WO2022222388A1 - L-山梨糖在制备治疗肿瘤的药物中的应用 - Google Patents

L-山梨糖在制备治疗肿瘤的药物中的应用 Download PDF

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WO2022222388A1
WO2022222388A1 PCT/CN2021/123204 CN2021123204W WO2022222388A1 WO 2022222388 A1 WO2022222388 A1 WO 2022222388A1 CN 2021123204 W CN2021123204 W CN 2021123204W WO 2022222388 A1 WO2022222388 A1 WO 2022222388A1
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sorbose
cells
cancer
tumor
preparation
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高晓冬
李子杰
徐慧琳
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江南大学
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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
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the invention belongs to the anti-tumor drug technology, in particular to the application of the rare sugar L-sorbose in the preparation of a drug for treating tumors.
  • Cancer is the second leading cause of death in the world, and its mortality rate is second only to cardiovascular and cerebrovascular diseases.
  • the burden of cancer continues to increase year by year worldwide, causing huge physical, emotional and economic burdens to individuals, families, communities and health systems.
  • Cancer belongs to a large category of diseases in which cells grow abnormally uncontrollably and become malignant cells beyond their normal range, invading adjacent parts of the body and spreading to other organs. Tumor metastasis is the main cause of cancer death.
  • Cancer arises from a multistage transformation process of normal cells to tumor cells, which often progresses from precancerous lesions to malignant tumors. These changes are the result of the interaction of human genetic factors and three types of external factors, including: physical carcinogens, such as ultraviolet and ionizing radiation; chemical carcinogens, such as asbestos, components of tobacco smoke, aflatoxins, and arsenic; biological Infections with carcinogens, such as certain viruses, bacteria or parasites.
  • the main treatment methods for cancer include surgery, radiotherapy, chemotherapy and biological therapy, but there are still many limitations of treatment options for various cancers. Since most patients have lost the best time for surgery at the time of diagnosis, radiotherapy and chemotherapy are the main treatment methods for most cancer patients in clinical practice. However, the resistance of tumor cells to chemotherapy drugs is often an important reason for chemotherapy failure.
  • Cancer cells consume large amounts of glucose as an energy source, allowing the use of large amounts of amino acids and nucleotides as cells overproliferate to synthesize DNA. Taking advantage of this feature, by supplying excess glucose, the aerobic glycolytic metabolism of tumor cells can reach a very high degree, excreting a large amount of lactic acid, resulting in an acidic microenvironment of tumor cells. Therefore, interfering with the glucose metabolism of tumor cells can also be used as an anti-tumor strategy.
  • L-Sorbitose is a type of rare sugar.
  • the International Rare Sugar Society defines "rare sugars and their derivatives as monosaccharides that hardly exist in nature", and despite their low natural abundance, rare sugars exhibit a variety of biological functions.
  • rare sugars have been recognized as supplements and non-nutritive sweeteners for low-calorie foods. It also has great development potential in industries such as synthesis, cosmetics and pharmaceuticals. For example, D-psicose can inhibit liver lipase activity, thereby reducing abdominal fat accumulation, and can inhibit the rise of blood sugar and relieve type 2 diabetes; D-tagatose is certified by the U.S. Food and Drug Administration (FDA).
  • FDA U.S. Food and Drug Administration
  • D-allose has various physiological functions as an anticancer drug, anti-inflammatory and antioxidant;
  • L-fructose is a non-nutritive sweetener, glycosidase inhibitor, as well as housefly and ants of pesticides.
  • Current research shows that L-sorbose can be used to synthesize glycosidase inhibitors (1-deoxygalactosidomycin) and L-ascorbate. But there is no report about its application in antitumor pharmacy.
  • the present application provides the application of L-sorbose in the preparation of a medicament for treating tumors.
  • This application discloses the application of L-sorbose in the preparation of medicines for treating tumors.
  • the present application uses a certain dose of L-sorbose to act on human tumor cells, and the experimental results show that L-sorbose has an inhibitory effect on tumor cell proliferation, inhibits tumor cell metabolism, blocks cell cycle progression and promotes cell apoptosis.
  • the application also discloses the application of L-sorbose combined with chemotherapeutic drugs in the preparation of antitumor drugs.
  • the present application adopts L-sorbose and tumor chemotherapeutic drugs to be used in combination on human tumor cells.
  • the results show that L-sorbose has a synergistic effect on chemotherapeutic drugs, and in vivo experiments have confirmed that the combined use has no toxic and side effects.
  • the chemotherapeutic drugs include but are not limited to commonly used clinical drugs such as sorafenib, lenvatinib, cisplatin, doxorubicin, and paclitaxel.
  • the preferred chemotherapeutic agent is sorafenib.
  • liver cancer examples include, but are not limited to, liver cancer, breast cancer, lung cancer, cervical cancer, lymphoma, bladder cancer, melanoma, and the like. Especially liver cancer.
  • the present application also discloses an antitumor pharmaceutical composition
  • an antitumor pharmaceutical composition comprising L-sorbose and a pharmaceutically acceptable carrier.
  • anti-tumor drug composition in the preparation of anti-tumor drugs is also within the protection scope of the present application.
  • L-sorbose can be administered in linear or cyclic form without affecting the therapeutic effect.
  • Various methods of administration can be used in therapy, including oral administration, intravenous injection, intramuscular injection, and intratumoral injection.
  • This application discloses for the first time the application of L-sorbose in the preparation of anti-tumor drugs, which inhibits the proliferation of tumor cells by inhibiting the metabolism of tumor cells, blocks the progress of its cell cycle and promotes cell apoptosis .
  • the combined use of L-sorbose and chemotherapeutic drugs can significantly enhance the therapeutic effect of chemotherapeutic drugs, reduce the dosage of chemotherapeutic drugs, and reduce toxic and side effects.
  • Figure 1 is a graph showing the effect of different rare sugars on the growth of cancer cells
  • Fig. 2 is a graph showing the growth inhibition curve of L-sorbose on hepatoma cell line
  • Figure 3 is the effect of L-sorbose on the cell cycle of liver cancer cells
  • Figure 4 is the effect of L-sorbose on apoptosis of liver cancer cells
  • Figure 5 shows the effect of L-sorbose on the expression of HIF-1 ⁇ and its energy metabolism-related target genes in cancer cells
  • Figure 6 is the effect of different rare sugars in combination with Sorafenib on the growth of cancer cells
  • Figure 7 is the effect of L-sorbose combined with sorafenib on enhancing the growth inhibition of cancer cells by sorafenib in vitro;
  • Figure 8 shows that L-sorbose in combination with sorafenib enhances tumor therapy in vivo.
  • Example 1 Inhibition of cancer cell growth by L-sorbose in vitro
  • CCK-8 experiment was performed. First, a certain volume of CCK-8 reagent was diluted with the complete medium at a ratio of 1:10, and then the old medium containing the drug was aspirated, and 100 ⁇ L of diluted medium was added to each well. CCK-8 working solution, select 3 blank wells, discard the old D-PBS, add the prepared CCK-8 working solution as Blank (used to deduct the background value of the well), and then put the 96-well plate back into the culture Incubate for 45min-1h. After incubation, the OD450nm value was detected at a wavelength of 450nm, and the cell viability was calculated according to the formula [(medicated group-Blank)/(control group-Blank)] ⁇ 100%.
  • the IC 50 of L-sorbose on Huh7 cells was 33.82mM (24h), 27.32mM (48h) and 30.88mM (72h); the IC 50 of L-sorbose on HepG2 cells was 27.68mM (24h) ), 34.89 mM (48h) and 22.60 mM (72h).
  • Hepatoma cells Huh7 and HepG2 were stimulated by different concentrations of L-Sorbose, and the effect of L-Sorbose on cell cycle was detected by flow cytometry.
  • Cells were seeded into 6-well plates with a volume of 2 mL per well at a density of 2 ⁇ 10 5 cells/mL, and cultured overnight in a cell incubator. After the cells adhered for 24 h, the cells were stimulated with different doses (0, 12.5, 25 and 50 mM) of L-Sorbose for 24 h. After the administration and incubation, the cells in each group were digested with trypsin, centrifuged at 1400 rpm for 3 min, and 1 mL of D-PBS was added to gently wash the cells, centrifuged at 1400 rpm for 3 min, and the washing was repeated twice.
  • Preparation of Working Solution 1 sample: 500 ⁇ L Assay Buffer+25 ⁇ L PI Solution+2.5 ⁇ L RNase Solution. Add 0.5mL Working Solution to the cell pellet to resuspend, and incubate at 4°C for 30min in the dark. After the endowment, mediate shaking to disperse the cells, and incubate at 37°C for 30 min in the dark. Then mediate shaking, and the cell cycle was detected by flow cytometry.
  • Example 3 L-sorbose promotes apoptosis of cancer cells
  • Hepatoma cells Huh7 and HepG2 were stimulated by different concentrations of L-Sorbose, and the effect of L-Sorbose on apoptosis was detected by flow cytometry.
  • the cells were seeded in a 6-well plate with a volume of 2 mL per well at a density of 2 ⁇ 10 5 cells/mL, and placed in a cell incubator for overnight culture. After the cells adhered for 24 h, the cells were stimulated with different doses (0, 12.5, 25 and 50 mM) of L-Sorbose for 24 h. After the administration and incubation, the cells in each group were digested with trypsin without EDTA, centrifuged at 1400 rpm for 3 min, and 1 mL of D-PBS was added to gently wash the cells, centrifuged at 1400 rpm for 3 min, and the washing was repeated twice.
  • Preparation of 1 ⁇ Annexin V Binding Solution Dilute 10 ⁇ Annexin V Binding Solution with ultrapure water to 1 ⁇ Annexin V Binding Solution. Add 500 ⁇ L Annexin V Binding Solution to the cell pellet. Take 100 ⁇ L of the suspension into a new EP tube, add 5 ⁇ L Annexin V, FITC conjugate, and then add 5 ⁇ L PI Solution. Incubate at room temperature for 15 min in the dark. 400 ⁇ L Annexin V Binding Solution was added to each EP tube, and flow cytometry was performed within 1 h. Set the control group: 1 unstained cells; 2 Annexin V, FITC-stained cells (without PI); 3 PI-stained cells (without Annexin V, FITC).
  • Example 4 The effect of L-sorbose on the expression of hypoxia-inducible factor HIF-1 ⁇ and its metabolism-related target genes in cancer cells
  • L-sorbose Different doses (0, 12.5, 25 and 50 mM) of L-sorbose were applied to liver cancer cells for 24 h, and Western Blot was used to detect HIF-1 ⁇ and its downstream metabolism-related target genes (HIF-1 ⁇ , HK2, PKM2) in liver cancer cells. , LDHA) protein expression changes.
  • the cells were seeded in a 6-well plate with a volume of 2 mL per well at a density of 2 ⁇ 10 5 cells/mL, and placed in a cell incubator for overnight culture. After the cells adhered for 24 h, the cells were stimulated with different doses (0, 12.5, 25 and 50 mM) of L-Sorbose for 24 h.
  • the cells in each group were digested with trypsin without EDTA, centrifuged at 1400 rpm for 3 min, washed with D-PBS three times, and added to the L-sorbose-treated human hepatoma cells by adding protein lysate and Protease inhibitors were lysed at low temperature on ice for 30 min, centrifuged at 15,000 rpm for 10 min at 4°C, and the supernatant was collected. After quantification, an equal amount of protein samples were taken for SDS-PAGE electrophoresis separation, semi-dry transfer membrane, and 5% nonfat milk powder was blocked for 2 h. Antibodies were added and incubated overnight at 4°C. After washing with PBST, HRP-labeled secondary antibody was added, incubated at room temperature for 1 h, and after washing, chemiluminescent solution was added to develop color, and the cells were imaged and photographed.
  • Example 5 Combined use of L-sorbose and sorafenib inhibits cancer cell growth in vitro
  • Human hepatoma cells Huh7 and HepG2 were stimulated with 25mM different rare sugars in combination with 2 ⁇ M sorafenib to detect the changes of cell viability.
  • Human hepatoma cells Huh7 and HepG2 were stimulated with 12.5mM and 25mM L-sorbose in combination with 2 ⁇ M and 4 ⁇ M sorafenib to detect changes in cell viability.
  • Cells were seeded in a 96-well plate at a density of 5 ⁇ 10 4 cells/mL in a volume of 100 ⁇ L per well, and cultured overnight in a cell incubator. After 24h of cell attachment, 25mM rare sugar standard D-Tagatose, L-Tagatose, D-Sorbose, L-Sorbose, D-Allose, L-Fructose and D-Allulose were given in combination with 2 ⁇ M sorafenib, Or use 12.5mM and 25mM L-sorbose in combination with 2 ⁇ M and 4 ⁇ M sorafenib to stimulate human hepatoma cells Huh7 and HepG2 for 24h.
  • CCK-8 experiment was carried out. First, a certain volume of CCK-8 reagent was diluted with the complete medium at a ratio of 1:10, and then the old medium containing the drug was removed from the well, and 100 ⁇ L of diluted CCK-8 was added to each well. CCK-8 working solution, select 3 blank wells and add the prepared CCK-8 working solution as Blank (used to deduct the background value of the wells), then put the 96-well plate back into the incubator and continue to incubate for 45min-1h. After the incubation with CCK-8 working solution, take out the 96-well plate to be tested from the incubator, and detect the OD450nm value of each well at a wavelength of 450nm. ⁇ 100% to calculate cell viability.
  • Example 6 Combined use of L-sorbose and sorafenib enhances tumor therapy
  • mice Four-week-old male SPF-grade Balb/c nu mice were adaptively reared for one week and maintained in a state of free water and food intake.
  • Human hepatoma cell line Huh7 cells were cultured, and the cell concentration was adjusted to 1.0 ⁇ 10 7 /100 ⁇ L with D-PBS. The cell suspension was gently mixed before inoculation, and 100 ⁇ L of Huh7 cell suspension was subcutaneously inoculated under the armpit near the back of each nude mouse. liquid. Nude mice were reared normally and the tumor size at the subcutaneous inoculation site was closely observed every day.
  • the nude mice were randomly divided into 4 groups, and L-sorbose and sorafenib were administered by gavage.
  • the model group was intragastrically administered with normal saline, the L-sorbose group was intragastrically administered 20% L-sorbose (200 ⁇ L/20g), and the sorafenib group was intragastrically administered 5 mg/mL sorafenib (50 mg/kg).
  • Groups were gavaged with 20% L-sorbose (200 ⁇ L/20 g) and 5 mg/mL sorafenib (50 mg/kg).
  • the growth state of the nude mice was observed every day, the body weight of the nude mice was recorded every other day, and the size of the tumor was precisely measured with a vernier caliper, and the graph of the tumor change of the nude mice and the change of the body weight of the nude mice were drawn.
  • the animals were sacrificed, the intact tumor was carefully peeled off and weighed, and the tumor tissue was divided into four parts, one part was fixed in 10% formalin, and the three parts were quick-frozen in liquid nitrogen and then transferred to -80°C refrigerator for storage. .

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Abstract

L-山梨糖或其与化疗药物如索拉非尼、乐伐替尼、顺铂、多柔比星和紫杉醇的组合在制备治疗肿瘤的药物中的应用，含有山梨糖的药物组合物，以及该药物组合物在制备肿瘤药物中的应用。

Description

L-山梨糖在制备治疗肿瘤的药物中的应用 技术领域
本发明属于抗肿瘤药物技术，特别是涉及稀有糖L-山梨糖在制备治疗肿瘤的药物中的应用。
背景技术
癌症是全球第二大死亡原因，其死亡率仅次于心脑血管疾病，癌症负担在全球范围内逐年继续增长，给个人、家庭、社区和卫生系统造成巨大的身体、情感和经济负担。癌症属于一大类疾病，细胞无法控制地异常生长而超出其正常范围变成恶性肿瘤细胞，侵入身体的邻近部位及扩散到其他器官，肿瘤转移是癌症死亡的主要原因。
癌症来自正常细胞向肿瘤细胞的多阶段转化过程，该过程通常从癌前病变发展为恶性肿瘤。这些变化是人的遗传因素以及三类外部因素相互作用的结果，包括：物理致癌物，如紫外线和电离辐射等；化学致癌物，如石棉，烟草烟雾的成分，黄曲霉毒素和砷等；生物致癌物，如某些病毒，细菌或寄生虫的感染。目前癌症的主要治疗方式包括手术治疗、放疗、化疗和生物治疗等，但应对各种癌症依旧存在许多治疗方案的局限性。由于多数患者在确诊时已丧失手术最佳时机，因此放、化疗是临床上大部分肿瘤患者接受的主要治疗手段。然而，肿瘤细胞对化疗药物的耐受性常常是化疗失败的重要原因。
癌细胞消耗大量葡萄糖作为能量来源，从而允许在细胞过度增殖合成DNA时使用大量氨基酸和核苷酸。利用这一特点，通过提供过量的葡萄糖，肿瘤细胞的有氧糖酵解代谢可以达到非常高的程度，排出大量乳酸，导致肿瘤细胞的酸性微环境。因此，干扰肿瘤细胞的葡萄糖代谢亦可作为一种抗肿瘤的策略。
L-山梨糖是稀有糖的一种。国际稀有糖协会(ISRS)定义“稀有糖及其衍生物是自然界中几乎不存在的单糖”，尽管其天然丰度很低，但稀有糖表现出多种生物学功能。在食品工业领域，稀有糖已被公认为是低热量食品的补充剂和非营养性甜味剂。在合成、化妆品和制药等行业也具有巨大的开发潜力。例如，D-阿洛酮糖能够抑制肝脂肪酶活性，从而减少腹部脂肪堆积，且可以抑制血糖的上升，缓解2型糖尿病；D-塔格糖是美国食品和药物管理局(FDA)认证的食品添加剂；D-阿洛糖具有多种生理功能，可作为抗癌药，抗炎药和抗氧化剂；L-果糖是一种非营养性甜味剂、糖苷酶抑制剂，以及家蝇和蚂蚁的杀虫剂。目前研究显示，L-山梨糖可用于合成糖苷酶抑制剂(1-脱氧半乳糖苷霉素)和L-抗坏血酸盐。但是关于其在抗肿瘤制药中的应用未见报道。
发明内容
发明目的：针对上述现有技术，本申请提供了L-山梨糖在制备治疗肿瘤的药物中的应用。
技术方案：本申请公开了L-山梨糖在制备治疗肿瘤的药物中的应用。
本申请采用一定剂量的L-山梨糖作用于人肿瘤细胞，实验结果显示L-山梨 糖对肿瘤细胞增殖有抑制作用，通过抑制肿瘤细胞代谢，阻滞细胞周期进展并促进细胞凋亡。
本申请还公开了L-山梨糖联合化疗药物在制备抗肿瘤药物中的应用。
本申请采用L-山梨糖和肿瘤化疗药物联合使用于人肿瘤细胞，结果显示L-山梨糖对化疗药物有协同增效的作用，并经体内实验证实联用增效的同时无毒副作用。
所述的化疗药物包括但不限于索拉非尼、乐伐替尼、顺铂、多柔比星、紫杉醇等常用临床药物。优选化疗药物是索拉非尼。
本申请所述的肿瘤包括但不限于肝癌、乳腺癌、肺癌、宫颈癌、淋巴瘤、膀胱癌、黑色素瘤等。特别是肝癌。
本申请还公开了一种抗肿瘤药物组合物，其包含L-山梨糖，以及药学上可接受的载体。
所述抗肿瘤药物组合物在制备抗肿瘤药物中的应用也在本申请保护范围内。
此外，L-山梨糖可以直链形式或环状形式施用而不影响治疗效果。治疗中可使用多种给药方法，包括口服给药、静脉注射、肌肉内给药注射和瘤内注射等。
有益效果：本申请首次公开了L-山梨糖在制备治疗抗肿瘤药物中的应用，其通过抑制肿瘤细胞的代谢，抑制了肿瘤细胞的增殖，阻滞其细胞周期的进展并促进细胞的凋亡。并且，L-山梨糖与化疗药物联用能明显增强化疗药物的治疗作用，减少化疗药物用药剂量，降低毒副作用。
附图说明
图1是不同稀有糖对癌细胞生长的影响结果图；
图2是L-山梨糖对肝癌细胞株生长抑制曲线图；
图3是L-山梨糖对肝癌细胞细胞周期的影响；
图4是L-山梨糖对肝癌细胞细胞凋亡的影响；
图5是L-山梨糖对癌细胞HIF-1α及其能量代谢相关靶基因表达的影响；
图6是不同稀有糖与索拉非尼联用对癌细胞生长影响；
图7是L-山梨糖与索拉非尼联用增强索拉非尼体外抑制癌细胞生长的作用；
图8是L-山梨糖与索拉非尼联用增强体内肿瘤治疗作用。
具体实施方式
下面结合实施例对本申请技术方案作详细说明。
实施例所用所有稀有糖均购于梯希爱(上海)化成工业发展有限公司，细胞系来源于中国医学科学院基础医学研究所细胞资源中心。
实施例1：L-山梨糖对体外癌细胞生长的抑制
选用不同的稀有糖分别刺激人肝癌细胞Huh7和HepG2、乳腺癌细胞MCF7和肺癌细胞A549后考察细胞活力变化。选用不同浓度的L-山梨糖分别刺激人肝癌细胞Huh7和HepG2，并计算IC 50。
按5×10 4个细胞/mL的密度将细胞按每孔100μL体积接种于96孔板内，将96孔板放置于细胞培养箱内培养过夜。细胞贴壁24h后给予50mM的稀有糖标 准品D-塔格糖(D-Tagatose)、L-塔格糖(L-Tagatose)、D-山梨糖(D-Sorbose)、L-山梨糖(L-Sorbose)、D-阿洛糖(D-Allose)和L-果糖(L-Fructose)刺激细胞24h或不同剂量(0、5、10、25、50和100mM)的L-山梨糖刺激细胞24h、48h和72h。给药孵育结束后，进行CCK-8实验，先用完全培养基按1:10的比例稀释一定体积的CCK-8试剂，之后吸去孔内的含药旧培养基，每孔加入100μL稀释后的CCK-8工作液，选择3个空白孔，弃去含有旧D-PBS，加入配好的CCK-8工作液，作为Blank(用于扣除孔背景值)，之后将96孔板放回培养箱孵育45min-1h。孵育结束后，在450nm波长下检测OD450nm值，按照公式[(加药组-Blank)/(对照组-Blank)]×100％计算细胞存活率。
实验结果如图1所示，根据图示可见，50mM的L-山梨糖对人肝癌细胞Huh7、HepG2、乳腺癌细胞MCF-7和肺癌细胞A549均有一定程度的生长抑制作用，且除MCF7细胞外，L-山梨糖在其余细胞上的抑制作用均强于D-Allose。如图2所示，L-山梨糖作用于Huh7细胞IC 50为33.82mM(24h)、27.32mM(48h)和30.88mM(72h)；L-山梨糖作用于HepG2细胞IC 50为27.68mM(24h)、34.89mM(48h)和22.60mM(72h)。
实施例2：L-山梨糖对癌细胞的细胞周期阻滞
给予不同浓度的L-Sorbose刺激肝癌细胞Huh7和HepG2，利用流式细胞术检测L-Sorbose对细胞周期的影响。
按2×10 5个细胞/mL的密度分别将细胞按每孔2mL体积接种于6孔板内，放置于细胞培养箱内培养过夜。细胞贴壁24h后，给于不同剂量(0、12.5、25和50mM)L-Sorbose刺激细胞24h。给药孵育结束后，将各组细胞分别用胰酶消化，1400rpm，离心3min，加入1mL D-PBS轻柔清洗细胞，1400rpm，离心3min，重复清洗两次。Working Solution的配制：1sample：500μL Assay Buffer+25μL PI Solution+2.5μL RNase Solution。在细胞团块中加入0.5mL Working Solution重悬，4℃避光孵育30min。赋予结束后斡旋振荡使细胞分散，37℃避光孵育30min。然后斡旋振荡，流式细胞仪检测细胞周期。
实验结果如图3所示，根据图示可见，L-Sorbose对Huh7和HepG2有细胞周期阻滞作用，将细胞周期阻滞于S期，且呈现剂量依赖性。
实施例3：L-山梨糖促进癌细胞凋亡
给予不同浓度的L-Sorbose刺激肝癌细胞Huh7和HepG2，利用流式细胞术检测L-Sorbose对细胞凋亡的影响。
按2×10 5个细胞/mL的密度将细胞按每孔2mL体积接种于6孔板内，放置于细胞培养箱培养过夜。细胞贴壁24h后，给于不同剂量(0、12.5、25和50mM)L-Sorbose刺激细胞24h。给药孵育结束后，将各组细胞分别用不含EDTA的胰酶消化后，1400rpm离心3min，加入1mL D-PBS轻柔地清洗细胞，1400rpm，离心3min，重复清洗两次。1×Annexin V Binding Solution的配制：将10×Annexin V Binding Solution用超纯水稀释为1×Annexin V Binding Solution。在细胞团块中加入500μL Annexin V Binding Solution。取100μL悬液于新的EP管中，加入5 μL Annexin V，FITC结合物，再加入5μL PI Solution。室温避光培养15min。各EP管中分别加入400μL Annexin V Binding Solution，1h内流式细胞仪检测。设置对照组：①未染色细胞；②Annexin V，FITC染色细胞(无PI)；③PI染色细胞(无Annexin V，FITC)。
实验结果如图4所示，根据图示可见，L-Sorbose能促进肝癌细胞Huh7和HepG2的细胞凋亡。
实施例4：L-山梨糖对癌细胞缺氧诱导因子HIF-1α及其代谢相关靶基因表达的影响
不同剂量(0、12.5、25和50mM)的L-山梨糖分别作用于肝癌细胞24h后，采用Western Blot方法检测肝癌细胞中HIF-1α及其下游代谢相关靶基因(HIF-1α、HK2、PKM2、LDHA)的蛋白表达变化。
按2×10 5个细胞/mL的密度将细胞按每孔2mL体积接种于6孔板内，放置于细胞培养箱培养过夜。细胞贴壁24h后，给于不同剂量(0、12.5、25和50mM)L-Sorbose刺激细胞24h。给药孵育结束后，将各组细胞分别用不含EDTA的胰酶消化后，1400rpm离心3min，D-PBS清洗细胞3次，在L-山梨糖处理后的人肝癌细胞中加入蛋白裂解液和蛋白酶抑制剂，于冰上低温裂解30min，4℃，15000rpm离心10min，收集上清液，定量后取等量蛋白样品进行SDS-PAGE电泳分离，半干法转膜，5％脱脂奶粉封闭2h，加入抗体，4℃过夜孵育。PBST洗涤后加入HRP标记的二抗，室温孵育1h，洗涤后加入化学发光液显色，成像拍照。
实验结果如图5所示，根据图示可见，不同剂量的L-山梨糖刺激肝癌细胞24h后，能够不同程度地抑制HIF-1α、HK2、PKM2、LDHA的蛋白表达，且呈剂量依赖性。
实施例5：L-山梨糖与索拉非尼联用体外抑制癌细胞生长
选用25mM不同稀有糖与2μM索拉非尼联用刺激人肝癌细胞Huh7和HepG2后检测细胞活力变化。选用12.5mM和25mM的L-山梨糖与2μM和4μM索拉非尼联用刺激人肝癌细胞Huh7和HepG2后检测细胞活力变化。
按5×10 4个细胞/mL的密度将细胞按每孔100μL体积接种于96孔板内，置于细胞培养箱培养过夜。细胞贴壁24h后，给于25mM的稀有糖标准品D-Tagatose、L-Tagatose、D-Sorbose、L-Sorbose、D-Allose、L-Fructose和D-Allulose与2μM索拉非尼联用，或选用12.5mM和25mM的L-山梨糖与2μM和4μM索拉非尼联用联用刺激人肝癌细胞Huh7和HepG2 24h。给药孵育结束后，进行CCK-8实验，先用完全培养基按1:10的比例稀释一定体积的CCK-8试剂，之后去除孔内的含药旧培养基，每孔加入100μL稀释后的CCK-8工作液，选择3个空白孔加入配好的CCK-8工作液，作为Blank(用于扣除孔背景值)，之后将96孔板放回培养箱继续孵育45min-1h。CCK-8工作液孵育结束后，从培养箱中取出待检测96孔板，在450nm波长下检测每个孔的OD450nm值，按照公式[(加药组-Blank)/(对照组-Blank)]×100％计算细胞存活率。
实验结果如图6所示，根据图示可见，除L-山梨糖外，其他稀有糖在25mM 时与2μM索拉非尼联用无增效作用。如图7所示，12.5mM和25mM的L-山梨糖与2μM和4μM索拉非尼联用均能明显增强索拉非尼对癌细胞生长的抑制作用，且在低剂量12.5mM的L-山梨糖与2μM索拉非尼联用已具有明显的抑制癌细胞生长的作用。
实施例6：L-山梨糖与索拉非尼联用增强肿瘤治疗作用
体内药效学研究通过裸鼠皮下接种Huh7肝癌细胞构建裸鼠肝癌移植瘤小鼠模型考察L-山梨糖体内对肝癌细胞的抑制作用。
四周龄雄性SPF级Balb/c nu小鼠，先适应性饲养一周，保持自由摄水摄食状态。培养人肝癌细胞株Huh7细胞，用D-PBS调整细胞浓度为1.0×10 7/100μL，接种前将细胞悬液轻轻混匀，每只裸鼠腋下靠近背部一侧皮下接种100μL Huh7细胞悬液。正常饲养并每天密切观察裸鼠皮下接种处肿瘤大小。接种四天左右，肿瘤体积达到约50mm 3时，将裸鼠随机分为4组，采用灌胃的方式给予L-山梨糖和索拉非尼。模型组每天灌胃生理盐水，L-山梨糖组每天灌胃20％的L-山梨糖(200μL/20g)，索拉非尼组灌胃5mg/mL索拉非尼(50mg/kg)，联用组灌胃20％的L-山梨糖(200μL/20g)和5mg/mL索拉非尼(50mg/kg)。每天观察裸鼠生长状态，每隔一天记录一次裸鼠体重，并用游标卡尺精密测定肿瘤大小，绘制裸鼠肿瘤变化曲线图和裸鼠体重变化曲线图。给药四周结束后处死动物，小心剥离完整肿瘤并称瘤重，并将肿瘤组织分成四份，一份置于10％福尔马林中固定，三份用液氮速冻后转移至-80℃冰箱保存。
实验结果如图8所示，根据图示可见，由裸鼠肿瘤体积变化曲线及肿瘤重量变化可知，与模型组相比，L-山梨糖组和索拉非尼组均能不同程度的抑制肿瘤体积的生长，抑制肿瘤重量，且联用组抑制效果最好。由裸鼠体重变化曲线可知，在整个实验过程中小鼠体重变化较为平缓，状态良好。

Claims (10)

  1. L-山梨糖在制备治疗肿瘤的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，L-山梨糖抑制肿瘤细胞增殖，促进肿瘤细胞凋亡。
  3. L-山梨糖联合化疗药物在制备抗肿瘤药物中的应用。
  4. 根据权利要求3所述的应用，其特征在于，L-山梨糖能增强化疗药物对肿瘤细胞、肿瘤组织的毒性作用，抑制肿瘤细胞增殖，促进肿瘤细胞凋亡。
  5. 根据权利要求3所述的应用，其特征在于，所述化疗药物包括索拉非尼、乐伐替尼、顺铂、多柔比星和紫杉醇。
  6. 根据权利要求1或3所述的应用，其特征在于，所述肿瘤包括肝癌、宫颈癌、肺癌、乳腺癌、淋巴瘤、膀胱癌和黑色素瘤。
  7. 根据权利要求6所述的应用，其特征在于，所述肿瘤为肝癌。
  8. 一种抗肿瘤药物组合物，其特征在于，包含L-山梨糖，以及药学上可接受的载体。
  9. 权利要求8所述药物组合物在制备治疗肿瘤的药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述肿瘤包括肝癌、宫颈癌、肺癌、乳腺癌、淋巴瘤、膀胱癌和黑色素瘤。
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