WO2020087937A1 - 治疗伴有纤维化肝癌的组合物 - Google Patents

治疗伴有纤维化肝癌的组合物 Download PDF

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WO2020087937A1
WO2020087937A1 PCT/CN2019/091265 CN2019091265W WO2020087937A1 WO 2020087937 A1 WO2020087937 A1 WO 2020087937A1 CN 2019091265 W CN2019091265 W CN 2019091265W WO 2020087937 A1 WO2020087937 A1 WO 2020087937A1
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liver cancer
fibrosis
liver
syk
mice
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洪健
蒋煜川
陈鹏
戴冠齐
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南方医科大学中西医结合医院
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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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
    • A61K31/395—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
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • the invention relates to a composition for treating liver cancer accompanied by fibrosis.
  • liver fibrosis caused by various reasons is closely related to the occurrence of liver cancer. Most liver cancer patients are accompanied by different degrees of liver fibrosis. And the current research found that the liver microenvironment in the background of fibrosis promoted the proliferation and invasion of liver cancer. In addition, liver fibrosis also reduced the choice of treatment for liver cancer and reduced the therapeutic effect. However, there is currently no treatment strategy for liver cancer in the context of liver fibrosis.
  • liver cancer At present, surgery and interventional therapy are the main treatments for liver cancer, but a considerable number of patients with liver cancer have entered the advanced stage of the tumor when they are diagnosed, and lose the opportunity for treatment. In addition, due to the characteristics of easy metastasis and recurrence of liver cancer, the effect of surgery and interventional therapy is not very ideal.
  • chemotherapy, radiotherapy and targeted therapy are mostly used as adjuvant treatments, and their practical applications are limited.
  • the only liver cancer targeting drugs Sorafenib and Regorafenib although approved for the treatment of inoperable or distant metastatic hepatocellular carcinoma, but its effect on the extension of the survival of liver cancer patients is very limited. And it does not have a good therapeutic effect on liver cancer with fibrosis. Therefore, the development of new treatment strategies and drugs to treat liver cancer in the context of liver fibrosis is of great significance.
  • Spleen tyrosine kinase (SYK) gene was first cloned from porcine spleen cDNA in 1991 and encodes a non-receptor protein tyrosine kinase.
  • the human SYK gene is located in the q22 region of chromosome 9, and the SYK protein contains 635 amino acids. It is an effective target for the treatment of inflammation and hematological malignancies.
  • SYK inhibitors have been used in rheumatoid arthritis, chronic lymphocytic leukemia, etc. The clinical phase II / III experiment is encouraging, and the drug safety is good.
  • SYK small molecule compound inhibitors include Entospletinib (GS-9973), Fostamatinib (R788), R406, and PRT062607 (P505-15, BIIB057) HCl, these SYK inhibitors
  • the biological effect trend of the agent is similar, with Entospletinib (GS-9973) inhibiting SYK kinase activity the most stable and significant.
  • SYK small molecule compound inhibitors on the market that have been used in clinical phase II / III studies of various diseases; GS-9973 is the latest reported clinical phase II experiment for chronic lymphocytic leukemia, with an effective rate of up to 91 %, And the drug SYK has strong targeting specificity, low off-target rate, and good biological safety.
  • An object of the present invention is to provide a composition for treating liver cancer accompanied by fibrosis.
  • Another object of the present invention is to provide the use of the composition in the preparation of a medicament for treating liver cancer accompanied by fibrosis.
  • Another method of the present invention is to provide a method for treating liver cancer accompanied by fibrosis.
  • a composition for treating liver cancer accompanied by fibrosis the active ingredient of which includes at least one SYK inhibitor and at least one multi-target kinase inhibitor against liver cancer.
  • the multi-target kinase inhibitors against liver cancer are Sorafenib and Reforafenib.
  • the SYK inhibitor is selected from GS-9973, R788, R406, PRT062607.
  • composition in the preparation of a medicine for treating liver cancer accompanied by fibrosis, wherein the active ingredient of the composition includes at least one SYK inhibitor and at least one multi-target kinase inhibitor against liver cancer.
  • the multi-target kinase inhibitors for liver cancer are Sorafenib and Reforafenib.
  • SYK inhibitors are selected from GS-9973, R788, R406, PRT062607.
  • a method for treating liver cancer associated with fibrosis includes treating a patient with a therapeutic amount of at least one SYK inhibitor and at least one multi-target kinase inhibitor against liver cancer.
  • the multi-target kinase inhibitors against liver cancer are Sorafenib and Reforafenib.
  • the SYK inhibitor is selected from GS-9973, R788, R406, and PRT062607.
  • the inventors found through experiments that the combined application of SYK inhibitors against the liver microenvironment and multi-target kinase inhibitors Sorafenib or Reforafenib against liver cancer enhanced the cancer suppressive effects of Sorafenib or Reforafenib and further extended the model mice
  • the survival time indicates that the combined application of SYK inhibitors and multi-target kinase inhibitors against liver cancer has a good therapeutic effect on liver cancer with fibrosis.
  • Figure 1 is the results of ELISA experiments based on human liver stellate cell lines LX-2 and TWNT-4;
  • Figure 2 is a statistical diagram of the survival period of liver fibrosis mouse liver cancer orthotopic transplantation tumor model after treatment
  • Figure 3 is the survival curve of liver fibrosis mice liver cancer orthotopic transplantation tumor model in different treatment groups
  • Figure 4 is the size of primary foci of liver transplanted tumors and the number of intrahepatic metastases after two weeks of treatment for liver fibrosis liver cancer orthotopic transplanted mice;
  • Figure 5 is a physical picture of representative liver orthotopic tumor metastasis of mice in each group after two weeks of treatment of liver fibrotic liver cancer orthotopic tumor mice;
  • Figure 6 is representative HE staining and Sirius red staining of livers of mice in each group after two-week treatment of orthotopic transplanted mice with liver fibrosis and liver cancer;
  • Figure 7 is the statistical results of the survival period of non-fibrotic liver mice liver cancer orthotopic transplantation tumor model after treatment
  • Fig. 9 is the size of primary foci of liver transplanted tumors and the number of intrahepatic metastases after a two-week treatment of the liver cancer orthotopic transplanted tumor model of non-fibrotic liver mice.
  • a composition for treating liver cancer accompanied by fibrosis the active ingredient of which includes at least one SYK inhibitor and at least one multi-target kinase inhibitor against liver cancer.
  • the multi-target kinase inhibitors against liver cancer are Sorafenib and Reforafenib.
  • the SYK inhibitor is selected from GS-9973, R788, R406, PRT062607.
  • composition in the preparation of a medicine for treating liver cancer accompanied by fibrosis, wherein the active ingredient of the composition includes at least one SYK inhibitor and at least one multi-target kinase inhibitor against liver cancer.
  • the multi-target kinase inhibitors for liver cancer are Sorafenib and Reforafenib.
  • SYK inhibitors are selected from GS-9973, R788, R406, PRT062607.
  • a method for treating liver cancer associated with fibrosis includes treating a patient with a therapeutic amount of at least one SYK inhibitor and at least one multi-target kinase inhibitor against liver cancer.
  • the multi-target kinase inhibitors against liver cancer are Sorafenib and Reforafenib.
  • the SYK inhibitor is selected from GS-9973, R788, R406, and PRT062607.
  • mice Four-week-old male C57BL / 6 mice were selected and gavaged with 40% CCl 4 solution, 7 times for 2 weeks for 6 weeks, and liver cancer cells derived from mice were used for liver cancer in situ from 4 weeks
  • Transplantation to construct orthotopic xenograft model of liver cancer in liver fibrosis mice -9973 & Regorafenib combination treatment group began to give medication on the second day after transplantation, the dosage of GS-9973 5mg / kg, Sorafenib 30mg / kg, Regorafenib 20mg / kg.
  • half of the mice in each group were taken, and the liver was removed after anesthesia and sacrifice. The primary tumor volume and the number of intrahepatic metastases were evaluated. The other half of the mice in each group continued to be treated to observe the effect of the treatment drugs on survival.
  • the median survival of the control group was 27 days.
  • GS-9973 and Regorafenib monotherapy significantly prolonged the median survival of mice (32 days and 33 days, respectively), while Sorafenib or Regorafenib Combined with GS-9973 treatment, the survival time of mice was further extended (36 days and 39 days respectively),
  • GS GS-9973
  • Sora Sorafenib
  • Regora Regorafenib
  • Figure 3 shows the survival curves of the liver fibrosis mouse liver cancer orthotopic transplantation tumor model, the control group, the GS-9973 treatment group, the Regorafenib treatment group and the GS-9973 combined with Regorafenib treatment group, and the GS-9973 combined with Regorafenib treatment group. Bit survival time was significantly increased compared with Regorafenib treatment group.
  • mice with orthotopic transplantation of liver fibrosis and liver cancer were treated for two weeks. The mice were sacrificed the next day after the last treatment to evaluate the size of the primary foci of liver transplantation tumors and the number of intrahepatic metastases (Figure 4): GS-9973 significantly inhibited The growth and metastasis of orthotopic transplantation tumors of liver cancer in liver fibrosis mice were enhanced, and the inhibitory effect of Sorafenib and Regorafenib on liver cancer was enhanced.
  • A the primary tumor volume of transplanted tumors in model mice of each group
  • B the number of intrahepatic metastases of model mice in each group
  • mice with orthotopic transplantation of liver fibrosis and liver cancer were treated for two weeks. After the last treatment, the mice were sacrificed the next day and the liver was taken to take pictures. The physical map of representative liver orthotopic transplantation of mice in each group ( Figure 5). The results showed that, compared with Sorafenib or Regorafenib alone, the combined GS-9973 treatment significantly enhanced the anti-hepatoma effect of Sorafenib and Regorafenib. After combined treatment, the tumor size and the number of intrahepatic metastases were significantly improved.
  • mice with orthotopic transplantation of liver fibrosis and liver cancer were treated for two weeks, and the mice were sacrificed the next day after the last treatment.
  • A control group, GS-9973 treatment group, Regorafenib treatment group and GS-9973 combined with Regorafenib treatment group Representative HE staining and Sirius red staining of tumor-bearing liver in mice ( Figure 6);
  • B control group, GS-9973 treatment group , Ishak score of tumor-bearing liver in mice treated with Regorafenib and GS-9973 combined with Regorafenib treatment. Ishak score in mice with tumor-bearing liver suggested that GS-9973 monotherapy and combination treatment significantly reduced tumor-bearing liver fibrosis in mice.
  • * Means P ⁇ 0.05, ** means P ⁇ 0.01.
  • mice Four-week-old male C57BL / 6 mice were selected for orthotopic transplantation of liver cancer using mouse-derived liver cancer cells to construct a mouse liver cancer orthotopic transplantation tumor model, and then the model mice were divided into a control group and GS-9973 single drug Group, Sorafenib single drug group, Regorafenib single drug group, GS-9973 & Sorafenib combination treatment group and GS-9973 & Regorafenib combination treatment group, the drug treatment was started on the second day after transplantation, and the dosage was GS-9973 5mg / kg, Sorafenib 30mg / kg, Regorafenib 20mg / kg.
  • mice in each group were taken, and the liver was removed after anesthesia and sacrifice. The primary tumor volume and the number of intrahepatic metastases were evaluated. The other half of the mice in each group continued to be treated to observe the effect of the treatment drugs on survival.
  • Non-fibrotic liver mouse liver cancer orthotopic transplantation tumor model survival statistics after treatment showed that the median survival of the control group was 33 days.
  • GS-9973 failed to further increase Sorafenib or Regorafenib to prolong the survival of tumor-bearing mice (The median survival time of Sorafenib and Regorafenib monotherapy is 42 days and 47 days, respectively, and the median survival time of Sorafenib and Regorafenib combination therapy is 45 days and 46 days, respectively), * means P ⁇ 0.05.
  • the non-fibrotic liver mouse liver cancer orthotopic transplantation tumor model was treated for two weeks, and the mice were sacrificed the next day after the last treatment to evaluate the size of the primary liver transplantation tumor and the number of intrahepatic metastases (Figure 9): GS-9973
  • the inhibitory effect of Sorafenib and Regorafenib on liver cancer is not obvious, which further shows that the combined use of medicine has no significance for liver cancer in nonfibrotic liver mice.
  • A the primary tumor volume of transplanted tumors in model mice of each group
  • B the number of intrahepatic metastases of model mice in each group

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Abstract

本发明公开了一种治疗伴有纤维化肝癌的组合物，其活性成分包括至少一种SYK抑制剂和至少一种针对肝癌的多靶点激酶抑制剂。发明人通过实验发现，联合应用针对肝脏微环境的SYK抑制剂和针对肝癌的多靶点激酶抑制剂Sorafenib或Reforafenib，SYK抑制剂增强了Sorafenib或Reforafenib的抑癌作用，并进一步延长了模型小鼠的生存期，说明SYK抑制剂和针对肝癌的多靶点激酶抑制剂联合应用对伴有纤维化肝癌具有很好的治疗作用。

Description

治疗伴有纤维化肝癌的组合物 技术领域
本发明涉及一种治疗伴有纤维化肝癌的组合物。
背景技术
各种原因导致的肝炎肝纤维化与肝癌的发生密切相关。大部分的肝癌患者均伴有不同程度的肝纤维化。并且目前研究发现纤维化背景下的肝脏微环境促进了肝癌的增殖与侵袭，此外肝纤维化也减少了肝癌治疗方法的选择，降低了治疗效果。但目前并没有针对肝纤维化背景下肝癌的治疗策略。
目前，手术与介入治疗是肝癌的主要治疗手段，但相当一部分肝癌患者确诊时已进入肿瘤晚期阶段，失去治疗机会。此外由于肝癌易转移易复发的特性，手术与介入治疗的效果也并不是非常理想。对于肝癌而言，化疗、放疗与靶向治疗多作为辅助治疗手段，实际应用有限。其中目前仅有的肝癌靶向药物Sorafenib和Regorafenib，虽被批准应用于治疗无法手术或远处转移的肝细胞癌，但其对肝癌患者生存期的延长作用十分有限。并且其对伴有纤维化的肝癌并不具有很好的治疗作用。因此发展新的治疗策略和药物，治疗肝纤维化背景下的肝癌具有重要的意义。
脾酪氨酸激酶(spleen tyrosine kinase，SYK)基因是1991年首次从猪脾cDNA克隆出来，编码一种非受体型蛋白酪氨酸激酶。人类SYK基因定位于9号染色体q22区，SYK蛋白含635个氨基酸，为治疗炎症与血液恶性肿瘤的有效靶点，目前，SYK抑制剂目前已用于类风湿性关节炎、慢性淋巴细胞白血病等的临床II/III期实验，结果令人鼓舞，且药物安全性好。
在细胞学实验和动物体内实验中，已经开发了多种SYK小分子化合物抑制剂，包括Entospletinib(GS-9973)、Fostamatinib(R788)、R406和PRT062607(P505-15，BIIB057)HCl，这些SYK抑制剂的生物学效应趋势相似，以Entospletinib(GS-9973)抑制SYK激酶活性最为稳定、显著。目前市场上已有多种SYK小分子化合物抑制剂已应用于多种疾病的临床II/III期研究；其中GS-9973是最新报道用于慢性淋巴细胞白血病的临床II期实验，有效率高达91％，且药物SYK靶向特异性强、脱靶率低、生物安全性好。
CN 105664178A及Qu C，Zheng D，Li S，et al.Tyrosine Kinase SYK is a Potential Therapeutic Target for Liver Fibrosis.[J].Hepatology，2018.公开了SYK作为肝纤维化/硬化治疗靶点的应用，具体是发现SYK基因在肝纤维化/硬化过程中表达升高，通过促进肝星状细胞活化，加速肝纤维化的进程；使用SYK抑制剂或者干扰SYK基因的表达，可以有效地减缓肝纤维化/硬化的进程，具有很好地治疗作用。
胡青钢，刘小卫，郑启昌.酪氨酸激酶Syk在肝癌中的表达及与血管生成的关系[J].中华肝胆外科杂志，2007，13(7)：463-465.采用R27-PCR检测Syk mRNA在肝细胞性肝癌及癌旁正常组织中的表达，免疫组化SABC法检测标本中CD34的表达反映肿瘤的微血管密度(MVD).结果24例癌旁正常组织中Syk mRNA均表达阳性，32例肝细胞性肝癌中Syk mRNA表达率为46.9％(15/32)，其中低分化组阳性表达率23.1％(3/13)， 明显低于高分化组阳性表达率63.2％(12/19)(P＜0.05).肿瘤微血管密度(MVD)检测：低分化组(III级IV级为49.2±3.6，54.9±4.3)，明显高于高分化组(I级II级为13.6±4.5，32.3±3.2)与正常组织(5.9±1.7)，有显著统计学意义(P＜0.05).Syk mRNA的表达与CD34的表达明显负相关(r＝-0.97).结论肝细胞性肝癌中Syk基因的缺失对癌组织血管的生成起重要的作用。换言之，抑制SYK基因的表达会促进癌组织血管生成，进而促进肝癌的进展。
如何有效治疗伴有纤维化肝癌，对提高病人的生存质量，具有非常重要的意义。
发明内容
本发明的一个目的在于提供一种治疗伴有纤维化肝癌的组合物。
本发明的另一个目的在于提供组合物在制备治疗伴有纤维化肝癌药物中的应用。
本发明的另一个方法在于提供一种治疗伴有纤维化肝癌的方法。
本发明所采取的技术方案是：
一种治疗伴有纤维化肝癌的组合物，其活性成分包括至少一种SYK抑制剂和至少一种针对肝癌的多靶点激酶抑制剂。
作为上述组合物的进一步改进，针对肝癌的多靶点激酶抑制剂为Sorafenib和Reforafenib。
作为上述组合物的进一步改进，SYK抑制剂选自GS-9973、R788、R406、PRT062607。
组合物在制备治疗伴有纤维化肝癌药物中的应用，其中，组合物的活性成分包括至少一种SYK抑制剂和至少一种针对肝癌的多靶点激酶抑制剂。
作为上述应用的进一步改进，针对肝癌的多靶点激酶抑制剂为Sorafenib和Reforafenib。
作为上述应用的进一步改进，SYK抑制剂选自GS-9973、R788、R406、PRT062607。
一种治疗伴有纤维化肝癌的方法，包括给病人治疗量的至少一种SYK抑制剂和至少一种针对肝癌的多靶点激酶抑制剂。
作为上述方法的进一步改进，针对肝癌的多靶点激酶抑制剂为Sorafenib和Reforafenib。
作为上述方法的进一步改进，SYK抑制剂选自GS-9973、R788、R406、PRT062607。
本发明的有益效果是：
发明人通过实验发现，联合应用针对肝脏微环境的SYK抑制剂和针对肝癌的多靶点激酶抑制剂Sorafenib或Reforafenib，SYK抑制剂增强了Sorafenib或Reforafenib的抑癌作用，并进一步延长了模型小鼠的生存期，说明SYK抑制剂和针对肝癌的多靶点激酶抑制剂联合应用对伴有纤维化肝癌具有很好的治疗作用。
附图说明
图1是基于人肝星状细胞系LX-2和TWNT-4的ELISA实验结果；
图2是肝纤维化小鼠肝癌原位移植瘤模型治疗后生存期统计图；
图3是肝纤维化小鼠肝癌原位移植瘤模型中不同处理组的生存曲线图；
图4是对肝纤维化肝癌原位移植瘤小鼠进行为期两周的治疗后，肝脏移植瘤原发灶大小及肝内转移数情况；
图5是对肝纤维化肝癌原位移植瘤小鼠进行为期两周的治疗后，各组小鼠代表性肝脏原位移植瘤转移实物图；
图6是对肝纤维化肝癌原位移植瘤小鼠进行为期两周的治疗后，各组小鼠肝脏代表性HE染色与天狼星红染色；
图7是非纤维化肝小鼠肝癌原位移植瘤模型治疗后生存期统计结果；
图8是非纤维化肝小鼠肝癌原位移植瘤模型不同处理组的生存期统计图；
图9是对非纤维化肝小鼠肝癌原位移植瘤模型进行为期两周的治疗后，肝脏移植瘤原发灶大小及肝内转移数情况。
具体实施方式
一种治疗伴有纤维化肝癌的组合物，其活性成分包括至少一种SYK抑制剂和至少一种针对肝癌的多靶点激酶抑制剂。
作为上述组合物的进一步改进，针对肝癌的多靶点激酶抑制剂为Sorafenib和Reforafenib。
作为上述组合物的进一步改进，SYK抑制剂选自GS-9973、R788、R406、PRT062607。
组合物在制备治疗伴有纤维化肝癌药物中的应用，其中，组合物的活性成分包括至少一种SYK抑制剂和至少一种针对肝癌的多靶点激酶抑制剂。
作为上述应用的进一步改进，针对肝癌的多靶点激酶抑制剂为Sorafenib和Reforafenib。
作为上述应用的进一步改进，SYK抑制剂选自GS-9973、R788、R406、PRT062607。
一种治疗伴有纤维化肝癌的方法，包括给病人治疗量的至少一种SYK抑制剂和至少一种针对肝癌的多靶点激酶抑制剂。
作为上述方法的进一步改进，针对肝癌的多靶点激酶抑制剂为Sorafenib和Reforafenib。
作为上述方法的进一步改进，SYK抑制剂选自GS-9973、R788、R406、PRT062607。
下面结合实验，进一步说明本发明的技术方案。
不同SYK抑制剂对人肝星状细胞系的抑制情况
以特定浓度不同SYK抑制剂GS-9973(1μM)、R406(2μM)和PRT062607(2μM)分别处理人肝星状细胞系LX-2或TWNT-4细胞48小时后，通过ELISA实验检测细胞培养上清液中相关肝星状细胞标志物水评，评估肝星状细胞激活情况。
实验结果如图1所示，ELISA实验显示，包括GS-9973(图A)、R406(图B)和PRT062607(图C) 在内的多种SYK抑制剂能显著抑制肝星状细胞(肝脏微环境细胞)的活性。特定浓度的3种抑制剂分别处理相应细胞后，相应细胞肝星状细胞激活标志物的表达显著降低，且三种抑制剂抑制肝星状细胞活性的效果相近。
不同处理对肝纤维化小鼠肝癌原位移植瘤模型的治疗结果
选取4周龄，雄性C57BL/6小鼠，应用40％的CCl 4溶液进行灌胃，两周7次，共6周，在灌胃至4周时应用小鼠源性肝癌细胞进行肝癌原位移植，构建肝纤维化小鼠肝癌原位移植瘤模型，后将模型小鼠分为对照组、GS-9973单药组、Sorafenib单药组、Regorafenib单药组、GS-9973&Sorafenib联合治疗组和GS-9973&Regorafenib联合治疗组，在移植后第二天开始给予药物治疗，用药剂量分别为GS-9973 5mg/kg、Sorafenib 30mg/kg、Regorafenib 20mg/kg。治疗两周后取各组一半小鼠，麻醉并牺牲后开腹取出肝脏，评估原发灶体积与肝内转移数量，各组另一半小鼠继续治疗，观察治疗药物对生存期的影响。
实验结果如图2～6所示，
如图2所示，对照组中位生存期为27天，GS-9973和Regorafenib单药治疗都显著延长了小鼠的生存期中位生存期(分别为32天与33天)，而Sorafenib或Regorafenib联合GS-9973治疗后进一步延长了小鼠的生存期(分别为36天与39天)，GS＝GS-9973，Sora＝Sorafenib，Regora＝Regorafenib，*表示P＜0.05。
图3显示肝纤维化小鼠肝癌原位移植瘤模型中，对照组，GS-9973治疗组，Regorafenib治疗组和GS-9973联合Regorafenib治疗组的生存曲线图，GS-9973联合Regorafenib治疗组的中位生存期较Regorafenib治疗组显著增加。
对肝纤维化肝癌原位移植瘤小鼠进行为期两周的治疗，末次治疗后隔天牺牲小鼠，评估肝脏移植瘤原发灶大小及肝内转移数(图4)：GS-9973显著抑制了肝纤维化小鼠肝癌原位移植瘤的生长与转移，并增强了Sorafenib和Regorafenib对肝癌的抑制作用。A，各组模型小鼠移植瘤原发灶体积；B，各组模型小鼠肝内转移数量；C各组模型小鼠标准化肝内转移数量(标准化肝内转移数量＝肝内转移数量/移植瘤原发灶体积)。*表示P＜0.05，**表示P＜0.01。
对肝纤维化肝癌原位移植瘤小鼠进行为期两周的治疗，末次治疗后隔天牺牲小鼠，取肝拍照，各组小鼠代表性肝脏原位移植瘤转移实物图(图5)。结果显示：相对单独使用Sorafenib或Regorafenib，联合GS-9973治疗显著增强了Sorafenib和Regorafenib的抗肝癌效果，联合治疗后肿瘤大小和肝内转移灶数量情况均明显改善。
对肝纤维化肝癌原位移植瘤小鼠进行为期两周的治疗，末次治疗后隔天牺牲小鼠。A，对照组，GS-9973治疗组，Regorafenib治疗组和GS-9973联合Regorafenib治疗组小鼠荷瘤肝脏代表性HE染色与天狼星红染色(图6)；B，对照组，GS-9973治疗组，Regorafenib治疗组和GS-9973联合Regorafenib治疗组小鼠荷瘤肝脏Ishak评分，小鼠荷瘤肝脏Ishak评分提示GS-9973单药治疗及联合治疗组显著降低了小鼠荷瘤 肝脏纤维化，*表示P＜0.05，**表示P＜0.01。
非纤维化肝小鼠肝癌原位移植瘤模型治疗后生存情况
选取4周龄，雄性C57BL/6小鼠，应用小鼠源性肝癌细胞进行肝癌原位移植，构建小鼠肝癌原位移植瘤模型，后将模型小鼠分为对照组、GS-9973单药组、Sorafenib单药组、Regorafenib单药组、GS-9973&Sorafenib联合治疗组和GS-9973&Regorafenib联合治疗组，在移植后第二天开始给予药物治疗，用药剂量分别为GS-9973 5mg/kg、Sorafenib 30mg/kg、Regorafenib 20mg/kg。治疗两周后取各组一半小鼠，麻醉并牺牲后开腹取出肝脏，评估原发灶体积与肝内转移数量，各组另一半小鼠继续治疗，观察治疗药物对生存期的影响。
非纤维化肝小鼠肝癌原位移植瘤模型治疗后生存期统计(图7)显示，对照组中位生存期为33天GS-9973未能进一步增加Sorafenib或Regorafenib延长荷瘤小鼠的生存期(Sorafenib和Regorafenib单药治疗中位生存期分别为42天和47天，Sorafenib和Regorafenib联合治疗中位生存期分别为45天和46天)，*表示P＜0.05。
非纤维化肝小鼠肝癌原位移植瘤模型中，对照组，GS-9973治疗组，Regorafenib治疗组和GS-9973联合Regorafenib治疗组的生存曲线图如图8所示，显示GS-9973联合Regorafenib治疗组的中位生存期与Regorafenib治疗组无显著差别。说明联合用药对非纤维化肝小鼠肝癌没有意义。
对非纤维化肝小鼠肝癌原位移植瘤模型进行为期两周的治疗，末次治疗后隔天牺牲小鼠，评估肝脏移植瘤原发灶大小及肝内转移数(图9)：GS-9973对Sorafenib和Regorafenib关于肝癌的抑制作用影响不明显，进一步说明联合用药对非纤维化肝小鼠肝癌没有意义。A，各组模型小鼠移植瘤原发灶体积；B，各组模型小鼠肝内转移数量；C各组模型小鼠标准化肝内转移数量(标准化肝内转移数量＝肝内转移数量/移植瘤原发灶体积)。*表示P＜0.05，**表示P＜0.01。

Claims (9)

  1. 一种治疗伴有纤维化肝癌的组合物，其活性成分包括至少一种SYK抑制剂和至少一种针对肝癌的多靶点激酶抑制剂。
  2. 根据权利要求1所述的组合物，其特征在于：针对肝癌的多靶点激酶抑制剂为Sorafenib和Reforafenib。
  3. 根据权利要求1或2所述的组合物，其特征在于：SYK抑制剂选自GS-9973、R788、R406、PRT062607。
  4. 组合物在制备治疗伴有纤维化肝癌药物中的应用，其中，组合物的活性成分包括至少一种SYK抑制剂和至少一种针对肝癌的多靶点激酶抑制剂。
  5. 根据权利要求4所述的应用，其特征在于：针对肝癌的多靶点激酶抑制剂为Sorafenib和Reforafenib。
  6. 根据权利要求4或5所述的应用，其特征在于：SYK抑制剂选自GS-9973、R788、R406、PRT062607。
  7. 一种治疗伴有纤维化肝癌的方法，包括给病人治疗量的至少一种SYK抑制剂和至少一种针对肝癌的多靶点激酶抑制剂。
  8. 根据权利要求7所述的方法，其特征在于：针对肝癌的多靶点激酶抑制剂为Sorafenib和Reforafenib。
  9. 根据权利要求7或8所述的方法，其特征在于：SYK抑制剂选自GS-9973、R788、R406、PRT062607。
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WO2017223433A1 (en) * 2016-06-24 2017-12-28 Ohio State Innovation Foundation Methods and compositions for the treatment of cancer
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