WO2022110568A1 - Use of vitamin c and disulfiram in preparing anti-tumor combination drug - Google Patents

Use of vitamin c and disulfiram in preparing anti-tumor combination drug Download PDF

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WO2022110568A1
WO2022110568A1 PCT/CN2021/079565 CN2021079565W WO2022110568A1 WO 2022110568 A1 WO2022110568 A1 WO 2022110568A1 CN 2021079565 W CN2021079565 W CN 2021079565W WO 2022110568 A1 WO2022110568 A1 WO 2022110568A1
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dsf
disulfiram
vitamin
tumor
concentration
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李为民
何杨
吴琼
黄日东
陈海
罗雨蕉
夏贞强
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四川大学华西医院
成都华西精准医学产业技术研究院有限公司
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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
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    • 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 invention belongs to the field of antitumor drugs.
  • Tumor treatment methods include surgery, radiotherapy and chemotherapy, molecular targeted therapy, and immunotherapy. Simple surgery has a low cure rate; radiotherapy and chemotherapy with obvious curative effects have strong toxic and side effects; molecular targeted therapy and immunotherapy have good curative effects and low toxic and side effects, but there are also problems such as drug resistance, genomic instability and response rate. This is also the reason why the prognosis and long-term survival rate of lung cancer patients are still very low.
  • vitamin C As an antioxidant at physiological concentration ( ⁇ M level), vitamin C (VC) can protect cells from oxidative stress, and at the same time, it has the functions of enhancing human immunity and preventing scurvy.
  • Clinical trials and case studies in recent years have shown that intravenous injection of high-dose (mM grade) VC can selectively inhibit the growth of various tumors such as lung cancer, and at the same time, it has no obvious toxicity to normal tissues.
  • mM grade high-dose
  • DSF Disrlfiram
  • DEDTC N,N-diethyldithiocarbamate
  • the chemical formula is C 10 H 20 N 2 S 4
  • the structural formula is as follows.
  • DSF has been used to treat alcohol dependence since its discovery in 1930.
  • DSF has been shown to have certain anticancer activity in various cancer models such as breast cancer, prostate cancer, glioblastoma, lung cancer, cervical cancer, liver cancer, myeloma, melanoma, neuroblastoma, etc. It has natural targeting to tumor tissue, but its specific mechanism of action is not yet clear.
  • DSF can produce a variety of side effects, currently reported are: optic neuritis and other neuropathies, hepatitis, rash, headache and so on. Therefore, the dose of DSF must be strictly controlled when it is used.
  • the problem to be solved by the present invention is to provide a combination drug and an antitumor drug composition with excellent antitumor effect.
  • An anti-tumor combined drug the combined drug is
  • the molar ratio of disulfiram and vitamin C is 1:2.5 ⁇ 4000.
  • the molar ratio of the disulfiram and vitamin C is 1:2.5-400.
  • the molar ratio of disulfiram and vitamin C is 1:5-400; preferably, it is 1:20-400.
  • An antitumor pharmaceutical composition is composed of vitamin C and disulfiram;
  • the molar ratio of disulfiram and vitamin C is 1:2.5 ⁇ 4000.
  • the molar ratio of the disulfiram and vitamin C is 1:2.5-400.
  • the molar ratio of disulfiram and vitamin C is 1:5-400; preferably, it is 1:20-400.
  • vitamin C and disulfiram in the preparation of anti-tumor combined drugs, or the use of vitamin C and disulfiram as anti-tumor active ingredients in the preparation of anti-tumor drugs.
  • the tumor is lung cancer, preferably, non-small cell lung cancer.
  • the tumor is lung cancer, preferably, non-small cell lung cancer.
  • vitamin C and disulfiram have a synergistic effect in inhibiting tumor cell activity.
  • the anti-tumor combination drug or the anti-tumor drug composition of the invention has significantly better anti-tumor effect than vitamin C or disulfiram, and has good application prospect.
  • Figure 1 Cell viability results of H1299 cells inhibited by VC or DSF alone.
  • Figure 2 Results of the combined use of VC and SDF to inhibit cell viability of H1299 cells.
  • Figure 3 Cell viability results of H1975 cells inhibited by VC or DSF alone.
  • Figure 4 Results of cell viability inhibition of H1975 cells by combined use of VC and SDF.
  • Example 1 VC combined with DSF inhibits H1299 cells
  • the H1299 cell line (a non-small cell lung cancer cell line) was cultured in vitro in a complete medium (RPMI1640+10% fetal bovine serum), and the cells were cultured in a 37°C, 5% CO 2 incubator.
  • a complete medium RPMI1640+10% fetal bovine serum
  • the adherent cells were digested with 0.25% trypsin, and different cells were prepared into a single cell suspension with complete medium, and seeded in a 96-well plate at a density of 15,000 cells per well. , the volume of each well is 100 ⁇ L. Transfer the culture plate into a CO 2 incubator and incubate for 36h at 37°C and 5% CO 2 .
  • the original culture medium was aspirated and discarded, and the cells were divided into normal control group and drug groups with different concentrations.
  • the normal control group was replaced with fresh culture medium; the other groups were replaced with different concentrations of VC, DSF, VC+DSF, and the volume of each well was 100 ⁇ L. Then continue to cultivate for 24h.
  • the inventors analyzed the synergy index (CI, CI ⁇ 1, indicating the existence of synergistic effect) of DSF and VC at each concentration by using CompuSyn software widely used in the art. The results are shown in Table 2.
  • DSF and VC have obvious synergistic effects
  • DSF concentration is 1 mM
  • DSF and VC also had obvious synergistic effects.
  • the synergy index of DSF concentration of 1 ⁇ M and VC concentration of 2 to 3 mM in Table 2 is higher than 1, because the DSF and/or VC concentration is too low in a single case, with the increase of DSF (including DSF concentration exceeding 200 ⁇ M) ), DSF can generally synergize with VC (including when the concentration of VC exceeds 4mM).
  • results of this example show that the combined use of VC and DSF can produce a significant anti-tumor synergistic effect, which is beneficial to the treatment of lung cancer.
  • Example 2 VC combined with DSF inhibits H1975 cells
  • H1975 cells (a non-small cell lung cancer cell line) as the test object, they were treated by the method of Example 1.
  • the results of using VC or DSF alone are shown in Figure 3.
  • the cells were not inhibited when the VC concentration was 0.5-3 mM, and the cells were significantly inhibited when the VC concentration was 4 mM.
  • the cells were not inhibited when the DSF concentration was 1-100 ⁇ M, and the cells were significantly inhibited when the concentration was 200 ⁇ M.
  • the above ratios are molar ratios.
  • VC combined with DSF can produce significant anti-tumor synergistic effects; the use of VC and DSF as active ingredients to prepare anti-tumor drugs, or the use of VC and DSF to prepare anti-tumor combination drugs, will have good anti-tumor effects. prospect.

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Abstract

A use of vitamin C and disulfiram in preparing a combination drug, which belongs to the field of anti-tumor drugs. The combination drug can achieve a vitamin C and disulfiram anti-tumor synergistic effect, an inhibitory effect on tumor cells is superior to using vitamin C or disulfiram in isolation, and said drug has good application prospects.

Description

维生素C和双硫仑在制备抗肿瘤联合用药物中的用途The use of vitamin C and disulfiram in the preparation of anti-tumor combination drugs 技术领域technical field
本发明属于抗肿瘤药物领域。The invention belongs to the field of antitumor drugs.
背景技术Background technique
肿瘤的治疗手段包括外科手术、放化疗以及分子靶向治疗、免疫治疗等。单纯的外科手术治愈率低；疗效明显的放化疗，毒副作用也强；分子靶向治疗和免疫治疗疗效好，毒副作用低，但也存在耐药性、基因组不稳定和应答率等问题。这也是肺癌患者的预后及长期生存率仍旧很低的原因。Tumor treatment methods include surgery, radiotherapy and chemotherapy, molecular targeted therapy, and immunotherapy. Simple surgery has a low cure rate; radiotherapy and chemotherapy with obvious curative effects have strong toxic and side effects; molecular targeted therapy and immunotherapy have good curative effects and low toxic and side effects, but there are also problems such as drug resistance, genomic instability and response rate. This is also the reason why the prognosis and long-term survival rate of lung cancer patients are still very low.
维生素C(VC)在生理浓度下(μM级)作为一种抗氧化剂，可保护细胞免受氧化应激，同时具有增强人体免疫力，阻止坏血病的等作用。近几年的临床试验及病例研究表明，静脉注射高剂量(mM级)VC时，可选择性抑制肺癌等多种肿瘤生长，同时对正常组织无明显毒性。但是单独使用VC的抗肿瘤效果有限，且容易产生耐药性。As an antioxidant at physiological concentration (μM level), vitamin C (VC) can protect cells from oxidative stress, and at the same time, it has the functions of enhancing human immunity and preventing scurvy. Clinical trials and case studies in recent years have shown that intravenous injection of high-dose (mM grade) VC can selectively inhibit the growth of various tumors such as lung cancer, and at the same time, it has no obvious toxicity to normal tissues. However, the anti-tumor effect of VC alone is limited, and it is prone to drug resistance.
双硫仑(Disrlfiram，DSF)是N,N-二乙基二硫代氨基甲酸酯(DEDTC)的二硫化衍生物，化学式C 10H 20N 2S 4，结构式如下。 Disrlfiram (DSF) is a disulfide derivative of N,N-diethyldithiocarbamate (DEDTC), the chemical formula is C 10 H 20 N 2 S 4 , and the structural formula is as follows.
Figure PCTCN2021079565-appb-000001
Figure PCTCN2021079565-appb-000001
自1930年发现以来，DSF一直用于治疗酒精依赖。近年来，DSF在乳腺癌、***癌、成胶质细胞瘤、肺癌、***、肝癌、骨髓瘤、黑色素瘤、神经母细胞瘤等多种癌症模型中被证明具有一定的抗癌活性，并对肿瘤组织具有天然的靶向性，但其具体作用机制尚不明确。DSF has been used to treat alcohol dependence since its discovery in 1930. In recent years, DSF has been shown to have certain anticancer activity in various cancer models such as breast cancer, prostate cancer, glioblastoma, lung cancer, cervical cancer, liver cancer, myeloma, melanoma, neuroblastoma, etc. It has natural targeting to tumor tissue, but its specific mechanism of action is not yet clear.
DSF可产生多种副作用，目前报道的有：视神经炎等神经性病变，肝炎，皮疹，头痛等等。因此，DSF在使用时必须严格控制剂量。DSF can produce a variety of side effects, currently reported are: optic neuritis and other neuropathies, hepatitis, rash, headache and so on. Therefore, the dose of DSF must be strictly controlled when it is used.
目前尚未见VC和DSF联合使用以***的报道。There is no report on the combined use of VC and DSF to treat tumors.
发明内容SUMMARY OF THE INVENTION
本发明要解决的问题是：提供一种抗肿瘤效果优良的联合用药物以及抗肿瘤药物组合物。The problem to be solved by the present invention is to provide a combination drug and an antitumor drug composition with excellent antitumor effect.
本发明的技术方案如下：The technical scheme of the present invention is as follows:
一种抗肿瘤联合用药物，所述联合用药物为An anti-tumor combined drug, the combined drug is
双硫仑和维生素C；disulfiram and vitamin C;
双硫仑和维生素C的摩尔比为1:2.5～4000。The molar ratio of disulfiram and vitamin C is 1:2.5～4000.
进一步地，所述双硫仑和维生素C的摩尔比为1:2.5～400。Further, the molar ratio of the disulfiram and vitamin C is 1:2.5-400.
进一步地，所述双硫仑和维生素C的摩尔比为1:5～400；优选地，为1:20～400。Further, the molar ratio of disulfiram and vitamin C is 1:5-400; preferably, it is 1:20-400.
一种抗肿瘤药物组合物，所述组合物由维生素C和双硫仑组成；An antitumor pharmaceutical composition, the composition is composed of vitamin C and disulfiram;
双硫仑和维生素C的摩尔比为1:2.5～4000。The molar ratio of disulfiram and vitamin C is 1:2.5～4000.
进一步地，所述双硫仑和维生素C的摩尔比为1:2.5～400。Further, the molar ratio of the disulfiram and vitamin C is 1:2.5-400.
进一步地，所述双硫仑和维生素C的摩尔比为为1:5～400；优选地，为1:20～400。Further, the molar ratio of disulfiram and vitamin C is 1:5-400; preferably, it is 1:20-400.
维生素C和双硫仑在制备抗肿瘤联合用药物的用途、或者维生素C和双硫仑作为抗肿瘤活性成分在制备抗肿瘤药物中的用途。The use of vitamin C and disulfiram in the preparation of anti-tumor combined drugs, or the use of vitamin C and disulfiram as anti-tumor active ingredients in the preparation of anti-tumor drugs.
进一步地，所述肿瘤为肺癌，优选地，为非小细胞肺癌。Further, the tumor is lung cancer, preferably, non-small cell lung cancer.
前述的抗肿瘤药物组合物在制备抗肿瘤药物中的用途。Use of the aforementioned antitumor drug composition in the preparation of an antitumor drug.
进一步地，所述肿瘤为肺癌，优选地，为非小细胞肺癌。Further, the tumor is lung cancer, preferably, non-small cell lung cancer.
实验表明，维生素C和双硫仑在抑制肿瘤细胞活性方面具有协同增效作用。本发明的抗肿瘤联合用药物或抗肿瘤药物组合物具有显著优于维生素C或双硫仑的抗肿瘤效果，应用前景良好。Experiments show that vitamin C and disulfiram have a synergistic effect in inhibiting tumor cell activity. The anti-tumor combination drug or the anti-tumor drug composition of the invention has significantly better anti-tumor effect than vitamin C or disulfiram, and has good application prospect.
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.
附图说明Description of drawings
图1：单独使用VC或DSF抑制H1299细胞的细胞存活率结果。Figure 1: Cell viability results of H1299 cells inhibited by VC or DSF alone.
图2：联合使用VC和SDF抑制H1299细胞的细胞存活率结果。Figure 2: Results of the combined use of VC and SDF to inhibit cell viability of H1299 cells.
图3：单独使用VC或DSF抑制H1975细胞的细胞存活率结果。Figure 3: Cell viability results of H1975 cells inhibited by VC or DSF alone.
图4：联合使用VC和SDF抑制H1975细胞的细胞存活率结果。Figure 4: Results of cell viability inhibition of H1975 cells by combined use of VC and SDF.
具体实施方式Detailed ways
实施例1 VC联合DSF抑制H1299细胞Example 1 VC combined with DSF inhibits H1299 cells
1.方法1. Method
体外培养H1299细胞系(一种非小细胞肺癌细胞系)，培养体系为完全培养基(RPMI1640+10％胎牛血清)，细胞置于37℃，5％CO 2培养箱中培养。待细胞汇合至80％左右，用0.25％胰蛋白酶将贴壁细胞消化下来，用含完全培养基将不同的细胞配成单个细胞悬液，以每孔15000个细胞的密度接种于96孔板中，每孔体积100μL。将培养板移入CO 2孵箱中，在37℃、5％CO 2条件下，孵育36h。吸弃原培养液，将细胞分为正常对照组和不同浓度药物组。正常对照组换为新鲜培养液；其余各组换为不同浓度的VC、DSF、VC+DSF，每孔体积100μL。然后继续培养24h。 The H1299 cell line (a non-small cell lung cancer cell line) was cultured in vitro in a complete medium (RPMI1640+10% fetal bovine serum), and the cells were cultured in a 37°C, 5% CO 2 incubator. When the cells were confluent to about 80%, the adherent cells were digested with 0.25% trypsin, and different cells were prepared into a single cell suspension with complete medium, and seeded in a 96-well plate at a density of 15,000 cells per well. , the volume of each well is 100 μL. Transfer the culture plate into a CO 2 incubator and incubate for 36h at 37°C and 5% CO 2 . The original culture medium was aspirated and discarded, and the cells were divided into normal control group and drug groups with different concentrations. The normal control group was replaced with fresh culture medium; the other groups were replaced with different concentrations of VC, DSF, VC+DSF, and the volume of each well was 100 μL. Then continue to cultivate for 24h.
按操作手册每孔加入CCK8 5μL，继续孵育1-2h，终止培养。一组不含细胞的组为空白组。选择450nm波长，在酶联免疫检测仪上调定各孔吸光度值，记录结果。以时间为横轴，吸光度值为纵轴绘图。按式1-1计算细胞存活率。每个实验至少重复3次。Add 5 μL of CCK8 to each well according to the operation manual, continue to incubate for 1-2 h, and terminate the culture. A group without cells is a blank group. Select 450nm wavelength, adjust the absorbance value of each well on the enzyme-linked immunosorbent assay instrument, and record the results. Plot the time as the horizontal axis and the absorbance value on the vertical axis. Calculate the cell viability according to formula 1-1. Each experiment was repeated at least 3 times.
细胞存活率＝(A 药液-A 空白)/(A 正常对照-A 空白)×100％(1-1) Cell viability = (A drug solution -A blank )/(A normal control -A blank ) × 100% (1-1)
2.结果2. Results
单独使用VC或DSF的结果如图1所示。VC浓度在0.5～2mM时细胞未被抑制，在3～4mM时细胞受到较明显的抑制。而DSF在1～200μM时细胞均未受到抑制。The results of using VC or DSF alone are shown in Figure 1. The cells were not inhibited when the VC concentration was 0.5-2 mM, and the cells were significantly inhibited when the VC concentration was 3-4 mM. However, the cells were not inhibited when DSF was 1-200μM.
VC和DSF联合使用的结果如图2和表1所示。The results of the combined use of VC and DSF are shown in Figure 2 and Table 1.
DSF浓度为1μM、VC浓度4mM时(DSF：VC＝1:4000)，细胞活性受到显著抑制。When the DSF concentration was 1 μM and the VC concentration was 4 mM (DSF:VC=1:4000), the cell activity was significantly inhibited.
DSF浓度为10μM时，VC浓度在2～4mM(DSF：VC＝1:200～400)，细胞活性受到显著抑制。When the DSF concentration was 10 μM, and the VC concentration was 2-4 mM (DSF:VC=1:200-400), the cell activity was significantly inhibited.
DSF浓度为50μM时，VC浓度在2～4mM以上(DSF：VC＝1:40～80)，细胞活性受到显著抑制。When the DSF concentration was 50 μM, the cell activity was significantly inhibited when the VC concentration was above 2-4 mM (DSF:VC=1:40-80).
DSF浓度为100μM时，VC浓度在2～4mM以上(DSF：VC＝1:20～40)，细胞活性受到显著抑制。When the DSF concentration was 100 μM, the cell activity was significantly inhibited when the VC concentration was above 2-4 mM (DSF:VC=1:20-40).
DSF浓度为200μM时，VC浓度在2～4mM以上(DSF：VC＝1:10～20)，细胞活性受到显著抑制。When the DSF concentration was 200 μM, the cell activity was significantly inhibited when the VC concentration was above 2-4 mM (DSF:VC=1:10-20).
DSF浓度为10～200μM且VC浓度为4mM时(DSF：VC＝1:20～400)，细胞活性受到十分明显的限制，活性低于10％。可见，DSF：VC＝1:20～400是DSF和VC联用抑制H1299细胞的优选摩尔比范围。When the concentration of DSF was 10-200 μM and the concentration of VC was 4 mM (DSF:VC=1:20-400), the cell viability was significantly restricted, and the activity was lower than 10%. It can be seen that DSF:VC=1:20～400 is the preferred molar ratio range for the combined use of DSF and VC to inhibit H1299 cells.
表1不同浓度VC和/或双硫仑作用H1299细胞24h的细胞存活率，结果以平均值±标准偏差(％)表示。Table 1. The cell viability of H1299 cells treated with different concentrations of VC and/or disulfiram for 24 h, the results are expressed as mean ± standard deviation (%).
Figure PCTCN2021079565-appb-000002
Figure PCTCN2021079565-appb-000002
为了进一步分析DSF和VC的协同效果，发明人通过本领域广泛使用的CompuSyn软件对各浓度下DSF和VC的协同指数(CI，CI<1则表明存在协同增效作用)进行分析。结果如表2所示，在DSF浓度10～200μM、VC浓度0.5～4mM时(DSF：VC＝1:2.5～400)，DSF和VC具有明显的协同增效作用；DSF浓度1mM且VC浓度0.5～1mM(DSF：VC＝1:500～1000)或4mM(DSF：VC＝1:4000)时，DSF和VC也具有明显的协同增效作用。值得注意的是，表2中DSF浓度1μM且VC浓度2～3mM的协同指数高于1，是因为DSF和/或VC浓度过低产生的个例，随着DSF的提高(包括DSF浓度超过200μM时)，DSF普遍能与VC(包括VC浓度超过4mM时)进行协同增效。In order to further analyze the synergistic effect of DSF and VC, the inventors analyzed the synergy index (CI, CI<1, indicating the existence of synergistic effect) of DSF and VC at each concentration by using CompuSyn software widely used in the art. The results are shown in Table 2. When the DSF concentration is 10-200 μM and the VC concentration is 0.5-4 mM (DSF:VC=1:2.5-400), DSF and VC have obvious synergistic effects; DSF concentration is 1 mM and VC concentration is 0.5 At ~1 mM (DSF:VC=1:500-1000) or 4mM (DSF:VC=1:4000), DSF and VC also had obvious synergistic effects. It is worth noting that the synergy index of DSF concentration of 1 μM and VC concentration of 2 to 3 mM in Table 2 is higher than 1, because the DSF and/or VC concentration is too low in a single case, with the increase of DSF (including DSF concentration exceeding 200 μM) ), DSF can generally synergize with VC (including when the concentration of VC exceeds 4mM).
上述比例均为摩尔比。The above ratios are all molar ratios.
表2使用CompuSyn软件计算的VC联合DSF作用H1299细胞24h的联合药物指数值(Combination Index,CI value)Table 2 Combination Index (CI value) of VC combined with DSF on H1299 cells for 24 hours calculated by CompuSyn software
Figure PCTCN2021079565-appb-000003
Figure PCTCN2021079565-appb-000003
本实施例的结果表明：VC和DSF联合使用，能产生显著的抗肿瘤协同增效作用，利于肺癌的治疗。The results of this example show that the combined use of VC and DSF can produce a significant anti-tumor synergistic effect, which is beneficial to the treatment of lung cancer.
实施例2 VC联合DSF抑制H1975细胞Example 2 VC combined with DSF inhibits H1975 cells
1.方法1. Method
以H1975细胞(一种非小细胞肺癌细胞系)为试验对象，采用实施例1 的方法对其进行处置。Taking H1975 cells (a non-small cell lung cancer cell line) as the test object, they were treated by the method of Example 1.
2.结果2. Results
单独使用VC或DSF的结果如图3所示。VC浓度在0.5～3mM时细胞未被抑制，在4mM时细胞受到较明显的抑制。而DSF浓度在1～100μM时细胞未受到抑制，在200μM时，细胞受到明显抑制。The results of using VC or DSF alone are shown in Figure 3. The cells were not inhibited when the VC concentration was 0.5-3 mM, and the cells were significantly inhibited when the VC concentration was 4 mM. However, the cells were not inhibited when the DSF concentration was 1-100 μM, and the cells were significantly inhibited when the concentration was 200 μM.
表3不同浓度VC和/或DSF作用H1975细胞24h的细胞存活率，结果以平均值±标准偏差(％)表示。Table 3. The cell viability of H1975 cells treated with different concentrations of VC and/or DSF for 24 h, the results are expressed as mean ± standard deviation (%).
Figure PCTCN2021079565-appb-000004
Figure PCTCN2021079565-appb-000004
VC和DSF联合使用的结果如图4和表3所示。The results of the combined use of VC and DSF are shown in Figure 4 and Table 3.
当DSF为1μM时，VC用量3～4mM(DSF：VC＝1:3000～4000)即可产生显著的抑制H1975细胞的作用。When DSF is 1 μM, the dosage of 3-4mM (DSF:VC=1:3000-4000) of VC can significantly inhibit H1975 cells.
当DSF为10μM时，VC用量2～4mM(DSF：VC＝1:200～400)即可产生显著的抑制H1975细胞的作用。When DSF is 10μM, the dosage of VC is 2～4mM (DSF:VC=1:200～400), which can significantly inhibit H1975 cells.
当DSF为50μM时，VC用量为0.5～4mM(DSF:VC＝1:10～80)即可产生显著的抑制H1975细胞的作用。When DSF is 50μM, the dosage of VC is 0.5～4mM (DSF:VC=1:10～80), which can significantly inhibit H1975 cells.
当DSF为100μM时，VC用量为0.5～4mM(DSF:VC＝1:5～40)即可产生显著的抑制H1975细胞的作用。When DSF is 100μM, the dosage of VC is 0.5～4mM (DSF:VC=1:5～40), which can significantly inhibit H1975 cells.
当DSF为200μM时，VC用量为0.5～4mM(DSF:VC＝1:2.5～10)即可产生很显著的抑制H1975细胞的作用。When DSF is 200μM, the dosage of VC is 0.5～4mM (DSF:VC=1:2.5～10), which can significantly inhibit H1975 cells.
当DSF为200μM且VC为1～4mM时(DSF：VC＝1:5～20)，以及DSF为10～200μM且VC为4mM时(DSF：VC＝1：20～400)，以及DSF为50μM且VC为3mM(DSF：VC＝1:60)，抑制效果极其显著，细胞活性为3％以下。合并区间可知，DSF:VC＝1:5～400是DSF和VC联用抑制H1975细胞的优选摩尔比范围。When DSF is 200 μM and VC is 1-4 mM (DSF:VC=1:5-20), and when DSF is 10-200 μM and VC is 4 mM (DSF:VC=1:20-400), and DSF is 50 μM In addition, when VC was 3 mM (DSF:VC=1:60), the inhibitory effect was extremely significant, and the cell activity was 3% or less. The combined range shows that DSF:VC=1:5～400 is the optimal molar ratio range for the combined use of DSF and VC to inhibit H1975 cells.
为了进一步分析DSF和VC的协同效果，发明人通过本领域广泛使用的CompuSyn软件对各浓度下DSF和VC的协同指数(CI，CI<1则表明存在协同增效作用)进行分析。结果如表4所示，在DSF为10～200μM且VC浓度为0.5～4时(DSF：VC＝1:2.5～400)，DSF和VC具有明显的协同增效作用；DSF浓度1mM且VC浓度3～4mM(DSF：VC＝1:3000～4000)时，DSF和VC也具有明显的协同增效作用。值得注意的是，表4中DSF浓度1μM且VC浓度在2mM 以下时，协同指数高于1，是因为DSF和/或VC浓度过低产生的个例，随着DSF的提高(包括DSF浓度超过200μM时)，DSF普遍能与VC(包括VC浓度超过4mM时)进行协同增效。In order to further analyze the synergistic effect of DSF and VC, the inventors analyzed the synergy index (CI, CI<1, indicating the existence of synergistic effect) of DSF and VC at each concentration by using CompuSyn software widely used in the art. The results are shown in Table 4. When DSF is 10-200 μM and VC concentration is 0.5-4 (DSF:VC=1:2.5-400), DSF and VC have obvious synergistic effect; DSF concentration is 1 mM and VC concentration is 1 mM. At 3～4mM (DSF:VC=1:3000～4000), DSF and VC also have obvious synergistic effect. It is worth noting that in Table 4, when the DSF concentration is 1 μM and the VC concentration is below 2 mM, the synergy index is higher than 1, which is because the DSF and/or VC concentration is too low. 200μM), DSF can generally synergize with VC (including when the VC concentration exceeds 4mM).
上述比例为摩尔比。The above ratios are molar ratios.
表4使用CompuSyn软件计算的VC联合DSF作用H1975细胞24h的联合药物指数值(Combination Index,CI value)Table 4 Combination Index (CI value) calculated by VC combined with DSF on H1975 cells for 24 hours using CompuSyn software
Figure PCTCN2021079565-appb-000005
Figure PCTCN2021079565-appb-000005
本实施例的结果表明：VC和DSF联合使用能产生显著的抗肿瘤协同增效作用。The results of this example show that the combined use of VC and DSF can produce a significant anti-tumor synergistic effect.
综上，将VC联合DSF使用，可产生显著的抗肿瘤协同增效作用；将VC和DSF作为活性成分制备抗肿瘤药物，或者将VC和DSF用于制备抗肿瘤联合用药物，将具有良好的前景。In summary, the use of VC combined with DSF can produce significant anti-tumor synergistic effects; the use of VC and DSF as active ingredients to prepare anti-tumor drugs, or the use of VC and DSF to prepare anti-tumor combination drugs, will have good anti-tumor effects. prospect.

Claims (10)

  1. 一种抗肿瘤联合用药物，其特征在于：所述联合用药物为An anti-tumor combined drug, characterized in that: the combined drug is
    双硫仑和维生素C；disulfiram and vitamin C;
    双硫仑和维生素C的摩尔比为1:2.5～4000。The molar ratio of disulfiram and vitamin C is 1:2.5～4000.
  2. 如权利要求1所述的联合用药物，其特征在于：所述双硫仑和维生素C的摩尔比为1:2.5～400。The combined medicine of claim 1, wherein the molar ratio of the disulfiram and vitamin C is 1:2.5-400.
  3. 如权利要求1所述的联合用药物，其特征在于：所述双硫仑和维生素C的摩尔比为1:5～400；优选地，为1:20～400。The combined medicine of claim 1, wherein the molar ratio of the disulfiram to vitamin C is 1:5-400; preferably, it is 1:20-400.
  4. 一种抗肿瘤药物组合物，其特征在于：所述组合物由维生素C和双硫仑组成；An antitumor pharmaceutical composition, characterized in that: the composition is composed of vitamin C and disulfiram;
    双硫仑和维生素C的摩尔比为1:2.5～4000。The molar ratio of disulfiram and vitamin C is 1:2.5～4000.
  5. 如权利要求4所述的组合物，其特征在于：所述双硫仑和维生素C的摩尔比为1:2.5～400。The composition of claim 4, wherein the molar ratio of the disulfiram and vitamin C is 1:2.5-400.
  6. 如权利要求5所述的组合物，其特征在于：所述双硫仑和维生素C的摩尔比为1:5～400；优选地，为1:20～400。The composition of claim 5, wherein the molar ratio of the disulfiram to vitamin C is 1:5-400; preferably, it is 1:20-400.
  7. 维生素C和双硫仑在制备抗肿瘤联合用药物的用途、或者维生素C和双硫仑作为抗肿瘤活性成分在制备抗肿瘤药物中的用途。The use of vitamin C and disulfiram in the preparation of anti-tumor combined drugs, or the use of vitamin C and disulfiram as anti-tumor active ingredients in the preparation of anti-tumor drugs.
  8. 如权利要求7所述的用途，其特征在于：所述肿瘤为肺癌，优选地，为非小细胞肺癌。The use according to claim 7, wherein the tumor is lung cancer, preferably non-small cell lung cancer.
  9. 权利要求4～6任意一项所述的抗肿瘤药物组合物在制备抗肿瘤药物中的用途。Use of the antitumor pharmaceutical composition according to any one of claims 4 to 6 in the preparation of an antitumor drug.
  10. 如权利要求9所述的用途，其特征在于：所述肿瘤为肺癌，优选地，为非小细胞肺癌。The use according to claim 9, wherein the tumor is lung cancer, preferably non-small cell lung cancer.
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