WO2020087938A1 - 脾酪氨酸激酶作为肝内胆管细胞癌治疗靶点的应用 - Google Patents

脾酪氨酸激酶作为肝内胆管细胞癌治疗靶点的应用 Download PDF

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WO2020087938A1
WO2020087938A1 PCT/CN2019/091268 CN2019091268W WO2020087938A1 WO 2020087938 A1 WO2020087938 A1 WO 2020087938A1 CN 2019091268 W CN2019091268 W CN 2019091268W WO 2020087938 A1 WO2020087938 A1 WO 2020087938A1
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syk
intrahepatic cholangiocarcinoma
cholangiocarcinoma
expression
intrahepatic
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洪健
袁辉
林泽龙
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南方医科大学中西医结合医院
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/538—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with carbocyclic ring systems
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Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to the application of spleen tyrosine kinase as a therapeutic target for intrahepatic cholangiocarcinoma.
  • 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.
  • the SYK protein contains 635 amino acids. It is a class of specific protein kinases that can catalyze the phosphorylation of substrate protein tyrosine residues, including receptor-type tyrosine kinases and cytoplasm Type tyrosine kinase and nuclear tyrosine kinase.
  • Tyrosine protein kinase can regulate signal transduction within and between cells, plays an important role in cell differentiation, proliferation and spread, and is closely related to the occurrence and development of tumors. It plays an important role in autoimmune diseases and hematological malignancies. For example, high expression of SYK gene can inhibit the proliferation and migration of malignant tumor cells such as breast cancer, melanoma and liver cancer. Numerous studies have shown that the SYK gene has cancer suppressing effects. These studies include but are not limited to:
  • SYK (L) The expression of SYK (L) gene in hepatocellular carcinoma and its clinical significance [D]. Sun Yat-sen University, 2012. It is pointed out: 1. SYK (L) can inhibit the proliferation, migration and metastasis of liver cancer cells; 2. The expression of SYK (L) protein in liver cancer tissue is reduced, which is related to the clinical and pathological factors related to tumor differentiation, vascular invasion and other invasion and metastasis; 3. SYK (L) can be used as an effective molecular marker for evaluating invasion and metastasis of liver cancer.
  • the expression rate of Syk mRNA in cellular liver cancer was 46.9% (15/32), of which the positive expression rate in the poorly differentiated group was 23.1% (3/13), which was significantly lower than the positive expression rate in the highly differentiated group of 63.2% (12/19) (P ⁇ 0.05).
  • Syk gene in hepatocellular carcinoma has a significant impact on cancer.
  • the formation of tissue blood vessels plays an important role.
  • inhibiting the expression of the SYK gene will promote angiogenesis in cancer tissues, and then promote the progress of liver cancer.
  • SYK inhibitors have been used in clinical phase II / III trials of rheumatoid arthritis, chronic lymphocytic leukemia, etc. The results are 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.
  • Intrahepatic cholangiocarcinoma is a malignant tumor that originates from bile duct epithelial cells. It has the differentiation characteristics of cholangiocarcinoma cells. It can be divided into intrahepatic cholangiocarcinoma (ICC), hilar cholangiocarcinoma, and extrahepatic bile duct according to different anatomical locations. Cellular carcinoma (ECC). Over the past four decades, the overall incidence of intrahepatic cholangiocarcinoma has gradually increased globally.
  • ICC intrahepatic cholangiocarcinoma
  • ECC Cellular carcinoma
  • cholangiocarcinoma The incidence of intrahepatic cholangiocarcinoma accounts for 10% to 15% of the primary malignant tumors of the liver, and the morbidity and mortality have increased year by year in recent years.
  • the cholangiocarcinoma has an insidious onset and no obvious symptoms and signs in the early stage. It develops rapidly in the later stage, and it is easy to invade and metastasize to the surrounding tissues. Due to the complex anatomical relationship around the bile duct, early resection of tumors after surgical resection is prone to local recurrence and distant metastasis, resulting in a poor prognosis of intrahepatic cholangiocarcinoma.
  • cholangiocarcinoma lacks suitable early diagnosis methods and effective treatment methods. The prognosis of patients is poor, and most patients die within a few months after a clear diagnosis.
  • intrahepatic cholangiocarcinoma mainly includes surgical resection, local treatment (hepatic artery perfusion chemotherapy, radiofrequency ablation, microwave ablation, etc.), chemotherapy and radiotherapy.
  • surgery alone cannot effectively treat most patients (the pathogenesis is unknown, the lack of early diagnostic markers, and the opportunity for surgery has been lost when found), and the postoperative survival rate is low (5-year survival rate is 8% to 47%), recurrence High rate (postoperative intrahepatic recurrence rate of 46% to 68%); non-surgical patients lack effective treatment, local treatment for single lesions ⁇ 3cm, and residual or local recurrence after surgery may benefit (overall efficacy Worrying);
  • the chemotherapy regimen of gemcitabine combined with platinum antitumor drugs can prolong the survival of patients with cholangiocarcinoma, but is limited by the toxicity and drug resistance of the drug; radiotherapy alone
  • the curative effect is
  • FOXM1 is highly expressed in cholangiocarcinoma and is related to prognosis.
  • FOXM1 inhibitors inhibit the proliferation of cholangiocarcinoma by increasing apoptosis.
  • FOXM1 knockdown can increase the sensitivity of cholangiocarcinoma to cisplatin, and FOXM1 inhibitors combined with cisplatin Inhibit the proliferation of cholangiocarcinoma. It can be seen that FOXM1 plays an important role in the occurrence and development of cholangiocarcinoma, and provides new ideas for the diagnosis and treatment of cholangiocarcinoma.
  • the purpose of the present invention is to overcome the deficiencies of the prior art and provide splenic tyrosine kinase as a therapeutic target for intrahepatic cholangiocarcinoma.
  • SYK gene or SYK protein as a therapeutic target for intrahepatic cholangiocarcinoma.
  • the SYK antagonist is selected from GS-9973, Fostamatinib, R406, PRT062607.
  • the SYK expression inhibitor is selected from SYK-siRNA, miRNA targeting SYK or antisense nucleotides and other nucleic acids that down-regulate SYK expression.
  • intrahepatic cholangiocarcinoma is intrahepatic cholangiocarcinoma with significantly higher expression of SYK than normal intrahepatic cholangiocarcinoma cells.
  • Figure 1 is the expression of SYK in human normal intrahepatic bile duct cells and intrahepatic bile duct cell carcinoma;
  • Figure 2 is the inhibition results of SYK antagonist GS-9973 on cholangiocarcinoma cells (HuCCT-1, RBE);
  • Fig. 3 shows the therapeutic effect of SYK antagonist GS-9973 on a model of subcutaneously transplanted tumor of bile duct cancer cells in nude mice.
  • SYK gene or SYK protein as a therapeutic target for intrahepatic cholangiocarcinoma.
  • the SYK antagonist is selected from GS-9973, Fostamatinib, R406, PRT062607.
  • the SYK expression inhibitor is selected from SYK-siRNA, miRNA targeting SYK or antisense nucleotides and other nucleic acids that down-regulate SYK expression.
  • intrahepatic cholangiocarcinoma is intrahepatic cholangiocarcinoma with significantly higher expression of SYK than normal intrahepatic cholangiocarcinoma cells.
  • the change in SYK expression level can be determined using methods commonly used in the art, such as evaluating the change in expression level by comparing immunohistochemical staining scores. By comparing the changes in its expression level, it is determined whether its expression level is significantly high.
  • Immunohistochemical staining was used to detect human normal intrahepatic cholangiocarcinoma and intrahepatic cholangiocarcinoma, and compare the expression of SYK.
  • HuCCT-1 and RBE Two cholangiocarcinoma cell lines, HuCCT-1 and RBE, were selected, treated with GS-9973 for 24h, 48h and 72h respectively, and CCK8 reagent was used to detect the effect of GS-9973 on the proliferation of cholangiocarcinoma cells at different time points.
  • HuCCT-1 and RBE cells were made into a single cell suspension, and 100 ⁇ l of about 3000 cells were added to each well in a 96-well plate. After the cells adhered (approximately 6h), the medium containing GS-9973 (concentration: 2 ⁇ M) was replaced and cultured for 24h, 48h, and 72h, respectively. Add 10 ⁇ l of CCK8 reagent to each well in a 96-well plate, and continue incubating in a 37 ° C incubator for 2h and 30min. Select 450nm to measure absorbance.
  • HuCCT-1 cells in logarithmic growth phase after trypsin digestion and other treatments, resuspend the cells in PBS to prepare a single cell suspension, and adjust the cell concentration to 1 * 10 ⁇ 7 / ml.
  • 200 ⁇ l of the cell mixture was sucked with a 1 ml syringe and injected subcutaneously into the right armpit of nude mice.
  • SYK antagonist GS-9973 is administered by intragastric administration, 5 mg / kg, once daily, since the 9th day At the beginning, dosing continued until the 30th day.

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Abstract

提供了脾酪氨酸激酶（SYK）作为肝内胆管细胞癌治疗靶点的应用。该SYK在肝内胆管细胞癌中表达升高；在体外细胞和体内动物实验中均发现，使用SYK拮抗剂GS-9973可以有效抑制肝内胆管细胞癌的增殖。

Description

脾酪氨酸激酶作为肝内胆管细胞癌治疗靶点的应用 技术领域
本发明属于生物医药领域，具体涉及脾酪氨酸激酶作为肝内胆管细胞癌治疗靶点的应用。
背景技术
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，是一类能催化底物蛋白酪氨酸残基磷酸化的特异性蛋白激酶，包括受体型酪氨酸激酶、胞质型酪氨酸激酶和核内酪氨酸激酶。酪氨酸蛋白激酶可调节细胞内部及之间的信号转导，在细胞分化、增殖和扩散中起重要作用，且与肿瘤的发生发展存在密切关系。在自身免疫性疾病和血液恶性肿瘤中作用重要，如SYK基因高表达可抑制乳腺癌、黑色素瘤和肝癌等恶性肿瘤细胞的增殖与迁移。众多研究表明SYK基因具有抑癌作用，这些研究包括但不限于：
郭嘉仲.脾酪氨酸激酶、血管内皮生长因子-C在非小细胞肺癌淋巴转移中作用的研究[D].山东大学，2013.
寇昌伟.鼻咽癌组织脾酪氨酸激酶表达与其启动子甲基化关系和临床意义[D].2009.
杨星.脾酪氨酸激酶在结直肠癌中的表达及其对浸润转移的影响[D].2006.
张倩.脾酪氨酸激酶Syk在食管癌中的表达及对其生物学特征影响的研究[D].河北医科大学，2011.
董尚文.DNA启动子甲基化在脾酪氨酸激酶Syk肺癌表达中的影响[D].天津医科大学，2009.
吉海龙.脾源性酪氨酸激酶基因启动子甲基化与髓母细胞瘤侵袭转移的关系[D].2016.等。
进一步的，杨龙君.肝细胞癌中SYK(L)基因的表达及其临床意义[D].中山大学，2012.指出：1、SYK(L)可抑制肝癌细胞的增殖、迁移与转移；2、肝癌组织中SYK(L)蛋白表达降低，与肿瘤的分化、血管侵犯等侵袭转移有关的临床病理因素相关；3、SYK(L)可作为评价肝癌侵袭转移的有效分子标志物。
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。
CN105664178A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。
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。
肝内胆管细胞癌是一种起源于胆管上皮细胞的恶性肿瘤，具有胆管细胞的分化特性，可以根据解剖位置不同，分为肝内胆管细胞癌(ICC)，肝门部胆管癌以及肝外胆管细胞癌(ECC)。在过去的四十年里，肝内胆管细胞癌的整体发病率在全球范围内逐步增加。
肝内胆管细胞癌的发病率占肝脏原发恶性肿瘤的10％～15％，并且近年来的发病率和死亡率逐年升高。胆管细胞癌起病隐匿，早期无明显症状和体征，后期发展迅速，易向周围组织侵犯和转移。由于胆管周围解剖关系复杂，外科切除后早期肿瘤易出现局部复发和远处转移发，造成肝内胆管细胞癌预后较差。目前，胆管癌缺乏合适的早期诊断方法和有效的治疗手段，患者预后不良，大部分患者在明确诊断后的几个月内死亡。
目前临床上肝内胆管细胞癌主要有外科手术切除、局部治疗(肝动脉灌注化疗、射频消融、微波消融等)、化疗和放疗等治疗方法。但是，单纯外科手术不能有效治疗大多数患者(发病机制不明，缺少早期诊断标志物，发现时已失去手术机会)，且术后生存率低(5年生存率 为8％～47％)、复发率高(术后肝内复发率达46％～68％)；而非手术患者缺少有效的治疗手段，局部治疗对＜3cm的单个病灶，以及手术后残留或局部复发者可能获益(整体疗效堪忧)；对不能手术切除或伴有转移的进展期胆管癌，吉西他滨联合铂类抗肿瘤药的化疗方案能延长胆管癌患者生存期，但受限于药物的毒性和耐药性；单纯放疗的疗效非常有限，常常作为一种辅助治疗方式，尚无证据表明术中放疗及导管内短距放疗对进展期胆管癌的疗效优于标准化疗、放化疗联合。到目前为止，胆管癌尚无标准的靶向药物，仅处于探索和临床试验阶段，尚无确定疗效的结论。
CN108273062A公开了FOXMl在胆管细胞癌中高表达并和预后相关，FOXMl抑制剂通过增加凋亡抑制胆管细胞癌增殖，FOXMl敲低可以增加胆管细胞癌对于顺铂的敏感性，且FOXMl抑制剂联合顺铂抑制胆管细胞癌增殖。由此可知，FOXMl在胆管细胞癌的发生和发展过程中扮演着重要的角色，为胆管细胞癌的诊断与治疗提供了新思路。
开发出一种可以有效治疗或改善肝内胆管细胞癌的药物对肝内胆管细胞癌患者具有非常重要的意义。
发明内容
本发明的目的在于克服现有技术的不足，提供脾酪氨酸激酶作为肝内胆管细胞癌治疗靶点的应用。
本发明所采取的技术方案是：
SYK基因或SYK蛋白作为肝内胆管细胞癌治疗靶点的应用。
SYK拮抗剂在制备治疗肝内胆管细胞癌药物中的应用。
进一步的，SYK拮抗剂选自GS-9973、Fostamatinib、R406、PRT062607。
SYK表达抑制剂在在制备治疗肝内胆管细胞癌药物中的应用。
进一步的，SYK表达抑制剂选自SYK-siRNA、以SYK为靶标的miRNA或反义核苷酸等调降SYK表达的核酸。
进一步的，肝内胆管细胞癌为SYK表达量显著高于正常肝内胆管细胞的肝内胆管细胞癌。
本发明的有益效果是：
发明人通过实验发现，SYK在肝内胆管细胞癌中表达升高；在体外细胞和体内动物实验中均发现，使用SYK拮抗剂GS-9973可以有效抑制肝内胆管细胞癌的增殖，抑制效果接近于一线化疗药物吉西他滨。
附图说明
图1是人正常肝内胆管细胞和肝内胆管细胞癌的SYK表达情况；
图2是SYK拮抗剂GS-9973对胆管癌细胞(HuCCT-1、RBE)的抑制结果；
图3是SYK拮抗剂GS-9973对胆管癌细胞裸鼠皮下移植瘤模型的治疗效果。
具体实施方式
SYK基因或SYK蛋白作为肝内胆管细胞癌治疗靶点的应用。
SYK拮抗剂在制备治疗肝内胆管细胞癌药物中的应用。
进一步的，SYK拮抗剂选自GS-9973、Fostamatinib、R406、PRT062607。
SYK表达抑制剂在在制备治疗肝内胆管细胞癌药物中的应用。
进一步的，SYK表达抑制剂选自SYK-siRNA、以SYK为靶标的miRNA或反义核苷酸等调降SYK表达的核酸。
进一步的，肝内胆管细胞癌为SYK表达量显著高于正常肝内胆管细胞的肝内胆管细胞癌。SYK表达量变化，可以使用本领域常用的方法确定，如通过比较免疫组化染色评分的方法评价表达量变化。通过比较其表达量的变化，确定其表达量是否显著高。
下面结合实验，进一步说明本发明的技术方案。
SYK在胆管癌组织中的表达情况
采用免疫组织化学染色法检测人正常肝内胆管细胞和肝内胆管细胞癌，比较两者SYK的表达情况。
实验如果如图1所示。从图1中可知，对目前收集的5例正常胆管组织与43例肝内胆管细胞癌组织的SYK免疫组化染色后进行评分分析，所有肝内胆管细胞癌组织中SYK均着色，且与肝内正常胆管细胞相比，SYK表达显著升高，平均免疫组化强度评分增高超过1分(＊＊＊，＜0.001)。
SYK对胆管癌细胞增殖的影响
选用HuCCT-1和RBE两种胆管癌细胞株，分别使用GS-9973处理24h、48h和72h，并使用CCK8试剂检测不同时间点GS-9973对胆管癌细胞增殖的影响。将HuCCT-1和RBE细胞制成单细胞悬液，在96孔板中每孔加入100μl约3000个细胞。待细胞贴壁后(约6h)更换含有GS-9973(浓度为2μM)的培养基，分别培养24h、48h、72h。在96孔板中每孔加入10μl的CCK8试剂，继续在37℃孵箱中培养2h 30min。选择450nm测定吸光度。
实验如果如图2所示。从图2中可知，GS-9973(2μM)可以显著抑制胆管癌细胞HuCCT-1和RBE的增殖。
使用SYK特异性siRNA敲低HuCCT-1和RBE两种胆管癌细胞中的SYK表达，并检测敲低SYK表达对这两种细胞增殖的影响，结果发现敲低SYK能明显抑制这两种胆管细胞的 增殖，其结果与使用SYK抑制剂的效果类似。
裸鼠皮下移植瘤的靶向SYK治疗
对数生长期的HuCCT-1细胞，经过胰酶消化等处理后用PBS重悬细胞，制备成单细胞悬液，调整细胞浓度为1*10^7/ml。Balb/c裸鼠常规消毒后，以1ml注射器吸取200μl细胞混合液，于裸鼠右侧腋窝皮下注射。每3天观察裸鼠，测量并记录瘤体的长短径和体重，绘制生长曲线；SYK拮抗剂GS-9973采用灌胃方式给药治疗，5mg/kg，每日给药一次，自第9天开始，连续给药至第30天。
实验结果如图3所示。从图3中可知，GS-9973可以显著降低肿瘤的生长速度和肿瘤大小，抑制效果略逊于一线化疗药物吉西他滨(在治疗前期抑制效果强于吉西他滨)。

Claims (6)

  1. SYK基因或SYK蛋白作为肝内胆管细胞癌治疗靶点的应用。
  2. SYK拮抗剂在制备治疗肝内胆管细胞癌药物中的应用。
  3. 根据权利要求2所述的应用，其特征在于：SYK拮抗剂选自GS-9973、Fostamatinib、R406、PRT062607。
  4. SYK表达抑制剂在在制备治疗肝内胆管细胞癌药物中的应用。
  5. 根据权利要求4所述的应用，其特征在于：SYK表达抑制剂选自SYK-siRNA、以SYK为靶标的miRNA或反义核苷酸等调降SYK表达的核酸。
  6. 根据权利要求1～5任一项所述的应用，其特征在于：肝内胆管细胞癌为SYK表达量显著高于正常肝内胆管细胞的肝内胆管细胞癌。
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