WO2022236963A1 - 聚普瑞锌在制备用于治疗去势抵抗性前列腺癌药物中的应用 - Google Patents

聚普瑞锌在制备用于治疗去势抵抗性前列腺癌药物中的应用 Download PDF

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WO2022236963A1
WO2022236963A1 PCT/CN2021/108355 CN2021108355W WO2022236963A1 WO 2022236963 A1 WO2022236963 A1 WO 2022236963A1 CN 2021108355 W CN2021108355 W CN 2021108355W WO 2022236963 A1 WO2022236963 A1 WO 2022236963A1
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drug
pharmaceutical composition
enza
prostate cancer
dtp
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French (fr)
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陈永泉
王荣
朱升龙
王小英
糜远源
吴升
孙健
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • 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
    • A61K31/075—Ethers or acetals
    • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
    • A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4166—1,3-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. phenytoin
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/502—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with carbocyclic ring systems, e.g. cinnoline, phthalazine
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • 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

Definitions

  • the invention belongs to the technical field of biomedicine, and specifically relates to the application of polyprezinc 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
  • Serum testosterone is maintained at the castration level ( ⁇ 50ng/dL or 1.7nmol/L)
  • Biochemical progress three consecutive detections of PSA values rising by more than 50% of the lowest value at intervals of one week and The absolute value of the increase is >2ng/ml; or imaging progress: two or more new lesions appear on bone scan or enlargement of soft tissue lesions is evaluated by RECIST. At present, it is believed that the progression of symptoms alone is not enough to diagnose CRPC.
  • CRPC patients had no effective treatment and could only accept some palliative care. Since 2004, when docetaxel was proven to prolong the overall survival of patients with metastatic castration-resistant prostate cancer (mCRPC), In recent years, drugs targeting mCRPC disease stages such as abiraterone acetate, enzalutamide, and cabazitaxel have emerged, which have changed the treatment status of these patients, but in the end, it is difficult to completely reverse CRPC. Therefore, finding an effective combined drug treatment strategy is another research hotspot for the treatment of CRPC.
  • mCRPC metastatic castration-resistant prostate cancer
  • tumor cell drug resistance also known as tumor cell plasticity, minimal residual disease, or drug-tolerant residue. persisters, DTP
  • the characteristics of this mechanism are: in the drug-resistant state, tumor cells no longer rely on the pathway targeted by the drug, but survive through other pathways, but the target gene does not undergo any mutation; drug sensitivity recovers after a period of withdrawal of the drug.
  • 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.
  • Polaprezinc is a chelated form of zinc and L-carnosine. This is the first zinc-related drug approved in Japan and has been clinically used to treat gastric ulcers. The results suggest that Pola may be effective in treating pressure ulcers. A 2013 study suggested that a combination of pola may be effective against damage to the small intestinal mucosa caused by long-term aspirin use.
  • the technical problem to be solved by the present invention is to overcome the drug resistance existing in the above-mentioned existing drugs, and provide a drug for effectively treating CRPC, that is, the combination of Enza and Pola, which can significantly improve the curative effect on CRPC and exert excellent synergistic growth. effect.
  • the first object of the present invention is to provide the application of polyprezinc in the preparation of drugs for treating castration-resistant prostate cancer.
  • the application includes: combining polyprezinc and 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 paprezinc and an androgen receptor antagonist.
  • the mass ratio of the androgen receptor antagonist to polyprezinc is (1-5):1. Among them, preferably, the mass ratio of Enza and Pola is 1 ⁇ 2:1.
  • 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, foaming 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 has found a drug for treating CRPC, that is, the combination of Enza and Pola.
  • a drug for treating CRPC that is, the combination of Enza and Pola.
  • 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 Pola alone, , Enza and Pola 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 Pola 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 based on the combination of Enza and Pola to prepare a drug for treating CRPC by using Pola, and clarifies its mechanism of action, which will promote the application of Enza and Pola 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 solution of the present invention can realize "new use of old drugs", and can greatly shorten the time from drug discovery to clinical transformation.
  • Figure 1 is a description of the recovery characteristics of DTP (drug-tolerant persisters) after drug withdrawal; among them, Figure 1A is a process diagram of DTP and cell recovery after drug withdrawal; Figure 1B is the third generation of DTP-EPI drug withdrawal and DTP-Enza drug withdrawal After 3 generations, the sensitivity to the corresponding drugs regained its characteristics.
  • DTP drug-tolerant persisters
  • Figure 2 is the characteristics of the expression changes of AR-related genes in DTP and drug withdrawal (Recovery Cell) cells; among them, Figure 2A is the change of protein expression detected by WB; Figure 2B is the change of transcript expression detected by q-PCR.
  • Figure 3 is the in vitro pharmacodynamic diagram of EPI, Enza combined with Pola respectively in L-DTP cells; wherein, Figure 3A is a histogram of the relative survival rate of cells combined with L-DTP-EPI and L-DTP-Enza cells; Figure 3B is a histogram of the CI values of EPI, Enza and Pola respectively on L-DTP-EPI and L-DTP-Enza cells.
  • Figure 4 is a graph showing the effect of Enza, Pola and their combination on the C-MYC overexpression prostate cancer mouse model after the continuous drug Enza produces resistance to the prostate weight change of the combined drug; among them, Figure 4A is the progress of each group of mice with drug administration Changes in prostate weight; Figure 4B is a photographic comparison of stripped prostates of mice in each group; Figure 4C is a change in body weight of mice in each group with administration.
  • Figure 5 is the effect of Enza, Pola 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 5A is the HE of the prostate tissue sections of mice in each group Staining diagram; Fig. 5B is the immunohistochemical diagram of PRDX5 and AR of the prostate tissue sections of mice in each group and the histogram of 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 EPI, Enza are used for the process of producing DTP by prostate cancer LNCaP cells
  • EPI Enza-resistant prostate cancer L-DTP cell lines L-DTP-EPI, L-DTP-Enza have recoverable properties.
  • LNCaP cells 1x106 were planted in a 10cm cell culture dish, and treated with EPI and Enza for 9 days after adherence on the second day, during which the fresh medium containing the drug was changed every three days; after 9 days, the drug was withdrawn and the medium was replaced with a fresh medium
  • the cells were normally cultured in the incubator, passaged on the 6th day, 12th day, and 17th day respectively, and the cell morphology of DTP (9th day), R5 (5th day after drug withdrawal), R10, and R20 were photographed under an inverted microscope picture. After producing DTP and cloned DTP cells, the cells were digested and counted, and the percentage of DTP cells to the total L-parent cells was calculated.
  • L-parent cells and cells of the third generation (R20) after drug withdrawal were respectively spread in 96-well plates, and after adhering to the wall overnight, a series of EPI and Enza from high to low concentrations were prepared, and a control group was added to the wells.
  • Set up 3 duplicate wells add CCK8 after incubation in the cell culture incubator for 48 hours, use a full-wavelength multifunctional microplate reader to detect the OD value of the cells at a wavelength of 450nm after 4 hours, count the survival rate and draw a survival curve.
  • Figure 1A is the process diagram of DTP and cell recovery after withdrawal of the drug; Sensitivity reinstatement characteristics for corresponding drugs are indicated.
  • LNCaP cells were placed in a 10cm dish, after overnight adherence to the wall, EPI and Enza were added to treat the cells for 9 days, and then the cells were collected. During this period, the fresh medium added with drugs was replaced every 3 days, and the remaining DTP cells were collected 20 days after drug dispensing.
  • Figure 2A is the change of protein expression detected by WB
  • Figure 2B is the change of transcript expression detected by q-RTPCR.
  • Example 3 EPI, Enza respectively combined with Pola drug.
  • CCK8 in drug-resistant L-DTP cells was used alone or in combination to illustrate the in vitro anti-tumor effect of Pola 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 Pola drug concentrations from high to low were prepared to find the optimal concentration.
  • the CI value was calculated in L-DTP cells using Calcusyn software.
  • Figure 3A 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 Pola combination on L-DTP (EPI) and L-DTP (Enz) drug-resistant cells respectively.
  • Example 4 The effect of the combination regimen of Enza and Pola on the C-MYC overexpression prostate cancer mouse model after continuous drug Enza 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, Pola single-drug group, and Enza and Pola combined medication group were treated accordingly, and all were intragastrically administered once every three days, keeping Enza at 10mg/Kg and Pola at 20mg/Kg each time. 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 4A is a graph showing the weight change of the prostate of each group of mice along with the administration; Body weight changes of mice in each group after administration.
  • Fig. 5A is the HE staining diagram of the prostate tissue sections of mice in each group;
  • Fig. 5B is the AR, PRDX5 immunohistochemical diagram and positive cell quantification histogram of the mouse prostate tissue sections of each group.
  • the average prostate weight of mice was 41.2 mg, while the average weight of the NC control group was 85.3 mg.
  • the average prostate weight of mice in the Enza group changed to 78.4 mg.
  • the average weight of the NC control group It is 95.6 mg, indicating that drug resistance has relapsed, causing CRPC.
  • the drug should be grouped immediately to explain the effect of combined drug use.
  • medication mouse prostate weight mean NC control group 100.1mg Continuous Enza single-drug group after drug-resistant relapse 83.3mg Pola monotherapy group after drug-resistant relapse 80.625mg Combination of Enza and Pola after relapse of drug resistance 53.14mg

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Abstract

聚普瑞锌或其与雄激素受体拮抗剂联合在制备用于治疗去势抵抗性前列腺癌药物中的应用。聚普瑞锌与雄激素受体拮抗剂联合使用可显著提高雄激素受体拮抗剂如恩杂鲁胺的抑制去势抵抗性前列腺癌的作用。

Description

聚普瑞锌在制备用于治疗去势抵抗性前列腺癌药物中的应用 技术领域
本发明属于生物医药技术领域，具体涉及聚普瑞锌在制备用于治疗去势抵抗性前列腺癌药物中的应用。
背景技术
雄激素剥夺治疗是晚期前列腺癌的标准治疗方法，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前列腺癌(Castration-resistant prostate cancer，CRPC)。所谓CRPC，2014年CUA指南：经过初次持续雄激素剥夺治疗(ADT)后疾病依然进展的前列腺癌。应同时具备以下条件：(1)血清睾酮维持在去势水平(＜50ng/dL或1.7nmol/L)；(2)生化进展：间隔一周，连续三次检测PSA值上升超过最低值的50％并且升高绝对值＞2ng/ml；或影像学进展：骨扫描出现两个或两个以上新病灶或用RECIST评估软组织病灶增大。目前认为，仅有症状上的进展并不足以诊断为CRPC。
曾经CRPC患者并无有效的治疗手段而只能接受一些姑息治疗，自从2004年多西他赛被证明能延长转移性去势抵抗前列腺癌(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。因此寻找有效的联合用药治疗策略是治疗CRPC的另一研究热点。
近来有很多学者提出一种新的肿瘤细胞抗药机制—存留细胞(persister cell)，也叫肿瘤细胞可塑性(tumor cell plasticity)、微小残留疾病(minimal residual disease)、或耐药残留(drug-tolerant persisters,DTP)等。此机制的特点是：在抗药状态下肿瘤细胞不再依赖药物靶向的通路，而是通过其它通路生存，但靶点的基因未发生任何突变；撤药一段时间后药物敏感性恢复。目前提出建立此机制的三种假说：①预存在少数药物容忍细胞(drug-tolerant cells)，经药物处理后通过达尔文进化论增多；②少数药物难治性的细胞被小部分癌细胞通过表观遗传修饰产生药物容忍细胞，与残余病灶共存；③肿瘤细胞动态表达各种抗性基因，在药物处理时高表达这些抗性基因，从而进一步重建抗性表达系统，产生药物容忍细胞。近几年，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。阻断新的抗药通路有效的抑制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产生的前列腺癌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。
聚普瑞锌(Polaprezinc，Pola)是锌和左旋肌肽的螯合形式。这是日本首次批准的一种与锌有关的药物，已被临床用于治疗胃溃疡。结果表明，Pola可能对治疗压迫溃疡有效。2013年的一项研究表明，联合服用Pola可能对长期服用阿司匹林导致的小肠黏膜损伤有效。
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Pola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。
本发明的第一个目的是提供聚普瑞锌在制备用于治疗去势抵抗性前列腺癌药物中的应用。
在本发明的一种实施方式中，所述应用包括：将聚普瑞锌与雄激素受体拮抗剂联合制备用于治疗去势抵抗性前列腺癌的药物。
本发明的第二个目的是提供一种用于治疗去势抵抗性前列腺癌的药物组合物，所述组合物包括聚普瑞锌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聚普瑞锌质量比为(1～5)：1。其中优选地，Enza和Pola的质量比为1～2:1。
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Pola联用。研究结果显示，通过建立耐EPI、Enza的前列腺癌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Pola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过表达前列腺癌小鼠模型，比较动物体内Enza和Pola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Pola，Enza和Pola体外联用具有协同抗CRPC作用。通过建立C-MYC过表达前列腺癌小鼠模型，在长期用药Enza产生抗性的模型下确定动物体内Enza和Pola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Pola制备治疗CRPC药物，以及基于Enza和Pola药物联用的新策略，并阐明其作用机制，将推动Enza和Pola在前列腺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“老药新用”，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)撤药后可恢复特性说明；其中，图1A为DTP及撤药后细胞恢复的过程图；图1B为DTP-EPI撤药3代、DTP-Enza撤药3代后对相应药物的敏感性重新恢复特点。
图2是DTP及撤药(Recovery Cell)细胞AR相关基因表达量变化的特征；其中，图2A为WB检测蛋白表达量的变化；图2B为q-PCR检测转录本表达量的变化。
图3是EPI、Enza分别与Pola联合用药在L-DTP细胞中的体外药效图；其中，图3A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3B为EPI、Enza分别与Pola联用分别在L-DTP-EPI、L-DTP-Enza细胞上的CI值柱状图。
图4是Enza、Pola及其联合用药对C-MYC过表达前列腺癌小鼠模型在持续用药Enza产生抗性后联合用药前列腺重量变化效果图；其中，图4A为随给药进行各组小鼠前列腺重量变化图；图4B为剥取各组小鼠前列腺拍照对比图；图4C为随给药进行各组小鼠体重变化图。
图5是Enza、Pola及其联合用药方案对C-MYC过表达前列腺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5A为各组小鼠前列腺组织切片的HE染色图；图5B为各组小鼠前列腺组织切片的PRDX5、AR免疫组化图及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用于前列腺癌LNCaP细胞产生DTP的过程
耐EPI、Enza的前列腺癌L-DTP细胞株L-DTP-EPI、L-DTP-Enza具有可恢复特性。
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；9天后撤药，换新鲜培养基并于培养箱中正常培养细胞，在第6天、12天、17天分别传代，并于倒置显微镜拍下DTP(第9天)、R5(撤药第5天)、R10、R20的细胞形态图。在产生DTP及clone的DTP细胞后，消化细胞并计数，算出DTP细胞占总L-parent细胞的百分比。L-parent细胞及撤药后三代(R20)的细胞分别铺96孔板中，过夜贴壁后，配置一系列从高到低浓度的EPI、Enza，并设置对照组加入孔中，每个浓度设置3个复孔，在细胞培养箱中孵育48h后加入CCK8，4h后利用全波长多功能酶标仪检测450nm波长的细胞OD值，计数生存率并绘制生存曲线。
2、结果如图1和表1所示：图1中，图1A为DTP及撤药后细胞恢复的过程图；图1B为DTP-EPI撤药3代、DTP-Enza撤药3代后对相应药物的敏感性重新恢复特点说明。
表1 LNCaP细胞产生DTP模型后细胞计数
Cell line DTP(％) S.D.(N＝3)
LNCCaP-1 0.82 0.08
LNCaP-1cl.A 0.18 0.12
LNCaP-1cl.B 0.22 0.06
LNCaP-1cl.C 0.54 0.03
LNCCaP-a 0.33 0.94
LNCaP-a cl.A 0.15 0.11
LNCaP-a cl.B 0.24 0.21
LNCaP-a cl.C 0.13 0.18
结果显示，通过EPI、Enza产生的LNCaP-DTP细胞为数极少，这些耐药的细胞均对EPI、Enza产生抗性，但是均可在撤药3代后细胞形态恢复、对药物的敏感性也恢复。
实施例2 DTP及撤药(Recovery Cell)细胞AR相关基因表达量变化的特征建立
DTP细胞在撤药3代后细胞形态恢复，此时的细胞AR相关基因表达量变化也可恢复。
1、实验方法：
LNCaP细胞中在10cm皿中，贴壁过夜之后分别加入EPI、Enza处理细胞9天后收取细胞，期间每3天更换加药的新鲜培养基，剩下的DTP细胞撤药配药20天后同样收取细胞。设置LNCaP细胞DMSO处理9天为NC对照组，加EPI、Enza处理9天产生L-DTP-EPI、L-DTP-Enza的耐药细胞为处理组，此处理组在撤药20天后的细胞为恢复组，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。同时，此三组细胞进行q-RTPCR测定，检测AR-FL及其相关靶基因、AR-Vs及其相关靶基因的表达量变化。
2、结果如图2所示，图2中，图2A为WB检测蛋白表达量的变化；图2B为q-RTPCR检测转录本表达量的变化。
结果显示，AR其相关靶蛋白PSA、TMPRSS2；AR-Vs及其相关靶蛋白UBE2C、CDC20；均不同程度地在DTP阶段表达量下调，而在撤药R20后表达量恢复；同样地，AR-Vs及其相关靶基因、AR-Vs及其相关靶基因、以及生长marker：AKT1、C-MYC表达量也在DTP阶段下调，R20表达量不同程度的恢复。
实施例3 EPI、Enza分别与Pola药物联用作用。
进一步运用CCK8，在耐药L-DTP细胞中分别单用、联用，说明Pola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Pola药物的浓度，从而找到最佳的Pola药物浓度，接着用此浓度测定单用(L-DTP(EPI)-Pola)、联用【L-DTP(EPI)-combination(EPI+Pola)】、【L-DTP(Enza)-combination(Enza+Pola)】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3所示，图3中，图3A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3B为EPI、Enza分别与Pola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Pola仍无明显抑制率，但是当联用(EPI+Pola)时可达到55.74％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Pola仍无明显抑制率，但是联用(Enza+Pola)则可达到60.765％的抑制率。通过计算CI值，发现Pola在L-DTP-EPI细胞中可达到0.525的高协同作用；在L-DTP(Enza)细胞可达到0.695的高协同作用。
实施例4 Enza和Pola联合用药方案对C-MYC过表达前列腺癌小鼠模型在持续用药Enza产生抗性后联合用药的作用
进一步在前列腺癌小鼠模型中说明化学阉割(即持续用药Enza)后复发的小鼠通过联合用药Enza和Pola后的效果。
1、实验方法
构建C-MYC(Hi-Myc)过表达的自发前列腺癌小鼠模型，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前列腺癌进行拍照、称重，发现Enza可明显减轻病症，前列腺重相比于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、Pola单药组以及Enza和Pola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，Pola为20mg/Kg，共给药30天。之后断颈小鼠并取其前列腺癌经行拍照、称重及免疫组化等实验。
2、结果如图4和图5所示，图4中，图4A为随给药进行各组小鼠前列腺重量变化图；图4B为剥取各组小鼠前列腺拍照对比图；图4C为随给药进行各组小鼠体重变化图。图5中，图5A为各组小鼠前列腺组织切片的HE染色图；图5B为各组小鼠前列腺组织切片的AR、 PRDX5免疫组化图及阳性细胞量化柱状图。
在持续用药Enza 30天的小鼠前列腺重均值为41.2mg，此时NC对照组均值为85.3mg，继续用药，达到60天后发现Enza组小鼠前列腺重均值变78.4mg，此时NC对照组均值为95.6mg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2所示：
表2不同给药(用药30d)对耐药复发后的用药效果
给药 小鼠前列腺重均值
NC对照组 100.1mg
耐药复发后持续Enza单药组 83.3mg
耐药复发后Pola单药组 80.625mg
耐药复发后Enza和Pola联合用药组 53.14mg
结合图4和表2可见，Enza和Pola联用相比于Enza单用和Pola单用均有极显著作用，前列腺重可降为53.14mg左右，同时可看出Pola单用与Enza单用效果几乎一致，说明单用Pola对耐药的CRPC无显著效果。
从组织切片HE染色结果(图5)可以看出：联合用药后CRPC的前列腺肿瘤有明显的退化和纤维化。从免疫组化可看出Enza和Pola联用相比于Enza单用和Pola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3：
表3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 94 61.7
耐药复发后持续Enza单药组 71.5 93.6
耐药复发后Pola单药组 70.4 74.2
耐药复发后Enza和Pola联合用药组 20.5 15

Claims (10)

  1. 聚普瑞锌在制备用于治疗去势抵抗性前列腺癌药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述应用包括：将聚普瑞锌与雄激素受体拮抗剂联合制备用于治疗去势抵抗性前列腺癌的药物。
  3. 一种用于治疗去势抵抗性前列腺癌的药物组合物，其特征在于，所述组合物包括聚普瑞锌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聚普瑞锌质量比为(1～5)：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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