer A549.
  • the drug was administered for 7 days, once a day for 10 times, and the capecitabine was administered in the same manner as DPM+BEN+DEX, and was sacrificed at 17 days. Compared with the control group *P ⁇ 0. 01
  • Table 22 Tumor growth inhibition effect of dipyridamole, umbrel and dexamethasone on transplanted human pancreatic adenocarcinoma MPAC (28d)
  • DPM+BEN+DEX was administered 5 times a week for 3 weeks; capecitabine was administered in the same manner as DPM+BEN+DEX. Compared with the control group *P ⁇ 0.01.
  • Fig. 7 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human pancreatic MPAC.
  • Table 23 Tumor growth inhibition effect of dipyridamole, umbrel and dexamethasone on transplanted human pancreatic cancer SW1990 (35d) Dosage Animals Body Weight Change Tumor Weight (g) Group Tumor Suppression Rate % (mg/kg) Start/End (g) SSD
  • the drug is administered 7 days after the tumor is inoculated, and DPM+BEN+DEX is administered 5 times a week. A total of 3 weeks; capecitabine administration is the same as DPM+BEN+DEX. Compared with the control group *P ⁇ 0.01.
  • the main organs were taken for pathological examination, and the bone marrow nucleated cells of the rats were counted.
  • the results are shown in Table 24, Fi g. 9. The results showed that there were no obvious pathological changes in the main organs such as heart, lung, liver, spleen, stomach, small intestine, kidney and femur (bone marrow).
  • the number of bone marrow nucleated cells was comparable to that of normal mice without tumor inoculation. Significant differences. It can be seen that the combination of dipyridamole, umbrel and dexamethasone did not lead to toxicity changes of cytotoxic drugs, indicating that the composition has good safety.
  • Table 24 Effect of dipyridamole, umbrel and dexamethasone on nucleated cells of bone marrow of human hepatoma HepG2 mice (pathological examination, average number of cells per field of view)
  • the tumor tissue was added 1:9 to the lysate (50 mM Tris ⁇ CI (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 100 ⁇ g / ml PMSF, lug/ml Aprotinin, 0.02% sodium azide), low temperature homogenate, 4.
  • C is cleaved for 1 h, 10000 rpm 4 .
  • FLK1 and N0S3 decreased, and other detected proteins: EGF, The changes of VEGF, TGF, Beb2, k-Ras, P27, P21 and NF-kB were not obvious.
  • FLK1 is closely related to angiogenesis.
  • N0S is closely related to inflammation and plays an important role in the development of tumors (Clinical Chemother Pharmacol. 2011, 67 (6): 1211-24). This study shows dipyridamole.
  • the pharmaceutical composition of molybdenum, umbrel and dexamethasone may affect tumor angiogenesis and be associated with inflammatory responses.
  • Fig. 10 Wes tern blot was used to detect the effect of drugs on the expression of tissue protein in transplanted tumor of mouse liver cancer H 22 mice.
  • Tumor tissue proteomics study Two-dimensional electrophoresis: Two-dimensional electrophoresis was used to detect the effect of dipyridamole, umbrel and dexamethasone on the expression of human hepatocarcinoma in the transplanted tumor tissue of human liver cancer.
  • the sample was ground with liquid nitrogen through a pre-cooled metal sampler.
  • the TCA acetone method extracts whole proteins from tumor tissues and removes salts and impurities.
  • the extracted whole protein was quantified by Bradford, and each sample was taken with an equal amount of protein, and subjected to IEF isoelectric focusing (glue: PH3 - 10), and each was equilibrated with an equilibrium solution of 1% DTT and 2.5% IAM. 15 min, the strip was removed and transferred to a two-way gel, and the gel was run on a vertical plate polyacrylamide gel with a gel of 24 cm (adhesion of 12.5%).
  • Glue, fixed for 30min, sensitized for 30min washed with water 3 (each lOmin), silver stained, washed twice (each time lmin), color development, termination.
  • Glue scanning, glue analysis, three parallel glues for each sample were combined into a virtual glue, and the two sample virtual shares were compared and analyzed by GE's Imagemaster 2D version 5. 0.
  • Fig. 11 Two-dimensional electrophoresis detection of drug on the expression of human hepatoma Bel-7402 mouse transplanted tumor tissue protein. The figure shows a total of 17 change points: 4 more than 3 times change; 13 with or without, 12 of which are down, 1 on The results of the analysis are shown in Table 25, Table 26
  • the dexamethasone composition has a wide range of effects on protein metabolism, nucleoside metabolism, etc., and exerts anti-tumor effects through various effects.
  • Table 25 Proteins with more than three times the change and with or without.
  • Example 10 Acute toxicity test in mice with anti-tumor composition composed of dipyridamole, umbrel and dexamethasone
  • mice 60 Kunming mice were divided into 3 dose groups, 20 in each group, half male and half female. The difference ratio of each group was 0.8, the oral administration, the dose was 2. O g / kg, 1. 6 g / kg, 1. 28g / kg, observed for 14 days after the stomach administration and recorded the toxicity Including general indicators, death, weight changes, determination of toxicity, death, and calculation of median lethal dose in mice, pathological examination at the end of the experiment.
  • mice The symptoms of poisoning in the mice were normal, and the general indicators were normal. There were no deaths, weight loss and other symptoms. The body weight increased to 28 ⁇ 38 grams before the death on the 14th day after administration, and the appearance was healthy. Pathological examination was performed at the end of the experiment, and no obvious pathological changes were observed in the liver, kidney, lung and other major organs of the animals. The results showed that the anti-tumor new composition was very low in toxicity, especially for oral administration, and there was no significant difference between male and female animals. The oral LD50 (half lethal dose) of mice is greater than 2 g/kg. Through the acute toxicity test, it was proved that at a very high dose (mouse 2.

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Abstract

The present invention belongs to the field of medical chemistry, and relates to an antitumour pharmaceutical composition and use thereof. In particular, the pharmaceutical composition to which the present invention relates comprises two or three ingredients of the following three ingredients A, B and C: A. dipyridamole and/or pharmaceutically acceptable derivatives thereof; B. ubenimex and/or pharmaceutically acceptable derivatives thereof; C. dexamethasone and/or pharmaceutically acceptable derivatives thereof; optionally, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier or excipient. The present invention also relates to a use of the pharmaceutical composition in preparing the antitumour drug and an antitumour method. The antitumour pharmaceutical composition in the present invention achieves the purpose of treating tumours effectively primarily by acting on the tumour microenvironment, and the features of the drug adjusting the tumour microenvironment are that it is acting on multiple targets and multiple paths, which can be expected to relieve the toxic and side effects caused by cytotoxic drugs.

Description

一种抗肿瘤药物组合物及其用途 技术领域  Antitumor pharmaceutical composition and use thereof
本发明属于药物化学领域， 具体地， 涉及一种抗肿瘤药物组 合物及其用途。 背景技术  The present invention is in the field of medicinal chemistry, and in particular, relates to an antitumor drug composition and use thereof. Background technique
恶性肿瘤是严重威胁人类健康的疾病。 目前研制抗肿瘤新药的 策略包括两个方面： 寻找作用于肿瘤细胞的药物和调节肿瘤微环境 的药物， 其中作用于肿瘤细胞的药物主要通过抑制肿瘤细胞增殖， 诱导肿瘤细胞凋亡， 诱导肿瘤细胞分化起作用。 而调节肿瘤微环境 的药物主要通过以下几个方面起作用：抑制肿瘤血管新生成或干扰 肿瘤血管网络； 调节肿瘤内浸润的免疫细胞及相关细胞因子； 调节 生长因子分泌与生长因子受体表达；抑制特定酶的分泌与调节相应 抑制因子； 干扰肿瘤细胞内与细胞外的物质传输、 摄取与外排。  Malignant tumors are diseases that seriously threaten human health. At present, the strategy for developing new anti-tumor drugs includes two aspects: finding drugs for tumor cells and drugs for regulating tumor microenvironment. Among them, drugs acting on tumor cells mainly induce tumor cell proliferation, induce tumor cell apoptosis, and induce tumor cells. Differentiation works. The drugs that regulate the tumor microenvironment mainly play the role of inhibiting tumor angiogenesis or interfering with tumor vascular network; regulating immune cells and related cytokines in tumor infiltration; regulating growth factor secretion and growth factor receptor expression; Inhibition of specific enzyme secretion and regulation of corresponding inhibitors; Interfering with the transport, uptake and efflux of intracellular and extracellular substances in tumor cells.
无论是哪种策略研发药物， 寻找低毒， 高效的抗肿瘤药物或者 药物组合物都是临床肿瘤治疗的终极目标。  Regardless of the strategy of developing drugs, finding low-toxic, highly effective anti-tumor drugs or pharmaceutical compositions is the ultimate goal of clinical oncology treatment.
双嘧达莫（Dipyr idamole, DPM )是上世纪 80 年代合成的一 种非肿瘤治疗药物，其药学上可接受的衍生物或类似物如单哌潘生 丁（mopidamole )、 BIBW22BS, RA25或其药学上可接受的盐为非硝 酸酯类冠状动脉扩张剂， 具有扩张冠状血管、 促进侧支循环形成的 作用。 双嘧达莫还具有抑制血小板凝聚， 预防血栓形成的作用。  Dipyr idamole (DPM) is a non-tumor therapeutic drug synthesized in the 1980s. Its pharmaceutically acceptable derivatives or analogues such as mopidamole, BIBW22BS, RA25 or its pharmacy An acceptable salt is a non-nitrate coronary artery dilator that has the effect of expanding the coronary vessels and promoting the formation of collateral circulation. Dipyridamole also has the effect of inhibiting platelet aggregation and preventing thrombosis.
双嘧达莫化学名为： 2, 2 ' ，2 "， 2" ' - [ (4, 8 - 二哌啶基嘧啶 并 [5，4，- d] 嘧啶- 2, 6-二基）双次氮基] -四乙醇， 分子式为 C24H40N804 , 分子量为 504. 63, 化学结构式如下所示：
Figure imgf000003_0001
Dipyridamole chemical name: 2, 2 ' , 2 ", 2"' - [ (4, 8 - Dipiperidylpyrimido[5,4,-d]pyrimidine-2,6-diyl) Nitrilo]-tetraethanol, the molecular formula is C24H40N804, the molecular weight is 504.63, and the chemical structural formula is as follows:
Figure imgf000003_0001
该药物作为心血管扩张剂载入中华人民共和国药典（ 2000 年 版）和美国药典（XXI — XXI I I) ， 但因其存在所谓 "冠状动脉偷 窃"现象， 即与肝素、 香豆素类及纤维蛋白溶解药合用时可引起出 血倾向， 故临床上已趋不用。  The drug is included as a cardiovascular dilating agent in the Pharmacopoeia of the People's Republic of China (2000 edition) and the United States Pharmacopoeia (XXI-XXI II), but because of its so-called "coronary theft" phenomenon, ie heparin, coumarins and fibrin When dissolved drugs are combined, they can cause bleeding tendency, so they have become clinically unnecessary.
在药物作用机理方面， 双嘧达莫是一种有效的核苷转运抑制 剂，通过阻断平衡型核苷转运蛋白 hENTl( Nat Med， 1997, 3: 89-93 ) 和 hENT2 ( Biochem. J， 1997， 328: 739-43 ) 而抑制核苷转运。  Dipyridamole is a potent nucleoside transport inhibitor in the mechanism of drug action by blocking the balanced nucleoside transporter hENTl (Nat Med, 1997, 3: 89-93) and hENT2 (Biochem. J, 1997, 328: 739-43) inhibits nucleoside transport.
鉴于这种作用机理， 已有科研人员将双嘧达莫与典型的细胞毒 药物（具体为抗代谢药物）相组合， 进行了抗肿瘤作用的研究， 结 果表明该药物在细胞水平能够增强 5-氟尿嘧啶、甲氨喋呤、阿霉素、 依托泊苷、 长春花碱、 顺铂等的细胞毒活性。 但是在体内疗效并不 明显。  In view of this mechanism of action, researchers have combined dipyridamole with typical cytotoxic drugs (specifically antimetabolites) to conduct anti-tumor studies, and the results indicate that the drug can be enhanced at the cellular level 5- Cytotoxic activity of fluorouracil, methotrexate, doxorubicin, etoposide, vinblastine, cisplatin, and the like. However, the efficacy in the body is not obvious.
乌苯美司（Ubenimex, Bes tat in, BEN)及其药学上可接受的衍 生物或类似物包括 AHPA-VaK Bestat in Hydrochloride 等。 乌 苯美司是 1976 年由日本学者 Umezawa 等从橄榄网状链霉菌 (Streptomyces ol ivoret icul i)的发酵液中分离得到的小分子二 肽化合物， 能竟争性抑制多种氨肽酶活性， 通过促进机体的免疫功 能和直接作用于肿瘤组织而发挥双重抗肿瘤作用。 1987年，作为具 有免疫調节功能的抗癌新药在日本上市，用于在恶性肿瘤的辅助治 疗。  Ubenimex (Bes tat in, BEN) and pharmaceutically acceptable derivatives or analogs thereof include AHPA-VaK Bestat in Hydrochloride and the like. Ubumex is a small molecule dipeptide compound isolated from the fermentation broth of Streptomyces ol ivoret icul i by Japanese scholar Umezawa in 1976. It can competitively inhibit various aminopeptidase activities. It exerts a dual anti-tumor effect by promoting the immune function of the body and directly acting on tumor tissues. In 1987, it was marketed as a new anticancer drug with immunomodulatory function in Japan for the adjuvant treatment of malignant tumors.
乌苯美司化学名为： N- [ (2S, 3R) -4-苯基- 3-氨基- 2-羟基丁 酰] - L-亮氨酸, 分子式为 308. 38， 结构式为:
Figure imgf000004_0001
Ubumex chemical name: N- [ (2S, 3R) -4-phenyl-3-amino-2-hydroxybutyrate Acyl]-L-leucine, the molecular formula is 308. 38, and the structural formula is:
Figure imgf000004_0001
乌苯美司显示出多方面的免疫活性， 不仅能够增强淋巴细胞的 功能， 而且能够激活单核巨噬细胞， 使 NK细胞的杀伤活力增强。 分子机理研究表明其作用靶点是位于免疫细胞表面的亮氨酸氨肽 酶（ Leu- AP )及氨肽酶 B (AP-B)， 这两种酶可以催化底物氨基端的 裂解，使抗原分子灭活。乌苯美司通过与酶活性中心的锌离子螯合， 抑制了氨肽酶的活性。 由于具有免疫增强功能， 乌苯美司常用于肿 瘤化疗、 放疗及手术后的辅助治疗， 可配合化疗、 放疗及联合用于 白血病、 多发性骨髓瘤以及肺癌、 乳腺癌等实体瘤的治疗。  Ubumex exhibits a multi-faceted immune activity that not only enhances lymphocyte function, but also activates monocyte macrophages to enhance the killing activity of NK cells. Molecular mechanism studies have shown that its target is leucine aminopeptidase (Leu-AP) and aminopeptidase B (AP-B) located on the surface of immune cells, which can catalyze the cleavage of the amino terminus of the substrate to make the antigen The molecule is inactivated. Ubumex inhibits the activity of aminopeptidase by chelation with zinc ions in the active center of the enzyme. Due to its immune-enhancing function, Ubumex is often used in the treatment of tumor chemotherapy, radiotherapy and postoperative surgery. It can be combined with chemotherapy, radiotherapy and combined therapy for leukemia, multiple myeloma and solid tumors such as lung cancer and breast cancer.
乌苯美司还能通过直接作用于肿瘤组织而发挥抗肿瘤活性。氨 肽酶 N ( CD13 )在肿瘤新生血管内皮细胞高表达， 通过降解细胞外 基质、 促进内皮细胞的侵袭及调节某些生长因子和细胞因子的活 性， 促进肿瘤血管的形成 ( Int J Cancer, 1993， 54: 137-43 ) 。 某些肿瘤细胞如人纤维肉瘤 HT- 1080细胞氨肽酶 N也呈现高表达， 与肿瘤细胞的侵袭转移密切相关 （ Int J Cancer, 1993, 54: 137-43 )。 乌苯美司能够抑制氨肽酶 N的活性， 从而有效的抑制肿 瘤血管的形成及预防肿瘤细胞的转移。 此外， 乌苯美司还能够直接 i秀导肿瘤细胞的凋亡（Biomed Pharmacother. 1996; 50: 283-9 ) ， 从而发挥抗肿瘤作用。  Ubumex can also exert anti-tumor activity by directly acting on tumor tissues. Aminopeptidase N (CD13) is highly expressed in tumor neovascular endothelial cells, which promotes tumor vascular formation by degrading extracellular matrix, promoting endothelial cell invasion, and regulating the activity of certain growth factors and cytokines (Int J Cancer, 1993) , 54: 137-43). Certain tumor cells, such as human fibrosarcoma HT-1080 cell aminopeptidase N, also exhibit high expression and are closely related to invasion and metastasis of tumor cells (Int J Cancer, 1993, 54: 137-43). Ubumex can inhibit the activity of aminopeptidase N, thereby effectively inhibiting the formation of tumor blood vessels and preventing the metastasis of tumor cells. In addition, Ubumex can also directly induce tumor cell apoptosis (Biomed Pharmacother. 1996; 50: 283-9), thereby exerting an anti-tumor effect.
糖皮质激素， 如地塞米松（Dexamethason, DEX)及其药学上可 接受的衍生物或类似物， 如醋酸地塞米松、 地塞米松磚酸钠、 地塞 米松棕榈酸钠、 氢化可的松、 可的松、 强的松、 强的松龙、 甲基强 的松龙、 去炎松、 倍他米松等具有消炎、 免疫抑制、 抗毒素、 抗休 克等药理作用。 至今， 上市的地塞米松衍生物已达 12种以上。 地 塞米松广泛应用于多种疾病的治疗， 如自身免疫性疾病、 过敏、 炎 症、 哮喘及皮肤科、 眼科疾病等。 Glucocorticoids, such as dexamethasone (Dexamethason, DEX) and pharmaceutically acceptable derivatives or analogs thereof, such as dexamethasone acetate, dexamethasone sodium silicate, dexamethasone palmitate, hydrocortisone , cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone and other pharmacological effects such as anti-inflammatory, immunosuppressive, anti-toxin, anti-shock. To date, more than 12 dexamethasone derivatives have been marketed. Ground Dexamethasone is widely used in the treatment of various diseases such as autoimmune diseases, allergies, inflammation, asthma and dermatology, and ophthalmology diseases.
地塞米松化学名为： 9-氟- 11， 17， 21-三羟基 -16-甲基（ 11 β , 16 α ) -孕笛 -1, 4-二烯 -3， 20-二酮， 分子式为 C22H29F05, 分子量为 392. 46 , 结构式如下所 Dexamethasone chemical name: 9-fluoro- 11, 17, 21-trihydroxy-16-methyl ( 11 β , 16 α ) - pregnancy flute-1, 4-diene-3, 20-dione, molecular formula Is C 22 H 29 F0 5 , molecular weight is 392. 46, the structural formula is as follows
Figure imgf000005_0001
Figure imgf000005_0001
糖皮质激素的基本结构特征包括肾上腺皮质激素所具有的 C3 的羰基、 Δ 4和 17 β 酮醇侧链以及糖皮质激素独有的 17 α - 0Η和 11 β - 0Η。 目前糖皮质激素概念上不仅包括具有上述特征和活性的 内源性物质，还包括很多经过结构优化的具有类似结构和活性的人 工合成药物， 目前糖皮质激素类药物是临床应用较多的一类药物。 分子药理学研究表明，地塞米松主要通过细胞内的糖皮质激素受体 (Glucocort icoid Receptor, GR)发挥作用。 激素与受体的结合导 致分子伴侣（如 hsp90 )从 GR解离， GR活化； 活化的 GR进入核内， 通过与糖皮质激素应答元件或其他转录因子如 NF-kB相互作用调节 基因的转录。 糖皮质激素 -受体复合物位于炎症调节网络的顶端， 能够抑制多条炎症通路。 以前列腺素的合成为例， 糖皮质激素通过 诱导和激活 Annexin I， 诱导 MKP- 1以及抑制 C0X- 2的转录， 抑制 前列腺素的合成， 控制炎症反应。 近来的研究表明糖皮质激素-受 体复合物还能通过非转录途径快速的调节炎症反应 ( Nat Med, 2002 8: 473-9 ) 。 肿瘤与炎症关系密切， 炎症的发生促进了肿瘤的发生 发展。 地塞米松通过多条通路控制炎症反应， 抑制肿瘤的发展。  The basic structural features of glucocorticoids include the C3 carbonyl group, the Δ 4 and 17 β ketol side chains of the adrenocortical hormone, and the 17 α - 0Η and 11 β - Η unique to glucocorticoids. At present, glucocorticoids not only include endogenous substances with the above characteristics and activities, but also many synthetic drugs with similar structure and activity optimized by structure. Currently, glucocorticoid drugs are a kind of clinical application. drug. Molecular pharmacology studies have shown that dexamethasone mainly acts through the intracellular Glucocort icoid Receptor (GR). Binding of hormones to receptors leads to the dissociation of molecular chaperones (such as hsp90) from GR, GR activation; activated GR enters the nucleus and regulates gene transcription by interacting with glucocorticoid response elements or other transcription factors such as NF-kB. The glucocorticoid-receptor complex is located at the apex of the inflammatory regulatory network and is capable of inhibiting multiple inflammatory pathways. Taking prostaglandin synthesis as an example, glucocorticoids induce and activate Annexin I, induce MKP-1 and inhibit C0X-2 transcription, inhibit prostaglandin synthesis, and control inflammatory responses. Recent studies have shown that glucocorticoid-receptor complexes can also rapidly regulate inflammation through non-transcriptional pathways (Nat Med, 2002 8: 473-9). Tumors are closely related to inflammation, and the occurrence of inflammation promotes the development of tumors. Dexamethasone controls the inflammatory response through multiple pathways and inhibits tumor development.
地塞米松还能够抑制肿瘤血管的生成， Yano A等运用前列腺癌 进行的研究显示地塞米松能够抑制肿瘤血管 （Cl in Cancer Res, 2006， 12: 3003-9 ) 及淋巴管的生成 ( Cl in Cancer Res, 2006， 12: 6012-7 ) , Wi lson C等的研究显示地塞米松能够增强多西紫杉 醇抗肿瘤血管生成的活性（Br J Cancer, 2008, 99: 2054-64 ) 。 Dexamethasone also inhibits tumor angiogenesis, and Yano A uses prostate cancer. Studies conducted have shown that dexamethasone inhibits tumor blood vessels (Cl in Cancer Res, 2006, 12: 3003-9) and lymphangiogenesis (Cl in Cancer Res, 2006, 12: 6012-7), Wilson C et al. Studies have shown that dexamethasone enhances the anti-tumor angiogenesis activity of docetaxel (Br J Cancer, 2008, 99: 2054-64).
在肿瘤治疗方面，地塞米松可减轻癌症患者化疗时的某些副作 用， 减轻化疗后的恶心呕吐症状。 地塞米松也常用于一些恶性血液 病的治疗， 有方案将地塞米松与沙利度胺 （ thal idomide )联合 ( thal/dex )用于新确诊的多发性骨髓瘤的治疗 （M. D. Anderson Cancer Center ) , 也有方案将地塞米松与环磷酰胺、 长春新碱和 阿霉素联合（Hyper- CVAD)用于难治复发性急性淋巴细胞白血病的 治疗 （M. D. Anderson Cancer Center ) 。 脑肿瘤治疗方面， 地塞 米松常用来抑制肿瘤相关的血管的通透性， 以减轻水肿的发展。  In the treatment of cancer, dexamethasone can reduce some side effects of chemotherapy in cancer patients, and reduce the symptoms of nausea and vomiting after chemotherapy. Dexamethasone is also commonly used in the treatment of some hematological malignancies. There are protocols for the combination of dexamethasone and thal idomide (thal/dex) for the treatment of newly diagnosed multiple myeloma (MD Anderson Cancer Center). There are also protocols for the combination of dexamethasone with cyclophosphamide, vincristine and doxorubicin (Hyper-CVAD) for the treatment of refractory relapsed acute lymphoblastic leukemia (MD Anderson Cancer Center). In the treatment of brain tumors, dexamethasone is commonly used to inhibit the permeability of tumor-associated blood vessels to reduce the development of edema.
然而， 在临床上， 双嘧达莫与地塞米松都是作为非抗肿瘤药物 用于心血管以及抗炎，地塞米松只有在治疗恶性血液病中作为辅助 药物， 而乌苯美司也只是辅助抗肿瘤药物， 均没有作为一线抗肿瘤 药物在使用。 发明内容  However, clinically, both dipyridamole and dexamethasone are used as non-antitumor drugs for cardiovascular and anti-inflammatory drugs. Dexamethasone is only used as an adjunct to hematologic malignancies, and Ubmen is only The adjuvant anti-tumor drugs are not used as first-line anti-tumor drugs. Summary of the invention
本发明人希望寻找低毒、 高效而且能够综合调节肿瘤微环境 的新型药物组合物， 经过深入地研究和创造性的劳动， 得到了一 种新型的药物组合物， 并且惊奇地发现， 所述药物组合物具有良 好的抗肿瘤效果（显著优于其中任意一种成分单独使用的效果）， 并且毒副作用小。 由此提供了下述发明：  The present inventors hope to find a novel pharmaceutical composition which is low in toxicity, high in efficiency, and capable of comprehensively regulating the tumor microenvironment. After intensive research and creative labor, a novel pharmaceutical composition is obtained, and it is surprisingly found that the pharmaceutical composition The substance has a good anti-tumor effect (significantly superior to the effect of any one of the components alone), and the toxic side effect is small. The following invention is thus provided:
本发明的一个方面涉及一种药物组合物， 其包含如下的 A、 B 和 C三种成分（有效成分） 中的两种或三种：  One aspect of the present invention relates to a pharmaceutical composition comprising two or three of the following three components (active ingredients) of A, B and C:
A.双嘧达莫和 /或其可药用衍生物； B.乌苯美司和 /或其可药用衍生物； A. dipyridamole and/or a pharmaceutically acceptable derivative thereof; B. Ubumex and/or a pharmaceutically acceptable derivative thereof;
C.地塞米松和 /或其可药用衍生物；  C. Dexamethasone and / or a pharmaceutically acceptable derivative thereof;
可选地，所述药物组合物还包含药学上可接受的载体或辅料。 在本发明的一个实施方案中， 所述有效成分的重量含量为 0. 5 - 99. 9%„  Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or adjuvant. 9% „ „ „ „ „ „ „ „ „ „ „ „
具体地， 所述药物组合物为抗肿瘤药物组合物。 更具体地， 所述肿瘤选自肝癌、 鱗癌、 肺癌以及胰腺癌中的一种或多种。  Specifically, the pharmaceutical composition is an antitumor pharmaceutical composition. More specifically, the tumor is selected from one or more of liver cancer, squamous cell carcinoma, lung cancer, and pancreatic cancer.
根据本发明任一项所述的药物组合物， 其中， 所述双嘧达莫 的可药用衍生物选自单哌潘生丁（mopidamole ) 、 BIBW BS以及 RA25中的一种或多种。所述双嘧达莫的可药用衍生物还包括双嘧 达莫的可药用盐。  The pharmaceutical composition according to any one of the present invention, wherein the pharmaceutically acceptable derivative of dipyridamole is one or more selected from the group consisting of mopidamole, BIBW BS and RA25. The pharmaceutically acceptable derivative of dipyridamole also includes a pharmaceutically acceptable salt of dipyridamole.
根据本发明任一项所述的药物组合物， 其中， 所述乌苯美司 的可药用衍生物为 AHPA-Val和 /或 Bes tat in Hydrochlor ide。 所 述乌苯美司的可药用衍生物还包括乌苯美司的可药用盐。  The pharmaceutical composition according to any one of the present invention, wherein the pharmaceutically acceptable derivative of umbrel is AHPA-Val and/or Bes tat in Hydrochloride. The pharmaceutically acceptable derivative of ursinide also includes a pharmaceutically acceptable salt of umbrel.
根据本发明任一项所述的药物组合物， 其中， 所述地塞米松 的可药用衍生物选自醋酸地塞米松、 地塞米松磷酸钠、 地塞米松 椋榈酸钠、 氢化可的松、 可的松、 强的松、 强的松龙、 甲基强的 松龙、 去炎松、 以及倍他米松中的一种或多种。 所述地塞米松的 可药用衍生物还包括地塞米松的可药用盐。  The pharmaceutical composition according to any one of the present invention, wherein the pharmaceutically acceptable derivative of dexamethasone is selected from the group consisting of dexamethasone acetate, dexamethasone sodium phosphate, dexamethasone sodium sulphate, and hydrogenated One or more of pine, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, and betamethasone. The pharmaceutically acceptable derivative of dexamethasone also includes a pharmaceutically acceptable salt of dexamethasone.
根据本发明任一项所述的药物组合物，其特征在于如下的（ 1 ) - ( 13 ) 中的任一项或多项：  A pharmaceutical composition according to any one of the present invention, which is characterized by any one or more of the following (1) to (13):
( 1 )含有 A和 B, 重量比 A： B = 0. 001 ~ 200： 1 ;  (1) Containing A and B, weight ratio A: B = 0. 001 ~ 200: 1 ;
( 2 )含有 A和 B, 重量比 A： B = 0. 025 ~ 22. 5： 1 ;  (2) Containing A and B, weight ratio A: B = 0. 025 ~ 22. 5: 1 ;
( 3 )含有 B和 C, 重量比 B： C = 0. 001 ~ 3000： 1 ;  (3) Containing B and C, weight ratio B: C = 0. 001 ~ 3000: 1 ;
( 4 )含有 B和 C, 重量比 B： C = 0. 5 ~ 450： 1;  (4) Containing B and C, weight ratio B: C = 0. 5 ~ 450: 1;
( 5 )含有 A和 C, 重量比 A： C = 1： 0. 00001 ~ 100; ( 6 )含有 A和 C, 重量比 A : C = 1： 0· 0005 ~ 0. 05; (5) containing A and C, weight ratio A: C = 1: 0. 00001 ~ 100; (6) containing A and C, weight ratio A: C = 1: 0 · 0005 ~ 0. 05;
( 7 )含有 Α、 Β和 C,重量比 A : Β : C = 1： 0· 00001 ~ 32000： 0. 00001 ~ 1600;  (7) Containing Α, Β and C, weight ratio A : Β : C = 1: 0· 00001 ~ 32000: 0. 00001 ~ 1600;
( 8 )含有 Α、 Β和 C, 重量比 A : Β : C = 1 ： 0· 001 ~ 1000： 0. 0001 - 10;  (8) Containing lanthanum, cerium and C, weight ratio A : Β : C = 1 : 0· 001 ~ 1000: 0. 0001 - 10;
( 9 )含有 Α、 Β和 C, 重量比 A： B： C = 50 - 150： 20： 1; (9) Containing lanthanum, cerium and C, weight ratio A: B: C = 50 - 150: 20: 1;
( 10 )含有 A、 B和 C, 重量比 A： B： C = 100： 10 - 40： 1;(10) contains A, B and C, weight ratio A: B: C = 100: 10 - 40: 1;
( 11 )含有 A、 B和 C, 重量比 A： B： C = 200： 40： 1 ~ 3;(11) contains A, B and C, weight ratio A: B: C = 200: 40: 1 ~ 3;
( 12 )含有入、8和（，重量比 A： B： C = 100 ~ 400： 20 - 80：(12) Contains in, 8 and (, weight ratio A: B: C = 100 ~ 400: 20 - 80:
1 - 3; 1 - 3;
( 13 )含有 A、 B和 C, 重量比 A： B： C = 100： 20： 1。  (13) Contains A, B and C, weight ratio A: B: C = 100: 20: 1.
本发明所述的药物组合物， 可以采用药学领域常规的方法进 行制备。 例如常规的物理混合的方法， 将所述的 A、 B或 C中的两 种或三种混合即可。 本发明采用克隆形成法检测药物的体外细胞 毒性， 使用小鼠肿瘤模型和人体癌瘤棵鼠移植模型， 观察药物的 体内实验疗效。 同时使用细胞学和分子生物学方法， 研究有效药 物的作用机制， 用病理组织学方法研究其毒性作用特点。 动物试 验证明， 本发明的抗肿瘤药物， 在动物体内具有显著的抗肿瘤活 性， 且系统毒性低， 可望成为一种具有抗肿瘤活性的新药物。  The pharmaceutical composition of the present invention can be produced by a conventional method in the pharmaceutical field. For example, a conventional physical mixing method may be carried out by mixing two or three of the A, B or C. The invention adopts the clone formation method to detect the in vitro cytotoxicity of the drug, and uses the mouse tumor model and the human cancer tumor mouse transplantation model to observe the in vivo experimental effect of the drug. At the same time, cytology and molecular biology methods were used to study the mechanism of action of effective drugs, and histotoxicity was studied by histopathological methods. Animal Tests It has been confirmed that the antitumor drug of the present invention has remarkable antitumor activity in animals and has low systemic toxicity, and is expected to be a new drug having antitumor activity.
本发明的另一方面涉及本发明任一项所述的药物组合物在制 备抗肿瘤药物或者在体内或体外抑制肿瘤细胞的药物或试剂中的 用途。 在本发明具体的实施方式中， 所述肿瘤选自肝癌、 鳞癌、 肺癌以及胰腺癌中的一种或多种。  Another aspect of the invention relates to the use of a pharmaceutical composition according to any of the inventions in the manufacture of an antitumor drug or a medicament or agent for inhibiting tumor cells in vivo or in vitro. In a specific embodiment of the invention, the tumor is selected from one or more of liver cancer, squamous cell carcinoma, lung cancer, and pancreatic cancer.
本发明的再一方面涉及一种抗肿瘤的方法， 包括给予受试者 有效量的本发明任一项所述的药物组合物的步骤。 在本发明具体 的实施方式中， 所述肿瘤选自肝癌、 鳞癌、 肺癌以及胰腺癌中的 一种或多种。 A further aspect of the invention relates to a method of anti-tumor comprising the step of administering to a subject an effective amount of a pharmaceutical composition according to any one of the inventions. In a specific embodiment of the present invention, the tumor is selected from the group consisting of liver cancer, squamous cell carcinoma, lung cancer, and pancreatic cancer. One or more.
本发明的具有抗肿瘤作用的药物组合物，可以组合物的形式， 通过口服施加于需要治疗的患者， 剂量一般为 1 00 ~ 500 mg/人 / 天， 具体可根据患者的年龄、 体重、 病情等， 由医师决定。  The anti-tumor pharmaceutical composition of the present invention can be orally administered to a patient in need of treatment in the form of a composition, and the dosage is generally from 100 to 500 mg/person/day, depending on the age, weight and condition of the patient. Etc., determined by the physician.
本发明的再一方面涉及一种在体内或体外抑制肿瘤细胞的方 法， 包括使用有效量的本发明任一项所述的药物组合物的步骤。 在本发明具体的实施方式中， 所述肿瘤选自肝癌、 鳞癌、 肺癌以 及胰腺癌中的一种或多种。  A further aspect of the invention relates to a method of inhibiting tumor cells in vivo or in vitro, comprising the step of using an effective amount of a pharmaceutical composition according to any of the inventions. In a specific embodiment of the invention, the tumor is selected from one or more of liver cancer, squamous cell carcinoma, lung cancer, and pancreatic cancer.
在本发明中，  In the present invention,
术语 "药学上可接受的载体或辅料" 是指药学领域常规的药 物载体或辅料， 如稀释剂、 赋形剂如水等， 填充剂， 如淀粉、 蔗 糖等， 粘合剂， 如纤维素衍生物明胶、 聚乙烯吡咯烷酮等， 润滑 剂， 如滑石粉等。  The term "pharmaceutically acceptable carrier or adjuvant" means a conventional pharmaceutical carrier or adjuvant in the pharmaceutical field, such as a diluent, an excipient such as water, a filler, such as starch, sucrose, etc., a binder such as a cellulose derivative. Gelatin, polyvinylpyrrolidone, etc., lubricants, such as talc.
术语 "有效量"是指可在受试者中实现治疗、 预防、 减轻和 / 或緩解本发明所述疾病或病症的剂量。  The term "effective amount" refers to a dose that can achieve treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.
术语 "受试者" 可以指患者或者其它接受本发明组合物以治 疗、预防、 减轻和 /或緩解本发明所述疾病或病症的动物， 特别是 哺乳动物， 例如人、 狗、 猴、 牛、 马等。  The term "subject" can refer to a patient or other animal that receives the composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog, a monkey, a cow, Horse and so on.
发明的有益效果 Advantageous effects of the invention
本发明提供了一种新的抗肿瘤药物组合物， 其主要通过作用 于肿瘤微环境达到有效治疗肿瘤的目的。 本发明的药物组合物调 节肿瘤微环境药物的特点是作用于多靶点、 多通路； 由细胞毒类 药物引起的毒副作用可望减轻。  The present invention provides a novel antitumor pharmaceutical composition which is effective for the treatment of tumors mainly by acting on the tumor microenvironment. The pharmaceutical composition of the present invention is characterized by the regulation of tumor microenvironment drugs which act on multiple targets and multiple pathways; the side effects caused by cytotoxic drugs are expected to be alleviated.
本发明将双嘧达莫、 乌苯美司与地塞米松联合， 用于肿瘤的 治疗。 双嘧达莫、 乌苯美司与地塞米松这三种药物都不是细胞毒 药物， 因此在单独使用时， 在可耐受剂量也只有中等抑瘤作用， 约在 20-60%。 本发明人将三者二二合用， 发现其抑瘤率可以提升 到 60- 70%。 本发明人惊奇地发现地双嘧达莫、 乌苯美司与地塞米 松三者联合用药， 在动物体内具有显著的抗肿瘤活性， 抑瘤率可 达 90%以上， 对于人肝癌、 人肺癌以及人胰腺癌的治疗效果大大 优于相应的临床常用一线药物 5-氟尿嘧啶、 吉西他滨、 卡培他滨 等， 对于表皮生长因子受体（EGFR)高表达的人鳞癌 A431 , 其治疗 效果与临床常用一线药物吉非替尼疗效相当， 且系统毒性极低， 可望成为一种具有高效抗肿瘤活性的药物组合物。 本发明中双嘧 达莫、 乌苯美司与地塞米松二二合用以及三者合用用于肿瘤治疗 属于首创， 国内外未见相关报道。 附图说明 The invention combines dipyridamole, umbrel and dexamethasone for the treatment of tumors. Dipyridamole, umbrel and dexamethasone are not cytotoxic drugs, so when used alone, there is only moderate antitumor effect at tolerable doses. About 20-60%. The inventors combined the three and two, and found that the tumor inhibition rate can be increased to 60-70%. The present inventors have surprisingly discovered that the combination of dipyridamole, umbrel and dexamethasone has significant anti-tumor activity in animals, and the tumor inhibition rate can reach more than 90%, for human liver cancer, human lung cancer. And the treatment effect of human pancreatic cancer is much better than the corresponding clinical first-line drugs 5-fluorouracil, gemcitabine, capecitabine, etc., for human epidermal cancer A431 with high expression of epidermal growth factor receptor (EGFR), its therapeutic effect and clinical The first-line drug gefitinib is equally effective, and the system toxicity is extremely low, and it is expected to be a pharmaceutical composition with high anti-tumor activity. In the present invention, the combination of dipyridamole, umbrel and dexamethasone 22, and the combination of the three for tumor treatment are the first, and no relevant reports have been reported at home and abroad. DRAWINGS
Fig. 1： 双嘧达莫、 乌苯美司及地塞米松合用对小鼠肝癌 H22 的肿瘤生长抑制作用。 . Fig 1: dipyridamole, ubenimex combination of dexamethasone inhibition of tumor growth in mice liver cancer H 22.
Fig. 2： 双嘧达莫、乌苯美司与地塞米松合用对棵鼠移植人肝 癌 BEL- 7402的肿瘤生长抑制作用。  Fig. 2: Tumor growth inhibition effect of dipyridamole, umbrel and dexamethasone on transplanted human liver cancer BEL-7402.
Fig. 3: 双嘧达莫、乌苯美司与地塞米松合用对棵鼠移植人肝 癌 HepG2的肿瘤生长抑制作用。  Fig. 3: Inhibition of tumor growth by transplanting human hepatoma HepG2 into mice by dipyridamole, umbrel and dexamethasone.
Fig. 4： 双嘧达莫、乌苯美司与地塞米松合用对棵鼠移植人鳞 癌 A431的肿瘤生长抑制作用。  Fig. 4: Inhibition of tumor growth by transplanting human squamous cell carcinoma A431 with dipyridamole, umbrel and dexamethasone.
Fig. 5: 双嘧达莫、乌苯美司与地塞米松合用对棵鼠移植人肺 癌 PG的肿瘤生长抑制作用。  Fig. 5: Inhibition of tumor growth by transplanting human lung cancer PG with dipyridamole, umbrel and dexamethasone.
Fig. 6: 双嘧达莫、乌苯美司与地塞米松合用对棵鼠移植人肺 癌 A549的肿瘤生长抑制作用。  Fig. 6: Tumor growth inhibition effect of dipyridamole, umbrel and dexamethasone on transplanted human lung cancer A549.
Fig. 7: 双嘧达莫、乌苯美司与地塞米松合用对棵鼠移植人胰 腺 MPAC的肿瘤生长抑制作用。 Fig. 8: 双嘧达莫、乌苯美司与地塞米松合用对棵鼠移植人胰 腺 SW1990的肿瘤生长抑制作用。 Fig. 7: Inhibition of tumor growth by transplanting human pancreatic MPAC with dipyridamole, umbrel and dexamethasone. Fig. 8: Inhibition of tumor growth by transplanting human pancreas SW1990 with dipyridamole, umbrel and dexamethasone.
Fig. 9:双嘧达莫、乌苯美司与地塞米松合用对荷人肝癌 HepG2 棵鼠的各器官组织的病理学检查的病理切片。 Fig. 9A - Fig. 9P所 用的组织和样本给药情况如下：  Fig. 9: Pathological section of pathological examination of various organ tissues of HepG2 mice bearing dipyridamole, umbrel and dexamethasone. The tissue and sample administration used in Fig. 9A - Fig. 9P are as follows:
编号 组织 样本 编号 组织 样本  Number organization sample number organization sample
A 心 对照 B 心 DPM+BEN+DEX A heart control B heart DPM+BEN+DEX
C 肺 对照 D 肺 DPM+BEN+DEXC lung control D lung DPM+BEN+DEX
E 肝 对照 F 肝 DPM+BEN+DEXE liver control F liver DPM+BEN+DEX
G 脾 对照 H 脾 DPM+BEN+DEXG spleen control H spleen DPM+BEN+DEX
I 肾 对照 J 肾 DPM+BEN+DEXI kidney control J kidney DPM+BEN+DEX
K 胃 对照 L 胃 DPM+BEN+DEXK stomach control L stomach DPM+BEN+DEX
M 小肠 对照 N 小肠 DPM+BEN+DEXM small intestine control N small intestine DPM+BEN+DEX
0 骨髓 对照 P 骨髓 DPM+BEN+DEX。 0 Bone marrow control P bone marrow DPM+BEN+DEX.
Fig. 10: Wes tern blot检测药物对鼠肝癌 H22小鼠移植瘤组织 蛋白表达的影响。 Fig. 10: Wes tern blot to detect the effect of drugs on the expression of tissue protein in transplanted tumor of mouse liver cancer H 22 mice.
Fig. 11： 双向电泳检测药物对人肝癌 Be卜 7402棵鼠移植瘤组 织蛋白表达的影响。 Fig. 11A为对照组， Fig. 11 B为 DPM+BEN+DEX 组。 具体实施方式  Fig. 11: Effect of two-dimensional electrophoresis on the expression of tissue protein in human liver cancer Be-Bad 7402 mice. Fig. 11A is the control group, and Fig. 11 B is the DPM+BEN+DEX group. detailed description
下面将结合实施例对本发明的实施方案进行详细描述， 但是 本领域技术人员将会理解， 下列实施例仅用于说明本发明， 而不 应视为限定本发明的范围。 实施例中未注明具体技术或条件者， 按照本领域内的文献所描述的技术或条件 (例如参考 J.萨姆布鲁 克等著， 黄培堂等译的 《分子克隆实验指南》 ， 第三版， 科学出 版社以及甄永苏主编的 《抗肿瘤药物研究与开发》 ， 化学工业出 版社）或者按照产品说明书进行。 所用试剂或仪器未注明生产厂 商者， 均为可以通过市购获得的常规产品。 The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, however, In the examples, the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Scientific The Press and the "Anti-Tumor Drug Research and Development" edited by Yan Yongsu, Chemical Industry Press) or in accordance with the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained commercially.
实施例中， 各个药物的简称分别为： 双嘧达莫（DPM ) , 乌苯 美司 （BEN ) , 地塞米松 ( DEX ) 。 实施例 1 : 包含双嘧达莫和乌苯美司的组合物样品的制备 配方： 双嘧达莫分另 'J为 lmg、 10mg、 100mg、 200mg、 300mg、 400mg、 900mg或 1600mg ,乌苯美司分另1 J为 lmg、 10mg、 20mg、 40mg、 60mg、 80mg、 100mg、 300mg、 900mg、 1600mg 或 3200 mg , 将双 嘧达莫和乌苯美司搅拌混合， 即可。 如表 1所示， 表中交叉点即 为配方。 In the examples, the abbreviations of the respective drugs are: dipyridamole (DPM), umbrel (BEN), and dexamethasone (DEX). Example 1: Preparation of a sample of a composition comprising dipyridamole and umbrelzine: Dipyridamole, 'J is 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 900 mg or 1600 mg, Uzbek Division 1 Another J is 1mg, 10mg, 20mg, 40mg, 60mg, 80mg, 100mg, 300mg, 900mg, 1600mg or 3200mg, dipyridamole and umbrel can be mixed and mixed. As shown in Table 1, the intersection in the table is the recipe.
表 1 : 包含双嘧达莫和乌苯美司的组合物的配方  Table 1: Formulations of compositions containing dipyridamole and umbrel
Figure imgf000012_0001
实施例 2: 包含双嘧达莫和地塞米松的组合物样品的制备 配方： 双嘧达莫分另 'J为 lmg、 10mg、 100mg、 200mg、 300mg、 400mg、 900mg或 1600mg， 地塞米松分另 'J为 0. lmg、 lmg、 1. 5mg、 2mg、 3mg、 4mg、 5mg、 10mg、 20mg、 40mg、 80mg或 160mg。 将双 嘧达莫和地塞米松搅拌混合， 即可。 如表 2所示， 表中交叉点即 为配方。
Figure imgf000012_0001
Example 2: Preparation of a sample of a composition comprising dipyridamole and dexamethasone: Dipyridamole, 'J is 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 900 mg or 1600 mg, dexamethasone Further, 'J is 0.1 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 20 mg, 40 mg, 80 mg or 160 mg. Mix dipyridamole and dexamethasone, and mix. As shown in Table 2, the intersection in the table is For the recipe.
表 2: 包含双嘧达莫和地塞米松的组合物的配方  Table 2: Formulations of compositions containing dipyridamole and dexamethasone
Figure imgf000013_0001
实施例 3: 包含乌苯美司和地塞米松的组合物样品的制备 配方： 乌苯美司分别为 lmg、 10mg、 20mg、 40mg、 60mg、 80mg、 100mg、 300mg、 900mg、 1600mg或 3200 mg ,地塞米松分另 'J为 0. lmg、 lmg、 1. 5mg、 2mg、 3mg、 4mg、 5mg、 10mg、 20mg、 40mg、 80mg或 160mg。 将乌苯美司和地塞米松搅拌混合， 即可。 如表 3所示， 表 中交叉点即为配方。
Figure imgf000013_0001
Example 3: Preparation of a sample of a composition comprising umbrel and dexamethasone: Ubimetrex is 1 mg, 10 mg, 20 mg, 40 mg, 60 mg, 80 mg, 100 mg, 300 mg, 900 mg, 1600 mg or 3200 mg, respectively. Dexamethasone is divided into '1 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 20 mg, 40 mg, 80 mg or 160 mg. Mix uzumex with dexamethasone and mix. As shown in Table 3, the intersection in the table is the recipe.
表 3: 包含乌苯美司和地塞米松的组合物的配方  Table 3: Formulations containing compositions of umbrel and dexamethasone
乌苯美司和地 乌苯美司 （mg ) Ubumex and dexamethasone (mg)
塞米松的组合 Combination of dexamethasone
1 10 20 40 60 80 100 300 900 1600 3200 物  1 10 20 40 60 80 100 300 900 1600 3200
0. 1 + + + + + + + + + + + 0. 1 + + + + + + + + + + +
1 + + + + + + + + + + +1 + + + + + + + + + + +
1. 5 + + + + + + + + + + +1. 5 + + + + + + + + + + +
2 + + + + + + + + + + +2 + + + + + + + + + + +
3 + + + + + + + + + + +3 + + + + + + + + + + +
4 + + + + + + + + + + + 地塞 4 + + + + + + + + + + +
5 + + + + + + + + + + + 米松  5 + + + + + + + + + + +
10 + + + + + + + + + + + 10 + + + + + + + + + + +
( mg ) ( mg )
20 + + + + + + + + + + + 20 + + + + + + + + + + +
40 + + + + + + + + + + +40 + + + + + + + + + + +
80 + + + + + + + + + + +80 + + + + + + + + + + +
160 + + + + + + + + + + + 实施例 4: 包含双嘧达莫、 乌苯美司和地塞米松的组合物样 品的制备 160 + + + + + + + + + + + Example 4: Preparation of a sample containing a composition of dipyridamole, umbrel and dexamethasone
配方 1：将实施例 1中的配方分别与 0. lmg、 lmg、 1. 5mg、 2mg、 3mg、 4mg、 5mg、 10mg、 20mg、 40mg、 80mg或 160mg的地塞米松 搅拌混合， 即可。  Formulation 1: The formulation of Example 1 is mixed with 0.1 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 20 mg, 40 mg, 80 mg or 160 mg of dexamethasone, respectively.
配方 2:将实施例 2中的配方分别与 lmg、 10mg、 20mg、 40mg、 60mg、 80mg、 100mg、 300mg、 900mg、 1600mg或 3200 mg的乌苯 美司搅拌混合， 即可。  Formulation 2: The formulation of Example 2 was mixed with 1 mg, 10 mg, 20 mg, 40 mg, 60 mg, 80 mg, 100 mg, 300 mg, 900 mg, 1600 mg or 3200 mg of urinium, respectively.
配方 3:将实施例 3中的配方分别与 lmg、10mg、100mg、200mg、 300mg、 400mg、 900mg或 1600mg的双嘧达莫搅拌混合， 即可。 实施例 5: 药物的体外细胞毒性实验  Formulation 3: The formulation of Example 3 was mixed with 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 900 mg or 1600 mg of dipyridamole, respectively. Example 5: In vitro cytotoxicity test of drugs
1.材料与方法：  1. Materials and methods:
1. 1 药品与试剂： 双嘧达莫： 标准品， 购自中国药品生物制 品检定所； 乌苯美司： 原料药， 浙江普洛康裕制药有限公司； 地 塞米松： 标准品， 购自中国药品生物制品检定所； 5-氟尿嘧啶注 射液为上海旭东海普药业有限公司生产；  1. 1 Drugs and Reagents: Dipyridamole: Standard, purchased from China National Institute for the Control of Pharmaceutical and Biological Products; Ubumex: API, Zhejiang Plo Kangyu Pharmaceutical Co., Ltd.; Dexamethasone: Standard, purchased from China Pharmaceutical and Biological Products Laboratory; 5-fluorouracil injection for Shanghai Xudong Haipu Pharmaceutical Co., Ltd.;
1. 2实验细胞： 肝癌 BEL- 7402细胞， 购自中国医学科学院基 础医学研究所细胞中心。  1. 2 Experimental cells: Liver cancer BEL-7402 cells were purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.
1. 3受试药物： 选自实施例 1 - 4的制备的药物组合物样品。 1. 3 Test drug: A sample of the pharmaceutical composition selected from the preparation of Examples 1 - 4.
1. 4 实验方法： 采用克隆形成法检测。 取对数生长期的 BEL- 7402细胞， 按每孔 50个细胞 / 0. 2ml加入 96孔板， 培养 24 h。 加入不同浓度待测样品， 每个浓度设三个平行孔， 每孔 50ul , 无药对照孔加 50 μ 1无血清 1640 , 继续培养 7-10天， 镜下记数 细胞集落，以大于等于 30个细胞计为一个克隆，数出每孔克隆数， 计算克隆形成百分率及 IC5。。 2.结果： 检测结果见表 4。 1. 4 Experimental method: Detection by clone formation method. BEL-7402 cells in logarithmic growth phase were added to a 96-well plate at 50 cells/0.2 ml per well for 24 h. Add different concentrations of the sample to be tested, each concentration is set to three parallel holes, 50ul per well, no drug control well plus 50 μ 1 serum-free 1640, continue to culture for 7-10 days, count cell colonies under the microscope, to be greater than or equal to 30 One cell was counted as one clone, and the number of clones per well was counted, and the percentage of clone formation and IC 5 were calculated. . 2. Results: The test results are shown in Table 4.
表 4: 药物的体外细胞毒性实验  Table 4: In vitro cytotoxicity experiments of drugs
Figure imgf000015_0001
Figure imgf000015_0001
体外实验结果表明： 单独使用 BEN 及 DEX对细胞克隆形成 的 IC5。均大于 100 μ M，未显示细胞毒作用。单独使用 DPM的 IC5。 为 13. 34 μ Μ , 有一定的细胞毒作用。 实施例 1, 2 , 4的药物组 合物对肿瘤细胞有一定的抑制作用。 实施例 6: 药物的体内抗肿瘤活性研究一对小鼠肿瘤生长的 抑制作用 The results of in vitro experiments showed that IC 5 was formed by cell clones using BEN and DEX alone. Both were greater than 100 μM and showed no cytotoxic effects. Use IC 5 for DPM alone. It is 13. 34 μ Μ and has a certain cytotoxic effect. The pharmaceutical compositions of Examples 1, 2, and 4 have a certain inhibitory effect on tumor cells. Example 6: In vivo antitumor activity of drugs The inhibition of tumor growth in a pair of mice
1材料与方法：  1 Materials and methods:
1. 1药品与试剂： 受试药物选自实施例 1 - 4的制备的药物组 合物样品。 双嘧达莫： 标准品， 购自中国药品生物制品检定所； 乌苯美司： 原料药， 浙江普洛康裕制药有限公司； 地塞米松： 标 准品， 购自中国药品生物制品检定所； 双嘧达莫片： 山西亚宝药 业集团股份有限公司生产； 醋酸地塞米松片： 天津力生制药股份 有限公司生产； 乌苯美司 （百士欣）胶嚢： 浙江普洛康裕制药有 限公司生产； 小鼠肝癌 Η22细胞以腹水形式在昆明小鼠体内传代。 1.2动物： 昆明小鼠（清洁 II级）为雌性， 6~8周龄， 体重 18 ~ 22 g,由军事医学科学院实验动物中心提供，许可证编号 SCXK (军） 2007-004。 1. 1 Drugs and Reagents: The test drug is selected from the samples of the pharmaceutical compositions prepared in Examples 1-4. Dipyridamole: Standard, purchased from China National Institute for the Control of Pharmaceutical and Biological Products; Ubumex: API, Zhejiang Plo Kangyu Pharmaceutical Co., Ltd.; Dexamethasone: Standard, purchased from China National Institute for the Control of Pharmaceutical and Biological Products; Pyramidazole tablets: Shanxi Yabao Pharmaceutical Group Co., Ltd. production; dexamethasone acetate tablets: Tianjin Lisheng Pharmaceutical Co., Ltd. production; Ubimex (Bai Shixin) plastic bottles: Zhejiang Plo Kangyu Pharmaceutical Co., Ltd. Production; Mouse liver cancer Η 22 cells were passaged in Kunming mice as ascites. 1.2 Animals: Kunming mice (clean grade II) are female, 6-8 weeks old, weighing 18-22 g, provided by the Experimental Animal Center of the Academy of Military Medical Sciences, license number SCXK (Army) 2007-004.
1.3实验方法：  1.3 Experimental methods:
1.3.1 根据剂量初筛结果， 设计动物实验治疗的给药方式和 剂量。 取体重为 18-22 g的昆明种小鼠， 随机分组， 每组 10只。 取小鼠肝癌 H22腹水， 以生理盐水稀释成细胞数为 7.5X106个 /ml 的悬液， 0.2ml/只， 接种于小鼠腋窝皮下。 接种肿瘤后第 3天开 始治疗， 每天濯胃给药共 10次， 给药方案以 "qdxlO"表示。 对 照组给予生理盐水，其余各组分别给予相应实施例 1-4中的药物。 短期观察的实验于第 14天处死小鼠称体重，分离肿瘤称瘤重，计 算抑瘤率。 1.3.1 According to the results of the initial screening of the dose, the mode of administration and dosage of the experimental treatment of the animal are designed. Kunming mice weighing 18-22 g were randomly divided into groups of 10 each. The mouse liver cancer H 22 ascites was taken and diluted with physiological saline into a suspension of 7.5× 10 6 cells/ml, 0.2 ml/mouse, and inoculated into the axilla of the mouse. The treatment was started on the third day after the tumor was inoculated, and the stomach administration was performed 10 times a day, and the administration schedule was expressed by "qdxlO". The control group was given physiological saline, and the remaining groups were administered with the drugs of the respective Examples 1-4. In the short-term observation experiment, the mice were sacrificed on the 14th day, and the tumors were weighed and the tumor inhibition rate was calculated.
1.3.2长期观察的实验期间， 每周测量 2次肿瘤的长径 a和 短径 b, 并记录动物体重。 以公式 V= ab2/2计算瘤体积， 绘制肿 瘤生长曲线，计算抑瘤率，采用 Student' s ί检验比较组间差异。 观察动物死亡情况，绘制动物生存曲线，并以 Kaplan-Meier法求 出中位生存时间。 1.3.2 During the long-term observation period, the long diameter a and the short diameter b of the tumor were measured twice a week, and the animal body weight was recorded. In the formula V = ab 2/2 calculated tumor volume, tumor growth curve inhibition rate was calculated, using inter Student 's ί comparative test group differences. Animal mortality was observed, animal survival curves were plotted, and median survival time was determined by Kaplan-Meier method.
2.结果： 结果见表 5- 13和 Fig.1。  2. Results: The results are shown in Tables 5-13 and Fig.1.
2.1 不同剂量的双嘧达莫与乌苯美司合用用于治疗小鼠肝癌 H22, 实验结果表明： 二者合用对小鼠肝癌 H22肿瘤生长有中度抑瘤 作用。 结果见表 5。 2.1 Different doses of dipyridamole combined with umbrelzine for the treatment of mouse liver cancer H 22 , the experimental results show that: the combination of the two has a moderate anti-tumor effect on mouse liver cancer H 22 tumor growth. The results are shown in Table 5.
表 5: 不同剂量的双嘧达莫与乌苯美司对小鼠肝癌 H22肿瘤生 长抑制作用 Table 5: Inhibition of tumor growth of mouse liver cancer H 22 by different doses of dipyridamole and umbrel
剂量 动物数 体重变化 瘤重（g)  Dosage number of animals weight change tumor weight (g)
组别 抑瘤率（  Group inhibition rate
( mg/kg ) 开始 /结束 (g) 士 SD  ( mg/kg ) Start / End (g) Shi SD
对照 - 10/10 2.10±0.47 - Control - 10/10 2.10 ± 0.47 -
DPM 1 10/10 +13.12 2.03±0.35 3.3¾ DPM 10 10/10 +12.68 1.90±0.49 9.5¾DPM 1 10/10 +13.12 2.03±0.35 3.33⁄4 DPM 10 10/10 +12.68 1.90±0.49 9.53⁄4
DPM 100 10/10 +12.65 1.66±0.59 21.0¾DPM 100 10/10 +12.65 1.66±0.59 21.03⁄4
DPM 300 10/10 +11.12 1.27±0.42 DPM 300 10/10 +11.12 1.27±0.42
DPM 900 10/10 +12.15 1.25±0.58 40.5¾* DPM 900 10/10 +12.15 1.25±0.58 40.53⁄4*
BEN 40 10/10 +11.02 1.35±0.49 BEN 40 10/10 +11.02 1.35±0.49
DPM+BEN 1+40 10/10 +10.23 1.40±0.31 33.3%*Δ Δ DPM+BEN 1+40 10/10 +10.23 1.40±0.31 33.3%*Δ Δ
DPM+BEN 10+40 10/10 +10.45 1.41±0.30 32.9%*Δ ΔDPM+BEN 10+40 10/10 +10.45 1.41±0.30 32.9%*Δ Δ
DPM+BEN 100+40 10/10 +8.23 1.09±0.14 48.1%*Δ ΔDPM+BEN 100+40 10/10 +8.23 1.09±0.14 48.1%*Δ Δ
DPM+BEN 300+40 10/10 0.93±0.18 55.7%*Δ▲DPM+BEN 300+40 10/10 0.93±0.18 55.7%*Δ▲
DPM+BEN 900+40 10/10 0.86±0.14 59.0%*Δ▲▲ DPM+BEN 900+40 10/10 0.86±0.14 59.0%*Δ▲▲
oo  Oo
注：与对照组比较 *Ρ<0.01;与相应的 DPM比较 ΔΡ<0.05, ΔΡ<0.01; 与 BEN比较 AP<0.05, A AP<0.01ο Note: Compared with the control group *Ρ<0.01; compared with the corresponding DPM ΔΡ<0.05, ΔΡ<0.01; compared with BEN AP<0.05, A AP<0.01 ο
2.2 不同剂量的乌苯美司与地塞米松合用用于治疗小鼠肝癌 Η22, 实验结果表明： 二者合用对小鼠肝癌 Η22肿瘤生长有较好抑瘤 作用。 结果见表 6。 2.2 Different doses of umbrel and dexamethasone were used to treat liver cancer in mice. 22 The experimental results show that the combination of the two has a good anti-tumor effect on the growth of mouse liver cancer Η 22 tumor. The results are shown in Table 6.
表 6: 不同剂量的乌苯美司和地塞米松对小鼠肝癌 Η22肿瘤生 长抑制作用 Table 6: Inhibition of tumor growth of mouse liver cancer Η 22 by different doses of umbrel and dexamethasone
剂量 动物数 体重变化 瘤重（g) Dosage number of animals weight change tumor weight (g)
组别 抑瘤率（W  Group inhibition rate (W
( mg/kg ) 开始 /结束 ( g) 士 SD  ( mg/kg ) start / end ( g ) Shi SD
对照 - 10/10 +14.37 2.13±0.92 - Control - 10/10 +14.37 2.13±0.92 -
BEN 1 10/10 +10.65 1.85±0.27 13.1¾BEN 1 10/10 +10.65 1.85±0.27 13.13⁄4
BEN 10 10/10 +11.22 1.66±0.18 22.1¾BEN 10 10/10 +11.22 1.66±0.18 22.13⁄4
BEN 100 10/10 +11.29 0.98±0.23 54.0¾*BEN 100 10/10 +11.29 0.98±0.23 54.03⁄4*
BEN 300 10/10 +11.15 0.99±0.16 53.5¾*BEN 300 10/10 +11.15 0.99±0.16 53.53⁄4*
BEN 900 10/10 +10.66 1.05±0.22 50.7¾*BEN 900 10/10 +10.66 1.05±0.22 50.73⁄4*
DEX 2 10/10 +6.84 1.08±0.17 49.3¾*DEX 2 10/10 +6.84 1.08±0.17 49.33⁄4*
BEN+DEX 1+2 10/10 +6.55 1.01±0.27 52.6¾* ΔBEN+DEX 1+2 10/10 +6.55 1.01±0.27 52.63⁄4* Δ
BEN+DEX 10+2 10/10 +6.72 0.84±0.09 60.6%* Δ▲BEN+DEX 10+2 10/10 +6.72 0.84±0.09 60.6%* Δ▲
BEN+DEX 100+2 10/10 +6.03 0.66±0.17 69.0%* Δ▲BEN+DEX 100+2 10/10 +6.03 0.66±0.17 69.0%* Δ▲
BEN+DEX 300+2 10/10 +6.03 0.68±0.07 68.1%* Δ▲BEN+DEX 300+2 10/10 +6.03 0.68±0.07 68.1%* Δ▲
BEN+DEX 900+2 10/10 +6.12 0.74±0.18 65.3%* Δ▲ 注： 与对照组比较 *P<0.01; 与相应的 BEN 比较 ΔΡ<0.01; 与 DEX 比较 A P<0.01。 BEN+DEX 900+2 10/10 +6.12 0.74±0.18 65.3%* Δ▲ Note: Compared with the control group *P<0.01; compared with the corresponding BEN ΔΡ<0.01; compared with DEX A P<0.01.
2.3 不同剂量的地塞米松与双嘧达莫合用用于治疗小鼠肝癌 H22, 实验结果表明： 二者合用对小鼠肝癌 H22肿瘤生长有较好抑瘤 作用。 结果见表 7。 2.3 Different doses of dexamethasone and dipyridamole were used to treat liver cancer H 22 in mice. The results showed that the combination of the two had a good anti-tumor effect on the growth of mouse liver cancer H 22 tumor. The results are shown in Table 7.
表 7: 双嘧达莫与不同剂量的地塞米松合用对小鼠肝癌 H22肿 瘤生长抑制作用 Table 7: Inhibition of tumor growth of mouse liver cancer H 22 by dipyridamole combined with different doses of dexamethasone
剂量 动物数 体重变化 瘤重 （g) Dosage number of animals weight change tumor weight (g)
组别 抑瘤率（¾) ( mg/kg ) 开始 /结束 (g) 士 SD  Group Tumor inhibition rate (3⁄4) (mg/kg) Start / end (g) Shi SD
对照 - 10/10 2.03±0.59 - Comparison - 10/10 2.03 ± 0.59 -
DEX 0.1 10/10 + 7.68 1.80±0.24 11.3¾DEX 0.1 10/10 + 7.68 1.80±0.24 11.33⁄4
DEX 1 10/10 + 7.02 1.03±0.30 DEX 1 10/10 + 7.02 1.03±0.30
DEX 5 10/10 + 5.33 0.88±0.15  DEX 5 10/10 + 5.33 0.88±0.15
DEX 10 10/10 + 4.12 0.78±0.11 61.6¾* DEX 10 10/10 + 4.12 0.78±0.11 61.63⁄4*
DPM 200 10/10 +12.02 1.27±0.23 DPM 200 10/10 +12.02 1.27±0.23
DPM+DEX 200+0.1 10/10 + 7.08 1.26±0.13 37.9%* Δ DPM+DEX 200+0.1 10/10 + 7.08 1.26±0.13 37.9%* Δ
DPM+DEX 200+1 10/10 + 6.82 0.91±0.15 55.2%* ADPM+DEX 200+1 10/10 + 6.82 0.91±0.15 55.2%* A
DPM+DEX 200+5 10/10 + 4.78 0.80±0.13 60.6%* ADPM+DEX 200+5 10/10 + 4.78 0.80±0.13 60.6%* A
DPM+DEX 200+10 10/10 + 4.15 0.62±0.13 69.5%*Δ▲ 注： 与对照组比较 *P<0.01; 与相应的 DEX 比较 ΔΡ<0.01; 与 DPM 比较 AP<0.01 DPM+DEX 200+10 10/10 + 4.15 0.62±0.13 69.5%*Δ▲ Note: Compared with the control group *P<0.01; compared with the corresponding DEX ΔΡ<0.01; compared with DPM AP<0.01
2.4 双嘧达莫、 乌苯美司与地塞米松合用用于治疗小鼠肝癌 H22, 实验结果表明： 三者合用对小鼠肝癌 H22肿瘤生长有显著抑瘤 作用。 结果见表 8 2.4 Dipyridamole, Ubumex and dexamethasone were used to treat liver cancer H 22 in mice. The results showed that the combination of the three drugs has a significant anti-tumor effect on the growth of mouse liver cancer H 22 tumor. The results are shown in Table 8.
表 8: 双嘧达莫、 乌苯美司与地塞米松合用对小鼠肝癌 H22的 肿瘤生长抑制作用 Table 8: Inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of mouse liver cancer H 22
Figure imgf000019_0001
Figure imgf000019_0001
与对照组比较 *P<0.01 2. 5 不同剂量的双嘧达莫与乌苯美司和地塞米松合用用于治 疗小鼠肝癌 H22 , 实验结果表明： 三者合用对小鼠肝癌 H22肿瘤生 长有显著抑瘤作用， 此作用比不同剂量的双嘧达莫以及乌苯美司 和地塞米松合用都强。 结果见表 9。 Compared with the control group *P<0.01 2. 5 different doses of dipyridamole combined with umbrel and dexamethasone for the treatment of liver cancer H 22 in mice, the experimental results show that: the combination of the three has a significant anti-tumor effect on the growth of mouse liver cancer H 22 tumor, This effect is stronger than the combination of different doses of dipyridamole and umbrel and dexamethasone. The results are shown in Table 9.
表 9: 不同剂量的双嘧达莫与乌苯美司和地塞米松合用对小 鼠肝癌 H22的生长抑制作用 Table 9: Growth inhibition of mouse liver cancer H 22 by different doses of dipyridamole combined with umbrel and dexamethasone
Figure imgf000020_0001
Figure imgf000020_0001
与对照组比较 *Ρ<0. 01 ;与相对应的 DPM比较 AP<0. 01 ;与 BEN+DEX 比较 ^P<0. 01。  Compared with the control group *Ρ<0. 01; compared with the corresponding DPM AP<0. 01; compared with BEN+DEX ^P<0.01.
2. 6 不同剂量的乌苯美司与双嘧达莫和地塞米松合用用于治 疗小鼠肝癌 H22， 实验结果表明： 三者合用对小鼠肝癌 H22肿瘤生 长有显著抑瘤作用， 此作用比不同剂量的乌苯美司以及双嘧达莫 和地塞米松合用都强。 结果见表 10。 2. 6 different doses of umbrel and dipyridamole combined with dexamethasone for the treatment of liver cancer H 22 in mice, the experimental results show that: the combination of the three has a significant anti-tumor effect on the growth of mouse liver cancer H 22 tumor, This effect is stronger than the combination of different doses of umbrel and dipyridamole and dexamethasone. The results are shown in Table 10.
表 10: 不同剂量的乌苯美司与双嘧达莫和地塞米松合用对小 鼠肝癌 H22的生长抑制作用 剂量 动物数 体重变 瘤重（g) Table 10: Inhibition of growth of mouse liver cancer H 22 by different doses of umbrel with dipyridamole and dexamethasone Dosage number of animals, body weight, tumor weight (g)
组别 抑瘤率 ¾ ( mg/kg ) 开始 /结束 化（g) X士 SD  Group tumor inhibition rate 3⁄4 (mg/kg) start / end (g) X Shi SD
对照 - 10/10 +13.72 1.95±1.01 - Control - 10/10 +13.72 1.95±1.01 -
BEN 20 10/10 + 9.95 0.97±0.38 50.3%*BEN 20 10/10 + 9.95 0.97±0.38 50.3%*
BEN 40 10/10 + 11.28 0.86±0.53 55.9¾**BEN 40 10/10 + 11.28 0.86±0.53 55.93⁄4**
BEN 80 10/10 + 11.36 1.15±0.65 BEN 80 10/10 + 11.36 1.15±0.65
DPM+DEX 200+2 10/10 + 6.79 0.69±0.38 64.6¾** DPM+DEX 200+2 10/10 + 6.79 0.69±0.38 64.63⁄4**
DPM+BEN+DEX 200+20+2 10/10 +5.58 0.41±0.17 79.0¾**ΔΑDPM+BEN+DEX 200+20+2 10/10 +5.58 0.41±0.17 79.03⁄4**ΔΑ
DPM+BEN+DEX 200+40+2 10/10 +5.58 0.35±0.16 82.1¾**ΔΑΑDPM+BEN+DEX 200+40+2 10/10 +5.58 0.35±0.16 82.13⁄4**ΔΑΑ
DPM+BEN+DEX 200+80+2 10/10 + 3.69 0.28±0.14 85.6¾**ΔΑΑ 与对照组比较 *Ρ<0.05, **Ρ<0.01;与相对应的 BEN比较 AP<0.01; 与 DPM+DEX比较 AP<0.05， AAP<0.01。 DPM+BEN+DEX 200+80+2 10/10 + 3.69 0.28±0.14 85.63⁄4**ΔΑΑ Compared with the control group *Ρ<0.05, **Ρ<0.01; compared with the corresponding BEN compared to AP<0.01; with DPM +DEX comparison AP<0.05, AAP<0.01.
2.7 不同剂量的地塞米松与双嘧达莫和乌苯美司合用用于治 疗小鼠肝癌 H22， 实验结果表明： 三者合用对小鼠肝癌 H22肿瘤生 长有显著抑瘤作用， 此作用比不同剂量的地塞米松以及双嘧达莫 和乌苯美司合用都强。 结果见表 11。 2.7 Different doses of dexamethasone combined with dipyridamole and umbrelzine for the treatment of liver cancer H 22 in mice, the results showed that: the combination of the three has a significant anti-tumor effect on the growth of mouse liver cancer H 22 tumor, this effect It is stronger than different doses of dexamethasone and dipyridamole and umbrel. The results are shown in Table 11.
寸  Inch
表 11: 不同剂量的地塞米松与双嘧达莫和乌苯美司合用对小 鼠肝癌 H22的生长抑制作用 Table 11: Growth inhibitory effects of different doses of dexamethasone combined with dipyridamole and umbrel on liver cancer H 22 in mice
动物数 Number of animals
剂量 体重变化 瘤重（g) 组别 开始 /结 抑瘤率 ¾ ( mg/kg ) (g)  Dosage Weight change Tumor weight (g) Group Start/nodule rate 3⁄4 (mg/kg) (g)
束 士 SD  束士 SD
对照 - 10/10 + 11.80 4.12±1.30 - Control - 10/10 + 11.80 4.12 ± 1.30 -
DEX 1 10/10 +7.22 2.38±0.85 42.2%*DEX 1 10/10 +7.22 2.38±0.85 42.2%*
DEX 2 10/10 +6.25 1.69±0.62 59.0%*DEX 2 10/10 +6.25 1.69±0.62 59.0%*
DEX 3 10/10 +6.12 1.29±0.41 68.7%*DEX 3 10/10 +6.12 1.29±0.41 68.7%*
DPM+BEN 200+40 10/10 + 10.24 1.17±0.33 DPM+BEN 200+40 10/10 + 10.24 1.17±0.33
DPM+BEN+DEX 200+40+1 10/10 +6.14 0.76±0.26 81.6¾*ΔΑ DPM+BEN+DEX 200+40+1 10/10 +6.14 0.76±0.26 81.63⁄4*ΔΑ
DPM+BEN+DEX 200+40+2 10/10 +5.65 0.60±0.25 85.4¾*ΔΑDPM+BEN+DEX 200+40+2 10/10 +5.65 0.60±0.25 85.43⁄4*ΔΑ
DPM+BEN+DEX 200+40+3 10/10 +5.97 0.44±0.19 89.3¾*ΔΑ 与对照组比较 *Ρ<0.01; 与相对应的 DEX 比较 AP<0.01; 与 DPM+BEN比较 AP<0.01。 DPM+BEN+DEX 200+40+3 10/10 +5.97 0.44±0.19 89.33⁄4*ΔΑ Compared with the control group *Ρ<0.01; AP<0.01 compared with the corresponding DEX; AP<0.01 compared with DPM+BEN.
2.8 长期观察双嘧达莫、 乌苯美司及地塞米松合用对小鼠肝 癌 H22的生长抑制作用。 结果见表 12、 表 13， Fig.1。 实验结果表 明： 三者合用对小鼠肝癌 H22有非常显著的抑瘤作用， 且呈明显的 量效关系， 此作用比单药 DPM、 BEN、 DEX以及二种药 DPM+BEN、 DPM+DEX、 BEN+DEX都强； 体内实验结果表明： 三者合用可明显延 长荷肝癌 H22的小鼠平均生存时间， 此作用比单药 DPM、 BEN, DEX 以及二种药 DPM+BEN、 DPM+DEX、 BEN+DEX都强。 2.8 Long-term observation of the growth inhibition effect of dipyridamole, umbrel and dexamethasone on liver cancer H 22 in mice. The results are shown in Table 12, Table 13, Fig. 1. The experimental results show that: the combination of the three has a very significant anti-tumor effect on mouse liver cancer H 22 , and has a significant dose-effect relationship, this effect is more than single drug DPM, BEN, DEX and two drugs DPM+BEN, DPM+DEX BEN+DEX is strong; the results of in vivo experiments show that: the combination of the three can significantly prolong the average survival time of mice bearing liver cancer H 22 , which is more effective than single drug DPM, BEN, DEX and two drugs DPM+BEN, DPM+DEX BEN+DEX is strong.
表 12: 双嘧达莫、 乌苯美司及地塞米松合用对小鼠肝癌 H22 Table 12: Dipyridamole, umbrel and dexamethasone combined with mouse liver cancer H 22
的生长抑制作用 （13d) Growth inhibition (13d)
动物数 Number of animals
剂量 体重变化 瘤体积 ( cm3) 组别 开始 /结 抑瘤率 ¾  Dosage Weight change Tumor volume (cm3) Group Start / Junction Tumor rate 3⁄4
( mg/kg ) ( g)  ( mg/kg ) ( g)
束 X士 SD  Bunch X Shi SD
对照 - 10/10 5.44±2.30 - Control - 10/10 5.44 ± 2.30 -
DPM 200 10/10 +8.66 3.25±1.46 40.3%*DPM 200 10/10 +8.66 3.25±1.46 40.3%*
BEN 40 10/10 +8.22 3.07±2.28 BEN 40 10/10 +8.22 3.07±2.28
DEX 1 10/10 +6.54 2.81±1.11  DEX 1 10/10 +6.54 2.81±1.11
DPM+DEX 200+1 10/10 +5.06 1.67±0.58 69.3¾**Δ ίΓ DPM+DEX 200+1 10/10 +5.06 1.67±0.58 69.33⁄4**Δ ίΓ
DPM+BEN 200+40 10/10 +10.21 2.96±1.50 45.6¾**DPM+BEN 200+40 10/10 +10.21 2.96±1.50 45.63⁄4**
BEN+DEX 40+1 10/10 +5.92 1.82±0.49 66.5¾** irO BEN+DEX 40+1 10/10 +5.92 1.82±0.49 66.53⁄4** irO
78.1%**ΔΑ ίΓ* 78.1%**ΔΑ ίΓ*
DPM+BEN+DEX 200+40+1 10/10 + 3.84 1.19±0.47 DPM+BEN+DEX 200+40+1 10/10 + 3.84 1.19±0.47
◊◊♦ ◊◊♦
DPM+BEN+DEX 300+60+1.5 10/10 + 3.56 0.96±0.25 82.4¾**DPM+BEN+DEX 300+60+1.5 10/10 + 3.56 0.96±0.25 82.43⁄4**
DPM+BEN+DEX 400+80+2 10/10 + 3.75 0.50±0.17 90.8¾**D 接种 3天给药， 每天一次， 共 10次。 观察肿瘤生长情况以及 动物生存时间。 DPM+BEN+DEX 400+80+2 10/10 + 3.75 0.50±0.17 90.83⁄4**D Inoculation 3 days administration, once a day for 10 times. Observe tumor growth and animal survival time.
与对照组比较 *P<0.05 **P<0.01; 与相对应的 DPM 比较 △Ρ<0· 01; 与相对应的 BEN比较 P<0.05; 与相对应的 DEX比较 ☆P<0.01; 与相对应的 DPM+DEX 比较★?<().05; 与相对应的 DPM+BEN比较 P<0.05 P<0.01; 与相对应的 BEN+DEX比较 ♦ P<0.01; 与 DPM200+BEN40+DEX 1比较 CIP<0.01  Compared with the control group *P<0.05 **P<0.01; compared with the corresponding DPM △Ρ<0· 01; compared with the corresponding BEN P<0.05; compared with the corresponding DEX ☆P<0.01; Corresponding DPM+DEX comparison ★?<().05; P<0.05 P<0.01 compared with the corresponding DPM+BEN; compared with the corresponding BEN+DEX ♦ P<0.01; compared with DPM200+BEN40+DEX 1 CIP<0.01
Fig.1双嘧达莫、 乌苯美司及地塞米松合用对小鼠肝癌 H22的 肿瘤生长抑制作用。 Fig. 1 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of mouse liver cancer H 22 .
表 13: 双嘧达莫、 乌苯美司及地塞米松合用对荷肝癌 H22的 小鼠平均生存时间的影响 剂量 中位生存时间 Table 13: Effect of dipyridamole, umbrel and dexamethasone on mean survival time of mice bearing liver cancer H 22 Dose median survival time
组别 T/C  Group T/C
( mg/kg ) 士 SD  ( mg/kg ) Shi SD
对照 - 31 - Control - 31 -
DPM 200 38 1.23DPM 200 38 1.23
BEN 40 32 1.03BEN 40 32 1.03
DEX 1 42* 1.35DEX 1 42* 1.35
DPM+DEX 200+1 38 1.23DPM+DEX 200+1 38 1.23
DPM+BEN 200+40 36 1.16DPM+BEN 200+40 36 1.16
BEN+DEX 40+1 50**Air 1.61BEN+DEX 40+1 50**Air 1.61
DPM+BEN+DEX 200+40+1 59**ΔΑ ίηίΓ* 1.90DPM+BEN+DEX 200+40+1 59**ΔΑ ίηίΓ* 1.90
DPM+BEN+DEX 300+60+1.5 63** 2.03DPM+BEN+DEX 300+60+1.5 63** 2.03
DPM+BEN+DEX 400+80+2 57** 1.84 DPM+BEN+DEX 400+80+2 57** 1.84
与对照组比较 *P<0.05， **P<0.01; 与相对应的 DPM 比较 △Ρ<0· 01; 与相对应的 BEN比较 P<0.01; 与相对应的 DEX比较 ☆P<0.05， ☆☆?<().01; 与相对应的 DPM+DEX比较 *P<0.01; 与 相对应的 DPM+BEN 比较 P<0.01; 与相对应的 BEN+DEX 比较 Compared with the control group *P<0.05, **P<0.01; compared with the corresponding DPM △Ρ<0· 01; compared with the corresponding BEN P<0.01; compared with the corresponding DEX ☆P<0.05, ☆ ☆?<().01; Compared with the corresponding DPM+DEX*P<0.01; P<0.01 compared with the corresponding DPM+BEN; compared with the corresponding BEN+DEX
Ρ<0· 01 实施例 7: 药物的体内抗肿瘤活性研究一对棵鼠移植的人肿 瘤的生长抑制作用 Ρ<0· 01 Example 7: In vivo antitumor activity of drugs The growth inhibition of human tumors transplanted in a pair of mice
1材料与方法  1 Materials and methods
1.1 药品与试剂： 受试药物选自实施例 4的制备的药物组合 物样品。 双嘧达莫： 标准品， 购自中国药品生物制品检定所； 乌 苯美司： 原料药， 浙江普洛康裕制药有限公司； 地塞米松： 标准 品， 购自中国药品生物制品检定所； 双嘧达莫片： 山西亚宝药业 集团股份有限公司生产； 醋酸地塞米松片： 天津力生制药股份有 Ρ艮公司生产； 乌苯美司 （百士欣）胶嚢： 浙江普洛康裕制药有限 公司生产； 5-氟尿嘧啶注射液为上海旭东海普药业有限公司生产； 吉西他滨（健泽）： 法国礼来有限公司生产； 卡培他滨（希罗达）： 罗氏制药生产； 吉非替尼（Gefitinib，Iressa易瑞沙）： 阿斯利 康制药有限公司生产； 人肝癌 BEL- 7402， 人肝癌 HepG2, 人鱗癌 A431,人肺癌 PG细胞，人肺癌 A549,人胰腺 MPAC,人胰腺 SW1990, 均常规体外细胞培养。 1.1 Drugs and Reagents: The test drug is selected from the sample of the pharmaceutical composition prepared in Example 4. Dipyridamole: Standard, purchased from China National Institute for the Control of Pharmaceutical and Biological Products; Ubumex: API, Zhejiang Plo Kangyu Pharmaceutical Co., Ltd.; Dexamethasone: Standard, purchased from China National Institute for the Control of Pharmaceutical and Biological Products; Pyramidazole tablets: Shanxi Yabao Pharmaceutical Group Co., Ltd. production; dexamethasone acetate tablets: Tianjin Lisheng Pharmaceutical Co., Ltd. is produced by the company; Ubumex (Bai Shixin) plastic bottles: Zhejiang Plo Kangyu Pharmaceutical Limited Company production; 5-fluorouracil injection for Shanghai Xudong Haipu Pharmaceutical Co., Ltd.; Gemcitabine (Jianze): produced by French Lilly Co., Ltd.; Capecitabine (Xeloda): Roche Pharmaceutical Production; Gefitinib (Gefitinib, Iressa Iressa): AstraZeneca Pharmaceutical Co., Ltd.; human liver cancer BEL-7402, human liver cancer HepG2, human squamous cell carcinoma A431, human lung cancer PG cells, human lung cancer A549, human pancreas MPAC, human pancreas SW1990, Conventional in vitro cell culture.
1.2动物： NIHnu/nu 棵小鼠为雌性， 6~8周龄， 体重 18~ 22 g,购自北京维通利华实验动物技术有限公司，许可证编号 SCXK (京） 2006-0009。  1.2 Animals: NIHnu/nu mice were female, 6-8 weeks old, weighing 18-22 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., license number SCXK (Beijing) 2006-0009.
1.3实验方法： 取体外培养的人肿瘤细胞， 接种于 NIHnu/nu 棵小鼠一侧腋窝皮下 ,传 2-3代后 ,取腋下传代肿瘤，切成 1.5腿 3 左右的小块， 接种于棵小鼠一侧腋窝皮下， 待肿瘤生长至 100 ~ 300腿 3后根据肿瘤大小随机分组， 并开始口服濯胃给药。 对照组 给予生理盐水， 其余各组分别给予相应实施例中的药物。 1.3 Experimental methods: Human tumor cells cultured in vitro were inoculated into the armpits of one side of NIHnu/nu mice. After 2-3 passages, the tumors were subcultured under the armpits and cut into small pieces of 1.5 legs and 3 pieces. The mice were subcutaneously on one side of the armpits. After the tumors were grown to 100 to 300 legs 3, they were randomly grouped according to tumor size, and oral administration was started. The control group was given physiological saline, and the other groups were administered with the drugs in the respective examples.
1.3. 1短期观察的实验于第 17天处死棵鼠称体重， 分离肿 瘤称瘤重， 计算抑制率。  1.3. 1 Short-term observation experiment The rats were sacrificed on the 17th day, and the tumors were weighed and the inhibition rate was calculated.
1.3.2长期观察的实验期间， 每周测量 2次肿瘤的长径 a和 短径 b, 并记录动物体重。 以公式 V= ab2/2计算瘤体积， 绘制肿 瘤生长曲线， 计算抑瘤率。 1.3.2 During the long-term observation period, the long diameter a and the short diameter b of the tumor were measured twice a week, and the animal body weight was recorded. The tumor volume was calculated by the formula V = ab 2 /2, the tumor growth curve was drawn, and the tumor inhibition rate was calculated.
2.结果： 结果见表 14- 23、 Fig.2 - 8。  2. Results: The results are shown in Tables 14-23 and Fig. 2-8.
2.1 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人肝癌 BEL- 7402的肿瘤生长抑制作用。  2.1 The combination of dipyridamole, umbrel and dexamethasone inhibited tumor growth in transplanted human hepatoma BEL-7402.
2.1.1 短期观察： 双嘧达莫、 乌苯美司与地塞米松合用用于 治疗棵鼠移植的人肝癌 BEL- 7402, 实验结果表明： 三者合用对棵 鼠移植的人肝癌 BEL- 7402肿瘤生长有显著抑瘤作用，且呈明显的 量效关系。 结果见表 14。 表 14: 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植的人 肝癌 BEL- 7402的生长抑制作用 （ 17天） 2.1.1 Short-term observation: Dipyridamole, umbrel and dexamethasone are used in the treatment of human liver cancer BEL-7402, and the results of the experiment show that: the three combined human liver cancer BEL-7402 Tumor growth has a significant anti-tumor effect, and it has a significant dose-effect relationship. The results are shown in Table 14. Table 14: Growth inhibition of human hepatocellular carcinoma BEL-7402 transplanted with dipyridamole, umbrel and dexamethasone (17 days)
Figure imgf000026_0001
Figure imgf000026_0001
接种 7天给药， 每天一次， 共 10次， 17天处死。 与对照组 比较 *P<0. 01。  The inoculation was administered for 7 days, once a day for 10 times and sacrificed for 17 days. Compared with the control group *P<0.01.
2. 1. 2 长期观察： 双嘧达莫、 乌苯美司与地塞米松合用用于 治疗棵鼠移植的人肝癌 BEL- 7402， 实验结果表明： 三者合用对棵 鼠移植的人肝癌 BEL- 7402肿瘤生长有非常显著抑制作用，且呈明 显的量效关系。作用持久，对实验动物体重无影响。结果见表 15， Fig. 2。  2. 1. 2 Long-term observation: Dipyridamole, umbrel and dexamethasone are used in the treatment of human liver cancer BEL-7402. The results of the experiment show that: the three combined human liver cancer BEL transplanted to the mouse - 7402 tumor growth has a very significant inhibitory effect, and has a significant dose-effect relationship. Long-lasting effect, no effect on the weight of experimental animals. The results are shown in Table 15, Fig. 2.
表 15: 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人肝 癌 BEL- 7402的生长抑制作用 （ 17d )  Table 15: Growth inhibition of dipyridamole, umbrel and dexamethasone on transplanted human liver cancer BEL-7402 (17d)
Figure imgf000026_0002
Figure imgf000026_0002
接种 7天给药， 每天一次， 共 10次， 观察 60天。 N=6， 60 天时各组动物未有死亡。 与对照组比较 *P<0. 01。  The inoculation was administered for 7 days, once a day for 10 times, and observed for 60 days. N=6, 60 days, no animals died in each group. Compared with the control group *P<0.01.
Fig. 2 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人肝 癌 BEL- 7402的肿瘤生长抑制作用。 Fig. 2 Dipyridamole, Ubumex and Dexamethasone for transplantation of human liver Tumor growth inhibition of cancer BEL-7402.
2. 2 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人肝癌 HepG2的肿瘤生长抑制作用。  2. 2 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human hepatoma HepG2.
长期观察： 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植 人肝癌 HepG2的肿瘤生长有非常显著的抑制作用， 每周连续给药 5次， 共 3周， 第 28天处理， 抑瘤率可达 93. 7%, , 且呈明显的 量效关系， 疗效优于氟尿嘧啶。 结果见表 16 , Fig. 3。  Long-term observation: The combination of dipyridamole, umbrel and dexamethasone has a very significant inhibitory effect on the tumor growth of transplanted human hepatoma HepG2, which is administered 5 times a week for 3 weeks, 28 days. The tumor inhibition rate can reach 93.7%, and it has obvious dose-effect relationship, and the curative effect is better than that of fluorouracil. The results are shown in Table 16, Fig. 3.
表 16: 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人肝 癌 HepG2的生长抑制作用 （28d )  Table 16: Growth inhibition of dipyridamole, umbrel and dexamethasone on human hepatoma HepG2 transplanted (28d)
Figure imgf000027_0001
Figure imgf000027_0001
接种 7天给药， 每周连续给药 5次， 共 3周， 第 28d处理， 各组动物未有死亡。 与对照组比较 *P<0. 01。  The cells were administered for 7 days, and were administered 5 times a week for 3 weeks and 28 days, and no animals died in each group. Compared with the control group *P<0.01.
Fig. 3 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人肝 癌 HepG2的肿瘤生长抑制作用。  Fig. 3 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human hepatic carcinoma HepG2.
2. 3 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人鳞癌 A431的肿瘤生长抑制作用。  2. 3 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human squamous cell carcinoma A431.
长期观察： 双嘧达莫、 乌苯美司与地塞米松合用用于治疗棵 鼠移植的人鳞癌 A431 , 结果见表 17， Fig. 4。 实验结果表明： 三 者合用对棵鼠移植的人鳞癌 A431肿瘤生长有非常显著抑制作用， 且呈明显的量效关系， 其效果与吉非替尼相当。 作用持久， 对实 验动物体重无影响。 表 17: 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人鳞 癌 A431的生长抑制作用（17d) Long-term observation: Dipyridamole, umbrel and dexamethasone were used to treat human squamous cell carcinoma A431 transplanted in rats. The results are shown in Table 17, Fig. 4. The results showed that the combination of the three groups had a significant inhibitory effect on the growth of human squamous cell carcinoma A431, and it showed a dose-effect relationship. The effect was similar to that of gefitinib. Long-lasting effect, no effect on the weight of experimental animals. Table 17: Inhibition of growth of human squamous cell carcinoma A431 by dipyridamole, umbrel and dexamethasone (17d)
Figure imgf000028_0001
Figure imgf000028_0001
接种 7天给药，每天给药一次，共 10次。观察至 35天。 N=6， 35天时各组动物未有死亡。 与对照组比较 *P<0. 05。  The inoculation was administered for 7 days and administered once a day for 10 times. Observed until 35 days. N=6, 35 days, no deaths in each group of animals. Compared with the control group *P<0.05.
Fig. 4 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人鳞 癌 A431的肿瘤生长抑制作用。  Fig. 4 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human squamous cell carcinoma A431.
2. 4 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人肺癌 PG的肿瘤生长抑制作用。  2. 4 The effect of dipyridamole, umbrel and dexamethasone on tumor growth inhibition of transplanted human lung cancer PG.
长期观察： 双嘧达莫、 乌苯美司与地塞米松合用用于治疗棵 鼠移植的人肺癌 PG, 结果见表 18， Fig. 5。 实验结果表明： 三者 合用对棵鼠移植的人肺癌 PG肿瘤生长有非常显著抑制作用，且呈 明显的量效关系， 其效果优于吉西他滨。  Long-term observation: Dipyridamole, umbrel and dexamethasone were used in the treatment of human lung cancer PG transplanted in rats. The results are shown in Table 18, Fig. 5. The results showed that the combination of the three drugs had a significant inhibitory effect on the growth of human lung cancer PG tumors, and it showed a significant dose-effect relationship, which was superior to gemcitabine.
表 18: 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人肺 癌 PG的肿瘤生长抑制作用（17d)  Table 18: Inhibition of tumor growth by transplanting human lung cancer PG with dipyridamole, umbrel and dexamethasone (17d)
Figure imgf000028_0002
Figure imgf000028_0002
接种 7天给药， 每天给药一次， 共 10次， 吉西他滨分别于第 7， 10, 13天给药共 3次。 观察至 35天。 N=6， 35天时各组动物 未有死亡。 与对照组比较 *P<0. 05。 Fig. 5 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人肺 癌 PG的肿瘤生长抑制作用。 The cells were administered for 7 days, once a day for 10 times, and gemcitabine was administered 3 times on days 7, 10, and 13, respectively. Observed until 35 days. N=6, 35 days, no deaths in each group of animals. Compared with the control group *P<0.05. Fig. 5 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human lung cancer PG.
2. 5 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人肺癌 A549的肿瘤生长抑制作用。  2. 5 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human lung cancer A549.
2. 5. 1短期观察： 三者合用对棵鼠移植的人肺癌 A549肿瘤生 长有非常显著抑制作用， 其效果优于吉西他滨。 结果见表 19。  2. 5. 1 Short-term observation: The combination of the three drugs has a very significant inhibitory effect on the growth of human lung cancer A549 tumor, which is better than gemcitabine. The results are shown in Table 19.
表 19: 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植的人 肺癌 A549的生长抑制作用 （17 d )  Table 19: Growth inhibition of lung cancer A549 in mice transplanted with dipyridamole, umbrel and dexamethasone (17 d )
Figure imgf000029_0001
Figure imgf000029_0001
接种 7天给药， 每天一次， 共 10次， 吉西他滨分别于第 7， 10， 13天给药共 3次。 17天处死。 与对照组比较 *Ρ<0. 01。  The drug was administered for 7 days, once a day for 10 times, and gemcitabine was administered 3 times on days 7, 10, and 13, respectively. He died in 17 days. Compared with the control group *Ρ<0. 01.
2. 5. 2长期观察： 双嘧达莫、 乌苯美司与地塞米松合用对棵 鼠移植人肺癌 Α549的肿瘤生长有显著的抑制作用，每周连续给药 5次， 共 3周， 第 28d处理。 结果见表 20， Fig. 6。 实验结果表明： 三者合用对棵鼠移植的人肺癌 A549 肿瘤生长有非常显著抑制作 用， 且呈明显的量效关系， 其效果优于吉西他滨。  2. 5. 2 Long-term observation: The combination of dipyridamole, umbrel and dexamethasone significantly inhibited the growth of tumor in human lung cancer Α549, which was administered 5 times a week for 3 weeks. Processing on page 28d. The results are shown in Table 20, Fig. 6. The results showed that the combination of the three drugs had a significant inhibitory effect on the growth of human lung cancer A549 tumors, and showed a significant dose-effect relationship, which was superior to gemcitabine.
表 20: 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人肺 癌 A549的肿瘤生长抑制作用 （28 d ) Table 20: Inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human lung cancer A549 (28 d)
剂量 动物数 体重变化 瘤重（g ) 组别 抑瘤率 ¾ Dosage Number of animals Weight change Tumor weight (g) Group Tumor inhibition rate 3⁄4
( mg/kg ) 开始 /结束 ( g ) 士 SD 对照 - 6/6 + 1. 31 0. 37 ± 0. 15 - 吉西他滨 200 6/6 + 0. 47 0. 13 ± 0. 11  ( mg/kg ) start / end ( g ) SD control - 6/6 + 1. 31 0. 37 ± 0. 15 - gemcitabine 200 6/6 + 0. 47 0. 13 ± 0. 11
DPM+BEN+DEX 100+20+1 6/6 -0. 54 0. 04 ± 0. 03 89. 5%** DPM+BEN+DEX 100+20+1 6/6 -0. 54 0. 04 ± 0. 03 89. 5%**
DPM+BEN+DEX 200+40+2 6/6 -0. 02 0. 02 ± 0. 01 94. 9%**DPM+BEN+DEX 200+40+2 6/6 -0. 02 0. 02 ± 0. 01 94. 9%**
DPM+BEN+DEX 400+80+4 6/6 -0. 93 0. 01 ± 0. 01 96. 9%** 肿瘤接种后 7天开始给药， DPM+BEN+DEX每周连续给药 5次， 共 3周； 吉西他滨于每周一腹腔注射给药一次， 共三次。 28天处 理。 与对照组比较 *P<0. 05 **P<0. 01 DPM+BEN+DEX 400+80+4 6/6 -0. 93 0. 01 ± 0. 01 96. 9%** Dosing is started 7 days after tumor inoculation, DPM+BEN+DEX is administered continuously 5 times a week. Times, 3 weeks; Gemcitabine was administered once a week intraperitoneally, three times in total. 28 days of processing. Compared with the control group *P<0. 05 **P<0. 01
Fig. 6 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人肺 癌 A549的肿瘤生长抑制作用。  Fig. 6 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human lung cancer A549.
2. 6 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人胰腺 MPAC的肿瘤生长抑制作用。  2. 6 The combination of dipyridamole, umbrel and dexamethasone inhibited tumor growth of transplanted human pancreas MPAC.
2. 6. 1短期观察： 三者合用对棵鼠移植的人胰腺 MPAC肿瘤生 长有非常显著抑制作用， 其效果优于卡培他滨。 结果见表 21 表 21: 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植的人 胰腺 MPAC的生长抑制作用 （17 d )  2. 6. 1 Short-term observation: The combination of the three drugs has a very significant inhibitory effect on the growth of pancreatic MPAC tumors in transplanted mice, and its effect is better than that of capecitabine. The results are shown in Table 21. Table 21: Growth inhibition of pancreatic MPAC in humans transplanted with dipyridamole, umbrel and dexamethasone (17 d )
Figure imgf000030_0001
Figure imgf000030_0001
接种 7天给药， 每天一次， 共 10次， 卡培他滨给药方式同 DPM+BEN+DEX, 17天处死。 与对照组比较 *P<0. 01  The drug was administered for 7 days, once a day for 10 times, and the capecitabine was administered in the same manner as DPM+BEN+DEX, and was sacrificed at 17 days. Compared with the control group *P<0. 01
2. 6. 2长期观察： 双嘧达莫、 乌苯美司与地塞米松合用对棵 鼠移植人胰腺癌 MPAC的肿瘤生长有显著的抑制作用，每周连续给 药 5次， 共 3周， 第 28d处理。 结果见表 22 , Fig. 7。 结果表明， 三者合用对棵鼠移植的人胰腺癌 MPAC 肿瘤生长有非常显著抑制 作用， 且呈明显的量效关系， 疗效优于卡培他滨。 2. 6. 2 Long-term observation: The combination of dipyridamole, umbrel and dexamethasone has a significant inhibitory effect on the tumor growth of transplanted human pancreatic cancer MPAC, which is given continuously every week. The drug was taken 5 times for 3 weeks and processed on the 28th day. The results are shown in Table 22, Fig. 7. The results showed that the combination of the three drugs had a significant inhibitory effect on the growth of human pancreatic cancer MPAC tumors, and showed a significant dose-effect relationship. The curative effect was better than that of capecitabine.
表 22: 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人胰 腺癌 MPAC的肿瘤生长抑制作用（28d)  Table 22: Tumor growth inhibition effect of dipyridamole, umbrel and dexamethasone on transplanted human pancreatic adenocarcinoma MPAC (28d)
Figure imgf000031_0001
Figure imgf000031_0001
肿瘤接种后 7天开始给药， DPM+BEN+DEX每周连续给药 5次， 共 3 周； 卡培他滨给药方式同 DPM+BEN+DEX。 与对照组比较 *P<0. 01。  Dosing was started 7 days after tumor inoculation. DPM+BEN+DEX was administered 5 times a week for 3 weeks; capecitabine was administered in the same manner as DPM+BEN+DEX. Compared with the control group *P<0.01.
Fig. 7 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人胰 腺 MPAC的肿瘤生长抑制作用。  Fig. 7 The inhibitory effect of dipyridamole, umbrel and dexamethasone on tumor growth of transplanted human pancreatic MPAC.
2. 7 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人胰腺 SW1990的肿瘤生长抑制作用。  2. 7 Inhibition of tumor growth by transplanting human pancreas SW1990 with dipyridamole, umbrel and dexamethasone.
长期观察双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人 胰腺癌 SW1990 的肿瘤生长有显著的抑制作用， 每周连续给药 5 次， 共 3周， 第 35天处理。 结果见表 23， Fig. 8。 结果表明， 三 者合用对棵鼠移植的人胰腺癌 SW1990 肿瘤生长有非常显著抑制 作用， 且呈明显的量效关系， 疗效优于卡培他滨。  Long-term observation of dipyridamole, umbrel and dexamethasone significantly inhibited tumor growth in transplanted human pancreatic cancer SW1990, and was administered 5 times a week for 3 weeks and 35 days. The results are shown in Table 23, Fig. 8. The results showed that the combination of the three had a significant inhibitory effect on the growth of human pancreatic cancer SW1990, and it showed a significant dose-effect relationship. The curative effect was better than that of capecitabine.
表 23: 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人胰 腺癌 SW1990的肿瘤生长抑制作用（35d) 剂量 动物数 体重变化 瘤重（g ) 组别 抑瘤率％ ( mg/kg ) 开始 /结束 ( g ) 士 SD Table 23: Tumor growth inhibition effect of dipyridamole, umbrel and dexamethasone on transplanted human pancreatic cancer SW1990 (35d) Dosage Animals Body Weight Change Tumor Weight (g) Group Tumor Suppression Rate % (mg/kg) Start/End (g) SSD
对照 - 6 /6 +0. 62 0. 84 ± 0. 27 - 卡培他滨 250 6 /6 +0. 25 0. 71 ± 0. 61 15. 3¾ Control - 6 /6 +0. 62 0. 84 ± 0. 27 - Capecitabine 250 6 /6 +0. 25 0. 71 ± 0. 61 15. 33⁄4
DPM+BEN+DEX 100+20+1 6 /6 -0. 63 0. 62 ± 0. 40 26. 4¾DPM+BEN+DEX 100+20+1 6 /6 -0. 63 0. 62 ± 0. 40 26. 43⁄4
DPM+BEN+DEX 200+40+2 6 /6 - 0. 7 0. 41 ± 0. 17 DPM+BEN+DEX 200+40+2 6 /6 - 0. 7 0. 41 ± 0. 17
DPM+BEN+DEX 400+80+4 6 /6 -0. 83 0. 25 ± 0. 06 70. 8¾* 肿瘤接种后 7天开始给药， DPM+BEN+DEX每周连续给药 5次， 共 3 周； 卡培他滨给药方式同 DPM+BEN+DEX。 与对照组比较 *P<0. 01。  DPM+BEN+DEX 400+80+4 6 /6 -0. 83 0. 25 ± 0. 06 70. 83⁄4* The drug is administered 7 days after the tumor is inoculated, and DPM+BEN+DEX is administered 5 times a week. A total of 3 weeks; capecitabine administration is the same as DPM+BEN+DEX. Compared with the control group *P<0.01.
Fi g. 8 双嘧达莫、 乌苯美司与地塞米松合用对棵鼠移植人胰 腺 SW1990的肿瘤生长抑制作用。 实施例 8: 病理组织检查  Fi g. 8 The effect of dipyridamole, umbrel and dexamethasone on tumor growth inhibition of transplanted human pancreatic gland SW1990. Example 8: Pathological examination
使用对棵鼠移植人肝癌 HepG2肿瘤生长有显著抑制作用的双 嘧达莫、 乌苯美司与地塞米松合用剂量（选自实施例 4的制备的 药物组合物样品， 其中 DPM: BEN: DEX=400: 80: 4 时的抑瘤率 93. 7%)， 取主要脏器进行病理组织检查， 并对棵鼠骨髓有核细胞 进行计数。 结果见表 24， Fi g. 9。 结果表明， 心、肺、 肝、 脾、 胃、 小肠、 腎以及股骨 （骨髓）等主要脏器未见明显的病理学改变， 骨髓有核细胞相对于未接种肿瘤的正常棵鼠数量相当， 无显著差 异。 由此可见： 双嘧达莫、 乌苯美司与地塞米松合用未导致出现 细胞毒类药物的毒性变化， 表明组合物具有较好的安全性。  A dose of dipyridamole, umbrel and dexamethasone used in a significant inhibition of tumor growth of human hepatoma HepG2 transplanted to a mouse (selected from a sample of the pharmaceutical composition prepared in Example 4, wherein DPM: BEN: DEX =400: At 80:4, the tumor inhibition rate was 93.7%). The main organs were taken for pathological examination, and the bone marrow nucleated cells of the rats were counted. The results are shown in Table 24, Fi g. 9. The results showed that there were no obvious pathological changes in the main organs such as heart, lung, liver, spleen, stomach, small intestine, kidney and femur (bone marrow). The number of bone marrow nucleated cells was comparable to that of normal mice without tumor inoculation. Significant differences. It can be seen that the combination of dipyridamole, umbrel and dexamethasone did not lead to toxicity changes of cytotoxic drugs, indicating that the composition has good safety.
Fi g. 9 双嘧达莫、乌苯美司与地塞米松合用对荷人肝癌 HepG2 棵鼠的各器官组织的病理学检查的病理切片。  Fi g. 9 Pathological sections of pathological examination of various organ tissues of HepG2 mice bearing dipyridamole, umbrel and dexamethasone.
表 24:双嘧达莫、乌苯美司与地塞米松合用对荷人肝癌 HepG2 棵鼠骨髓的有核细胞的影响 (病理组织检查， 每个视野平均细胞数） Table 24: Effect of dipyridamole, umbrel and dexamethasone on nucleated cells of bone marrow of human hepatoma HepG2 mice (pathological examination, average number of cells per field of view)
Figure imgf000033_0001
实施例 9: 本发明的抗肿瘤作用的机制研究
Figure imgf000033_0001
Example 9: Study on the mechanism of anti-tumor effect of the present invention
采用 Western blot检测肿瘤组织蛋白的表达变化。  Western blot was used to detect the expression changes of tumor tissue proteins.
1. 蛋白样品的制备  1. Preparation of protein samples
取小鼠肝癌 H22以及人肝癌 Be卜 7402棵鼠移植瘤新鲜瘤组织， 对照组以及受试药物双嘧达莫、 乌苯美司与地塞米松合用组（受 试 药 物 为 实施例 4 的 制 备的 药 物组合物样品 ， DPM: BEN: DEX=100: 20: 1 )各 5份， 生理盐水洗去血迹， - 70。C冻 存。提取蛋白时，取瘤组织按 1: 9加入裂解液（50mMTris · CI (pH 8.0)， 150 mM NaCl, 0.1% SDS， 1% NP-40, 0.5%去氧胆酸钠， 100 μ g /ml PMSF, lug/ml Aprotinin, 0.02%叠氮钠） ， 低温 匀浆， 4。C裂解 l h后， 10000 rpm 4。C离心 15 min， 收集上清液， 用 BCA试剂盒定量， 与适量 5X上样緩冲液混合， 沸水浴 5 min 变性蛋白， - 70eC存放。 Take mouse liver cancer H 22 and human liver cancer Be 7402 mouse transplant tumor fresh tumor tissue, control group and test drug dipyridamole, umbrel and dexamethasone combination group (test drug is Example 4 A sample of the prepared pharmaceutical composition, DPM: BEN: DEX = 100: 20: 1 ) 5 parts each, physiological saline was washed away, - 70. C frozen. When extracting the protein, the tumor tissue was added 1:9 to the lysate (50 mM Tris · CI (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 100 μg / ml PMSF, lug/ml Aprotinin, 0.02% sodium azide), low temperature homogenate, 4. After C is cleaved for 1 h, 10000 rpm 4 . C centrifuge for 15 min, collect the supernatant, quantify with BCA kit, mix with appropriate amount of 5X loading buffer, denatured protein in boiling water bath for 5 min, store at -70 e C.
2. Western检测肿瘤组织蛋白的表达变化  2. Western detection of tumor tissue protein expression changes
配制 10%的分离胶和 5%的浓缩胶， 濯制胶板。 将给药组和对 照组各 5只的蛋白等量混合， 以 50 ug的总蛋白量上样， 进行 SDS-PAGE电泳分析。 电泳完后进行转膜， 一抗孵育 2 h， 碱性磷 酸酶标记的二抗孵育 1 h， NBT/BCIP显色。 结果见 Fig.10。  Prepare 10% separation gel and 5% concentrated gel, and knead the rubber sheet. The protein of each of the drug-administered group and the control group was mixed in equal amounts, and the total amount of protein was loaded at 50 ug, and subjected to SDS-PAGE electrophoresis analysis. After electrophoresis, the membrane was transfected, the primary antibody was incubated for 2 h, and the alkaline phosphatase-labeled secondary antibody was incubated for 1 h, and NBT/BCIP was developed. The result is shown in Fig. 10.
结果显示 FLK1 和 N0S3的表达下降， 其他检测的蛋白： EGF、 VEGF、 TGF、 Be卜 2、 k- Ras、 P27、 P21、 NF-kB变化不明显。 FLK1 与血管生成密切相关， N0S 与炎症有密切的关系， 且在肿瘤的发 生 发展 过程 中 粉 演 重 要 角 色 （ Cancer Chemother Pharmacol. 2011, 67 (6)： 1211-24 ) , 本研究表明双嘧达莫、 乌苯 美司与地塞米松这一药物组合物可能影响了肿瘤的血管生成且与 炎症反应相关。 The results showed that the expression of FLK1 and N0S3 decreased, and other detected proteins: EGF, The changes of VEGF, TGF, Beb2, k-Ras, P27, P21 and NF-kB were not obvious. FLK1 is closely related to angiogenesis. N0S is closely related to inflammation and plays an important role in the development of tumors (Clinical Chemother Pharmacol. 2011, 67 (6): 1211-24). This study shows dipyridamole. The pharmaceutical composition of molybdenum, umbrel and dexamethasone may affect tumor angiogenesis and be associated with inflammatory responses.
Fig. 10 Wes tern blot检测药物对鼠肝癌 H22小鼠移植瘤组织 蛋白表达的影响。 Fig. 10 Wes tern blot was used to detect the effect of drugs on the expression of tissue protein in transplanted tumor of mouse liver cancer H 22 mice.
3. 肿瘤组织蛋白质组学研究 （双向电泳技术） ： 双向电泳检 测双嘧达莫、 乌苯美司与地塞米松合用组对人肝癌 Be卜 7402棵鼠 移植瘤组织蛋白表达的影响。  3. Tumor tissue proteomics study (two-dimensional electrophoresis): Two-dimensional electrophoresis was used to detect the effect of dipyridamole, umbrel and dexamethasone on the expression of human hepatocarcinoma in the transplanted tumor tissue of human liver cancer.
通过预冷后的金属砸样器， 液氮研磨样品。 TCA丙酮方法提取 肿瘤组织的全蛋白并去除盐和杂质。 将提取好的样品全蛋白通过 Bradford定量，每个样品取等量蛋白， 进行一向 IEF等电聚焦（胶 条： PH3 - 10 ) , 分别用 1% DTT和 2. 5%IAM的平衡液各平衡 15min， 去出胶条转入二向凝胶， 用 24cm (胶浓度 12. 5% )的胶跑垂直板聚 丙烯酰胺凝胶电泳。 下胶， 固定 30min，致敏 30min, 水洗 3 (每次 lOmin )，银染， 水洗 2次（每次 lmin ) , 显色， 终止。  The sample was ground with liquid nitrogen through a pre-cooled metal sampler. The TCA acetone method extracts whole proteins from tumor tissues and removes salts and impurities. The extracted whole protein was quantified by Bradford, and each sample was taken with an equal amount of protein, and subjected to IEF isoelectric focusing (glue: PH3 - 10), and each was equilibrated with an equilibrium solution of 1% DTT and 2.5% IAM. 15 min, the strip was removed and transferred to a two-way gel, and the gel was run on a vertical plate polyacrylamide gel with a gel of 24 cm (adhesion of 12.5%). Glue, fixed for 30min, sensitized for 30min, washed with water 3 (each lOmin), silver stained, washed twice (each time lmin), color development, termination.
胶图扫描， 胶图分析， 每个样品三块平行胶合成一块虛拟胶， 两个样品虛拟股之间进行比对，通过 GE公司的 Imagemaster 2D 5. 0 版本进行胶图分析。  Glue scanning, glue analysis, three parallel glues for each sample were combined into a virtual glue, and the two sample virtual shares were compared and analyzed by GE's Imagemaster 2D version 5. 0.
切点， 做酶解， 上质傳鉴定（BRUKER公司的 ul traf leXMALDI T0F/T0F II ) , 选择相应数据库进行搜索  Cut point, do enzymatic hydrolysis, quality identification (BRUKER's ul traf leXMALDI T0F/T0F II), select the appropriate database to search
双向电泳结果见 Fig. 11。 Fig. 11双向电泳检测药物对人肝癌 Bel-7402棵鼠移植瘤组织蛋白表达的影响。 图中显示总共 17个变 化点： 4个 3倍以上变化； 13个有或无， 其中 12个下调， 1个上 结果分析结果见表 25, 表 26 The results of two-dimensional electrophoresis are shown in Fig. 11. Fig. 11 Two-dimensional electrophoresis detection of drug on the expression of human hepatoma Bel-7402 mouse transplanted tumor tissue protein. The figure shows a total of 17 change points: 4 more than 3 times change; 13 with or without, 12 of which are down, 1 on The results of the analysis are shown in Table 25, Table 26
结果表明，双嘧达莫、 乌苯美司与地塞米松组合物可能通过影 L管生成及炎症反应， 从而影响肿瘤的微环境而发挥抗肿瘤活 双嘧达莫、 乌苯美司与地塞米松组合物对瘤组织存在广泛影 蛋白质代谢、核 ί苷代谢等，通过多方面的影响发挥抗肿瘤作用。  The results showed that the combination of dipyridamole, umbrel and dexamethasone may exert anti-tumor activity of dipyridamole, umbrel and ground through the formation of L-tube and inflammatory reaction, thereby affecting the microenvironment of the tumor. The dexamethasone composition has a wide range of effects on protein metabolism, nucleoside metabolism, etc., and exerts anti-tumor effects through various effects.
表 25: 三倍以上变化及有或无的蛋白。 Table 25: Proteins with more than three times the change and with or without.
¾^ ·¾ΐ!¾^ ^^i >¾S J¾ ¾L l JiL 3⁄4^ ·3⁄4ΐ!3⁄4^ ^^i >3⁄4S J3⁄4 3⁄4L l JiL
T r : r . SS ST nr i 1Έ . tP ― T r : r . SS ST nr i 1Έ . tP ―
ί: m , c¾ Ϊ: 4 3¾ .5  :: m , c3⁄4 Ϊ: 4 33⁄4 .5
a? (ϊ' : a? (ϊ' :
55
¾ "55 5
Figure imgf000036_0001
3⁄4 "55 5
Figure imgf000036_0001
Figure imgf000036_0002
Figure imgf000036_0002
实施例 10: ： 双嘧达莫、 乌苯美司与地塞米松组成的抗肿瘤 新组合物的小鼠急性毒性试验 Example 10: Acute toxicity test in mice with anti-tumor composition composed of dipyridamole, umbrel and dexamethasone
1材料与方法  1 Materials and methods
1.1 药品与试剂： 选自受试药物为实施例 4的制备的药物组 合物样品， 其中 DPM:BEN:DEX=100:20:1。 双嘧达莫： 标准品， 购 自中国药品生物制品检定所； 乌苯美司： 原料药， 浙江普洛康裕 制药有限公司； 地塞米松： 标准品， 购自中国药品生物制品检定 所。 1.1 Drugs and Reagents: A sample of the pharmaceutical composition selected from the test drug prepared in Example 4, wherein DPM: BEN: DEX = 100: 20:1. Dipyridamole: Standard, Purchase From China National Institute for the Control of Pharmaceutical and Biological Products; Ubumex: API, Zhejiang Plo Kangyu Pharmaceutical Co., Ltd.; Dexamethasone: Standard, purchased from China National Institute for the Control of Pharmaceutical and Biological Products.
1. 2动物： 雌雄各半， 6 ~ 8周龄， 体重 18 ~ 22 g , 由军事医 学科学院实验动物中心提供， 许可证编号 SCXK (军） 2007-004。 给药前驯养观察 2天， 观察动物的一般状态， 行为活动， 毛色。 测定体重等检查均未见明显异常。  1. 2 Animals: Male and female, 6-8 weeks old, weighing 18-22 g, provided by the Experimental Animal Center of the Academy of Military Medical Sciences, license number SCXK (Army) 2007-004. The animals were observed for 2 days before administration, and the general state of the animals, behavioral activities, and coat color were observed. No significant abnormalities were observed in the examinations such as measuring body weight.
1. 3实验方法： 昆明种小鼠 60只， 分 3个剂量组， 每组 20 只，雌雄各半。各组差比为 0. 8 ,—次口服给药，给药剂量 2. O g/kg , 1. 6 g/kg , 1. 28g/kg , 濯胃给药后观察 14天并记录毒性反应包括 一般指标、 死亡情况、 体重变化， 测定小鼠的毒性反应、 死亡情 况及计算半数致死量等， 实验结束时进行病理学检查。  1. 3 Experimental methods: 60 Kunming mice were divided into 3 dose groups, 20 in each group, half male and half female. The difference ratio of each group was 0.8, the oral administration, the dose was 2. O g / kg, 1. 6 g / kg, 1. 28g / kg, observed for 14 days after the stomach administration and recorded the toxicity Including general indicators, death, weight changes, determination of toxicity, death, and calculation of median lethal dose in mice, pathological examination at the end of the experiment.
2.结果： 小鼠未出的中毒表现， 一般指标均正常， 未出现死 亡、体重减轻及其他症状。给药后第 14天处死前体重增长至 28 ~ 38克左右， 外观健康。 实验结束时进行病理学检查， 动物的肝、 肾、 肺等主要脏器未见有明显病理变化。 结果表明， 抗肿瘤新组 合物毒性很低， 尤其是口服给药毒性小， 雌雄动物不存在显著差 异。 小鼠口服的 LD50 (半数致死量） 为大于 2g/kg。 通过急毒性 试验， 证明在很高的剂量下 （小鼠 2. Og/kg ) , 口服给药未见动 物死亡，动物也未见消瘦或其他症状，动物各项指示均未见异常， 可见其口服给药是安全的。 尽管本发明的具体实施方式已经得到详细的描述， 本领域技 术人员将会理解。 根据已经公开的所有教导， 可以对那些细节进 行各种修改和替换， 这些改变均在本发明的保护范围之内。 本发 明的全部范围由所附权利要求及其任何等同物给出。  2. Results: The symptoms of poisoning in the mice were normal, and the general indicators were normal. There were no deaths, weight loss and other symptoms. The body weight increased to 28 ~ 38 grams before the death on the 14th day after administration, and the appearance was healthy. Pathological examination was performed at the end of the experiment, and no obvious pathological changes were observed in the liver, kidney, lung and other major organs of the animals. The results showed that the anti-tumor new composition was very low in toxicity, especially for oral administration, and there was no significant difference between male and female animals. The oral LD50 (half lethal dose) of mice is greater than 2 g/kg. Through the acute toxicity test, it was proved that at a very high dose (mouse 2. Og/kg), no animal died after oral administration, and no signs of weight loss or other symptoms were observed in the animals. No abnormalities were observed in the animals. Oral administration is safe. Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions may be made to those details in light of the teachings of the invention, which are within the scope of the invention. The full scope of the invention is indicated by the appended claims and any equivalents thereof.

Claims

权利要求 Rights request
1. 一种药物组合物， 其包含如下的 A、 B和 C三种成分中的 两种或三种： A pharmaceutical composition comprising two or three of the following three components A, B and C:
A.双嘧达莫和 /或其可药用衍生物；  A. dipyridamole and / or a pharmaceutically acceptable derivative thereof;
B.乌苯美司和 /或其可药用衍生物；  B. Ubumex and / or its pharmaceutically acceptable derivatives;
C.地塞米松和 /或其可药用衍生物；  C. Dexamethasone and / or a pharmaceutically acceptable derivative thereof;
可选地， 所述药物组合物还包含药学上可接受的载体或辅料。 Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or adjuvant.
2. 根据权利要求 1所述的药物组合物， 其中， 所述双嘧达莫 的可药用衍生物选自单哌潘生丁（mopidamole ) 、 BIBW BS以及 RA25中的一种或多种。  The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable derivative of dipyridamole is one or more selected from the group consisting of mopidamole, BIBW BS and RA25.
3. 根据权利要求 1所述的药物组合物， 其中， 所述乌苯美司 的可药用衍生物为 AHPA-Val和 /或 Bes tat in Hydrochlor ide。 The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable derivative of umbrel is AHPA-Val and/or Bes tat in Hydrochloride.
4. 根据权利要求 1所述的药物组合物， 其中， 所述地塞米松 的可药用衍生物选自醋酸地塞米松、 地塞米松磷酸钠、 地塞米松 椋榈酸钠、 氢化可的松、 可的松、 强的松、 强的松龙、 甲基强的 松龙、 去炎松、 以及倍他米松中的一种或多种。 The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable derivative of dexamethasone is selected from the group consisting of dexamethasone acetate, dexamethasone sodium phosphate, dexamethasone sodium sulphate, and hydrogenated One or more of pine, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, and betamethasone.
5. 根据权利要求 1至 4中任一项所述的药物组合物， 其特征 在于如下的 （1 ) - ( 13 ) 中的任一项或多项：  The pharmaceutical composition according to any one of claims 1 to 4, which is characterized by any one or more of the following (1) - (13):
( 1 )含有 A和 B, 重量比 A： B = 0. 001 ~ 200： 1 ;  (1) Containing A and B, weight ratio A: B = 0. 001 ~ 200: 1 ;
( 2 )含有 A和 B, 重量比 A： B = 0. 025 ~ 22. 5： 1 ;  (2) Containing A and B, weight ratio A: B = 0. 025 ~ 22. 5: 1 ;
( 3 )含有 B和 C, 重量比 B： C = 0. 001 ~ 3000： 1 ;  (3) Containing B and C, weight ratio B: C = 0. 001 ~ 3000: 1 ;
( 4 )含有 B和 C, 重量比 B： C = 0. 5 ~ 450： 1;  (4) Containing B and C, weight ratio B: C = 0. 5 ~ 450: 1;
( 5 )含有 A和 C, 重量比 A： C = 1： 0. 00001 ~ 100;  (5) Containing A and C, weight ratio A: C = 1: 0. 00001 ~ 100;
( 6 )含有 A和 C, 重量比 A： C = 1： 0. 0005 ~ 0. 05;  (6) Containing A and C, weight ratio A: C = 1: 0. 0005 ~ 0. 05;
( 7 )含有 A、 B 和 C, 重量比 A： B： C = 1 ： 0. 00001 ~ 32000： (7) Contains A, B and C, weight ratio A: B: C = 1 : 0. 00001 ~ 32000:
0.00001 ~ 1600; 0.00001 ~ 1600;
( 8)含有 A、B和 C,重量比 A : B : C = 1： 0· 001 ~ 1000： 0.0001 10;  (8) Containing A, B and C, weight ratio A : B : C = 1: 0· 001 ~ 1000: 0.0001 10;
(9)含有 A、 B和 C, 重量比 A： B： C = 50 - 150： 20： 1;  (9) Containing A, B and C, weight ratio A: B: C = 50 - 150: 20: 1;
( 10)含有 A、 B和 C, 重量比 A： B： C= 100： 10-40： 1; (10) Containing A, B and C, weight ratio A: B: C= 100: 10-40: 1;
( 11 )含有 A、 B和 C, 重量比 A： B： C = 200： 40： 1 ~ 3; (11) contains A, B and C, weight ratio A: B: C = 200: 40: 1 ~ 3;
(12)含有入、 B和 C, 重量比 A： B： C=100~ 400： 20 - 80： 1 3;  (12) Inclusion, B and C, weight ratio A: B: C=100~ 400: 20 - 80: 1 3;
( 13)含有 A、 B和 C, 重量比 A： B： C= 100： 20： 1。  (13) Contains A, B and C, weight ratio A: B: C= 100: 20: 1.
6. 权利要求 1至 5中任一项所述的药物组合物在制备抗肿 药物或者在体内或体外抑制肿瘤细胞的药物或试剂中的用途。  6. Use of a pharmaceutical composition according to any one of claims 1 to 5 for the preparation of an anti-tumor drug or a medicament or agent for inhibiting tumor cells in vivo or in vitro.
7. 根据权利要求 6所述的用途， 其中， 所述肿瘤选自肝癌 鱗癌、 肺癌以及胰腺癌中的一种或多种。  The use according to claim 6, wherein the tumor is selected from one or more of liver cancer squamous cell carcinoma, lung cancer, and pancreatic cancer.
8. 一种抗肿瘤的方法， 包括给予受试者有效量的权利要求 至 5中任一项所述的药物组合物的步骤。  A method of anti-tumor comprising the step of administering to a subject an effective amount of the pharmaceutical composition according to any one of claims 5 to 5.
9. 一种在体内或体外抑制肿瘤细胞的方法， 包括使用有效 的权利要求 1至 5中任一项所述的药物组合物的步骤。  A method of inhibiting tumor cells in vivo or in vitro, comprising the step of using the pharmaceutical composition according to any one of claims 1 to 5.
10. 根据权利要求 8或 9所述的方法， 其中， 所述肿瘤选 肝癌、 鳞癌、 肺癌以及胰腺癌中的一种或多种。  The method according to claim 8 or 9, wherein the tumor is selected from one or more of liver cancer, squamous cell carcinoma, lung cancer, and pancreatic cancer.
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