WO2018184587A1 - 可利霉素及其药学上可接受的盐在制备治疗和/或预防肿瘤药物方面的应用 - Google Patents

可利霉素及其药学上可接受的盐在制备治疗和/或预防肿瘤药物方面的应用 Download PDF

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WO2018184587A1
WO2018184587A1 PCT/CN2018/082072 CN2018082072W WO2018184587A1 WO 2018184587 A1 WO2018184587 A1 WO 2018184587A1 CN 2018082072 W CN2018082072 W CN 2018082072W WO 2018184587 A1 WO2018184587 A1 WO 2018184587A1
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tumor
colimycin
group
cancer
cells
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PCT/CN2018/082072
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English (en)
French (fr)
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姜勋雷
姜勋东
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沈阳福洋医药科技有限公司
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Priority to EP18781541.0A priority Critical patent/EP3607952B1/en
Priority to MX2019011973A priority patent/MX2019011973A/es
Priority to KR1020197032624A priority patent/KR102327332B1/ko
Priority to AU2018247555A priority patent/AU2018247555B2/en
Priority to JP2019554848A priority patent/JP6959355B2/ja
Priority to RU2019135177A priority patent/RU2746047C1/ru
Priority to CN201880019117.0A priority patent/CN110545820B/zh
Priority to US16/500,967 priority patent/US11077126B2/en
Priority to CA3058935A priority patent/CA3058935A1/en
Publication of WO2018184587A1 publication Critical patent/WO2018184587A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the present invention relates to the field of pharmaceutical applications, and in particular to the use of colimycin and its pharmaceutically acceptable salts for the preparation of a medicament for the treatment and/or prevention of tumors.
  • Tumor is a common and frequently-occurring disease. It is a long-term action of various tumorigenic factors in the body and tissues of the body. The gene mutation loses its normal regulation of growth and differentiation, and causes new organisms formed by clonal abnormal proliferation and differentiation. Or a living creature. Tumors are classified into benign tumors and malignant tumors. Malignant tumors are subdivided into cancers derived from epithelial tissues, sarcomas derived from mesenchymal tissues, and carcinosarcomas. What is commonly referred to as "cancer” refers to all malignant tumors.
  • Malignant tumor is one of the most important malignant diseases that threaten human health. It is the number one cause of death in the global population. According to the latest statistics, in 2007, about 7.9 million people worldwide died of various types of cancer, accounting for 13% of the total number of deaths. More than 12 million tumor cases have been diagnosed, of which more than 72% of cancer patients and fatal cases have occurred in underdeveloped countries, and are on the rise. In 2015, the number of global cancer deaths increased to 9 million, which is expected to exceed 2030. At present, there are about 2.8 million cancer cases per year in China, and the number of deaths exceeds 400,000. It ranks first among the causes of death in various diseases in China, and it is on the rise.
  • Carrimycin clones the 4"-o-acyl-transferase of the carbomycin producing strain into the spiramycin producing strain by transgenic technology, and the oriented acylation helix 4'-OH, a novel antibiotic with a 4'-position isovalerylspiramycin as a main component formed by adding an isovaleryl side chain at the 4' position.
  • Colimycin is composed of a variety of spiramycin derivatives, the main active ingredient isopyryl spiromycin (I + II + III) total content of not less than 60%, is an acceptable pharmaceutical combination in pharmacy Things.
  • the central structure is a 16-membered lactone ring, which is formed by linking one molecule of felosamide, one molecule of carbamelamine sugar and one molecule of carbonaceous sugar. Its main components are isovalerylspiramycin I, II, III and spirochetes.
  • the difference in the structure of the prime is that the group attached to the 4' position of the carbonaceous sugar is isovaleryl rather than the hydroxyl group.
  • the drug is jointly declared by the Shenyang Tonglian and the like.
  • R H
  • R' COCH 2 CH (CH 3 ) 2 is isovaleryl spiromycin I;
  • R′ COCH 2 CH(CH 3 ) 2 is isovalerylspiramycin II;
  • R′ COCH 2 CH(CH 3 ) 2 is isovalerylspiramycin III;
  • the molecular structure contains two dimethylamine groups which are weakly basic and easily soluble in an acidic aqueous solution; and have a "negative solubility" property in which the solubility decreases with an increase in temperature. Since the main component of colimycin, isovalerylspiramycin, has a longer carbon chain at the 4' position and poor hydrophilicity, and its solubility in water is smaller than that of spiramycin and 4"-acetylspiramycin.
  • Colimycin is a white non-crystalline powder with slightly hygroscopicity, specific rotation of about -80.8 °, maximum ultraviolet absorption wavelength of 231 ⁇ 232 nm, itself with weak fluorescent chromophores, in case of concentrated sulfuric acid or hydrochloric acid It has a purple reaction and produces strong purple fluorescence with a maximum absorbance at 231 to 232 nm.
  • the drug has good lipophilicity, strong tissue penetration ability, rapid oral absorption, long body maintenance time, and sustained antibiotic effect. According to the relationship between pharmacodynamics and chemical conformation, the lipophilicity and in vivo activity of macrolide antibiotics after 4′ position acylation, the antibacterial activity and clinical therapeutic effect in vivo are significantly improved, and the stability of antibiotics in vivo. As the carbon chain of the 4" hydroxy ester grows, it is enhanced, that is, isovalerylspiramycin > butyrylspiramycin > propionylspiramycin > acetylspiramycin.
  • the preliminary in vitro and in vivo pharmacodynamic tests showed that the drug not only has good antibacterial activity against most G + bacteria, but also has certain effects on some G - bacteria.
  • the technical indicators are obviously superior to azithromycin, erythromycin, acetylspiramycin, Medicamycin, especially for Mycoplasma pneumoniae, has the strongest antibacterial activity against erythromycin-resistant bacteria, gonococcal bacteria, pneumococcus, Staphylococcus aureus, Pseudomonas aeruginosa, influenza bacillus, Haemophilus influenzae, Bacteroides fragilis Legionella, Pseudomonas aeruginosa and Clostridium perfringens also have certain antibacterial activity, and have little cross-resistance to clinical erythromycin-resistant Staphylococcus aureus.
  • Colimycin will be mainly used for the treatment of infectious diseases of Gram-positive bacteria, especially upper respiratory tract infections, and may be used for urinar
  • human breast cancer cells MCF-7 and MDA-MB-231 human hepatoma cells HepG2 or murine liver cancer cells H 22 , human non-small cell lung cancer cells A549, Lewis lung cancer cells Human large cell lung cancer H460 and H1299, human renal clear cell adenocarcinoma cell line 786-O, human renal cell adenocarcinoma cell line 769-P, human glioma cell line U251, human glioblastoma cell line A172, human tissue lymphoma cell line U937, human cervical cancer cell line HeLa, human prostate cancer cell line PC3, human pancreatic cancer cell line PANC-1, human esophageal cancer cell line TE-1, human gastric adenocarcinoma cell line SGC7901, human colon cancer cell line HT-29, human promyelocytic leukemia cell
  • the evaluation of in vitro antiproliferative activity of HL-60 against colimycin showed that the sample showed good anti-proliferative activity of HL-60
  • the present invention adopts the following technical solutions:
  • the present invention relates to the use of colimycin and its pharmaceutically acceptable salts for the preparation of a medicament for the treatment and/or prevention of tumors.
  • the tumor includes a solid tumor and a non-solid tumor.
  • the solid tumor includes a benign solid tumor and a malignant solid tumor
  • the non-solid tumor is a lymphoma or leukemia.
  • the malignant solid tumor is breast cancer, liver cancer, lung cancer, kidney cancer, brain tumor, cervical cancer, prostate cancer, pancreatic cancer, esophageal cancer, gastric cancer, colon cancer.
  • the brain tumor is a glioma or a meningioma
  • the gastric cancer is gastric adenocarcinoma.
  • the drug may be in various dosage forms made of colimycin and a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable adjuvant.
  • the medicament may also be various dosage forms made of colimycin and its pharmaceutically acceptable salts and antitumor drugs and pharmaceutically acceptable adjuvants.
  • colimycin and a pharmaceutically acceptable salt thereof can be formulated into various compound preparations together with a prior art antitumor drug such as oxaliplatin and the like with a pharmaceutically acceptable adjuvant.
  • the drug may further be a combination of a first agent containing colimycin and a pharmaceutically acceptable salt thereof and a second agent containing an antitumor drug.
  • the antitumor drug is an antitumor drug known in the prior art
  • a first agent containing colimycin and a pharmaceutically acceptable salt thereof can be used together with these antitumor agents.
  • the first agent containing colimycin and a pharmaceutically acceptable salt thereof may be used first, or the second agent containing the antitumor drug known in the prior art may be used first, or both use simultaneously.
  • the antitumor drug is a chemotherapy, a radiotherapy, a targeted therapy, and/or an immunotherapeutic drug.
  • the present invention shows that the first agent containing colamycin and its pharmaceutically acceptable salt has a good therapeutic effect on various cancers including lung cancer, liver cancer, cervical cancer, etc., and will contain colimycin and its pharmacy.
  • the first agent of the acceptable salt is contained with an antitumor drug known in the art such as cyclophosphamide, chlorambucil, ampoule, cyclohexyl nitrosourea, thiotepa, busulfan, cis chloride
  • an antitumor drug known in the art such as cyclophosphamide, chlorambucil, ampoule, cyclohexyl nitrosourea, thiotepa, busulfan, cis chloride
  • a combination of ammonia and platinum can achieve synergistic effects and have a better therapeutic effect on tumor patients.
  • Infected pathogens mainly include Gram-negative bacteria infection (45%-55%) and fungi (30%+) infections (results of throat swabs, sputum specimens, etc.), Gram Among the negative bacteria, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii were dominant. Some hospitals have even the highest fungal infection, probably because the malignant tumor itself is a wasting disease, and the radiotherapy and invasive operation inhibits the bone marrow hematopoietic function, weakens the defense ability of the body's monocyte phagocytic system and destroys the body. Immune barrier.
  • the colimycin of the invention has excellent anti-infective effect, is beneficial to help the patient to clear the infection and restore the immune function of the body, thereby achieving better therapeutic effect when combined with the anti-tumor drug.
  • the first agent containing colimycin and a pharmaceutically acceptable salt thereof is a pharmaceutically acceptable dosage form of colimycin and a pharmaceutically acceptable adjuvant, such as a tablet, a capsule, and an enteric solution. Preparation, injection, and the like.
  • the dose of the drug is 5 to 1500 mg; preferably 50 to 1000 mg; more preferably 100 to 400 mg.
  • the dose of the first agent is 5 to 1500 mg; preferably 50 to 1000 mg; more preferably 100 to 400 mg.
  • the present invention shows by experiments that colimycin and its pharmaceutically acceptable salts for human breast cancer cells MCF-7 and MDA-MB-231, human hepatoma cells HepG2 or murine liver cancer cells H 22 , human non-small cell lung cancer cells A549 , human large cell lung cancer H460 and H1299, human renal clear cell adenocarcinoma cell line 786-O, human kidney cell adenocarcinoma cell line 769-P, human glioma cell line U251, human glioblastoma cell line A172, human tissue lymphoma cell U937, human cervical cancer cell line HeLa, human prostate cancer cell line PC3, human pancreatic cancer cell line PANC-1, human esophageal cancer cell line TE-1, human gastric adenocarcinoma cell line SGC7901, human colon cancer cell line HT-29, human promyelocytic leukemia cell HL-60 showed good anti-proliferative activity, confirming that colimycin can be used for the
  • the present invention further show a pharmaceutically acceptable salt thereof and doxorubicin can benefit the human breast cancer cell line MCF-7 and MDA-MB-231, human hepatoma HepG2 cells or murine cells H 22, human small cell lung cancer by in vivo tests Cell A549, human large cell lung cancer H460 and H1299, human renal clear cell adenocarcinoma cell line 786-O, human renal cell adenocarcinoma cell line 769-P, human glioma cell line U251, human glioblastoma cell line A172, human tissue lymph U937, human cervical cancer cell line HeLa, human prostate cancer cell line PC3, human pancreatic cancer cell line PANC-1, human esophageal cancer cell line TE-1, human gastric adenocarcinoma cell line SGC7901, human colon cancer cell line HT-29, human early-young grain
  • the growth of leukemia cell HL-60 has obvious inhibitory effects.
  • the medicament can be prepared into various pharmaceutically acceptable dosage forms such as tablets, capsules and the like by a method conventional in the art.
  • the colimycin is composed of a plurality of spiramycin derivatives, and the main active ingredients are isovaleryl spiromycin I, II and III, and isovalerylspiramycin (I+II) +III) should be no less than 60%.
  • isovaleryl spiromycin III should be not less than 30%;
  • the total content of the acylated spiromycin in the colimycin of the present invention should be not less than 80%.
  • the amount of spiramycin III in the colimycin of the present invention should be not more than 1.0%.
  • the typical chromatogram of colimycin further comprises a selected from the group consisting of spiramycin III, monoacetylspiramycin II, monoacetylspiramycin III, propionylspiramycin II, propionyl spiromycin III, (iso) One or more of the butyryl spiromycin II and (iso)butyryl spiromycin III peaks.
  • the invention has the beneficial effects that the present invention proves that colimycin and its pharmaceutically acceptable salt have good anti-tumor effects, especially for breast cancer, liver cancer, lung cancer, renal clear cell adenocarcinoma, renal cell adenocarcinoma.
  • brain tumor, cervical cancer, prostate cancer, pancreatic cancer, esophageal cancer, gastric adenocarcinoma, colon cancer, lymphoma or leukemia and other tumors have good curative effect, not only colimycin and its pharmaceutically acceptable salts It provides a theoretical basis for the application and clinical promotion of anti-tumor drugs, and has important economic and social benefits.
  • Figure 1 is a photograph of tumors of each administration group in an in vivo test of the inhibitory effect of colimycin on a human non-small cell lung cancer nude mouse model.
  • the colimycin used is composed of a variety of spiramycin derivatives, the main active ingredients are isovaleryl spiromycin I, II and III, and isovalerylspiramycin (I+II) +III) should be no less than 60%.
  • isovaleryl spiromycin III should be not less than 30%;
  • the total content of the acylated spiromycin in the colimycin of the present invention should be not less than 80%.
  • the amount of spiramycin III in the colimycin of the present invention should be not more than 1.0%.
  • the typical chromatogram of colimycin further comprises a selected from the group consisting of spiramycin III, monoacetylspiramycin II, monoacetylspiramycin III, propionylspiramycin II, propionyl spiromycin III, (iso) One or more of the butyryl spiromycin II and (iso)butyryl spiromycin III peaks.
  • the main drug and the auxiliary material are respectively passed through a 100 mesh sieve, and the prescription amount of colimycin, microcrystalline cellulose and 1/2 prescription amount of sodium carboxymethyl starch are uniformly mixed, and then 5% povidone K 30 is added.
  • the soft material made of aqueous solution is granulated by 18 mesh sieve, the wet granules are dried under the condition of 60 ° C for 2 hours; after drying, the granules are sieved with 18 mesh, and then 1/2 prescription amount of sodium carboxymethyl starch and stearic acid are added.
  • the magnesium was uniformly mixed, it was tableted with a shallow concave die having a diameter of 11 mm to obtain a tablet core containing a tablet weight of 350 mg and a hardness of 6.5 kg.
  • Preparation of coating liquid Weigh the required Opadry II (white) into the liquid container, add the required amount of water, add in several portions, after all the addition, reduce the stirring speed, make the spiral disappear, continue to stir 30min, that's it.
  • Preparation of film coated tablets The core is placed in a coating pan to determine the coating conditions, the host speed is 20r/min, the inlet air temperature is 40 ° C, the outlet air temperature is 30 ° C, the spray pressure is 0.02 Mpa, and the spray flow rate is 1 ml. /min is coated, and the film is continuously sprayed for 1.5 hours, until the surface of the tablet is smooth and uniform in color, which is in compliance with the film coating inspection standard. The coating gains about 5%.
  • Preparation process weigh the appropriate amount of starch, dilute to 15% concentration, heat to a paste, to make a binder; main material can be lignin, adjuvant starch, low-substituted hydroxypropyl cellulose, sodium carboxymethyl starch, hard Magnesium citrate is passed through a 100 mesh sieve, and the required main ingredients and auxiliary materials are weighed according to the prescription amount; after the colimycin, starch and low-substituted hydroxypropyl cellulose are well mixed, the starch paste is made with 15% starch concentration.
  • 14 mesh sieve granules dry at 50-60 ° C, water control at 3-5%, 14 mesh sieve granules, add sodium carboxymethyl starch, magnesium stearate, determine the particle content; The content, the weight of the tablet is calculated, and the tablet is pressed ( ⁇ 9mm shallow concave punch), and the difference in the weight of the test piece is detected; after passing the test, the package is packaged.
  • Preparation process the main material of colamycin, auxiliary medicinal starch is weighed according to the process formula, and then mixed into the mixer for 1.5-2 hours; the data obtained by sampling and testing should be basically consistent with the theoretical data (each grain)
  • the weight of the capsule is about 0.105g), and the qualified medicinal No. 3 capsule and the mixed raw materials to be loaded are filled in the filling device according to the operation requirements of the automatic capsule machine, and the filled capsules are filled.
  • Difference test ( ⁇ 10% or less, ⁇ 0.3g), dissolution rate meets the requirements, the capsules that meet the requirements after inspection, put into the polishing machine, add liquid paraffin for 15-20 minutes of light, and then take out the finished packaging box test.
  • Preparation process the original powder of colimycin, citric acid and sucrose are respectively pulverized into 85% of the particles by 300 mesh, 15% through 180 mesh, and then the fine powder after crushing is weighed according to the prescription amount and fully mixed. 1-1.5 hours, measure the content, calculate the loading (theoretical capacity is 500mg per bag), then put the mixture into the bagging machine, install the aluminum foil paper, and pack according to the operation of the filling machine. The difference is ⁇ 5 Within %, after the installation, the outer packaging is carried out after passing the inspection.
  • Preparation process the original powder of colimycin, powdered sugar, dextrin passed through a 120 mesh sieve, and the colimycin, sugar powder and dextrin were uniformly mixed according to the prescription amount, and the above materials were uniformly mixed with 5% PVP-K.
  • 30 glue is made of soft material, the swinging granular mechanism is dried at 70 °C, and the whole grain is packed.
  • Example 6 lysine lyophilized powder injection
  • the purpose of the assay is to evaluate the in vitro cell proliferation inhibition or cytotoxic activity of the test sample.
  • Human breast cancer cells MCF-7 and MDA-MB-231 human hepatoma cells HepG2, human non-small cell lung cancer cells A549, human large cell lung cancer H460 and H1299, human renal clear cell adenocarcinoma cells 786-O, human renal cell adenocarcinoma Cell 769-P, human glioma cell U251, human glioblastoma cell line A172, human tissue lymphoma cell line U937, human cervical cancer cell line HeLa, human prostate cancer cell line PC3, human pancreatic cancer cell line PANC-1, human esophagus Cancer cell TE-1, human gastric adenocarcinoma cell line SGC7901, human colon cancer cell line HT-29, human promyelocytic leukemia cell HL-60.
  • RPMI1640 medium MEM medium, DMEM low sugar medium, fetal calf serum purchased from Gibco, USA, trypsin, glutamine, penicillin, streptomycin, dimethyl sulfoxide (DMSO), tetramethyl azo (MTT) was purchased from Sigma, USA.
  • Carbon dioxide incubator (Sanyo, Japan), enzyme-linked immunosorbent analyzer (Tecan, Austria), 96-well culture plate (Corning, USA), inverted microscope (Motic, China).
  • MCF-7, MDA-MB-231, HepG2, A549, H460, H1299, 786-O, 769-P, U251, A172, HeLa, PC3, PANC-1, TE-1, SGC7901, HT-29 are attached
  • the adherent tumor cells in the logarithmic growth phase were selected and digested with trypsin, and then mixed with a cell suspension containing 10% fetal bovine serum to prepare a cell suspension of 4 to 5 ⁇ 10 4 /ml, and inoculated into a 96-well culture plate.
  • the experimental group was replaced with a new culture medium containing different concentrations of the test sample, and the control group was replaced with a medium containing an equal volume of solvent.
  • Each group was set up with 3 parallel wells, and cultured at 37 ° C, 5% CO 2 for 48 h. The supernatant was discarded, washed carefully 3 times with PBS, 100 ⁇ L of freshly prepared medium containing 0.5 mg/ml MTT was added to each well, and incubation was continued at 37 ° C for 4 h. The supernatant was carefully discarded, and 150 ⁇ L of DMSO was added thereto, and after mixing for 10 minutes with a micro-oscillator, the optical density value was measured at 492 nm with a microplate reader.
  • U937 and HL-60 were suspension cells, and cells in logarithmic growth phase were selected, and RPMI l640 medium containing 10% calf serum was used to prepare 2 ⁇ 10 5 /ml cell suspension, which was inoculated into 96-well culture plates. 50 ⁇ L per well, cultured at 37 ° C, 5% CO 2 for 24 h. In the experimental group, 50 ⁇ L of the culture solution containing different concentrations of the test sample was added, and the control group was added with the culture medium containing the same volume of solvent. Each group was set up with 3 parallel wells, and cultured at 37 ° C, 5% CO 2 for 48 h, each well.
  • the inhibition rate of the drug on tumor cell growth was calculated as follows:
  • Tumor cell growth inhibition rate (%) [A 492 (negative control) - A 492 (dosing group)] / A 492 (negative control) ⁇ 100%
  • the half-inhibitory concentration (IC 50 ) of the sample was determined therefrom.
  • Human breast cancer cells MCF-7 and MDA-MB-231 human hepatoma cells HepG2, human non-small cell lung cancer cells A549, human lung cancer H460 and H1299, human renal clear cell adenocarcinoma cells 786-O, human renal cell glands Cancer cell 769-P, human glioma cell U251, human glioblastoma cell A172, human tissue lymphoma cell U937, human cervical cancer cell HeLa, human prostate cancer cell PC3, human pancreatic cancer cell PANC-1, human Esophageal cancer cell line TE-1, human gastric adenocarcinoma cell line SGC-7901, human colon cancer cell line HT-29, human promyelocytic leukemia cell line HL-60.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Fig. 1, Table 2, Table 3).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 54.46%, 66.07% and 75.89%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 54.55%, 45.57% and 29.21%, respectively.
  • the trypan blue anti-staining experiment showed that the living cells were >95%, trypsinized, centrifuged, and the supernatant was removed.
  • the cell concentration was adjusted to 1 ⁇ 10 7 /ml with matrigel, and the cells were seeded.
  • the nude right armpit of the nude mouse was subcutaneously 0.2 ml/head and recorded as the first day of inoculation.
  • the animals were randomly divided into 5 groups, 6 in each group: model group, cyclophosphamide group, colimycin 12.5, 25 and 50 mg/kg 3 dose groups.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 4, Table 5).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 11.73%, 25.13% and 45.55%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 57.37%, 47.65% and 33.46%, respectively.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 6, Table 7).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 61.08%, 65.94% and 70.50%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of low, medium and high doses of colimycin were 52.37%, 47.31% and 39.95%, respectively.
  • HeLa cells in logarithmic growth phase, trypan blue anti-staining experiment showed live cells >95%, trypsin digestion, centrifugation, de-clearing, adjust the cell concentration to 1 ⁇ 10 7 /ml with matrigel, inoculate cells with naked
  • the right axilla of the rat was subcutaneously 0.2 ml/head and recorded as the first day of inoculation.
  • the animals were randomly divided into 5 groups, 6 in each group: model group, cyclophosphamide group, colimycin 12.5, 25 and 50 mg/kg 3 dose groups. Each group of animals was administered orally for 30 days with a dose of 20 ml/kg. The mice were sacrificed the next day and the indicators were tested. Tumor long diameter, short diameter and body weight were recorded every 2 days from the start of drug administration to the time of sacrifice of nude mice.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 8, Table 9).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 28.75%, 46.28 and 56.18, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 61.04, 53.27 and 40.40, respectively.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 10, Table 11).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 25.92%, 34.67% and 60.32%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 55.93%, 43.45% and 30.02%, respectively.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 12, Table 13).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 40.55%, 60.68% and 73.16%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 49.31%, 42.30% and 30.96%, respectively.
  • the trypan blue anti-staining experiment showed that the living cells were >95%, trypsinized, centrifuged, and the supernatant was removed.
  • the cell concentration was adjusted to 1 ⁇ 10 7 /ml with matrigel, and the cells were seeded.
  • the nude right armpit of the nude mouse was subcutaneously 0.2 ml/head and recorded as the first day of inoculation.
  • the animals were randomly divided into 5 groups, 6 in each group: model group, cyclophosphamide group, colimycin 12.5, 25 and 50 mg/kg 3 dose groups.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 14, Table 15).
  • the tumor growth inhibition rates of the low, medium and high doses of colimycin were 40.26%, 70.92% and 83.35%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of low, medium and high doses of colimycin were 58.63%, 49.26% and 38.33%, respectively.
  • the trypan blue anti-staining experiment showed that the living cells were >95%, trypsinized, centrifuged, and the supernatant was removed.
  • the cell concentration was adjusted to 1 ⁇ 10 7 /ml with matrigel, and the cells were seeded.
  • the nude right armpit of the nude mouse was subcutaneously 0.2 ml/head and recorded as the first day of inoculation.
  • the animals were randomly divided into 5 groups, 6 in each group: model group, cyclophosphamide group, colimycin 12.5, 25 and 50 mg/kg 3 dose groups.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fats were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 16, Table 17).
  • the tumor growth inhibition rates of the low, medium and high doses of colimycin were 37.79%, 51.92% and 61.11%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 65.55%, 53.58% and 39.33%, respectively.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 18, Table 19).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 46.96%, 58.88% and 72.55%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 59.42%, 48.69% and 35.78%, respectively.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each administration group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 20, Table 21).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 37.79%, 51.92 and 61.11%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 73.83%, 61.83% and 49.82%, respectively.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 22, Table 23).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 19.57%, 49.58% and 59.65%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 78.57%, 63.62% and 49.71%, respectively.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 24, Table 25).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 30.32%, 47.24% and 63.71%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 80.81%, 67.16% and 42.82%, respectively.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 26, Table 27).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 38.40%, 53.67% and 69.53%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 62.29%, 43.16% and 31.34%, respectively.
  • U251 cells in logarithmic growth phase, trypan blue anti-staining experiment showed live cells >95%, trypsin digestion, centrifugation, supernatant, cell concentration to 1 ⁇ 10 7 /ml with matrigel, inoculate cells with naked
  • the right axilla of the rat was subcutaneously 0.2 ml/head and recorded as the first day of inoculation.
  • the animals were randomly divided into 5 groups, 6 in each group: model group, cyclophosphamide group, colimycin 12.5, 25 and 50 mg/kg 3 dose groups. Each group of animals was administered orally for 30 days with a dose of 20 ml/kg. The mice were sacrificed the next day and the indicators were tested. Tumor long diameter, short diameter and body weight were recorded every 2 days from the start of drug administration to the time of sacrifice of nude mice.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fats were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 28, Table 29).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 66.51%, 79.59% and 81.82%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 84.81%, 56.30% and 35.90%, respectively.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 30, Table 31).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 46.95%, 66.84% and 76.26%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of low, medium and high doses of colimycin were 68.62%, 55.91% and 38.53%, respectively.
  • PANC-1 cells in logarithmic growth phase trypan blue anti-staining experiment showed live cells >95%, trypsin digestion, centrifugation, de-clearing, cell concentration was adjusted to 1 ⁇ 10 7 /ml with matrigel, cells were seeded
  • the nude right armpit of the nude mouse was subcutaneously 0.2 ml/head and recorded as the first day of inoculation.
  • the animals were randomly divided into 5 groups, 6 in each group: model group, cyclophosphamide group, colimycin 12.5, 25 and 50 mg/kg 3 dose groups. Each group of animals was administered orally for 30 days with a dose of 20 ml/kg. The mice were sacrificed the next day and the indicators were tested. Tumor long diameter, short diameter and body weight were recorded every 2 days from the start of drug administration to the time of sacrifice of nude mice.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 32, Table 33).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 56.27%, 62.66% and 75.94%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 74.10%, 47.01% and 35.55%, respectively.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fat were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each drug-administered group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 34, Table 35).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 46.71%, 61.48% and 70.41%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 64.79%, 46.03% and 37.02%, respectively.
  • the body weight of the nude mice and the long diameter (a) and short diameter (b) of the transplanted tumor were measured every 2 days, and the tumor volume (v), relative tumor volume (RTV) and relative tumor growth rate (T/C) were calculated according to the following formulas.
  • V a ⁇ b 2 /2
  • mice were weighed and sacrificed. The tumors were completely removed, and the non-tumor tissues such as blood stains and fats were removed to call the tumor weight, and the tumor growth inhibition rate was calculated. The average tumor weight of each group of mice was used as an indicator of efficacy.
  • Tumor growth inhibition rate (%) (1 - treatment group mean tumor weight / model group mean tumor weight) ⁇ 100%.
  • each administration group had a certain degree of inhibition on tumor growth inhibition rate, tumor volume, relative tumor volume and relative tumor proliferation rate (Table 36, Table 37).
  • the tumor growth inhibition rates of low, medium and high doses of colimycin were 42.51%, 68.92% and 74.49%, respectively.
  • the tumor volume and relative tumor volume of the low, medium and high doses of colimycin were significantly lower than those of the model group (P ⁇ 0.05).
  • the relative tumor proliferation rates of the low, medium and high doses of colimycin were 69.12%, 47.88% and 38.35%, respectively.
  • the cryopreserved H 22 cell strain was resuscitated in Kunming mice. After 3 passages, ascites was taken and placed in a 50 ml centrifuge tube. 10 ml of 0.9% physiological saline was added, and the mixture was centrifuged at 1000 rpm for 5 min at room temperature, and the supernatant was discarded. Add 10 ml of 0.9% physiological saline, mix by pipetting, and then dilute to 5 ⁇ 10 6 viable cells/ml with physiological saline. Store in ice water and sterilize the right subcutaneous skin of the mice with 75% ethanol. Rapidly inoculated into the right forelimb of the Kunming mice, subcutaneously under the armpits, each inoculated with 0.2 ml.
  • Lewis lung cancer cells were cultured in RPMI 1640 medium containing 10% fetal calf serum in a 37 ° C, 5% CO 2 incubator.
  • the cells in the logarithmic growth phase were digested with 0.25% trypsin, the cells were collected, the supernatant was removed by centrifugation, washed twice with sterile physiological saline, and the cells were suspended in physiological saline.
  • the cell viability assay of trypan blue staining was greater than 95. % and perform cell counting.
  • the Lewis cell concentration was adjusted to 1 ⁇ 10 7 /mL and stored in ice water.
  • the right axillary skin of mice was sterilized with 75% ethanol, and rapidly inoculated into the right axilla of C57BL/6 mice by subcutaneous injection of 0.2 mL.
  • mice inoculated with the tumor were randomly divided into groups of 10 animals each. Including: model control group, positive drug cyclophosphamide control group (CTX, 26mg/kg), colimycin 13, 26 and 53mg/kg three dose groups. Each group of animals was administered intragastrically for 7 days with a dose of 20 ml/kg.
  • mice inoculated with the tumor were randomly divided into groups of 10 each in the next day. Including: model control group, positive drug cyclophosphamide control group (CTX, 30mg/kg), colimycin 13, 26 and 52mg/kg three dose groups. Each group of animals was administered orally for 15 days with a dose of 20 ml/kg.
  • CTX positive drug cyclophosphamide control group
  • the tumor-bearing mice were weighed and weighed the next day after the last administration.
  • the subcutaneous tumor masses were dissected and weighed.
  • the average tumor weight of each group was calculated and the tumor inhibition rate was calculated.
  • Tumor inhibition rate (1-T/C) ⁇ 100%
  • T mean tumor weight of the drug-administered group
  • C mean tumor weight of the blank control group.
  • the tumor suppressor ratio of the positive control drug cyclophosphamide to Kunming mice H 22 liver cancer was 47.25%.
  • Colimycin 26 and 52 mg/kg significantly inhibited the growth of mouse H 22 hepatocarcinoma.
  • the tumor inhibition rates were 50.67% and 79.50%, respectively.
  • the inhibition rate of colimycin 52 mg/kg was significantly lower.
  • Positive control group P ⁇ 0.05).
  • the positive drug cyclophosphamide group showed a slight decrease in body weight compared with the normal control group.
  • the body weight of the animals in each of the colimycin groups was increased compared with that before the administration, and there was no significant difference compared with the model control group.
  • the tumor suppressor rate of the positive control drug cyclophosphamide against mouse Lewis lung cancer was 49.14%.
  • Coriomycin 13 , 26 and 52 mg / kg significantly inhibited the growth of Lewis lung cancer in mice, the tumor inhibition rates were 50.30%, 55.88% and 76.23%, respectively, and the antibiotics in the 52 mg/kg dose of colimycin The rate was significantly lower than the positive control group (P ⁇ 0.05).
  • the body weight of the animals in each of the colimycin groups was increased compared with that before the administration, and there was no significant difference compared with the model control group.
  • the spleen and thymus were weighed and the spleen index and thymus index were calculated.
  • the serum-free RPMI 1640 medium was placed in a dish, placed on ice, and the spleen was aseptically taken, and the spleen was gently ground with a sterile glass slide to prepare a single cell suspension.
  • 2 mL of red blood cell lysate was added, allowed to stand for 2 min, 8 mL of RPMI 1640 culture solution was added, and centrifuged at 1500 rpm for 5 min. The supernatant was removed and washed twice with RPMI 1640 medium. Trypan blue staining live cell count, live cells >95%.
  • a single cell suspension was prepared using RPMI 1640 medium containing 10% fetal calf serum.
  • the spleen cell suspension was taken and the cell density was adjusted to 1 ⁇ 10 7 /mL.
  • A. Control well add 100 ⁇ L of RPMI 1640 medium
  • the above cells were added to a 96-well plate, and then 100 ⁇ L of spleen cell suspension was added to each of the above wells.
  • the spleen cell suspension was taken, and the cell density was adjusted to 1 ⁇ 10 7 /mL (effector cells).
  • a suspension of K562 cells was prepared at a cell density of 1 x 10 5 /mL (target cells).
  • target cells For each mouse: A. Effector cells: target cell wells (quantity ratio 20:1), add 20 ⁇ L spleen cell suspension and 100 ⁇ L K562 cell suspension; B. Effector cell control wells, add 100 ⁇ L spleen cell suspension and 100 ⁇ L RPMI 1640 medium; C. Target cell control wells, 100 ⁇ L of K562 cell suspension and 100 ⁇ L of RPMI 1640 medium.
  • NK cell activity (%) [TO-(SE)] / TO ⁇ 100%, where TO is the absorbance value of the target cell control well, S is the absorbance value of the control cell control well, and E is the absorbance value of the effector cell.
  • the thymus index and spleen index of the positive control drug cyclophosphamide group were significantly lower than those of the control group (P ⁇ 0.01).
  • the thymus index of the animals in the 13, 26 and 52 mg/kg groups had no significant change compared with the control group.
  • the spleen index of the 52 mg/kg group was significantly increased compared with the control group (P ⁇ 0.05).
  • the spleen index of the positive control drug cyclophosphamide group was significantly lower than that of the control group (P ⁇ 0.01).
  • the spleen index and thymus index of the animals in the colimycin 13, 12 and 52 mg/kg groups were not significantly different from those in the control group.
  • the NK cell activity of the positive control drug cyclophosphamide group was significantly lower than that of the control group (P ⁇ 0.05).
  • the activity of NK cells in the colimycin 13 and 26 mg/kg groups was significantly increased compared with the control group (P ⁇ 0.01).
  • the lymphocyte activity of the positive control drug cyclophosphamide group was significantly inhibited (P ⁇ 0.05).
  • the lymphocyte activity of colimycin 13 and 26 mg/kg group was significantly increased compared with the control group (P ⁇ 0.05, P ⁇ 0.01).
  • the spleen index of the positive control drug cyclophosphamide group was significantly lower than that of the control group (P ⁇ 0.01).
  • the spleen index of the animals in the colimycin 13, 26 and 52 mg/kg groups did not change significantly compared with the control group.
  • the invention collects a plurality of clinical cases, and some patients have breast cancer, and the pain symptoms are heavy. After taking the colimycin tablets (prepared in Example 1) for 2 courses (30 days for 1 course, oral, 2 tablets per day), the attending physician diagnosed the tumor to be small. The patient himself also felt pain relief and his mental condition was very good.
  • kidney tumors account for two-thirds of the left kidney area, the pathological results are renal clear cell adenocarcinoma, early.
  • the attending physician diagnosed that the tumor area of the kidney was reduced and improved significantly.
  • lymphoma diagnosed with enlarged lymph nodes in the neck, like jujube, medium hardness, and fullness.
  • the attending physician diagnosed that the lymph nodes were relieved and became soy, the patient also Feeling relieved, no longer feels very hard, and obviously improved.
  • Some patients have colon cancer, diagnosed as a tumor or infiltrated with the omentum, surrounding tissue, hard, irregular shape. After taking a dose of colimycin tablets (prepared in Example 1) for one course of treatment (30 days for one course of treatment, oral administration, 2 tablets per day), the attending physician diagnosed that the tumor was relieved and the patient felt a significant improvement.
  • Some patients have leukemia, varying degrees of anemia, bleeding, fever, and liver, spleen, lymphadenopathy and bone pain. After taking the colimycin tablets (prepared in Example 1) for 3 courses (30 days for 1 course of treatment, oral administration, 2 tablets per day), the attending physician diagnosed the symptoms and the patients felt obviously improved.
  • stomach adenocarcinoma Some patients have stomach adenocarcinoma, feeling the stomach is faint, the stomach seems to have something in the fermentation, it is very bloated, and the stomach acid water occasionally rushes into the throat, making the throat hot and disgusting, the body is hot and cold, go
  • the hospital examined stomach adenocarcinoma. After taking the colimycin tablets (prepared in Example 1) for 3 courses (30 days for 1 course of treatment, oral, 2 tablets per day), the attending physician diagnosed the symptoms and the patient felt a significant improvement.
  • CT examination peripheral lesions of the right upper lobe, 4.3*4.6 round agglomerates; then enhanced CT: mass 4.0*4.4, other changes are not significant.
  • 2 tablets of colimycin prepared in Example 1) / day * 14 days, 1 tablet / day to date; auxiliary calf thymosin 6 capsules / day.
  • CT examination the circular agglomeration was significantly reduced, and the patient's mental state was good and there was no discomfort.
  • CT examination of the lungs have a pleural effusion and multiple nodules, breathing difficulties, fever, treatment, radiotherapy, after taking Klebsiella After two courses of preparation of the tablets (prepared in Example 1), the CT examination revealed that the pleural effusion disappeared and the nodules became smaller.
  • Preparation method Weigh the appropriate amount into a powder, add an appropriate amount of 0.5% carboxymethyl cellulose solution, continue to stir and mix, orally administer 100mg/ml.
  • Preparation method Weigh the appropriate amount of colimycin fine powder in the mortar and grind it in the mortar, add appropriate amount of 0.5% sodium carboxymethyl cellulose solution to prepare a suspension, and store it in a refrigerator at 4 °C.
  • the negative control used an equal amount of 0.5% sodium carboxymethylcellulose solution.
  • the positive control weighed an appropriate amount of cyclophosphamide for injection, and dissolved in physiological saline at the time of use to prepare a solution of 60 mg/kg.
  • Solvent, excipient normal saline, 0.5% sodium carboxymethyl cellulose solution
  • Control Positive control: 60 mg/kg cyclophosphamide solution
  • mice Pre-experimentally selected dose, number of doses, and bone marrow sampling time.
  • the dose was reduced by 1/2 LD 50 as the standard, and the number of administrations was once. According to the pretest results, the bone marrow sampling time was selected as 24 hours sampling.
  • At least three dose groups respectively: 2000mg/kg, 1000mg/kg, 500mg/kg
  • the number of administrations one-time oral administration.
  • Bone marrow sampling time 24 hours after administration.
  • the animals were sacrificed by cervical dislocation, the sternum was taken, the muscles were removed, and the attachments were wiped with gauze. Direct smear of bone marrow was used to cut the sternum to expose the medullary cavity, and the extruded bone marrow was mixed on a slide with a drop of calf serum beforehand. After drying, it was fixed in methanol for 10 minutes, dried, and then subjected to Giemsa staining. Each mouse was microscopically examined for 1000 red blood cell contoured intact multi-stained red blood cells, and the frequency of occurrence of micronuclei and the ratio of red blood cells to red blood cells were counted.
  • Heddle et al reported that the spontaneous rate of micronuclei of 16 mouse bone marrow polychromatic erythrocytes was 3.1%.
  • the average value of the negative control group was 2.63 ⁇ , three dose groups of corticomycin drugs and the negative control. Compared with the group, the micronucleus rate of polychromatic erythrocytes did not increase significantly (P>0.05). The ratio of polychromatic erythrocytes to normal red blood cells was not significantly reduced, and fluctuations were in the normal range.
  • the cyclophosphamide-positive control group had a very significant increase in micronuclei of polychromatic erythrocytes (P ⁇ 0.01), and the micronucleus rate was 16.85 times that of the negative control group.
  • colimycin is not a chromosomal cleavage agent. At the doses used, it does not affect the normal mitosis of the cells and has no inhibitory effect on the bone marrow.

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Abstract

本发明提供了可利霉素及其药学上可接受的盐在制备治疗肿瘤药物方面的应用。所述的可利霉素及其药学上可接受的盐对乳腺癌、肝癌、肺癌、肾癌、脑瘤、宫颈癌、前列腺癌、胰腺癌、食管癌、胃腺癌、结肠癌、淋巴瘤或白血病等多种肿瘤具有较好的疗效，尤其是对人乳腺癌细胞MCF-7及MDA-MB-231，人肝癌细胞HepG2，人非小细胞肺癌细胞A549，人大细胞肺癌H460及H1299，人肾透明细胞腺癌细胞786-O，人肾细胞腺癌细胞769-P，人胶质瘤细胞U251等的生长均有明显的抑制作用。

Description

可利霉素及其药学上可接受的盐在制备治疗和/或预防肿瘤药物方面的应用 技术领域
本发明属于药物应用领域，具体地说，涉及可利霉素及其药学上可接受的盐在制备治疗和/或预防肿瘤药物方面的应用。
背景技术
肿瘤是一类常见病和多发病，是机体组织细胞在内外各种致瘤因素长期作用下，发生基因突变失去对其生长和分化的正常调控，引起克隆性异常增生和分化所形成的新生物或赘生物。肿瘤分为良性肿瘤和恶性肿瘤，恶性肿瘤又细分为来源于上皮组织的癌、来源于间叶组织的肉瘤和癌肉瘤三类。通常人们所说的“癌症”是泛指所有的恶性肿瘤。
恶性肿瘤是威胁人类健康最主要的恶性疾病之一，是目前全球人口的第一死因，最新数据统计，2007年，全球有约790万人死于各类癌症，占死亡总数的13％，有超过1200万个肿瘤病例被确诊，其中72％以上肿瘤患者和致死病例都发生在不发达国家，且呈不断上升的趋势，2015年，全球肿瘤致死人数增至900万人，预计2030年将超过1200万人；目前，我国每年癌症发病人数约280万，死亡人数超过40万人，位列我国各类疾病致死原因之首，并呈不断上升趋势。随着社会生活节奏加快，竞争压力增大，以及人类的生活方式和环境的改变，肿瘤发病率和死亡人数正逐年增长，已成为现代社会的常见病和高发病，不仅严重影响患者的生活质量，而且给家庭和社会带来沉重的经济和精神负担，也是困扰全球的重大社会问题，癌症的治疗和预防始终是全球范围内最为迫切的问题之一。目前，化学药物治疗是抗击肿瘤的主要手段，虽然有较好的疗效，但是常引起骨髓抑制、免疫功能低下等副反应，使患者难以坚持治疗，并且化疗药物在治疗过程中出现的耐药性已成为目前临床治疗中的难题之一。近年来，全球抗肿瘤药物市场呈快速增长态势，据美国FDA统计数据，全球抗癌药物市场销售总额由2004年的240亿美元，激增至2007年的396亿美元。虽然全球每年都不断有新型抗肿瘤药物问世，但至今人类仍然没有一种有效的手段战胜癌症，同时不断发现新的癌症种类，以及肿瘤抗/耐药性的产生和增强，使得对发现新型有效抗癌药物的需要显得尤为迫切。
可利霉素(Carrimycin)是通过转基因技术将碳霉素产生菌的4”异戊酰基转移酶基团 (4”-o-acyl-transferase)克隆至螺旋霉素产生菌中，定向酰化螺旋霉素4”-OH，在4”位加入异戊酰基侧链所形成的以4”位异戊酰基螺旋霉素为主要组分的新型抗生素。
可利霉素是由多种螺旋霉素衍生物组成，主要活性成分异戊酰螺旋霉素(I+II+III)总含量不低于60％，于药学上是一种可接受的药物组合物。中心结构为16元内酯环，与一分子福洛胺糖、一分子碳霉胺糖和一分子碳霉糖连接而成，其主要成分异戊酰螺旋霉素I、II、III与螺旋霉素结构不同之处在于碳霉糖4”位上连接的基团为异戊酰基而不是羟基。该药由沈阳同联等共同申报1.1类新药。
可利霉素主成分的化学结构，如式(1)所示：
Figure PCTCN2018082072-appb-000001
其中，当R＝H，R′＝COCH 2CH(CH 3) 2时为异戊酰螺旋霉素Ⅰ；
当R＝COCH 3，R′＝COCH 2CH(CH 3) 2时为异戊酰螺旋霉素Ⅱ；
当R＝COCH 2CH 3，R′＝COCH 2CH(CH 3) 2时为异戊酰螺旋霉素Ⅲ；
可利霉素属于16元大环内酯类抗生素，具有活性基团羧基、烷氧基、环氧基、酮基和醛基以及一对共轭的C＝C，分子量约为884～982。由于具有相似的化学结构，可利霉素与大环内酯类抗生素具有很多共性：易溶于酯类、丙酮、氯仿、醇类等大多数有机溶剂，微溶于石油醚，难溶于水；分子结构中含有两个二甲胺基而呈弱碱性，易溶于酸性水溶液；具有溶解度随温度的升高而降低的“负溶解度”性质。由于可利霉素主要组分异戊酰螺旋霉素4”位碳链较长，亲水性差，其水中溶解度比螺旋霉素及4”-乙酰螺旋霉素小。
可利霉素是一种白色非结晶粉末，略有引湿性，比旋度约为-80.8°，紫外最大吸收波长为231～232nm，本身带有弱荧光发色基团，遇浓硫酸或盐酸呈紫色反应，产生强紫色荧光，在231～232nm处有最大吸光值。
该药具有亲脂性好，组织渗透能力强，口服吸收快，体内维持时间长，有持续的抗生素后效应。根据药效与化学构象的关系，大环内酯类抗生素4”位酰化后，其亲脂性和体内活性 提高，体内抗菌活性与临床治疗效果均得到了显著提升，并且抗生素在体内的稳定性随着4”羟基酯的碳链增长而增强，即异戊酰螺旋霉素＞丁酰螺旋霉素＞丙酰螺旋霉素＞乙酰螺旋霉素。
初步体内外药效学试验表明，该药不仅对多数G +菌有较好抗菌活性，对部分G -菌也有一定作用，各项技术指标明显优于阿奇霉素、红霉素、乙酰螺旋霉素、麦迪霉素，尤其对肺炎支原体的抗菌活性最强，对红霉素耐药菌、淋球菌、肺炎球菌、金葡菌、绿脓假单胞菌、流感杆菌、流感嗜血杆菌、脆弱拟杆菌、军团菌、多行杆菌和产气荚膜梭菌也有一定抗菌活性，对临床耐红霉素的金葡球菌仅有极少交叉耐药性。可利霉素将主要用于治疗革兰氏阳性菌感染性疾病，尤其是上呼吸道感染，并可能用于泌尿系统感染等。
本申请人在近期的一项研究中发现，在以人乳腺癌细胞MCF-7及MDA-MB-231，人肝癌细胞HepG2或鼠肝癌细胞H 22，人非小细胞肺癌细胞A549、Lewis肺癌细胞、人大细胞肺癌H460及H1299，人肾透明细胞腺癌细胞786-O，人肾细胞腺癌细胞769-P，人胶质瘤细胞U251，人胶质母细胞瘤细胞A172，人组织淋巴瘤细胞U937，人宫颈癌细胞HeLa，人前列腺癌细胞PC3，人胰腺癌细胞PANC-1，人食管癌细胞TE-1，人胃腺癌细胞SGC7901，人结肠癌细胞HT-29，人早幼粒白血病细胞HL-60对可利霉素进行的体外抗增殖活性评价中发现样品对所测试细胞均显示了良好的抗增殖活性，表明可利霉素有望成为治疗肿瘤的新的药物，从而完成了本发明。
发明内容
本发明的目的在于提供一种可利霉素及其药学上可接受的盐在制备治疗和/或预防肿瘤药物方面的应用。
为实现上述目的，本发明采用如下技术方案：
本发明涉及可利霉素及其药学上可接受的盐在制备治疗和/或预防肿瘤药物方面的应用。
本发明中，所述的肿瘤包括实体瘤和非实体瘤。
具体地说，所述的实体瘤包括良性实体瘤和恶性实体瘤；
所述的非实体瘤为淋巴瘤或白血病。
进一步的，所述的恶性实体瘤为乳腺癌、肝癌、肺癌、肾癌、脑瘤、宫颈癌、前列腺癌、胰腺癌、食管癌、胃癌、结肠癌。
所述的脑瘤为胶质瘤或脑膜瘤，所述的胃癌为胃腺癌。
本发明中，所述的药物可以为可利霉素及其药学上可接受的盐与药学上可接受的辅料制成的各种剂型。
本发明中，所述的药物也可以为可利霉素及其药学上可接受的盐和抗肿瘤药物与药学上可接受的辅料制成的各种剂型。
本发明中，可以将可利霉素及其药学上可接受的盐与现有技术中的抗肿瘤药物如奥沙利铂等一起与药学上可接受的辅料制成各种复方制剂。
本发明中，所述的药物还可以为含有可利霉素及其药学上可接受的盐的第一药剂与含有抗肿瘤药物的第二药剂的组合。
本发明中，所述的抗肿瘤药物为现有技术中已知的抗肿瘤药物，在治疗肿瘤时，可以将含有可利霉素及其药学上可接受的盐的第一药剂与这些抗肿瘤药物联合应用。在联合治疗肿瘤时，可以先使用含有可利霉素及其药学上可接受的盐的第一药剂，也可以先使用含有现有技术已知的抗肿瘤药物的第二药剂，还可以两者同时使用。
本发明中，所述的抗肿瘤药物为化疗、放疗、靶向治疗和/或免疫治疗药物。
本发明表明，含有可利霉素及其药学上可接受的盐的第一药剂对包括肺癌、肝癌、宫颈癌等多种癌症具有良好的治疗作用，而将含有可利霉素及其药学上可接受的盐的第一药剂与含有现有技术已知的抗肿瘤药物如环磷酰胺、苯丁酸氮芥、消瘤芥、环己亚硝脲、噻替派、白消安、顺氯氨铂等联合使用，可以达到协同作用，对肿瘤患者具有更好的治疗效果。
恶性肿瘤患者由于反复接受放、化疗、手术、免疫制剂的治疗，受到各种侵入性操作和抗肿瘤药物等多重打击，身体免疫力整体下降，成为医院感染高危人群，极易导致各种感染的发生。分析发现，肿瘤病人医院感染发生率明显高于全院同期出院病人的医院感染率，引发肿瘤患者医院感染的危险因素主要有高龄、长期住院、侵入性操作和放化疗等，其中放化疗是肿瘤病人免疫力受损的重要特殊因素。由于老年患者机体的组织器官退行性变化，机体免疫防御功能降低，抵抗力差，伴有多种基础疾病，较易发生医院感染。住院时间与医院感染互为因果关系，随着住院时间的延长，增加感染机会。
所有感染中，合并呼吸道感染最多，其次是皮肤感染，胃肠道感染和泌尿道感染，血行感染较少见，但较凶险，死亡率高。肿瘤发生部位与感染率的关系：肺部、胃部和血液肿瘤发病率虽高，感染率却不算最高，为20％左右，胰腺、食道、肝胆、耳鼻喉等类型肿瘤中， 患者较少，但感染率较高，占40％以上。乳腺癌、甲状腺等均在10％左右。感染率最低的是泌尿系统肿瘤，只有5％左右。
感染的致病菌主要以革兰氏阴性菌感染(45％-55％左右)和真菌(30％以上)感染为主(以咽拭子、痰标本等为主要标本的结果)，革兰氏阴性菌中以大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌和鲍曼不动杆菌为主。部分医院真菌感染甚至居首位，可能是由于恶性肿瘤本身为一种消耗性疾病，加之放化疗及侵入性操作抑制了骨髓造血功能，削弱了机体单核细胞吞噬系统的防御能力和破坏了机体免疫屏障。另外，广谱抗菌药物、免疫抑制剂的大量使用，可影响人体蛋白代谢，甚至可造成对肝肾及骨髓等组织功能的损害，为真菌躲避或干扰宿主的防御提供了可能，这些是导致恶性肿瘤患者发生真菌等感染的重要原因。
而本发明的可利霉素具有优良的抗感染效果，有利于帮助患者清除感染，恢复机体免疫功能，从而使其与抗肿瘤药物联合使用时达到更好的治疗效果。
本发明中，含有可利霉素及其药学上可接受的盐的第一药剂为可利霉素与药学上可接受的辅料制成药学上可接受的剂型，如片剂、胶囊、肠溶制剂、注射剂等。
所用到的辅料及各剂型的制备方法可参照现有技术进行。
进一步的，所述的药物的剂量为5～1500mg；优选50～1000mg；更优选100～400mg。
或者，所述的第一药剂的剂量为5～1500mg；优选50～1000mg；更优选100～400mg。
本发明通过试验表明可利霉素及其药学上可接受的盐对于人乳腺癌细胞MCF-7及MDA-MB-231，人肝癌细胞HepG2或鼠肝癌细胞H 22，人非小细胞肺癌细胞A549，人大细胞肺癌H460及H1299，人肾透明细胞腺癌细胞786-O，人肾细胞腺癌细胞769-P，人胶质瘤细胞U251，人胶质母细胞瘤细胞A172，人组织淋巴瘤细胞U937，人宫颈癌细胞HeLa，人前列腺癌细胞PC3，人胰腺癌细胞PANC-1，人食管癌细胞TE-1，人胃腺癌细胞SGC7901，人结肠癌细胞HT-29，人早幼粒白血病细胞HL-60均显示了良好的抗增殖活性，从而证实了可利霉素可用于这些细胞引起的肿瘤或癌症疾病的治疗。
本发明进一步通过体内试验表明可利霉素及其药学上可接受的盐对于人乳腺癌细胞MCF-7及MDA-MB-231、人肝癌细胞HepG2或鼠肝癌细胞H 22、人非小细胞肺癌细胞A549、人大细胞肺癌H460及H1299、人肾透明细胞腺癌细胞786-O、人肾细胞腺癌细胞769-P、人胶质瘤细胞U251、人胶质母细胞瘤细胞A172、人组织淋巴瘤细胞U937、人宫颈癌细胞HeLa、人前列腺癌细胞PC3、人胰腺癌细胞PANC-1、人食管癌细胞TE-1、人胃腺癌细胞SGC7901、 人结肠癌细胞HT-29、人早幼粒白血病细胞HL-60的生长均有明显的抑制作用。
同时经多名患有各种肿瘤或癌症的患者试用，表明可利霉素及其药学上可接受的盐对肺癌、宫颈癌、子宫癌等多种肿瘤或癌症具有较好的治疗效果。
本发明中，所述的药物可采用本领域常规的方法制备成药学上可接受的各种剂型、如片剂、胶囊等。
本发明中，所述的可利霉素是由多种螺旋霉素衍生物组成的，主要活性成分为异戊酰螺旋霉素Ⅰ、Ⅱ和Ⅲ，其异戊酰螺旋霉素(Ⅰ+Ⅱ+Ⅲ)应不低于60％。
进一步，本发明的可利霉素中，异戊酰螺旋霉素Ⅲ应不低于30％；
更进一步本发明的可利霉素中酰化螺旋霉素总含量应不少于80％，
更进一步的，其他未知组分的总和应不大于5.0％。
再进一步的，本发明的可利霉素中螺旋霉素Ⅲ的量应不大于1.0％。
在可利霉素典型的色谱图中，除异戊酰螺旋霉素Ⅰ、Ⅱ和Ⅲ外，还包括至少包括(异)丁酰螺旋霉素Ⅱ和/或(异)丁酰螺旋霉素Ⅲ峰。
优选可利霉素典型的色谱图中还包括选自螺旋霉素III、单乙酰螺旋霉素II、单乙酰螺旋霉素III、丙酰螺旋霉素II、丙酰螺旋霉素III、(异)丁酰螺旋霉素II、(异)丁酰螺旋霉素III峰中的一种或多种。
本发明的有益效果是：本发明证明了可利霉素及其药学上可接受的盐具有较好的抗肿瘤作用，尤其对乳腺癌、肝癌、肺癌、肾透明细胞腺癌、肾细胞腺癌、脑瘤、宫颈癌、前列腺癌、胰腺癌、食管癌、胃腺癌、结肠癌、淋巴瘤或白血病等多种肿瘤具有较好的疗效，不仅为可利霉素及其药学上可接受的盐在制备抗肿瘤药物中的应用及其临床推广提供了理论依据，而且具有重要的经济效益和社会效益。
附图说明
图1为可利霉素对人非小细胞肺癌裸鼠模型的抑制作用的体内试验中各给药组肿瘤图片。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例，对实 施例中的技术方案进行清楚、完整地描述，以下实施例用于说明本发明，但不用来限制本发明的范围。
以下实施例中，所用的可利霉素是由多种螺旋霉素衍生物组成的，主要活性成分为异戊酰螺旋霉素Ⅰ、Ⅱ和Ⅲ，其异戊酰螺旋霉素(Ⅰ+Ⅱ+Ⅲ)应不低于60％。
进一步，本发明的可利霉素中，异戊酰螺旋霉素Ⅲ应不低于30％；
更进一步本发明的可利霉素中酰化螺旋霉素总含量应不少于80％，
更进一步的，其他未知组分的总和应不大于5.0％。
再进一步的，本发明的可利霉素中螺旋霉素Ⅲ的量应不大于1.0％。
在可利霉素典型的色谱图中，除异戊酰螺旋霉素Ⅰ、Ⅱ和Ⅲ外，还包括至少包括(异)丁酰螺旋霉素Ⅱ和/或(异)丁酰螺旋霉素Ⅲ峰。
优选可利霉素典型的色谱图中还包括选自螺旋霉素III、单乙酰螺旋霉素II、单乙酰螺旋霉素III、丙酰螺旋霉素II、丙酰螺旋霉素III、(异)丁酰螺旋霉素II、(异)丁酰螺旋霉素III峰中的一种或多种。
对不同批次的可利霉素也进行了下述试验，其获得的结果相似。
实施例1、可利霉素片
规格：200mg/350mg
片芯处方：
Figure PCTCN2018082072-appb-000002
包衣液处方：
欧巴代II    21g
蒸馏水      适量
制成        105ml
制备工艺：
片芯的制备：主药和辅料分别过100目筛，将处方量可利霉素、微晶纤维素与1/2处方量的羧甲淀粉钠混合均匀，然后加入5％聚维酮K 30水溶液制软材，以18目筛制粒，湿颗粒在60℃通风条件下干燥2h；干燥后以18目筛整粒，再加入1/2处方量的处方量羧甲淀粉钠与硬脂酸镁混合均匀后，用直径11mm的浅凹冲模压片，制得片重350mg、硬度6.5kg的含药片芯。
包衣液的配制：称好所需的欧巴代II(白色)在配液容器中加入所需量的水，分次加入，待全部加入后，降低搅拌速度，使蜗旋消失，继续搅拌30min，即得。
薄膜包衣片的制备：将片芯置包衣锅内，确定包衣条件，主机速度为20r/min，进风温度40℃，出风温度30℃，喷雾压力0.02Mpa，喷浆流量为1ml/min进行包衣，恒定后持续喷包1.5h，至片粒表面光滑、色泽均匀，符合薄膜衣检验标准为合格。包衣增重5％左右。
实施例2、可利霉素素片(按10000片计算)
处方：
Figure PCTCN2018082072-appb-000003
制备工艺：称取适量淀粉，稀释至15％浓度，加热至糊状，制成粘合剂；主料可利霉素、辅料淀粉、低取代羟丙基纤维素、羧甲基淀粉钠、硬脂酸镁分别过100目筛，按处方量，称取所需主料和辅料；可利霉素、淀粉、低取代羟丙基纤维素充分混合均匀后，用15％淀粉浓度的淀粉糊制成软材，14目筛制粒，50-60℃干燥，水份控制在3-5％，14目筛整粒，加羧甲基淀粉钠，硬脂酸镁混合，测定颗粒含量；根据颗粒含量，计算片重，压片(Φ9mm浅凹冲头)，检测片重差异；经检验合格后进行包装。
实施例3、可利霉素胶囊剂(按10000粒计算)
处方：
Figure PCTCN2018082072-appb-000004
制备工艺：将主料可利霉素、辅料药用淀粉按工艺配方量分别称取后，装入混合器充分混合后1.5-2小时；取样检测含量所得数据应和理论数据基本一致(每粒胶囊所装重量约为0.105g)，将经检验合格的药用3号胶囊及混合好的待装原料按全自动胶囊机操作要求，分别填入装料器进行填充，将填充好的胶囊进行差异检验(±10％以内，＜0.3g)，溶出度符合要求，将检验后符合要求的胶囊，放入打光机内加入液体石蜡进行15-20分钟的打光，然后取出进行成品包装盒检验。
实施例4、可利霉素干糖浆(按10000袋计算)
处方：
Figure PCTCN2018082072-appb-000005
制备工艺：可利霉素原粉，柠檬酸、蔗糖分别用高速气流粉碎机粉碎成颗粒85％通过300目，15％通过180目，然后将粉碎后的细粉按处方量称取后充分混合1-1.5小时，测其含量，计算装量(理论装量为每袋500mg)，然后将混合物装入袋装机中，装好铝箔纸，按分装机操作要求分装，装量差异在±5％以内，装好后进行检验合格后外包装。
实施例5、可利霉素颗粒剂(按10000袋计算)
处方：
Figure PCTCN2018082072-appb-000006
制备工艺：可利霉素原粉、糖粉、糊精过120目筛，按处方量称取可利霉素、糖粉、糊精混合均匀，将混合均匀的上述物料用5％PVP-K 30胶浆制成软材，摇摆式颗粒机制粒70℃干燥、整粒，送检合格后分装。
实施例6、可利霉素冻干粉针剂
称取可利霉素原粉500mg与等摩尔的丙二醇混合均匀后溶解于5ml水中，得到淡黄色澄明溶液，pH在4.6-5.6之间。再加入甘露醇40mg作为冻干支撑剂，低温快速冷冻9h后，冷冻干燥，获得淡黄色疏松块状物，使用前用10ml无菌水溶解。
试验例1、抗肿瘤活性的生物测定
所测定的目的是评价被测试样品的体外细胞增殖抑制作用或细胞毒活性。
细胞株：
人乳腺癌细胞MCF-7及MDA-MB-231，人肝癌细胞HepG2，人非小细胞肺癌细胞A549，人大细胞肺癌H460及H1299，人肾透明细胞腺癌细胞786-O，人肾细胞腺癌细胞769-P，人胶质瘤细胞U251，人胶质母细胞瘤细胞A172，人组织淋巴瘤细胞U937，人宫颈癌细胞HeLa，人前列腺癌细胞PC3，人胰腺癌细胞PANC-1，人食管癌细胞TE-1，人胃腺癌细胞SGC7901，人结肠癌细胞HT-29，人早幼粒白血病细胞HL-60。
试剂：
RPMI1640培养液、MEM培养液、DMEM低糖培养液、胎牛血清购于美国Gibco公司，胰蛋白酶、谷氨酰胺、青霉素、链霉素、二甲基亚砜(DMSO)、四甲基偶氮唑(MTT)购于美国Sigma公司。
仪器：
二氧化碳培养箱(Sanyo，Japan)、酶联免疫分析仪(Tecan，Austria)、96孔培养板(Corning，USA)、倒置显微镜(Motic，China)。
操作步骤如下：
贴壁细胞：
MCF-7，MDA-MB-231，HepG2，A549，H460，H1299，786-O，769-P，U251，A172，HeLa，PC3，PANC-1，TE-1，SGC7901，HT-29为贴壁细胞，选用对数生长期的贴壁肿瘤细胞，用胰酶消化后，用含10％胎牛血清的培养基配成4～5×10 4/ml的细胞悬液，接种在96孔培养板中，每孔100μL，37℃，5％CO 2培养24h。实验组更换新的含不同浓度被测样品可利 霉素的培养液，对照组则更换含等体积溶剂的培养液，每组设3个平行孔，37℃，5％CO 2培养48h。弃去上清液，用PBS小心洗3次，每孔加入100μL新鲜配制的含0.5mg/ml MTT的培养基，37℃继续培养4h。小心弃去上清，并加入150μL DMSO，用微型振荡器混匀10min后，用酶标仪在492nm处测定光密度值。
悬浮细胞：
U937、HL-60为悬浮细胞，选用对数生长期的细胞，用含10％小牛血清的RPMI l640培养基配成2×10 5/ml的细胞悬液，接种在96孔培养板中，每孔50μL，37℃，5％CO 2培养24h。实验组加入含不同浓度被测样品可利霉素的培养液50μL，对照组则加入含等体积溶剂的培养液，每组设3个平行孔，37℃，5％CO 2培养48h，每孔加入10μL新鲜配制的含5mg/ml MTT的培养基，37℃继续培养4h。用三联液(SDS 10g，10M HCl 0.1mL，异丁醇5mL，用蒸馏水稀释至100mL)100μL溶解结晶，37℃孵育12h。用酶标仪在492nm处测定光密度值。
结果评定：
按下式计算药物对肿瘤细胞生长的抑制率：
肿瘤细胞生长抑制率(％)＝[A 492(阴性对照)－A 492(加药组)]/A 492(阴性对照)×100％
从中求出样品的半数抑制浓度(IC 50)。
结果：
以人乳腺癌细胞MCF-7及MDA-MB-231，人肝癌细胞HepG2，人非小细胞肺癌细胞A549，人细胞肺癌H460及H1299，人肾透明细胞腺癌细胞786-O，人肾细胞腺癌细胞769-P，人胶质瘤细胞U251，人胶质母细胞瘤细胞A172，人组织淋巴瘤细胞U937，人宫颈癌细胞HeLa，人前列腺癌细胞PC3，人胰腺癌细胞PANC-1，人食管癌细胞TE-1，人胃腺癌细胞SGC-7901，人结肠癌细胞HT-29，人早幼粒白血病细胞HL-60。
对样品进行的体外抗增殖活性评价结果如下表1：
表1、可利霉素对肿瘤细胞的增殖抑制作用
Figure PCTCN2018082072-appb-000007
现有结果显示，样品对所测试细胞均显示了良好的抗增殖活性。
试验例2、体内试验
一、可利霉素对人非小细胞肺癌裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的A549细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(图1，表2、表3)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为54.46％、66.07％和75.89％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为54.55％、45.57％和29.21％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表2、可利霉素对裸鼠人非小细胞肺癌A549细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000008
Figure PCTCN2018082072-appb-000009
*p<0.05与模型组相比，**p<0.01与模型组相比，***p<0.001与模型组相比
表3、可利霉素对裸鼠人非小细胞肺癌A549细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000010
Figure PCTCN2018082072-appb-000011
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
二、可利霉素对人乳腺癌裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的MCF-7细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)， 相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表4、表5)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为11.73％、25.13％和45.55％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为57.37％、47.65％和33.46％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表4、可利霉素对裸鼠人乳腺癌MCF-7细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000012
Figure PCTCN2018082072-appb-000013
*p<0.05与模型组相比，**p<0.01与模型组相比，***p<0.001与模型组相比
表5、可利霉素对裸鼠人乳腺癌MCF-7细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000014
Figure PCTCN2018082072-appb-000015
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
三、可利霉素对人淋巴瘤裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的U937细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表6、表7)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为61.08％、65.94％和70.50％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为52.37％、47.31％和39.95％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表6、可利霉素对裸鼠人淋巴瘤U937细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000016
Figure PCTCN2018082072-appb-000017
*p<0.05与模型组相比，**p<0.01与模型组相比，***p<0.001与模型组相比
表7、可利霉素对裸鼠人淋巴瘤U937细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000018
Figure PCTCN2018082072-appb-000019
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
四、可利霉素对人宫颈癌裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的HeLa细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表8、表9)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为28.75％、46.28和56.18。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为61.04、53.27和40.40。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表8、可利霉素对裸鼠人宫颈癌HeLa细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000020
Figure PCTCN2018082072-appb-000021
*p<0.05与模型组相比，**p<0.01与模型组相比，***p<0.001与模型组相比
表9、可利霉素对裸鼠人宫颈癌HeLa细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000022
Figure PCTCN2018082072-appb-000023
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
五、可利霉素对人前列腺癌裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的PC3细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表10、表11)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为25.92％、34.67％和60.32％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为55.93％、43.45％和30.02％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表10、可利霉素对裸鼠人前列腺癌PC3细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000024
Figure PCTCN2018082072-appb-000025
*p<0.05与模型组相比，**p<0.01与模型组相比，***p<0.001与模型组相比
表11、可利霉素对裸鼠人前列腺癌PC3细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000026
Figure PCTCN2018082072-appb-000027
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
六、可利霉素对人结肠癌裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的HT-29细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰 胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表12、表13)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为40.55％、60.68％和73.16％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为49.31％、42.30％和30.96％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表12、可利霉素对裸鼠人结肠癌HT-29细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000028
Figure PCTCN2018082072-appb-000029
*p<0.05与模型组相比，**p<0.01与模型组相比
表13、可利霉素对裸鼠人结肠癌HT-29细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000030
Figure PCTCN2018082072-appb-000031
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
七、可利霉素对人白血病裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的HL-60细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表14、表15)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为40.26％、70.92％和83.35％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为58.63％、49.26％和38.33％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表14、可利霉素对裸鼠人白血病HL-60细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000032
Figure PCTCN2018082072-appb-000033
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
表15、可利霉素对裸鼠人白血病HL-60细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000034
Figure PCTCN2018082072-appb-000035
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
八、可利霉素对人肝癌裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的HepG-2细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤 生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表16、表17)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为37.79％、51.92％和61.11％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为65.55％、53.58％和39.33％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表16、可利霉素对裸鼠人肝癌HepG-2细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000036
Figure PCTCN2018082072-appb-000037
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
表17、可利霉素对裸鼠人肝癌HepG-2细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000038
Figure PCTCN2018082072-appb-000039
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
九、可利霉素对人乳腺癌裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的MDA-MB-231细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环 磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表18、表19)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为46.96％、58.88％和72.55％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为59.42％、48.69％和35.78％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表18、可利霉素对裸鼠人乳腺癌MDA-MB-231细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000040
Figure PCTCN2018082072-appb-000041
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
表19、可利霉素对裸鼠人乳腺癌MDA-MB-231细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000042
Figure PCTCN2018082072-appb-000043
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
十、可利霉素对人大细胞肺癌裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的H460细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表20、表21)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为37.79％、51.92和61.11％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为73.83％、61.83％和49.82％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表20、可利霉素对裸鼠人大细胞肺癌H460细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000044
Figure PCTCN2018082072-appb-000045
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
表21、可利霉素对裸鼠人大细胞肺癌H460细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000046
Figure PCTCN2018082072-appb-000047
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
十一、可利霉素对人大细胞肺癌裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的H1299细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表22、表23)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为19.57％、49.58％和59.65％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为78.57％、63.62％和49.71％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表22、可利霉素对裸鼠人大细胞肺癌H1299细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000048
Figure PCTCN2018082072-appb-000049
*p<0.05与模型组相比，**p<0.01与模型组相比，***p<0.001与模型组相比
表23、可利霉素对裸鼠人大细胞肺癌H1299细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000050
Figure PCTCN2018082072-appb-000051
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
十二、可利霉素对人肾透明细胞腺癌裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的786-O细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰 胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表24、表25)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为30.32％、47.24％和63.71％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为80.81％、67.16％和42.82％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表24、可利霉素对裸鼠人肾透明细胞腺癌786-O细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000052
Figure PCTCN2018082072-appb-000053
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
表25、可利霉素对裸鼠人肾透明细胞腺癌786-O细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000054
Figure PCTCN2018082072-appb-000055
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
十三、可利霉素对人肾细胞腺癌裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的769-P细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表26、表27)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为38.40％、53.67％和69.53％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为62.29％、43.16％和31.34％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表26、可利霉素对裸鼠人肾细胞腺癌769-P细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000056
Figure PCTCN2018082072-appb-000057
*p<0.05与模型组相比，**p<0.01与模型组相比，***p<0.001与模型组相比
表27、可利霉素对裸鼠人肾细胞腺癌769-P细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000058
Figure PCTCN2018082072-appb-000059
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
十四、可利霉素对人胶质瘤裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的U251细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤 生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表28、表29)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为66.51％、79.59％和81.82％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为84.81％、56.30％和35.90％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表28、可利霉素对裸鼠人胶质瘤细胞U251细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000060
Figure PCTCN2018082072-appb-000061
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
表29、可利霉素对裸鼠人胶质瘤细胞U251细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000062
Figure PCTCN2018082072-appb-000063
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
十五、可利霉素对人胶质母细胞瘤裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的A172细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为 20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表30、表31)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为46.95％、66.84％和76.26％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为68.62％、55.91％和38.53％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表30、可利霉素对裸鼠人胶质母细胞瘤A172细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000064
Figure PCTCN2018082072-appb-000065
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
表31、可利霉素对裸鼠人胶质母细胞瘤A172细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000066
Figure PCTCN2018082072-appb-000067
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
十六、可利霉素对人胰腺癌裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的PANC-1细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表32、表33)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为56.27％、62.66％和75.94％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为74.10％、47.01％和35.55％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表32、可利霉素对裸鼠人胰腺癌PANC-1细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000068
Figure PCTCN2018082072-appb-000069
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
表33、可利霉素对裸鼠人胰腺癌PANC-1细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000070
Figure PCTCN2018082072-appb-000071
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
十七、可利霉素对人食管癌裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的TE-1细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与模型组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表34、表35)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为46.71％、61.48％和70.41％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为64.79％、46.03％和37.02％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表34、可利霉素对裸鼠人食管癌TE-1细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000072
Figure PCTCN2018082072-appb-000073
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
表35、可利霉素对裸鼠人食管癌TE-1细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000074
Figure PCTCN2018082072-appb-000075
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
十八、可利霉素对人胃腺癌裸鼠模型的抑制作用
小鼠实体瘤模型的建立
取对数生长期的SGC7901细胞，台盼蓝抗染实验示活细胞>95％，胰蛋白酶消化，离心，去上清，用matrigel调细胞浓度至1×10 7/ml，将细胞接种与裸鼠右腋窝皮下0.2ml/只，并记为接种第1天。待肿瘤长至≥100mm 3时，将动物随机分为5组，每组6只：模型组、环磷酰胺组、可利霉素12.5、25及50mg/kg 3个剂量组。各组动物连续灌胃给药30天，给药体积为20ml/kg。停药次日处死小鼠，检测指标。从给药开始到处死裸鼠期间，每隔2天记录肿瘤长径、短径及体重。
瘤体积及相对肿瘤增殖率的计算
每隔2d测量裸鼠体重及移植瘤的长径(a)、短径(b)，按下列公式分别计算肿瘤体积(v)，相对肿瘤体积(RTV)和相对肿瘤增殖率(T/C)，其中V＝a×b 2/2；RTV＝V/V 0(V 0为给药前肿瘤体积，V为处死前肿瘤体积)，T/C(％)＝治疗组RTV/模型对照组RTV×100％。
瘤生长抑制率的计算
小鼠称重后处死，完整剥离肿瘤，去除血污、脂肪等非肿瘤组织称瘤体重量，计算肿瘤 生长抑制率。以每组小鼠的平均瘤重作为疗效指标。肿瘤生长抑制率(％)＝(1-治疗组平均瘤重/模型组平均瘤重)×100％。
试验结果表明，与对照组比较，各给药组对肿瘤生长抑制率、肿瘤体积、相对肿瘤体积和相对肿瘤增殖率均有一定程度的抑制作用(表36、表37)。
可利霉素低、中、高三个剂量组肿瘤生长抑制率分别为42.51％、68.92％和74.49％。
可利霉素低、中、高三个剂量组肿瘤体积及相对肿瘤体积明显低于模型组(P<0.05)。
可利霉素低、中、高三个剂量组相对肿瘤增殖率分别为69.12％、47.88％和38.35％。
可利霉素低、中、高三个剂量组动物体重与模型组相比均无明显变化。
表36、可利霉素对裸鼠人胃腺癌SGC7901细胞移植瘤抑制率的影响
Figure PCTCN2018082072-appb-000076
Figure PCTCN2018082072-appb-000077
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
表37、可利霉素对裸鼠人胃腺癌SGC7901细胞移植瘤瘤体积变化的影响
Figure PCTCN2018082072-appb-000078
Figure PCTCN2018082072-appb-000079
*p<0.05与模型组相比；**p<0.01与模型组相比，***p<0.001与模型组相比
十九、对小鼠H 22肝癌及小鼠Lewis肺癌移植瘤的抑制作用
小鼠实体瘤模型的建立：
取液氮冻存H 22细胞株于昆明种小鼠体内复苏，3代后取腹水，置于50ml离心管中，加入10ml 0.9％生理盐水，1000rpm室温离心5min，弃上清。加入10ml 0.9％生理盐水，吹打混匀，计数后用生理盐水稀释至5×10 6个活细胞/ml。置于冰水中保存，75％乙醇消毒小鼠右 腋下皮肤。迅速接种于昆明种小鼠右前肢腋下皮下，每只接种0.2ml。
Lewis肺癌细胞用含l0％胎牛血清的RPMI 1640培养基，于37℃、5％CO 2培养箱中培养。取对数生长期的细胞，以0.25％的胰酶消化，收集细胞，离心去上清，用无菌生理盐水洗涤两次，将细胞悬浮于生理盐水中，台盼蓝染色细胞活力测定大于95％，并进行细胞计数。将Lewis细胞浓度调至1×10 7/mL，置于冰水中保存。75％乙醇消毒小鼠右腋下皮肤，迅速接种于C57BL/6小鼠右腋部皮下注射0.2mL。
动物分组及给药方法
H 22肝癌模型中，接种次日，将接种肿瘤的小鼠随机分组，每组10只。包括：模型对照组，阳性药环磷酰胺对照组(CTX,26mg/kg)，可利霉素13、26及53mg/kg 3个剂量组。各组动物连续灌胃给药7天，给药体积为20ml/kg。
Lewis肺癌模型中，接种次日，将接种肿瘤的小鼠随机分组，每组10只。包括：模型对照组，阳性药环磷酰胺对照组(CTX,30mg/kg)，可利霉素13、26及52mg/kg 3个剂量组。各组动物连续灌胃给药15天，给药体积为20ml/kg。
计算抑瘤率：
将荷瘤小鼠于最后一次给药后次日称重后处死，解剖皮下瘤块并称重，分别计算各组的平均瘤重，计算抑瘤率。
抑瘤率＝(1－T/C)×100％
T：给药组平均瘤重；C：空白对照组平均瘤重。
结果：
1.可利霉素对小鼠H 22肝癌移植瘤的抑制作用
由表38结果可见，阳性对照药环磷酰胺对昆明种小鼠H 22肝癌的抑瘤率为47.25％。可利霉素26及52mg/kg对小鼠H 22肝癌的生长均有明显的抑制作用，抑瘤率分别为50.67％和79.50％，可利霉素52mg/kg剂量组抑瘤率明显低于阳性对照组(P<0.05)。
阳性药环磷酰胺组与正常对照组相比体重略有下降。可利霉素各给药组动物体重与给药前相比均有所增加，与模型对照组相比无明显区别。
表38、可利霉素对小鼠H22肝癌移植瘤的抑制作用
Figure PCTCN2018082072-appb-000080
Figure PCTCN2018082072-appb-000081
*p<0.05与模型组相比；**p<0.01与模型组相比； ##p<0.05与环磷酰胺组相比
2.可利霉素对小鼠Lewis肺癌移植瘤的抑制作用
由表39结果可见，阳性对照药环磷酰胺对小鼠Lewis肺癌的抑瘤率为49.14％。可利霉素13、26及52mg/kg对小鼠Lewis肺癌的生长均有明显的抑制作用，抑瘤率分别为50.30％、55.88％和76.23％，可利霉素52mg/kg剂量组抑瘤率明显低于阳性对照组(P<0.05)。可利霉素各给药组动物体重与给药前相比均有所增加，且与模型对照组相比无明显区别。
表39、可利霉素对小鼠Lewis肺癌移植瘤的抑制作用
Figure PCTCN2018082072-appb-000082
Figure PCTCN2018082072-appb-000083
*p<0.05与模型组相比；**p<0.01与模型组相比； ##p<0.05与环磷酰胺组相比
二十、可利霉素对荷瘤小鼠免疫功能的影响
方法
1、对荷瘤小鼠胸腺指数及脾脏指数的影响
荷瘤小鼠处死后，取脾脏及胸腺称重，计算脾脏指数及胸腺指数。
2、对荷瘤小鼠淋巴细胞增殖活性及自然杀伤(NK)细胞活性的影响
2.1脾淋巴细胞的制备
在平皿中放入无血清的RPMI 1640培养液，置于冰上，无菌取脾，用无菌载玻片将脾轻轻磨碎，制成单细胞悬液。用双层无菌纱布过滤，用无血清的RPMI1640培养液洗2次，1 500rpm离心5min，去上清液。加入2mL红细胞裂解液，静置2min，加入8mL RPMI 1640培 养液，1500rpm离心5min，去上清液，用RPMI 1640培养液洗两次。台盼蓝染色活细胞计数，活细胞>95％。用含体积分数为10％胎牛血清的RPMI1640培养液制成单细胞悬液。
2.2脾淋巴细胞增殖活性测定
取脾细胞悬液，将细胞密度调整为1×10 7/mL。每只鼠设：A.对照孔：加100μL RPMI 1640培养液；B.刀豆蛋白A(ConA)刺激孔：加刀豆蛋白A(ConA)溶液100μL(10mg/L)；C.细菌内毒素(LPS)刺激孔：加细菌内毒素(LPS)溶液100μL(20mg/L)。将上述细胞加入至96孔板中，然后以上各孔均加100μL脾细胞悬液。将培养板移入体积分数为5％CO 2、37℃、饱和湿度条件下孵育72h后，各孔加入MTT溶液(5g/L)10μL，同样条件继续孵育4h后终止培养。加入三联液(SDS 10g，10M HCl 0.1mL，异丁醇5mL，用蒸馏水稀释至100mL)100μl，振荡10min，使结晶物充分溶解，570nm处测各孔吸光度(OD)值，并计算淋巴细胞增殖率。淋巴细胞增殖率(％)＝[(T–C)/C]×100％，式中T为刺激孔吸光度值，C为对照孔吸光度值。
2.3自然杀伤(NK)细胞活性测定
取脾细胞悬液，将细胞密度调整为1×10 7/mL(效应细胞)。制备K562细胞混悬液，细胞密度1×10 5/mL(靶细胞)。每只鼠设：A.效应细胞：靶细胞孔(数量比为20：1)，加20μL脾细胞悬液和100μL K562细胞悬液；B.效应细胞对照孔，加100μL脾细胞悬液和100μL RPMI 1640培养液；C.靶细胞对照孔，加100μL K562细胞悬液和100μL RPMI 1640培养液。将上述细胞加入至96孔板中，将培养板移入体积分数为5％CO 2、37℃、饱和湿度条件下孵育22h后，各孔加入MTT溶液(5g/L)10μL，同样条件继续孵育4h后终止培养。加入三联液(SDS 10g，10M HCl 0.1mL，异丁醇5mL，用蒸馏水稀释至100mL)100μl，振荡10min，使结晶物充分溶解，490nm处测各孔吸光度值，并计算NK细胞活性。NK细胞活性(％)＝[TO-(S-E)]/TO×100％，式中TO为靶细胞对照孔吸光度值，S为效应细胞对照孔吸光度值，E为效应细胞吸光度值。
结果
1、对H 22肝癌荷瘤小鼠胸腺指数与脾指数的影响
由表40结果可见，阳性对照药环磷酰胺组动物胸腺指数和脾指数与对照组比较有显著下降(P<0.01)。可利霉素13、26及52mg/kg组动物胸腺指数与对照组比较无明显变化，52mg/kg组动物脾指数与对照组比较有明显增加(P<0.05)。
表40、可利霉素对H 22肝癌荷瘤小鼠胸腺指数和脾指数(w)的影响(
Figure PCTCN2018082072-appb-000084
n＝6)
Figure PCTCN2018082072-appb-000085
**p<0.01与对照组相比
2、对Lewis肺癌荷瘤小鼠胸腺指数与脾指数的影响
由表41结果可见，阳性对照药环磷酰胺组动物脾指数与对照组比较有显著下降(P<0.01)。可利霉素13、26及52mg/kg组动物脾指数及胸腺指数与对照组比较无明显变化。
表41、可利霉素对Lewis肺癌荷瘤小鼠胸腺指数和脾指数(w)的影响(
Figure PCTCN2018082072-appb-000086
n＝6)
Figure PCTCN2018082072-appb-000087
**p<0.01与对照组相比；*p<0.05与对照组相比；
3、对Lewis肺癌荷瘤小鼠NK细胞活性的影响
由表42结果可见，阳性对照药环磷酰胺组动物NK细胞活性与对照组比较有显著下降(P<0.05)。可利霉素13、26mg/kg组动物NK细胞活性与对照组相比有明显增加(P<0.01)。
表42、可利霉素对Lewis肺癌荷瘤NK细胞活性的影响
Figure PCTCN2018082072-appb-000088
n＝6)
Figure PCTCN2018082072-appb-000089
**p<0.01与对照组相比；*p<0.05与对照组相比；
4、对Lewis肺癌荷瘤小鼠淋巴细胞增殖活性的影响
由表43结果可见，阳性对照药环磷酰胺组动物淋巴细胞活性受到明显抑制(P<0.05)。可利霉素13、26mg/kg组动物淋巴细胞活性与对照组相比有明显增加(P<0.05，P<0.01)。
表43、可利霉素对Lewis肺癌小鼠移植肿瘤淋巴细胞增殖的影响
Figure PCTCN2018082072-appb-000090
n＝6)
Figure PCTCN2018082072-appb-000091
*p<0.05与对照组相比；**p<0.01与对照组相比；
5、对A549肺癌荷瘤小鼠脾指数的影响
由表44结果可见，阳性对照药环磷酰胺组动物脾指数与对照组比较有显著下降(P<0.01)。可利霉素13、26及52mg/kg组动物脾指数与对照组比较无明显变化。
表44、可利霉素对A549肺癌荷瘤小鼠脾指数的影响(
Figure PCTCN2018082072-appb-000092
n＝6)
Figure PCTCN2018082072-appb-000093
**p<0.01与对照组相比
试验例3、临床试验
本发明收集多起临床病例，有的患者患有乳腺癌，疼痛症状重。后配合服用可利霉素片(实施例1制备)2个疗程(30天为1个疗程，口服，每日2片)后，主治医师诊断其肿块见小。患者本人也感觉疼痛减轻，精神状况很好。
有的患者患有肾透明细胞腺癌，拍片子诊断肾部阴影，肾部肿瘤占左肾面积的三分之二，病理结果是肾透明细胞腺癌，早期。后配合服用可利霉素片(实施例1制备)2个疗程(30天为1个疗程，口服，每日2片)后，主治医师诊断其肾部肿瘤面积减小，有明显好转。
有的患者患有胶质瘤，诊断脑组织浸润破坏，周围脑水肿亦显著，有头痛，视力减退等症状。后配合服用可利霉素片(实施例1制备)2个疗程(30天为1个疗程，口服，每日2片)后，主治医师诊断其水肿减轻，患者也感觉症状减轻，有明显好转。
有的患者患有淋巴瘤，诊断颈部淋巴结肿大，似枣大，中等硬度，比较丰满。后配合服用可利霉素片(实施例1制备)3个疗程(30天为1个疗程，口服，每日2片)后，主治医师诊断其淋巴结肿大减轻，变为黄豆大，患者也感觉症状减轻，不再感觉很硬，有明显好转。
有的患者患有结肠癌，诊断为瘤体或与网膜、周围组织浸润肿块，质硬，形体不规则。后配合服用可利霉素片(实施例1制备)1个疗程(30天为1个疗程，口服，每日2片)后，主治医师诊断其肿块减轻，患者感觉有明显好转。
有的患者患有白血病，有不同程度的贫血、出血、感染发热以及肝、脾、淋巴结肿大和骨骼疼痛。后配合服用可利霉素片(实施例1制备)3个疗程(30天为1个疗程，口服，每日2片)后，主治医师诊断其症状减轻，患者感觉有明显很好转。
有的患者患有胃腺癌，感觉胃在隐隐作痛，胃里好像有东西在发酵一样，很胀，而且胃酸水偶尔还会冲上喉咙，使得喉间火辣辣的很恶心，身体发热冒冷汗，去医院检查得了胃腺癌。后配合服用可利霉素片(实施例1制备)3个疗程(30天为1个疗程，口服，每日2片)后，主治医师诊断其症状减轻，患者感觉有明显好转。
有的患者患有食管癌，食管钡餐X线片可见食管狭窄，壁管不光滑，黏膜破坏，患者感觉进行性咽下困难。后配合服用可利霉素片(实施例1制备)2个疗程(30天为1个疗程，口服，每日2片)后，主治医师诊断其症状减轻，患者感觉有明显好转。
有的被诊断为患有胰腺癌，四肢乏力、食欲不振还伴有恶心、呕吐、腹泻。后配合服用可利霉素片(实施例1制备)2个疗程(30天为1个疗程，口服，每日2片)后，主治医师诊断其症状减轻，患者感觉有明显好转。
有的被诊断患有前列腺癌，逐渐增大的前列腺腺体压迫患者尿道引起进行性排尿困难，表现为尿线细、射程短、尿流缓慢、尿流中断、尿后滴沥、排尿不尽、排尿费力，此外，还有尿频、尿急、夜尿增多、甚至尿失禁。肿瘤压迫直肠可引起大便困难或肠梗阻。后配合服用可利霉素片(实施例1制备)2个疗程后，主治医师诊断其症状减轻，患者感觉有明显好转。
有的患者肺癌、肝癌、转移至骨癌晚期，因呕吐、咳嗽、骨头疼入院，后服用可利霉素片(实施例1制备)4盒，经检查发现肺片肿瘤钙化、肺肿瘤消失。
有的患者患有肺癌，右肺上叶周围型占位病变，因呼吸困难，剧烈咳嗽就诊。CT检查：右肺上叶周围性病变，4.3*4.6类圆形团块影；而后又做增强CT：团块4.0*4.4，其他变化不大。后服用可利霉素，可利霉素2片(实施例1制备)/日*14天，以后1片/天至今；辅助小牛胸腺肽6粒/日。后经CT检查，类圆形团块影见明显减小，患者精神状态良好，无不适感。
有的患者患有宫颈癌，已转移至肺、骨、肠其他部位，CT检查肺部有胸积液及多个结节，呼吸困难，发烧，已做化、放疗治疗，后服用可利霉素片(实施例1制备)两个疗程以后，CT复查胸积液消失，结节变小。
试验例4、毒理学试验
1、急性毒性试验
1.1试验目的
测定犬一次灌服可利霉素的毒性反应严重程度、死亡情况和半数致死量LD 50，为长期毒性试验提供参考。
1.2受试药物：
名称：可利霉素
效价：927U/mg
配制方法：称取适量研磨成粉末，加适量0.5％羧甲基纤维素溶液，继续搅拌混匀，口服 给药配成100mg/ml。
溶剂：0.5％羧甲基纤维素溶液
1.3动物：犬
来源：中国医学科学院阜外心血管病医院动物饲养场
种属：杂种
合格证号：京动管犬字(96)第024号
体重：15-20公斤
性别：雄性
禁食时间：12小时
每组动物数：1-2只
1.4试验方法
经初步预试，犬口服灌胃2000mg/kg和3000mg/kg未见动物死亡，也未见严重毒性反应。按新药指导原则，增加50％药量，即增至4500mg/kg，按体重称取可利霉素用量，与0.5％CMC充分混悬，用胃管灌胃口服给药(试验前禁食过夜)，给药后观察毒性反应和死亡情况一周。
1.5犬口服急性毒性
1.5.1毒性反应
在预试验剂量为2000mg/kg和3000mg/kg，一次灌胃给药，仅呈现短暂呕吐反应，吐出药液残渣、胃液和食物残渣。除呕吐反应外，未见其他方面毒性反应。正式试验时两只犬分别口服4500mg/kg，呈现毒性反应仍是呕吐等消化道症状，未见腹泻或稀便，且呕吐反应后，未见明显其他毒性症状。
1.5.2半数致死量
经两次预试灌胃剂量为2000mg/kg和3000mg/kg，仅呈现呕吐等消化道症状。根据新药审批指导原则，按50％递增法要求，正式试验剂量为4500mg/kg。经灌胃给药后的两只犬，均呈现呕吐反应，未见其他毒性反应。犬口服给药可利霉素的LD 50＞4500mg/kg。
表45、犬口服可利霉素急性毒性(LD 50)
Figure PCTCN2018082072-appb-000094
2、可利霉素对啮齿类动物微核试验
2.1试验目的
检测药物对哺乳动物体细胞的致突变潜力，为临床用药提供依据。
2.2受试药物
名称：可利霉素
含量：效价927U/mg
配制方法：按剂量大小称取适量的可利霉素细粉在研钵中研磨，加入适量的0.5％羧甲基纤维素钠溶液，制成混悬液，置4℃冰箱中保存。阴性对照用等量的0.5％羧甲基纤维素钠溶液。阳性对照称取适量的注射用环磷酰胺，临用时溶于生理盐水中，配制成60mg/kg的溶液。
溶剂、赋形剂：生理盐水、0.5％羧甲基纤维素钠溶液
对照品：阳性对照：60mg/kg环磷酰胺溶液
阴性对照：0.5％羧甲基纤维素钠
2.3动物
选用性成熟雄性NIH小鼠，由中国药品生物制品检定所提供。
2.4试验方法
每组6只小鼠。预实验选出剂量、给药次数、骨髓采样时间。
表46、可利霉素对小鼠骨髓嗜多染红细胞微核试验预试结果
Figure PCTCN2018082072-appb-000095
剂量以1/2LD 50为标准递减，给药次数为一次，根据预试结果骨髓采样时间选定为24小时采样。
2.4.1剂量
至少三个剂量组，分别为：2000mg/kg、1000mg/kg、500mg/kg
剂距：0.5
给药次数：一次性口服给药。
2.4.2途径
口服(灌胃)给药。
2.4.3骨髓采样时间：给药后24小时。
2.4.4标本制作：处死动物、涂片、固定、染色。
2.4.5方法：
颈椎脱臼法处死动物，取胸骨，剔净肌肉，用纱布擦净附着物。采用骨髓直接涂片，将胸骨体剪断，露出骨髓腔，挤出骨髓于事先放有一滴小牛血清的载波片上混匀推片。晾干后放入甲醇中固定10分钟，晾干之后，进行Giemsa染色。每只小鼠镜检观察1000个左右红细胞轮廓完整的多染红细胞，计数微核出现的频率及多染红细胞占红细胞的比率。
2.5可利霉素对小鼠嗜多染红细胞微核试验结果见下表
2.5.1多染红细胞微核出现率
表47、多染红细胞微核出现率
Figure PCTCN2018082072-appb-000096
2.5.2多染红细胞与正常红细胞的比率
表48、多染红细胞与正常红细胞的比率
Figure PCTCN2018082072-appb-000097
2.5.3可利霉素对小鼠骨髓嗜多染红细胞微核的影响
表49、可利霉素对小鼠骨髓多染红细胞微核的影响
Figure PCTCN2018082072-appb-000098
*P/N正常红细胞/多染红细胞**P＜0.01
Heddle等报道16种小鼠骨髓嗜多染红细胞微核的自发率为3.1％，在本发明研究中，阴性对照组的平均值是2.63‰，可利霉素药物的三个剂量组与阴性对照组相比，嗜多染红细胞微核率无明显增加(P＞0.05)。嗜多染红细胞与正常红细胞比率无明显减少，波动在正常范围。环磷酰胺阳性对照组与阴性对照组相比较，嗜多染红细胞微核有非常显著的增加(P＜0.01)，其微核率为阴性对照组的16.85倍。
2.6结论
以上结果表明，可利霉素不是一个染色体断裂剂。在所用剂量下，不影响细胞的正常有丝分裂和对骨髓无抑制作用。
以上所述仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本发明的技术人员在不脱离本发明技术方案范围内，当可利用上述提示的技术内容作出些许变动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明方案的范围内。

Claims (10)

  1. 可利霉素及其药学上可接受的盐在制备治疗和/或预防肿瘤药物方面的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述的肿瘤包括实体瘤和非实体瘤。
  3. 根据权利要求2所述的应用，其特征在于，所述的实体瘤包括良性实体瘤和恶性实体瘤；所述的非实体瘤为淋巴瘤或白血病。
  4. 根据权利要求3所述的应用，其特征在于，所述的恶性实体瘤为乳腺癌、肝癌、肺癌、肾癌、脑瘤、宫颈癌、前列腺癌、胰腺癌、食管癌、胃癌或结肠癌；
    优选，所述的脑瘤为胶质瘤或脑膜瘤，所述的胃癌为胃腺癌。
  5. 根据权利要求1-4任意一项所述的应用，其特征在于，所述的药物为可利霉素及其药学上可接受的盐与药学上可接受的辅料制成的各种剂型。
  6. 根据权利要求1-4任意一项所述的应用，其特征在于，所述的药物为可利霉素及其药学上可接受的盐和抗肿瘤药物与药学上可接受的辅料制成的各种剂型。
  7. 根据权利要求1-4任意一项所述的应用，其特征在于，所述的药物为含有可利霉素及其药学上可接受的盐的第一药剂和含有抗肿瘤药物的第二药剂的组合。
  8. 根据权利要求6或7所述的应用，其特征在于，所述的抗肿瘤药物为化疗、放疗、靶向治疗和/或免疫治疗药物。
  9. 根据权利要求5或6所述的应用，其特征在于，所述的药物的剂量为5～1500mg；优选50～1000mg；更优选100～400mg。
  10. 根据权利要求5或6所述的应用，其特征在于，所述的第一药剂的剂量为5～1500mg；优选50～1000mg；更优选100～400mg。
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