WO2023272831A1 - 水苏糖在制备用于治疗去势抵抗性***癌药物中的应用 - Google Patents

水苏糖在制备用于治疗去势抵抗性***癌药物中的应用 Download PDF

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WO2023272831A1
WO2023272831A1 PCT/CN2021/108363 CN2021108363W WO2023272831A1 WO 2023272831 A1 WO2023272831 A1 WO 2023272831A1 CN 2021108363 W CN2021108363 W CN 2021108363W WO 2023272831 A1 WO2023272831 A1 WO 2023272831A1
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pharmaceutical composition
enza
stachyose
prostate cancer
drug
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PCT/CN2021/108363
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French (fr)
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陈永泉
王荣
许璐
王小英
朱升龙
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江南大学
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Priority to US17/860,451 priority Critical patent/US20220339175A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • 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 technical field of biomedicine, and specifically relates to the application of stachyose in the preparation of drugs for treating castration-resistant prostate cancer.
  • Androgen deprivation therapy is the standard treatment for advanced prostate cancer, but patients will eventually develop castration-resistant prostate cancer (CRPC) after an average of 1-3 years of treatment.
  • CRPC castration-resistant prostate cancer
  • ADT initial continuous androgen deprivation therapy
  • docetaxel was proven to prolong the overall survival of patients with metastatic castration-resistant prostate cancer (mCRPC) in 2004, abiraterone acetate, enzalutamide
  • Drugs targeting the disease stage of mCRPC such as cabazitaxel, have changed the current treatment status of these patients, but in the end, it is difficult to completely reverse CRPC. Therefore, finding other effective therapeutic targets or combined drug treatment strategies is another research hotspot for the treatment of CRPC.
  • EPI-001 is an inhibitor of AR and AR-splice variants (AR-Vs) awaiting clinical development with the potential to treat CRPC.
  • the target of EPI against CRPC is mainly the N-terminal domain (NTD).
  • Enzalutamide is the first approved second-generation AR antagonist, and its affinity for AR is 5-8 times higher than that of traditional antiandrogens. In 2012, the US FDA approved Enza for CRPC patients accordingly. However, whether it is EPI or Enza, the treatment of CRPC generally produces drug resistance in about 18 months. Therefore, other means are urgently needed to overcome drug resistance and delay CRPC.
  • the technical problem to be solved by the present invention is to overcome the drug resistance of the above-mentioned existing drugs, and provide a drug for effectively treating CRPC, that is, the combination of Enza and Stac, which can significantly improve the curative effect on CRPC and exert excellent synergistic growth. effect.
  • Stachyose is a sugar formed by combining two ⁇ -galactoses with 1,6-glycosidic bonds on the glucose side of sucrose, with a molecular formula of C 24 H 42 O 21 .
  • the first object of the present invention is to provide the application of Stac in the preparation of drugs for treating castration-resistant prostate cancer.
  • the application includes: combining Stac with an androgen receptor antagonist to prepare a drug for treating castration-resistant prostate cancer.
  • the second object of the present invention is to provide a pharmaceutical composition for treating castration-resistant prostate cancer, said composition comprising Stac and an androgen receptor antagonist.
  • the mass ratio of androgen receptor antagonist to Stac is (1-8):1. Wherein preferably, the mass ratio of Enza and Stac is 1-3:1.
  • the androgen receptor antagonist includes any one or more of the following: enzalutamide (Enza), EPI-001 (EPI), abiraterone, olaparib.
  • the pharmaceutical composition further includes pharmaceutical excipients.
  • the pharmaceutical excipients include solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, Osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integration agents, penetration enhancers, pH regulators, buffers, enhancers Plasticizers, surfactants, blowing agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and release retardants.
  • the dosage form of the preparation includes injection, lyophilized powder for injection, controlled release injection, liposome injection, suspension, implant, embolism, capsule, tablet pills, pills and liquids.
  • the pharmaceutical composition may further include a pharmaceutical carrier.
  • the drug carrier includes microcapsules, microspheres, nanoparticles and liposomes.
  • the present invention finds a drug for treating CRPC, that is, the combination of Enza and Stac.
  • a drug for treating CRPC that is, the combination of Enza and Stac.
  • L-DTP prostate cancer LNCaP-drug-tolerant persisters
  • the present invention establishes L-DTP recoverable drug-resistant cell lines, and uses the CCK8 method and Calcusyn software to calculate the CI value.
  • the results show that in this cell line, compared with Enza or Stac alone, , Enza and Stac combined in vitro have a synergistic anti-CRPC effect.
  • C-MYC overexpression prostate cancer mouse model it was determined that the combination of Enza and Stac in animals had a more significant in vivo anti-CRPC model effect than the single-drug group in the long-term Enza-resistant model.
  • the present invention proposes for the first time a new strategy of using Stac to prepare a drug for treating CRPC and a combination of Enza and Stac to treat CRPC, which will promote the application of Enza and Stac in the clinical treatment of prostate cancer, which is of great significance. It takes an average of 8-10 years for drug research to go from compound molecules to clinical practice, and requires a lot of manpower and material support, with huge time and economic costs.
  • the scheme of the present invention can realize the reuse of natural compound oligosaccharides, and can greatly shorten the time from drug discovery to clinical transformation.
  • Figure 1 is the formation process of DTP (drug-tolerant persisters) and DTEP (drug-tolerant expanded persisters) cells.
  • Figure 2 is the characteristics of the expression changes of AR-related proteins in DTP and DTEP cells.
  • Figure 3 is the cycle effect of DTP and DTEP cells
  • Figure 4 is the in vitro pharmacodynamic diagram of EPI, Enza combined with Stac respectively in L-DTP cells; wherein, Figure 4A is a histogram of the relative survival rate of cells combined with L-DTP-EPI and L-DTP-Enza cells; Figure 4B is a histogram of the CI values of EPI, Enza and Stac respectively on L-DTP-EPI and L-DTP-Enza cells.
  • Figure 5 is a graph showing the effect of Enza, Stac and their combination on the C-MYC overexpression prostate cancer mouse model after continuous administration of Enza to produce resistance to the prostate weight change of the combined drug; among them, Figure 5A is the progress of each group of mice with the administration Changes in prostate weight; Figure 5B is a photographic comparison of stripped prostates of mice in each group; Figure 5C is a change in body weight of mice in each group with administration.
  • Figure 6 is the effect of Enza, Stac and their combined drug regimen on the C-MYC overexpression prostate cancer mouse model after continuous drug use of Enza to produce resistance; among them, Figure 6A is the HE of the prostate tissue sections of mice in each group Staining diagram; Fig. 6B is the immunohistochemical diagram of PRDX5 and AR of the prostate tissue sections of mice in each group; Fig. 6C is the histogram of immunohistochemical quantification of positive cells.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • Example 1 The process of using EPI and Enza to produce DTP/DTEP in prostate cancer LNCaP cells and the characteristics of this model
  • Prostate cancer L-DTP cell lines resistant to EPI and Enza L-DTP-EPI and L-DTP-Enza can inhibit the protein expression of AR and its targets, and the cell growth inhibition is manifested by cycle arrest in G0/G1 phase.
  • LNCaP cells 1x106 were planted in a 10cm cell culture dish. After attaching to the wall on the second day, they were treated with EPI and Enza respectively for 9 days. The collected cells were used for follow-up experiments, and the rest continued to be treated with drugs. During this period, fresh drug-containing medium was changed every three days for cultivation, and after 33 days (ie, DTEP cells) were collected for subsequent experiments. After generating DTP and DTEP cells, the cells were digested and counted, and the percentage of cells accounting for 1 ⁇ 10 6 cells was calculated.
  • NC, DTP, and DTEP cells were used for subsequent Western Blot experiments: the three groups of cells were subjected to cell lysis, protein extraction and quantification, SDS-PAGE electrophoresis gel, transfer to membrane, blocking, incubation of primary antibody, incubation of secondary antibody, and development Then observe the expression changes of AR-FL and its related target proteins, AR-Vs and their related target proteins; and the expression changes of cell cycle related proteins.
  • Flow cytometry Dead cells were stained with a PI staining cycle kit, and cell cycle changes were measured by flow cytometry.
  • Figure 1 is the Giemsa staining diagram of DTP and DTEP cells
  • Figure 2 is the protein expression changes of AR&its targets and AR-Vs&its targets in NC, DTP, and DTEP cells
  • Figure 3 is Changes in the expression of cell cycle-related proteins in NC, DTP, and DTEP cells.
  • CCK8 was used alone and in combination in drug-resistant L-DTP cells to illustrate the in vitro anti-tumor effect of Stac drugs in drug-resistant L-DTP (EPI) and L-DTP (Enza) cells.
  • L-DTP including L-DTP (EPI) and L-DTP (Enza)
  • L-DTP including L-DTP (EPI) and L-DTP (Enza)
  • a series of concentrations of Stac drugs from high to low were prepared to find the optimal concentration.
  • the CI value was calculated in L-DTP cells using Calcusyn software.
  • Figure 4A is a histogram of the relative survival rate of the combined drug-resistant cells on L-DTP (EPI) and L-DTP (Enza) drug-resistant cells; Histogram of CI values of Stac combined with L-DTP (EPI) and L-DTP (Enz) drug-resistant cells respectively.
  • Example 3 The effect of the combined drug regimen of Enza and Stac on the C-MYC overexpression prostate cancer mouse model after continuous drug use of Enza to develop resistance
  • a spontaneous prostate cancer mouse model with overexpression of C-MYC (Hi-Myc) was constructed.
  • the mice developed into mPIN/Cancer transition.
  • the mice were randomly divided into NC control group (gavage solution), Enza drug After that, Enza was administrated once every three days, and Enza was 10mg/Kg for a total of 30 days. Afterwards, some mice were neck-broken and their prostate cancer was taken to take pictures and weighed. It was found that Enza could significantly alleviate the symptoms.
  • the NC control group was reduced by half, and the remaining mice were continued to use the drug for 30 days according to the above method.
  • the Enza group had a recurrence, and then (that is, the age of the mice was 6 months) were randomly divided into: NC control group (gastric administration of solvent all the time), Enza single-drug group, Stac single-drug group, and Enza and Stac combined medication group were administered accordingly, and all were intragastrically administered once every three days, each time keeping Enza at 10mg/Kg and Stac at 80mg/Kg, A total of 30 days of administration. Afterwards, the mice were neck-broken and the prostate cancer cells were taken for photographing, weighing and immunohistochemical experiments.
  • Figure 5A is a graph showing the weight change of the prostates of mice in each group along with administration; Body weight changes of mice in each group after administration.
  • Fig. 6A is the HE staining diagram of the prostate tissue sections of mice in each group;
  • Fig. 6B and C are the AR, PRDX5 immunohistochemical diagrams and positive cell quantification histograms of the mouse prostate tissue sections in each group.
  • the average weight of the prostate of the mice in the Enza group was 43.9 mg, and the average weight of the NC control group was 88.7 mg. It is 98.2 mg, indicating that drug resistance has relapsed, causing CRPC. At this time, the drug should be grouped immediately to explain the effect of combined drug use.
  • medication mouse prostate weight mean NC control group 102.012 mg Continuous Enza single-drug group after drug-resistant relapse 54.42 mg Stac monotherapy group after drug-resistant relapse 50.856mg Combination of Enza and Stac after relapse of drug resistance 37.068mg

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Abstract

本发明公开了水苏糖在制备用于治疗去势抵抗性***癌药物中的应用，属于生物医药技术领域。本发明首次提出了一种利用水苏糖联合雄激素受体拮抗剂制备用于治疗CRPC的药物这一新策略，并进行了多角度、多层次的验证研究。本发明水苏糖与雄激素受体联合的药物组合物能用于治疗去势抵抗性***癌，并显著提高恩杂鲁胺抑制去势抵抗性***癌的作用，将天然化合物应用于癌症晚期，具有重要临床治疗意义。

Description

水苏糖在制备用于治疗去势抵抗性***癌药物中的应用 技术领域
本发明属于生物医药技术领域，具体涉及水苏糖在制备用于治疗去势抵抗性***癌药物中的应用。
背景技术
雄激素剥夺治疗是晚期***癌的标准治疗方法，但是患者最终都会在平均1-3年的治疗后进展为去势抵抗性***癌(Castration-resistant prostate cancer，CRPC)。所谓CRPC，是指经过初次持续雄激素剥夺治疗(ADT)后疾病依然进展的***癌。自从2004年多西他赛被证明能延长转移性去势抵抗***癌(metastatic castration-resistant prostate cancer，mCRPC)患者总生存后，近几年涌现出了如醋酸阿比特龙、恩杂鲁胺、卡巴他赛等针对mCRPC疾病阶段的药物，改变了这些患者的治疗现状，但最终也均难以彻底逆转CRPC。因此寻找有效的其他治疗靶点或联合用药治疗策略是治疗CRPC的另一研究热点。
近年来，细胞可塑性作为靶向诊断逃逸模型出现，是很多癌症抗性的共性。阻断新的抗药通路可有效抑制存留细胞(persister cell)，如GPX4脂质过氧化通路是许多种persister cell状态下高表达的有效靶点。到目前为止，我们不清楚CRPC肿瘤是否有persister cell以及能否找到新的有效靶点。因此本研究着重从EPI-001和Enzalutamide产生的***癌LNCaP-persister cell出发，寻找到有效的联用药物以治疗CRPC。
EPI-001(EPI)是正在等待临床开发的、有可能用于治疗CRPC的AR及AR-剪接变体(AR-Vs)抑制剂。EPI抵抗CRPC的靶点主要针对N-末端结构域(NTD)。恩杂鲁胺(Enzalutamide，Enza)是第一个获得批准的第二代AR拮抗剂，对AR亲和力较传统抗雄激素高5-8倍。2012年，美国FDA据此批准Enza用于CRPC患者。但无论是EPI还是Enza，对CRPC的治疗一般在18个月左右均会产生耐药。因此急需其他手段去克服耐药，延缓CRPC。
发明内容
本发明要解决的技术问题是克服上述现有药物存在的耐药性，提供一种有效治疗CRPC的药物，即Enza和Stac联用，实现对CRPC的疗效得到显著的提升，发挥优异的协同增效作用。
水苏糖(Stachyose，Stac)是蔗糖的葡萄糖基一侧以1,6-糖苷键结合2个α-半乳糖而形 成的糖类，分子式C 24H 42O 21。
本发明的第一个目的是提供Stac在制备用于治疗去势抵抗性***癌药物中的应用。
在本发明的一种实施方式中，所述应用包括：将Stac与雄激素受体拮抗剂联合制备用于治疗去势抵抗性***癌的药物。
本发明的第二个目的是提供一种用于治疗去势抵抗性***癌的药物组合物，所述组合物包括Stac与雄激素受体拮抗剂。
在本发明的一种实施方式中，雄激素受体拮抗剂与Stac质量比为(1～8)：1。其中优选地，Enza和Stac的质量比为1～3:1。
在本发明的一种实施方式中，雄激素受体拮抗剂包括如下任意一种或多种：恩杂鲁胺(Enza)、EPI-001(EPI)、阿比特龙、奥拉帕尼。
在本发明的一种实施方式中，所述药物组合物还包括药用辅料。
在本发明的一种实施方式中，所述药用辅料包含溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏合剂、整合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂以及释放阻滞剂。
在本发明的一种实施方式中，所述制剂的剂型包括注射液、注射用冻干粉针、控释注射剂、脂质体注射剂、混悬剂、植入剂、栓塞剂、胶囊剂、片剂、丸剂和口服液。
在本发明的一种实施方式中，所述药物组合物还可以包括药物载体。
在本发明的一种实施方式中，所述药物载体包括微囊、微球、纳米粒和脂质体。
在本发明的一种实施方式中，本发明经过大量的研究探索，找到了治疗CRPC的药物，即Enza和Stac联用。研究结果显示，通过建立耐EPI、Enza的***癌LNCaP-drug-tolerant persisters(L-DTP)细胞株，此时LNCaP细胞对EPI、Enza产生可恢复性的耐药，联合用药Enza和Stac可显著抑制细胞生长，通过CCK8细胞增殖分析对药物联用效果进行验证，并通过CI值确定其协同作用。同时构建C-MYC过表达***癌小鼠模型，比较动物体内Enza和Stac单用及联用的治疗CRPC作用差异，药物联用所产生的协同作用大大提高了单独Enza或Stac药物对CRPC的抑制作用，两者协同作用得到体内外验证。
在本发明的一种实施方式中，本发明通过建立L-DTP可恢复性耐药细胞株，采用CCK8方法及Calcusyn软件计算CI值，结果显示在此细胞株中，相比单独给Enza或Stac，Enza和Stac体外联用具有协同抗CRPC作用。通过建立C-MYC过表达***癌小鼠模型，在长期用药Enza产生抗性的模型下确定动物体内Enza和Stac联用组，较单药组，具有更显著的体 内抗CRPC模型作用。
本发明具有以下有益效果：
本发明首次提出了一种利用Stac制备治疗CRPC药物，以及基于Enza和Stac药物联用治疗CRPC的新策略，将推动Enza和Stac在***癌临床治疗中的应用，具有重要意义。药物研究从化合物分子到真正走上临床平均需8-10年的时间，且需要大量的人力物力支持，时间成本和经济成本巨大。本发明的方案可实现天然化合物寡糖重利用，可大大缩短药物发现到临床转化的时间。
附图说明
图1是DTP(drug-tolerant persisters)和DTEP(drug-tolerant expanded persisters)细胞的形成过程。
图2是DTP和DTEP细胞中AR相关蛋白表达量变化的特征。
图3是DTP和DTEP细胞的周期影响
图4是EPI、Enza分别与Stac联合用药在L-DTP细胞中的体外药效图；其中，图4A为L-DTP-EPI、L-DTP-Enza细胞上联合用药细胞相对存活率柱状图；图4B为EPI、Enza分别与Stac联用分别在L-DTP-EPI、L-DTP-Enza细胞上的CI值柱状图。
图5是Enza、Stac及其联合用药对C-MYC过表达***癌小鼠模型在持续用药Enza产生抗性后联合用药***重量变化效果图；其中，图5A为随给药进行各组小鼠***重量变化图；图5B为剥取各组小鼠***拍照对比图；图5C为随给药进行各组小鼠体重变化图。
图6是Enza、Stac及其联合用药方案对C-MYC过表达***癌小鼠模型在持续用药Enza产生抗性后联合用药作用效果图；其中，图6A为各组小鼠***组织切片的HE染色图；图6B为各组小鼠***组织切片的PRDX5、AR免疫组化图；图6C为免疫组化的阳性细胞量化柱状图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1 EPI、Enza用于***癌LNCaP细胞产生DTP/DTEP的过程及此模型的特征
耐EPI、Enza的***癌L-DTP细胞株L-DTP-EPI、L-DTP-Enza具有抑制AR及其靶点的蛋白表达，细胞生长抑制表现为周期阻滞在G0/G1期。
1、实验方法：
LNCaP细胞1x10 6个种于10cm细胞培养皿种，第二天贴壁后分别加EPI、Enza处理9天，期间每三天换新鲜的含药培养基培养，一部分细胞9天后(即DTP细胞)收集用于后续试验，剩下的继续加药处理，期间每三天换新鲜的含药培养基培养，33天后(即DTEP细胞)收集用于后续试验。在产生DTP及DTEP细胞后，消化细胞并计数，算出细胞占1x10 6细胞量的百分比。收集好的NC、DTP、DTEP细胞用于后续的Western Blot试验：这三组细胞经细胞裂解、蛋白提取定量、SDS-PAGE电泳凝胶、转膜、封闭、孵育一抗、孵育二抗、显影后观察AR-FL及其相关靶蛋白、AR-Vs及其相关靶蛋白的表达量变化；以及细胞周期相关蛋白表达量变化。流式细胞术：通过PI染色的周期试剂盒将死细胞染色，通过流式细胞仪测定细胞周期变化。
2、结果如图1、2、3所示：图1为DTP及DTEP细胞Giemsa染色图；图2为NC、DTP、DTEP细胞中AR&its targets、AR-Vs&its targets的蛋白表达量变化；图3为NC、DTP、DTEP细胞中细胞周期相关蛋白表达量变化。
结果显示，通过EPI、Enza产生的LNCaP-DTP细胞数占初始种细胞量的103.11％，LNCaP-DTEP细胞占110.92％。这些耐药的细胞均对EPI、Enza产生抗性，在DTP时细胞成梭形，生长抑制；在DTEP时细胞克隆变多，细胞生长抑制得到抵抗，细胞重新增殖。这种细胞模型对雄激素受体(AR)及其靶的蛋白表达量影响：DTP状态抑制AR蛋白表达，DTEP时AR表达有一定的恢复；AR的靶TMPRSS2、PSA的表达与AR保持一致的趋势。雄激素受体剪接变体(AR-Vs)及其靶的蛋白表达量影响：在DTP及DTEP状态，AR-Vs及其靶UBE2C、CDC20蛋白表达量均表现为持续抑制。这种抑制是通过细胞周期阻滞引起的，具体表现为：P21(G1期marker)在DTP上调，DTEP有所恢复；Cyclin E1(G1-S期marker),CDC6(G1-S期marker)在DTP下调，DTEP有所恢复；CDC2(G1-S和G2-M期marker)在DTP下调，DTEP没有显著恢复。通过流式细胞术(FACS)分析发现，DTP时G0/G1期发生阻滞，而DTEP时恢复。
实施例2 EPI、Enza分别与Stac药物联用的体外效果
进一步运用CCK8，在耐药L-DTP细胞中分别单用、联用，说明Stac药物在耐药L-DTP(EPI)及L-DTP(Enza)细胞中的体外抗肿瘤作用。
1、实验方法
将耐药细胞L-DTP(包括L-DTP(EPI)和L-DTP(Enza))种在96孔板中贴壁后，通过配制一系列从高到低的Stac药物的浓度，从而找到最佳的Stac药物浓度，接着用此浓度测定单用(L-DTP(EPI)-Stac)、联用【L-DTP(EPI)-combination(EPI+Stac)】、【L-DTP(Enza)-combination(Enza+Stac)】分别在耐药细胞L-DTP中的生存率。最后利用Calcusyn软件，在L-DTP细胞中计算CI值。
2、结果如图4所示，图4中，图4A为L-DTP(EPI)、L-DTP(Enza)耐药细胞上联合用药细胞相对存活率柱状图；图4B为EPI、Enza分别与Stac联用分别在L-DTP(EPI)、L-DTP(Enz)耐药细胞上的CI值柱状图。
结果显示，在持续加药EPI 9天产生耐药L-DTP(EPI)细胞中，继续加EPI已无明显抑制效果，改为单加Stac时又起到了明显抑制效果，可达67.48％的抑制率，继而当联用(EPI+Stac)时可达到51.15％的抑制率。类似地，在持续加药Enza 9天产生耐药L-DTP(Enza)细胞中，继续加Enza已无明显抑制效果，改为单加Stac具有明显抑制率，可达62.47％的抑制率，继而联用(Enza+Stac)则可达到51.97％的抑制率。通过计算CI值，发现Stac在L-DTP-EPI细胞中可达到0.49的高协同作用；在L-DTP(Enza)细胞可达到0.54的高协同作用。
实施例3 Enza和Stac联合用药方案对C-MYC过表达***癌小鼠模型在持续用药Enza产生抗性后联合用药的作用
进一步在***癌小鼠模型中说明化学***(即持续用药Enza)后复发的小鼠通过联合用药Enza和Stac后的效果。
1、实验方法
构建C-MYC(Hi-Myc)过表达的自发***癌小鼠模型，在4个月时，小鼠发展为mPIN/Cancer transition，此时随机分组为NC对照组(灌胃溶剂)、Enza用药组，后每三天一次灌胃，Enza为10mg/Kg，共给药30天，后部分小鼠断颈并取其***癌进行拍照、称重，发现Enza可明显减轻病症，***重相比于NC对照组降低一半，后按上述方法继续用药剩余的小鼠30天，发现Enza组有复发，之后(即鼠龄为6个月)随机分组为：NC对照组(一直灌胃溶剂)、Enza单药组、Stac单药组以及Enza和Stac联合用药组，并进行相应给药处理，全部灌胃给药，每三天一次，每次保持Enza为10mg/Kg，Stac为80mg/Kg，共给药30天。之后断颈小鼠并取其***癌经行拍照、称重及免疫组化等实验。
2、结果如图5和图6所示，图5中，图5A为随给药进行各组小鼠***重量变化图；图5B为剥取各组小鼠***拍照对比图；图5C为随给药进行各组小鼠体重变化图。图6中，图6A为各组小鼠***组织切片的HE染色图；图6B、C为各组小鼠***组织切片的AR、 PRDX5免疫组化图及阳性细胞量化柱状图。
在持续用药Enza 30天的小鼠***重均值为43.9mg，此时NC对照组均值为88.7mg，继续用药，达到90天后发现Enza组小鼠***重均值变77.1mg，此时NC对照组均值为98.2mg说明已耐药复发，引起CRPC，此时立即分组用药说明联合用药效果。
分组联用和单用结果如表1所示：
表1不同给药(用药30d)对耐药复发后的用药效果
给药 小鼠***重均值
NC对照组 102.012mg
耐药复发后持续Enza单药组 54.42mg
耐药复发后Stac单药组 50.856mg
耐药复发后Enza和Stac联合用药组 37.068mg
结合图5和表1可见，Enza和Stac联用相比于Enza单用和Stac单用均有极显著作用，***重可降为37.068mg左右，同时可看出Stac单用比耐药后的Enza单用治疗效果要好，说明单用Stac对耐药的CRPC即有抑制效果。
从组织切片HE染色结果(图6)可以看出：联合用药后CRPC的***肿瘤有明显的退化和纤维化。从免疫组化可看出Enza和Stac联用相比于Enza单用和Stac单用均有显著的AR及PRDX5表达量的下降。证明联用效果显著。
分组联用和单用结果见表2：
表2不同给药(用药30d)对耐药复发后的用药效果
给药 AR表达量/％ PRDX5表达量/％
NC对照组 93.7 47.0
耐药复发后持续Enza单药组 89.4 78.4
耐药复发后Stac单药组 61.5 68.2
耐药复发后Enza和Stac联合用药组 46.3 39.2

Claims (10)

  1. 水苏糖在制备用于治疗去势抵抗性***癌药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述应用包括：利用水苏糖单独，或者与雄激素受体拮抗剂联合制备用于治疗去势抵抗性***癌的药物。
  3. 一种用于治疗去势抵抗性***癌的药物组合物，其特征在于，所述组合物包括水苏糖与雄激素受体拮抗剂。
  4. 根据权利要求3所述的药物组合物，其特征在于，雄激素受体拮抗剂与水苏糖质量比为(1～8)：1。
  5. 根据权利要求3所述的药物组合物，其特征在于，雄激素受体拮抗剂包括如下任意一种或多种：恩杂鲁胺、EPI、阿比特龙、奥拉帕尼。
  6. 根据权利要求3所述的药物组合物，其特征在于，所述药物组合物还包括药用辅料。
  7. 根据权利要求6所述的药物组合物，其特征在于，所述药用辅料包含溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏合剂、整合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂以及释放阻滞剂。
  8. 根据权利要求3所述的药物组合物，其特征在于，所述药物组合物还可以包括药物载体。
  9. 根据权利要求8所述的药物组合物，其特征在于，所述药物载体包括微囊、微球、纳米粒和脂质体。
  10. 根据权利要求3-9任一项所述的药物组合物，其特征在于，所述药物组合物的剂型包括注射液、注射用冻干粉针、控释注射剂、脂质体注射剂、混悬剂、植入剂、栓塞剂、胶囊剂、片剂、丸剂和口服液。
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