WO2020181802A1 - 一种有效抗恶性肿瘤的药物组合物及其应用 - Google Patents

一种有效抗恶性肿瘤的药物组合物及其应用 Download PDF

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WO2020181802A1
WO2020181802A1 PCT/CN2019/116993 CN2019116993W WO2020181802A1 WO 2020181802 A1 WO2020181802 A1 WO 2020181802A1 CN 2019116993 W CN2019116993 W CN 2019116993W WO 2020181802 A1 WO2020181802 A1 WO 2020181802A1
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cancer
tumor
metformin
chlorpromazine
malignant
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PCT/CN2019/116993
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French (fr)
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宁志丰
李文静
刘复兴
吴基良
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湖北科技学院
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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
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the present invention relates to the technical field of cancer treatment methods, in particular to an effective anti-malignant pharmaceutical composition and its application.
  • Metformin is an oral hypoglycemic drug widely used clinically to treat type 2 diabetes mellitus (T2DM). Preclinical studies have shown that metformin can inhibit the proliferation and apoptosis of a variety of cancer cells in vivo and in vitro. Some retrospective studies and prospective trials have also proved that metformin can reduce the incidence and mortality of tumors. Clinical trials using metformin as an auxiliary anticancer drug are being extensively carried out. A necessary summary of existing research can provide a basis for more in-depth research. Chlorpromazine is a classic antipsychotic of the phenothiazine class.
  • phenothiazine antipsychotic drug discovered in 1950 when studying the structural modification of the phenothiazine anti-allergic drug promethazine and started a psychotic drug
  • phenothiazines have also been found to have significant antimicrobial effects, and phenothiazines have also been shown to destroy cancer cells and make them sensitive to chemotherapy. From the point of view, there is no doubt that phenothiazine is one of the most functional compounds.
  • metformin activates adenosine mono-phosphate-activated protein kinase (AMPK) through liver kinase B1 (liver kinase B1, LKB1) and inhibits protein kinase B (protein kinase B, PKB) And downstream rapamycin target protein (mammalian target of rapamycin, mTOR) pathway; AMPK can also down-regulate the level of cyclin D1 (cyclin D1) to block cells in G0/G1 phase.
  • AMPK adenosine mono-phosphate-activated protein kinase
  • Metformin reduces the circulating levels of insulin and IGF-1 and blocks phosphati-dylinositol-3-kinase (PI3K)/Akt/mTOR and RAS/RAF/mitogen activation related to cell growth Protein kinase (mitogen-activated protein kinase, MAPK) pathway; through the MAPK pathway, it induces cell apoptosis and enhances the expression of growth arrest and DNA damage inducible gene 153 (GADD153).
  • metformin can also simultaneously regulate multiple miRNAs, exerting its biological functions such as inhibiting cancer cell proliferation and invasion and promoting apoptosis. Metformin can also maintain the activity of cytotoxic T cells and degrade programmed cell death 1 ligand 1 (PD-L1).
  • PD-L1 programmed cell death 1 ligand 1
  • chlorpromazine inducing apoptosis, inhibiting efflux pumps, inhibiting angiogenesis, and anti-cancer stem cell activity. The most important of which is the process of inducing apoptosis and targeted inhibition of cancer stem cells.
  • Such as inhibiting DNA repair mechanisms and signal transduction pathways, and phenothiazine drugs inhibit the binding of calcium to calcium-dependent enzymes such as calmodulin. Many calcium-mediated signal transduction pathways may be interfered, which may lead to cell apoptosis and inhibit proliferation.
  • Chlorpromazine can also target To suppress the dominant gene PELP1.
  • metformin inhibiting tumor is to inhibit tumor proliferation and invasion, while chlorpromazine mainly induces apoptosis and tumor stem cells. It can be seen that the combined application of the two drugs has a theoretical synergistic effect.
  • the purpose of the present invention is to provide an effective anti-malignant pharmaceutical composition and its application.
  • the present invention provides an effective anti-malignant medicinal composition.
  • the effective components of the medicinal composition consist of 100-400 parts of metformin and 1-10 parts of chlorpromazine in mole fractions.
  • the pharmaceutical composition is formed by mixing effective components and pharmaceutical excipients, and its dosage form is oral preparation, injection or sustained-release preparation.
  • the invention also provides the application of an effective anti-malignant pharmaceutical composition in the preparation of anti-tumor drugs.
  • the anti-tumor drug is for the treatment of lung adenocarcinoma, gastric cancer, lung squamous cell carcinoma and liver cancer, cervical cancer, breast cancer, esophageal cancer, colon cancer, pancreatic cancer, nasopharyngeal cancer, peritoneal cancer, mesothelioma, kidney Drugs for cancer, bladder cancer, prostate cancer, urethral cancer, endometrial cancer, ovarian cancer, malignant glioma, bone tumor, fibrosarcoma, retinoblastoma, leukemia, lymphoma.
  • the anti-tumor drug is a drug that can inhibit the proliferation of tumor cells.
  • the anti-tumor drug can inhibit lung adenocarcinoma cell A549, gastric adenocarcinoma cell AGS, lung squamous cell H520, liver cancer cell HepG2, cervical cancer Hela, breast cancer MCF-7, esophageal cancer EC109, colon cancer LOVO , Pancreatic cancer As-pc1, nasopharyngeal cancer CNE2, bladder cancer T24, kidney cancer OS-RC-2, prostate cancer PC3, endometrial cancer KLE, ovarian cancer Hey, malignant glioma U87, bone tumor U2-OS, Drugs for proliferation and metastasis of tumor cell lines such as fibrosarcoma Ag104, retinoblastoma y79, leukemia NOMO-1, and lymphoma Daudi.
  • tumor cell lines such as fibrosarcoma Ag104, retinoblastoma y79, leukemia NOMO-1, and lymphoma Daudi.
  • the anti-tumor drug is a drug capable of reducing tumor volume and weight.
  • the pharmaceutical composition with synergistic anti-tumor efficacy provided by the present invention whose effective components are composed of metformin and chlorpromazine, can be used to prepare anti-tumor drugs.
  • metformin and chlorpromazine have good anti-tumor effects after combined application, and the synergistic effect of the two drugs can maximize the anti-tumor efficacy, and has high scientific research value and good application prospects.
  • the present invention uses the MTT method and the drug combination index to study the effects of metformin and chlorpromazine on lung adenocarcinoma cells A549, gastric adenocarcinoma cells AGS, lung squamous cell carcinoma cells H520, and liver cancer cells HepG2, cervical cancer Hela, and breast cancer MCF-7.
  • the pharmaceutical composition provided by the present invention can significantly inhibit the proliferation of malignant tumor cells such as lung adenocarcinoma, gastric adenocarcinoma, lung squamous cell carcinoma and liver cancer, and subcutaneous tumor formation in nude mice. And treatment of malignant tumors such as liver cancer.
  • Figure 1 shows the inhibitory effect of metformin alone, chlorpromazine alone, and combined administration of metformin and chlorpromazine on the proliferation of lung adenocarcinoma cells A549;
  • Figure 2 shows the inhibitory effects of metformin alone, chlorpromazine, and combined administration of metformin and chlorpromazine on the proliferation of gastric adenocarcinoma cells AGS;
  • Figure 3 shows the inhibitory effects of metformin alone, chlorpromazine alone, and combined administration of metformin and chlorpromazine on the proliferation of lung squamous cell carcinoma cells H520;
  • Figure 4 shows the inhibitory effects of metformin alone, chlorpromazine alone, and combined administration of metformin and chlorpromazine on the proliferation of liver cancer cells HepG2.
  • Figure 5 shows the inhibitory effects of metformin alone, chlorpromazine alone, and combined administration of metformin and chlorpromazine on the migration ability of lung adenocarcinoma cells A549
  • Figure 6 shows the inhibitory effects of metformin alone, chlorpromazine alone, and combined administration of metformin and chlorpromazine on the invasive ability of lung adenocarcinoma cells A549
  • lung adenocarcinoma cells A549, gastric adenocarcinoma cells AGS, lung squamous cell carcinoma cells H520 and liver cancer cells HepG2 were from the cell bank of Wuhan University, in a medium containing 10% fetal bovine serum (containing 100U/ml penicillin and streptomycin) , Placed in a 37°C, 5% CO2 incubator for culture, passaged every 2-3d.
  • Metformin and chlorpromazine are composed according to the mole fraction (200): (1).
  • the molar ratio of metformin and chlorpromazine is 200:1 before being used.
  • Cell inoculation A single cell suspension is prepared with a culture medium containing 10% fetal calf serum, and 1,000-10,000 cells per well are inoculated into a 96-well plate with a volume of 100ul per well. After 24 hours of culture in a 5% CO 2 cell incubator, the drug was administered.
  • Drug preparation accurately weigh 35.53 mg of chlorpromazine hydrochloride and dissolve it in 10 ml of PBS to prepare a stock solution of 10 mmol/l. Set the dosing gradient 0, 2.5, 5, 10, 20, 40umol/l. Accurately weigh 16.56 mg of metformin hydrochloride and dissolve it in 10 ml of PBS to prepare 1 mol/l, and set the dose gradient 0, 0.5, 1, 2, 4, 8 mmol/l accordingly. The combined administration concentration is the sum of the administration concentration corresponding to the chlorpromazine and metformin administration gradient.
  • Colorimetry select the wavelength of 490nm, measure the light absorption value of each hole on the enzyme-linked immunosorbent monitor, record the result, draw the cell growth curve with the time as the abscissa and the absorbance as the ordinate.
  • the above-mentioned MTT experiment method was used to detect the cytostatic effect of the combination drug, and the Jinzhengjun method was used to judge whether the combination of the two drugs has a synergistic effect.
  • the MTT method was used to investigate the inhibitory effects of metformin and chlorpromazine on the proliferation of lung adenocarcinoma cells A549, gastric adenocarcinoma AGS, lung squamous cell carcinoma H520, and liver cancer cells HepG2.
  • the results showed that metformin and chlorpromazine alone had effects on lung adenocarcinoma cells A549 ( Figure 1), gastric adenocarcinoma cell line AGS ( Figure 2), lung squamous cell carcinoma cell line H520 ( Figure 3), and liver cancer cell line HepG2 ( Figure 4) have a good inhibitory effect.
  • the proliferation inhibitory effect of adenocarcinoma cell A549, gastric adenocarcinoma cell AGS, lung squamous cell carcinoma cell H520 and liver cancer cell HepG2 was obtained by calculation. It was found that when metformin (0, 0.5, 1, 2, 4, 8mmol/l) is used in combination with chlorpromazine (0, 2.5, 5, 10, 20, 40 ⁇ mol/L), it can show performance at certain concentrations Synergistic inhibition of lung adenocarcinoma cells A549, gastric adenocarcinoma cells AGS, lung squamous cell carcinoma cells H520 and liver cancer cells HepG2 (Figure 1 to Figure 4). The experimental results of the combined action index of the drug combination are shown in Tables 1 to 4.
  • Table 1 The combined effect index table (CI) of drug combination on lung adenocarcinoma cell A549
  • Table 4 The combined effect index table (CI) of drug combination on liver cancer cell HepG2
  • metformin 10mmol/L+chlorpromazine 5 ⁇ mol/L has a synergistic effect on lung adenocarcinoma cells A549; metformin 5mmol/L+chlorpromazine 2.5 ⁇ mol/L or metformin 1mmol/L+chlorpromazine 5 ⁇ mol/L combined use Gastric adenocarcinoma cells AGS showed a synergistic effect.
  • the other combinations of experimental concentrations all show simple additive effects or antagonistic effects.
  • mice Twenty BALB-C nude mice aged 24-28 days, male, weighing 10.01 ⁇ 0.73g.
  • control group equivalent PBS solution
  • metformin group 100mg/kg
  • chlorpromazine group 50mg/kg
  • the tumor inhibition rate (%) (1- average tumor weight of the experimental group/ The average tumor weight of the control group)*100%, and the statistics are shown in Table 5.
  • Table 5 The effect of combined medication on the tumor volume and quality of lung adenocarcinoma cell A549 inoculated with subcutaneous tumor
  • Group Control group 1 Metformin group Chlorpromazine group Metformin + Chlorpromazine Number of rats 5 5 5 5 Rat weight (g) 24.62 ⁇ 0.43 24.98 ⁇ 0.51 24.61 ⁇ 0.48 24.31 ⁇ 0.51 Tumor volume before treatment (cm3) 0.55 ⁇ 0.01 0.54 ⁇ 0.01 0.52 ⁇ 0.01 0.56 ⁇ 0.02 Tumor volume after one week (cm3) 0.72 ⁇ 0.02 0.69 ⁇ 0.01 0.7 ⁇ 0.02 0.64 ⁇ 0.01 Tumor volume after two weeks (cm3) 1.33 ⁇ 0.01 1.07 ⁇ 0.03 1.12 ⁇ 0.01 0.88 ⁇ 0.02 Tumor weight (g) 0.76 ⁇ 0.03 0.53 ⁇ 0.02 0.67 ⁇ 0.01 0.35 ⁇ 0.01 Tumor inhibition rate (%) / 61.2 43.5 84.9
  • the tumor inhibition rate of transplanted tumors is 61.2%; when chlorpromazine is administered alone, the tumor inhibition rate of transplanted tumors is 43.5%; when metformin and chlorpromazine are used in combination, transplantation
  • the tumor inhibition rate can reach 84.9%.
  • metformin group, chlorpromazine group, metformin + chlorpromazine the body weights of nude mice were 24.62 ⁇ 0.43, 24.98 ⁇ 0.51, 24.61 ⁇ 0.48 and 24.31 ⁇ 0.51, respectively.
  • the results showed that the combined treatment group significantly reduced mice At the same time as the tumor volume and tumor weight, the body weight of the mice did not decrease significantly, indicating that the combined medication had no obvious effect on the physiological state of the mice, and there were no toxic side effects on experimental animals.

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Abstract

一种有效抗恶性肿瘤的药物组合物及其应用，该药物组合物按摩尔份数计，有效组分由4～10份二甲双胍和1～3份氯丙嗪组成，具有协同抗肿瘤功效。二甲双胍和氯丙嗪联合应用后具有良好的抗肿瘤效果，通过两者的药物协同作用能够使抗肿瘤的疗效得以最大化，具有很高的科研价值和良好的应用前景。

Description

一种有效抗恶性肿瘤的药物组合物及其应用 技术领域
本发明涉及癌症治疗方法的技术领域，具体的说是涉及一种有效抗恶性肿瘤的药物组合物及其应用。
背景技术
二甲双胍为临床广泛用于治疗2型糖尿病(type 2diabetes mellitus，T2DM)的口服降糖药物。临床前研究表明，二甲双胍可以在体内外抑制多种癌细胞的增殖和凋亡。一些回顾性研究与前瞻性试验也证明二甲双胍可降低肿瘤的发病率及死亡率。将二甲双胍作为辅助抗癌药的临床试验正在广泛开展。对现有研究进行必要的归纳总结可为开展更深入的研究提供依据。氯丙嗪属于吩噻嗪类的经典抗精神病药，于1950年在研究吩噻嗪类抗过敏药异丙嗪的结构改造时发现的第一个吩噻嗪类抗精神病药物并开启了精神病药物治疗的新纪元，近些年除了它们的抗精神病活性外，还发现吩噻嗪具有显著的抗微生物作用，并且吩噻嗪类药物还被证明可以破坏癌细胞并使它们对化疗敏感,从生物活性的角度来看，毫无疑问吩噻嗪是功能最多的化合物之一。
二甲双胍的潜在抗癌机制是二甲双胍通过肝脏激酶B1(liver kinase B1，LKB1)激活腺苷酸活化蛋白激酶(adenosine mono-phosphate-activated protein kinase，AMPK)，抑制蛋白激酶B(protein kinase B，PKB)以及下游雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR)途径；还可通过AMPK下调细胞周期蛋白D1 (cyclin D1)水平，把细胞阻断在G0/G1期。二甲双胍降低了胰岛素和IGF-1的循环水平，阻断与细胞生长有关的磷脂酰肌醇-3激酶(phosphati-dylinositol-3-kinase，PI3K)/Akt/mTOR和RAS/RAF/丝裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)通路；通过MAPK途径诱导细胞凋亡并增强生长抑制和DNA损伤诱导基因153(growth arrest andDNA damage inducible gene 153，GADD153)的表达。除此之外二甲双胍还可同时调控多种miRNA，发挥其抑制癌细胞增殖与侵袭和促进凋亡等生物学功能。二甲双胍也可维持细胞毒性T细胞的活性，降解细胞程序性死亡-配体1(programmed cell death 1ligand 1，PD-L1)。而氯丙嗪的潜在抗肿瘤机制为诱导细胞凋亡作用、抑制外排泵、抑制血管生成、抗癌干细胞活性，其中最重要的是诱导细胞凋亡的过程和靶向抑制肿瘤干细胞的作用，如抑制DNA修复机制和信号转导途径，并且吩噻嗪类药物抑制钙与钙依赖性酶如钙调蛋白的结合。许多钙介导的信号转导途径可能受到干扰，可能导致细胞凋亡和抑制增殖，除此之外吩噻嗪类药物的直接DNA损伤和细胞膜去稳定作用也很突出，氯丙嗪还可以靶向抑制主导基因PELP1。其中二甲双胍抑制肿瘤的主要机制为抑制肿瘤增殖和侵袭而氯丙嗪主要诱导细胞凋亡和肿瘤干细胞，由此可见两药联合应用理论上有协同作用。
发明内容
为解决上述背景技术中提出的问题，本发明的目的在于提供一种有效抗恶性肿瘤的药物组合物及其应用。
为实现上述目的，本发明采取的技术方案为：
本发明提供了一种有效抗恶性肿瘤的药物组合物，按摩尔份数计，该药物组合物的有效组分由100-400份二甲双胍和1-10份氯丙 嗪组成。
进一步的，该药物组合物由有效组分和药用辅料混合而成，其剂型为口服制剂、针剂或者缓释制剂。
本发明还提供了一种有效抗恶性肿瘤的药物组合物在制备抗肿瘤药物中的应用。
进一步的，所述的抗肿瘤药物为治疗肺腺癌、胃癌、肺鳞癌和肝癌、宫颈癌、乳腺癌、食管癌、结肠癌、胰腺癌、鼻咽癌、腹膜癌、间皮瘤、肾癌、膀胱癌、前列腺癌、尿道癌、子宫内膜癌、卵巢癌、恶性胶质瘤、骨肿瘤、纤维肉瘤、视网膜母细胞瘤、白血病、淋巴瘤的药物。
进一步的，所述的抗肿瘤药物为能够抑制肿瘤细胞增殖的药物。
进一步的，所述的抗肿瘤药物为能够抑制肺腺癌细胞A549、胃腺癌细胞AGS、肺鳞癌细胞H520、肝癌细胞HepG2、宫颈癌Hela、乳腺癌MCF-7、食管癌EC109、结肠癌LOVO、胰腺癌As-pc1、鼻咽癌CNE2、膀胱癌T24、肾癌OS-RC-2、前列腺癌PC3、子宫内膜癌KLE、卵巢癌Hey、恶性胶质瘤U87、骨肿瘤U2-OS、纤维肉瘤Ag104、视网膜母细胞瘤y79、白血病NOMO-1、淋巴瘤Daudi等肿瘤细胞株增殖和转移的药物。
进一步的，所述的抗肿瘤药物为能够降低肿瘤体积和重量的药物。
与现有技术相比，本发明的有益效果是：
本发明提供的具有协同抗肿瘤功效的药物组合物，其有效组分由二甲双胍和氯丙嗪组成，可用于制备抗肿瘤药物。本发明发现了二甲双胍和氯丙嗪联合应用后具有良好的抗肿瘤效果，通过两者的药物协同作用能够使抗肿瘤的疗效得以最大化，具有很高的科研价值和良好的应用前景。
本发明分别采用MTT法和药物联合作用指数研究了二甲双胍和氯丙嗪对肺腺癌细胞A549、胃腺癌细胞AGS、肺鳞癌细胞H520、和肝癌细胞HepG2、宫颈癌Hela、乳腺癌MCF-7、食管癌EC109、结肠癌LOVO、胰腺癌As-pc1、鼻咽癌CNE2、膀胱癌T24、肾癌OS-RC-2、前列腺癌PC3、子宫内膜癌KLE、卵巢癌Hey、恶性胶质瘤U87、骨肿瘤U2-OS、纤维肉瘤Ag104、视网膜母细胞瘤y79、白血病NOMO-1、淋巴瘤Daudi的抑制作用，得到适合的二甲双胍和氯丙嗪的混合比例及浓度。结果证实本发明提供的药物组合物具有明显的抑制肺腺癌、胃腺癌、肺鳞癌和肝癌等恶性肿瘤细胞的增殖、裸鼠皮下成瘤，可用于肺腺癌、胃腺癌、肺鳞癌和肝癌等恶性肿瘤的治疗。
附图说明
图1为二甲双胍单独给药、氯丙嗪单独给药以及二甲双胍和氯丙嗪联合给药对肺腺癌细胞A549增殖的抑制作用；
图2为二甲双胍单独给药、氯丙嗪单独给药以及二甲双胍和氯丙嗪联合给药对胃腺癌细胞AGS增殖的抑制作用；
图3为二甲双胍单独给药、氯丙嗪单独给药以及二甲双胍和氯丙嗪联合给药对肺鳞癌细胞H520增殖的抑制作用；
图4为二甲双胍单独给药、氯丙嗪单独给药以及二甲双胍和氯丙嗪联合给药对肝癌细胞HepG2增殖的抑制作用。
图5为二甲双胍单独给药、氯丙嗪单独给药以及二甲双胍和氯丙嗪联合给药对肺腺癌细胞A549迁移能力的抑制作用
图6为二甲双胍单独给药、氯丙嗪单独给药以及二甲双胍和氯丙嗪联合给药对肺腺癌细胞A549侵袭能力的抑制作用
具体实施方式
为使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合附图和具体实施方式，进一步阐述本发明是如何实施的。
实施例1
本实施例提供的药物组合物的细胞活力实验及联合作用指数分析
1.材料
实验用肺腺癌细胞A549、胃腺癌细胞AGS、肺鳞癌细胞H520和肝癌细胞HepG2来自武汉大学细胞库，在含10％胎牛血清的培养基(含100U/ml青霉素和链霉素)中，放置于37℃，5％CO2培养箱中进行培养，每隔2-3d传代一次。
2.实验方法
二甲双胍与氯丙嗪按照摩尔份数(200)：(1)组成，本实施例中，按照二甲双胍与氯丙嗪摩尔比例200:1混合后待用。
通过MTT实验的方法，检测测试药物对肺腺癌细胞A549、胃腺癌细胞AGS、肺鳞癌细胞H520和肝癌细胞HepG2增殖的影响。
(1)接种细胞：用含10％胎小牛血清得培养液配成单个细胞悬液，以每孔1000-10000个细胞接种到96孔板，每孔体积100ul。在5％CO 2细胞培养箱中培养24h后给药。
(2)药物配制：精密称取盐酸氯丙嗪35.53mg溶于10mlPBS中配制成10mmol/l的原液。设置给药梯度0、2.5、5、10、20、40umol/l。精密称取盐酸二甲双胍16.56mg溶于10mlPBS中配制成1mol/l，对应设置给药梯度0、0.5、1、2、4、8mmol/l。联合给药浓度为氯丙嗪和二甲双胍单独给药梯度对应的给药浓度之和。
(3)给药后培养细胞：同一般培养条件，培养2天即48h。
(4)呈色：培养2天后，每孔加MTT溶液(5mg/ml用PBS配)20 ul。继续孵育4小时，终止培养，小心吸弃孔内培养上清液，对于悬浮细胞需要离心后再吸弃孔内培养上清液。每孔加150ul DMSO，振荡10分钟，使结晶物充分融解。
(5)比色：选择490nm波长，在酶联免疫监测仪上测定各孔光吸收值，记录结果，以时间为横坐标，吸光值为纵坐标绘制细胞生长曲线。
采用上述MTT实验方法检测联合用药的细胞抑制作用，用金正均法判断两药合用是否有协同作用。
for synergism or antagonism are recommended，as in Table 4.1：
Figure PCTCN2019116993-appb-000001
Table 4.1：Symbols for Synergism and Ant agonism usinf CI analysis
3.实验结果
分别用MTT法考察二甲双胍和氯丙嗪单独对肺腺癌细胞A549、胃腺癌细胞AGS、肺鳞癌细胞H520和肝癌细胞HepG2的增殖抑制作用，结果表明二甲双胍和氯丙嗪单独对肺腺癌细胞A549(图1)、胃腺癌细胞AGS(图2)、肺鳞癌细胞H520(图3)和肝癌细胞HepG2(图4)具有较好的抑制作用，进而考察二甲双胍和氯丙嗪联用对肺腺癌细胞A549、胃腺癌细胞AGS、肺鳞癌细胞H520和肝癌细胞HepG2的增殖抑制作用，通过计算获得药物相互作用指数CI。结果发现，其中二甲双胍(0、0.5、1、2、4、8mmol/l)与氯丙嗪(0、2.5、 5、10、20、40μmol/L)联合使用时，可以在部分浓度下表现出对肺腺癌细胞A549、胃腺癌细胞AGS、肺鳞癌细胞H520和肝癌细胞HepG2的协同抑制作用(图1至图4)。药物组合联合作用指数实验结果见表1至表4。
表1药物组合对肺腺癌细胞A549的联合作用指数表(CI)
Figure PCTCN2019116993-appb-000002
备注：叠加作用(0.9≤CI≤1.1)；协同作用(CI<0.9)拮抗作用(CI>1.1)
表2药物组合对胃腺癌细胞AGS的联合作用指数表(CI)
Figure PCTCN2019116993-appb-000003
备注：叠加作用(0.9≤CI≤1.1)；协同作用(CI<0.9)拮抗作用(CI>1.1)
表3药物组合对肺鳞癌细胞H520的联合作用指数表(CI)
Figure PCTCN2019116993-appb-000004
备注：叠加作用(0.9≤CI≤1.1)；协同作用(CI<0.9)拮抗作用(CI>1.1)
表4药物组合对肝癌细胞HepG2的联合作用指数表(CI)
Figure PCTCN2019116993-appb-000005
备注：叠加作用(0.9≤CI≤1.1)；协同作用(CI<0.9)拮抗作用(CI>1.1)表1至表4中，不同浓度的二甲双胍和氯丙嗪以摩尔比例为200：1联合使用，在大部分浓度下药物相互作用指数CI值小于0.9，表现出协同作用。如：二甲双胍10mmol/L+氯丙嗪5μmol/L联合使用对肺腺癌细胞A549表现出协同作用；二甲双胍5mmol/L+氯丙嗪2.5μmol/L或二甲双胍1mmol/L+氯丙嗪5μmol/L联合使用对胃腺癌细胞AGS表现出协同作用。其余实验浓度下的组合均表现为简单的相加作用，或者拮抗作用。
实施例2
本实施例提供的药物组合物在某种肿瘤裸鼠移植瘤模型实验：
1、24-28日鼠龄的BALB-C裸小鼠20只，雄性，鼠重10.01±0.73g。
2、培养肺腺癌细胞A549，待细胞处于指数级增长期时，消化收集细胞，用无菌生理盐水制2*10 7个/ml细胞悬液，皮下注射于雄性裸鼠右侧腋下位置处，每只0.2ml。每日观察小鼠状况，测量肿瘤长径和短径，当皮下瘤组织长至1*1cm大小时开始给药。20只裸鼠随机分成4组，每组5只。分别为：(1)对照组：等量PBS溶液；(2)二甲双胍组：100mg/kg；(3)氯丙嗪组：50mg/kg；(4)二甲双胍100mg/kg+50mg/kg氯丙嗪；4组每日一次灌胃给药，从开始给药后， 每周测量一次皮下肿瘤的长径(a)和短径(b)，按公式计算体积(V＝a*b 2/2)，再计算瘤体积的平均值，连续2周后处死小鼠，将肿瘤剥离，去掉非瘤组织后称重，计算抑瘤率，抑瘤率(％)＝(1-实验组平均瘤重/对照组平均瘤重)*100％，数据统计见表5所示。
表5联合用药对肺腺癌细胞A549接种皮下肿瘤的瘤体积、质量影响
组别 对照组1 二甲双胍组 氯丙嗪组 二甲双胍+氯丙嗪
鼠数 5 5 5 5
鼠重(g) 24.62±0.43 24.98±0.51 24.61±0.48 24.31±0.51
治疗前瘤体积(cm3) 0.55±0.01 0.54±0.01 0.52±0.01 0.56±0.02
一周后瘤体积(cm3) 0.72±0.02 0.69±0.01 0.7±0.02 0.64±0.01
二周后瘤体积(cm3) 1.33±0.01 1.07±0.03 1.12±0.01 0.88±0.02
瘤重量(g) 0.76±0.03 0.53±0.02 0.67±0.01 0.35±0.01
抑瘤率(％) / 61.2 43.5 84.9
如表5所示，二甲双胍单独给药时，移植瘤的抑瘤率为61.2％；氯丙嗪单独给药时，移植瘤的抑瘤率为43.5％；二甲双胍和氯丙嗪联合使用时，移植瘤的抑瘤率可达84.9％。同时，在对照组、二甲双胍组、氯丙嗪组、二甲双胍+氯丙嗪中，裸鼠体重分别24.62±0.43、24.98±0.51、24.61±0.48和24.31±0.51，结果表明联合用药组显著降低小鼠瘤体积和瘤重的同时，小鼠的体重并未出现明显的降低，说明联合用药对小鼠的生理状态没有明显的影响，未出现对实验动物的毒副作用。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围中。

Claims (9)

  1. 一种有效抗恶性肿瘤的药物组合物，其特征在于：按摩尔份数计，该药物组合物的有效组分由100-400份二甲双胍和1-5份氯丙嗪组成。
  2. 根据权利要求1所述的一种有效抗恶性肿瘤的药物组合物，其特征在于：按摩尔份数计，该药物组合物的有效组分由200-300份二甲双胍与1-2份氯丙嗪组成。
  3. 根据权利要求1所述的一种有效抗恶性肿瘤的药物组合物，其特征在于：按摩尔份数计，该药物组合物的有效组分由200份二甲双胍与1份氯丙嗪组成。
  4. 根据权利要求1-3中任一项所述的一种有效抗恶性肿瘤的药物组合物，其特征在于：该药物组合物由有效组分和药用辅料混合而成，其剂型为口服制剂、针剂或者缓释制剂。
  5. 根据权利要求4所述的一种有效抗恶性肿瘤的药物组合物在制备抗肿瘤药物中的应用。
  6. 根据权利要求5所述的应用，其特征在于：所述的抗肿瘤药物为治疗肺腺癌、胃腺癌、肺鳞癌和肝癌、宫颈癌、乳腺癌、食管癌、结肠癌、胰腺癌、鼻咽癌、腹膜癌、间皮瘤、肾癌、膀胱癌、前列腺癌、尿道癌、子宫内膜癌、卵巢癌、恶性胶质瘤、骨肿瘤、纤维肉瘤、视网膜母细胞瘤、白血病、淋巴瘤的药物。
  7. 根据权利要求5所述的应用，其特征在于：所述的抗肿瘤药物为能够抑制肿瘤细胞增殖和转移的药物。
  8. 根据权利要求5所述的应用，其特征在于：所述的抗肿瘤药物为能够抑制肺腺癌细胞A549、胃腺癌细胞AGS、肺鳞癌细胞H520、和肝癌细胞HepG2、宫颈癌Hela、乳腺癌MCF-7、食管癌EC109、结肠癌LOVO、胰腺癌As-pc1、鼻咽癌CNE2、膀胱癌T24、肾癌 OS-RC-2、前列腺癌PC3、子宫内膜癌KLE、卵巢癌Hey、恶性胶质瘤U87、骨肿瘤U2-OS、纤维肉瘤Ag104、视网膜母细胞瘤y79、白血病NOMO-1、淋巴瘤Daudi增殖的药物。
  9. 根据权利要求5所述的应用，其特征在于：所述的抗肿瘤药物为能够降低肿瘤体积和重量的药物。
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