WO2017050052A1 - Syk作为肝纤维化/肝硬化治疗靶点的应用 - Google Patents

Syk作为肝纤维化/肝硬化治疗靶点的应用 Download PDF

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WO2017050052A1
WO2017050052A1 PCT/CN2016/094618 CN2016094618W WO2017050052A1 WO 2017050052 A1 WO2017050052 A1 WO 2017050052A1 CN 2016094618 W CN2016094618 W CN 2016094618W WO 2017050052 A1 WO2017050052 A1 WO 2017050052A1
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syk
cirrhosis
hepatic
tyrosine kinase
liver fibrosis
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洪健
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洪健
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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
    • A61K31/535—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
    • A61K31/5383—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with heterocyclic ring systems
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • 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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/535—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
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/005—Enzyme inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

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  • the present invention relates to a therapeutic target for liver fibrosis/cirrhosis.
  • Liver fibrosis/cirrhosis is a major global health problem. Hepatic fibrosis is a necessary stage for the development of various chronic liver diseases to cirrhosis. When the liver is chronically damaged, the secretion and degradation of extracellular matrix of the liver are unbalanced, so that excessive or abnormal collagen fibers are deposited in the gap of the liver cells. Liver fibrosis is a pathological change in the early stage of cirrhosis. The structure of the liver changes, the supply of hepatocytes is affected, and the liver function is gradually lost. If liver fibrosis is not treated in time, 25%-40% of them eventually develop into cirrhosis or even liver cancer, causing liver failure, directly or indirectly causing death.
  • Liver fibrosis is a compensatory repair response, which is a chronic, gradual process.
  • the majority of chronic liver diseases caused by various causes are associated with liver fibrosis.
  • alcoholic liver damage is the main cause of liver fibrosis, while in Southeast Asia and China, the main infection is caused by hepatitis virus, especially hepatitis B virus.
  • 70%-90% of hepatitis B virus (HBV)-related liver cancer in China is associated with liver fibrosis/cirrhosis, and most of them have experienced the process of "hepatitis--hepatic fibrosis/cirrhosis-- liver cancer".
  • HBV hepatitis B virus
  • Hepatic stellate cells are the key cells for their occurrence and development. The activation of HSCs is the central link of liver fibrosis. Hepatic stellate cells are the main stromal cells in the liver microenvironment, accounting for about 10% of the liver's innate cells.
  • liver fibrosis there is no effective treatment for liver fibrosis; clinically classic liver and anti-fibrosis treatments have not brought about the desired therapeutic effect.
  • clinical and experimental research on the mechanism of liver fibrosis reversal has emerged, but no effective treatment has been found. Therefore, finding a treatment to reverse the progression of liver fibrosis is of great clinical significance for effectively preventing the formation of liver cirrhosis, and even the occurrence of liver cancer, and improving the quality of life of patients.
  • Tyrosine kinases are a broad class of kinases found in vertebrates and can be divided into three classes: receptor tyrosine kinases, cytoplasmic tyrosine kinases, and nuclear tyrosine kinases. There are a large number of tyrosine kinases, of which more than 50 receptor tyrosine kinases have been found in vertebrates, while cytoplasmic tyrosine kinases and nuclear tyrosine kinases include the Src family, the Tec family, the ZAP70 family, and the JAK family. , Abl and other gene superfamilies.
  • Tyrosine kinase-mediated phosphorylation of protein tyrosine residues is an important process regulating cell signaling pathways; the development of various human diseases is associated with abnormal expression or activity of tyrosine kinases, and its targeted targeting has been developed. Drugs are a hot trend in medical research in recent years.
  • Bcr-Abl inhibitor imatinib (Gleevec) is used to treat chronic myeloid leukemia and gastrointestinal stromal tumors, EGFR inhibitors gefitinib (Iressa), erlotinib (Troquet) EGFR monoclonal antibody cetuximab (Erbitux) and other malignant tumors such as breast cancer, non-small cell lung cancer and colorectal cancer.
  • the spleen tyrosine kinase (SYK) gene was first cloned from pig 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 plays an important role in autoimmune diseases and hematological malignancies. For example, SYK gene can inhibit the proliferation of malignant tumor cells such as breast cancer, melanoma and liver cancer. migrate. At present, SYK inhibitors have been used in clinical phase II/III trials of rheumatoid arthritis and chronic lymphocytic leukemia, and the results are encouraging and the drugs are safe.
  • SYK small molecule compound inhibitors In cytology experiments and in vivo experiments in animals, various SYK small molecule compound inhibitors have been developed, including Entospletinib (GS-9973), Fostamatinib (R788), R406 and PRT062607 (P505-15, BIIB057) HCl, which inhibit SYK.
  • the biological effects of the agents were similar, and the inhibition of SYK kinase activity by Entospletinib (GS-9973) was the most stable and significant.
  • a variety of SYK small molecule compound inhibitors have been applied to clinical phase II/III studies of various diseases.
  • GS-9973 is the latest clinical phase II trial for chronic lymphocytic leukemia with an efficiency of 91. %, and the drug SYK has strong specificity, low off-target rate and good biosafety.
  • the inventors found through experiments that the expression of SYK gene is elevated during liver fibrosis/cirrhosis, and accelerates the progression of hepatic fibrosis by promoting hepatic stellate cell activation; using SYK inhibitor or interfering with the expression of SYK gene can effectively It slows the progression of liver fibrosis/cirrhosis and has a good therapeutic effect.
  • Hepatitis C virus infection of hepatocytes can up-regulate the expression of SYK mRNA and protein levels in hepatocytes (A&B); compared with normal liver fibrosis-free liver tissue, hepatitis B virus (HBV) infection SYK protein expression was significantly increased in liver fibrosis tissue (C);
  • FIG. 2 In hepatic stellate cells LX-2 and TWNT-4, interference with SYK gene expression significantly reduced hepatic stellate cell activation markers ( ⁇ -SMA and PDGFR ⁇ ) and liver fibrosis-related markers (A&B); Overexpression of SYK gene promotes activation of hepatic stellate cells (C);
  • Figure 3 Effect of SYK inhibitor GS-9973 (1 ⁇ M) on mRNA levels of its activation markers ⁇ -SMA and PDGFR ⁇ after treatment of two human primary hepatic stellate cells for 48 hours, and at different time points Inhibition of cell proliferation (B) (*, P ⁇ 0.05; **, P ⁇ 0.01);
  • FIG. 4 SYK small molecule compound GS-9973 significantly inhibits hepatic collagen production (Sirius Red staining) and activation index ⁇ of hepatic stellate cells in a mouse model of liver fibrosis induced by carbon tetrachloride (CCl4). Expression of SMA protein (**, P ⁇ 0.01; ***, P ⁇ 0.001);
  • FIG. 5 SYK-targeted therapy inhibits hepatic collagen production, hepatic stellate cell activation, and inhibition of liver fibrosis in rats induced by diethylnitrosamine (DEN) in liver fibrosis/liver cancer
  • DEN diethylnitrosamine
  • hepatitis C virus can up-regulate the expression of SYK mRNA and protein levels in hepatocytes (A&B); compared with normal liver fibrosis-free liver tissue, hepatitis B virus (HBV) SYK protein expression was significantly increased in infected liver fibrosis tissue (C).
  • A&B hepatitis B virus
  • liver stellate cell lines LX-2 and TWNT-4, were used to knock down cells with SYK gene for 48 hours.
  • qRT-PCR and/or Western blot were used to detect hepatic stellate cell activation index ( ⁇ - SMA and PDGFR ⁇ ), and liver fibrosis related indicators COL1A1, TIMP-1, PAI-1 and TGF- ⁇ 1 (Fig. 2).
  • GS-9973 Two human primary hepatic stellate cells were treated with SYK small molecule compound inhibitor GS-9973 (1 ⁇ M) for 24, 48 and 72 hours. Promega's CellTiter-Glo Luminescent Cell Viability Assay kit was used to detect GS-9973 cells at different time points. Effect of proliferation; After 48 hours of treatment of the above cells, mRNA levels of hepatic stellate cell activation markers ( ⁇ -SMA and PDGFR ⁇ ) were detected by qRT-PCR (Fig. 3).
  • SYK inhibitor GS-9973 (1 ⁇ M) can significantly reduce the mRNA levels of its activation markers ⁇ -SMA and PDGFR ⁇ (A) after treatment of two human primary hepatic stellate cells for 48 hours. Prolonged treatment time significantly inhibited cell proliferation (B) (*, P ⁇ 0.05; **, P ⁇ 0.01).
  • Rat liver fibrosis/hepatoma model male Wistar rats (body weight 100-120g), 8 rats in each group, intraperitoneal injection of diethylnitrosamine (DEN), 50mg/kg once a week; control group abdominal cavity An equal volume of PBS was injected for 18 weeks.
  • the SYK inhibitor GS-9973 was administered by intragastric administration, 2.5 mg/kg once daily, starting from the 13th week and continuously administered to the 17th week for 5 weeks.
  • Mouse liver fibrosis model male C57BL/6 mice (8 weeks old), 8 rats in each group, each intraperitoneal injection of 10% CCl4 0.2ml (diluted with olive oil) twice a week; control group intraperitoneal injection An equal volume of olive oil for 18 weeks.
  • the SYK inhibitor GS-9973 was administered by intragastric administration, 5 mg/kg once daily, starting from the 13th week and continuously administered to the 18th week for 6 weeks.
  • FIG. 4 SYK small molecule compound GS-9973 significantly inhibits hepatic collagen production (Sirius Red staining) and activation index of hepatic stellate cells in a mouse model of liver fibrosis induced by carbon tetrachloride (CCl 4 ).
  • -SMA protein expression **, P ⁇ 0.01; ***, P ⁇ 0.001);
  • FIG. 5 SYK-targeted therapy inhibits hepatic collagen production, hepatic stellate cell activation, and inhibition of liver fibrosis in rats induced by diethylnitrosamine (DEN) in liver fibrosis/liver cancer
  • DEN diethylnitrosamine
  • nucleic acid interference technology can reduce the expression level of SYK gene or inhibit the expression of SYK gene, or make the transcribed DNA untransformable, and has therapeutic effects on liver fibrosis or cirrhosis.
  • SYK inhibitors also have therapeutic effects on liver fibrosis or cirrhosis.
  • SYK can be used as a therapeutic target for liver fibrosis or cirrhosis.

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Abstract

SYK作为肝纤维化/肝硬化治疗靶点的应用。SYK基因在肝纤维化/肝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/肝硬化的进程，具有很好地治疗作用。

Description

SYK作为肝纤维化/肝硬化治疗靶点的应用 技术领域
本发明涉及一种肝纤维化/肝硬化的治疗靶点。
背景技术
肝纤维化/肝硬化是一个全球重大的健康问题。肝纤维化是多种慢性肝脏疾病发展至肝硬化的必经阶段，是肝脏受到慢性损伤时，肝脏细胞外基质分泌与降解失衡，从而在肝细胞的间隙过多或异常的胶原纤维沉积。肝纤维化是肝硬化前期的一种病理变化，肝脏结构发生改变，肝细胞供应受到影响，使肝脏功能逐渐丧失。肝纤维化如果不及时治疗，其中25％-40％最终发展为肝硬化甚至肝癌，引起肝功能衰竭，直接或间接导致患者死亡。
肝纤维化是一种代偿修复反应，呈慢性、渐进性过程，各种病因所引起的慢性肝病绝大多数伴有肝纤维化。在西方国家，酒精性肝损害是造成肝纤维化的主要原因，而在东南亚和我国则以肝炎病毒尤其是乙型肝炎病毒的持续感染引起为主。中国70％-90％的乙型病毒肝炎(HBV)相关肝癌都伴发肝纤维化/肝硬化，且多数经历了“肝炎--肝纤维化/肝硬化--肝癌”进程。
肝硬化状态一旦形成，则无法逆转；然而其病理形成过程中必须经历肝纤维化阶段，肝纤维化的可逆转性已得到国内外众多学者的证实和认可。肝纤维化的形成机制较为复杂，其中肝星状细胞(hepatic stellate cells，HSCs)是其发生和发展的关键细胞，HSCs活化是肝纤维化发生的中心环节。肝星状细胞是肝脏微环境中的主要基质细胞，约占肝脏固有细胞10％。在去除损伤性因素(如抗肝炎病毒治疗、戒酒等)的前提下，下调肝星状细胞的激活或促进其凋亡，成为探索有效治疗肝纤维化的重要研究方向。
目前，肝纤维化尚无有效的治疗措施；临床上经典的护肝、抗肝纤维化治疗亦未能带来期望中的治疗效果。近年来，针对肝纤维化逆转机制的临床和实验研究不断涌现，但尚未找到切实有效的治疗方法。因此，寻找一种逆转肝纤维化进程的治疗手段，对于有效防止肝硬化形成、乃至肝癌的发生，提高患者生存质量具有重要临床意义。
酪氨酸激酶是脊椎动物中广泛存在的一类激酶，可分为三类：受体酪氨酸激酶、胞质酪氨酸激酶和核内酪氨酸激酶。酪氨酸激酶数量繁多，其中受体酪氨酸激酶在脊椎动物中已发现50余种，而胞质酪氨酸激酶与核内酪氨酸激酶包括Src家族、Tec家族、ZAP70家族、JAK家族、Abl等多个基因超家族。酪氨酸激酶介导的蛋白酪氨酸残基磷酸化是调控细胞信号通路的重要过程；人类多种疾病的发生发展与酪氨酸激酶的表达或活性异常相关，开发其针对性的靶向药物是近年来医学研究的一个热门方向。大量酪氨酸激酶抑制剂已应用于临床，如 Bcr-Abl抑制剂伊马替尼(格列卫)用于治疗慢性粒细胞白血病和胃肠间质瘤，EGFR抑制剂吉非替尼(易瑞沙)、厄洛替尼(特罗凯)、EGFR单抗西妥昔单抗(爱必妥)等治疗乳腺癌、非小细胞肺癌和结直肠癌等恶性肿瘤。
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，在自身免疫性疾病和血液恶性肿瘤中作用重要，如SYK基因可抑制乳腺癌、黑色素瘤和肝癌等恶性肿瘤细胞的增殖与迁移。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。
截止目前，尚未有实验研究结果表明SYK与肝纤维化/肝硬化相关，更未有相关实验数据证实SYK可以作为肝纤维化/肝硬化的治疗靶点。
发明内容
本发明的目的在于提供SYK作为肝纤维化/肝硬化的治疗靶点的应用。
发明人通过实验发现，SYK基因在肝纤维化/肝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/肝硬化的进程，具有很好地治疗作用。
附图说明
图1：丙型肝炎病毒感染肝细胞后，可上调肝细胞中SYK的mRNA和蛋白水平的表达(A&B)；与正常无肝纤维化的肝组织相比较，乙型肝炎病毒(HBV)感染的肝纤维化组织中SYK蛋白表达显著增高(C)；
图2：在肝星状细胞LX-2和TWNT-4中，干扰SYK基因表达可显著降低肝星状细胞激活指标(α-SMA和PDGFRβ)、以及肝纤维化相关指标(A&B)；相反，SYK基因过表达可促进肝星状细胞的活化(C)；
图3：SYK抑制剂GS-9973(1μM)处理两株人原代肝星状细胞48小时后，对其激活标志物α-SMA和PDGFRβ的mRNA水平的影响(A)，以及在不同时间点时对细胞增殖(B)的抑制作用(*，P＜0.05；**，P＜0.01)；
图4：在四氯化碳(CCl4)诱导的小鼠肝纤维化模型中，SYK小分子化合物GS-9973可显著抑制肝脏胶原生成(Sirius Red染色)，以及肝星状细胞的激活指标α-SMA蛋白的表达(**，P＜0.01；***，P＜0.001)；
图5：在二乙基亚硝胺(DEN)诱导的大鼠肝纤维化/肝癌模型中，SYK靶向治疗可抑制大鼠肝胶原生成、肝星状细胞激活状态，并抑制肝纤维化基础上肝癌的形成(##比较直径小于8mm的新生肝癌，P＜0.01)。
具体实施方式
下面结合实验，进一步说明本发明的技术方案。
HBV或HCV对肝细胞或肝组织中SYK表达的影响
选用易感染HCV病毒的肝细胞Huh7.5.1，感染HCV病毒复制子JFH1，采用实时定量PCR(qRT-PCR)及Western免疫印迹方法(Western blot)，分别检测HCV对细胞中SYK mRNA及蛋白水平的影响。采用免疫组织化学(IHC)方法检测人HBV相关肝纤维化组织、正常无HBV/HCV感染的正常无肝纤维化组织(来源于人肝血管瘤的瘤旁肝组织)，比较肝纤维化肝组织与正常肝组织中SYK蛋白的表达(图1)。
从图1中可知，丙型肝炎病毒感染肝细胞后，可上调肝细胞中SYK的mRNA和蛋白水平的表达(A&B)；与正常无肝纤维化的肝组织相比较，乙型肝炎病毒(HBV)感染的肝纤维化组织中SYK蛋白表达显著增高(C)。
SYK对肝星状细胞的活化及细胞增殖的影响
选用LX-2和TWNT-4两株人肝星状细胞系，采用SYK基因敲低或过表达处理细胞48小时，qRT-PCR和(或)Western blot方法检测肝星状细胞激活指标(α-SMA和PDGFRβ)、以及肝纤维化相关指标COL1A1、TIMP-1、PAI-1和TGF-β1(图2)。
从图2中可知，在肝星状细胞LX-2和TWNT-4中，干扰SYK基因表达可显著降低肝星状细胞激活指标(α-SMA和PDGFRβ)、以及肝纤维化相关指标(A&B)；相反，SYK基因过表达可促进肝星状细胞的活化(C)。
SYK小分子化合物抑制剂GS-9973(1μM)处理两株人原代肝星状细胞24、48和72小时，采用Promega公司的CellTiter-Glo Luminescent Cell ViabilityAssay试剂盒检测不同时间点GS-9973对细胞增殖的影响；抑制剂处理上述细胞48小时后，采用qRT-PCR法检测肝星状细胞激活指标(α-SMA和PDGFRβ)的mRNA水平(图3)。
从图3中可知，SYK抑制剂GS-9973(1μM)处理两株人原代肝星状细胞48小时后，可以显著降低其激活标志物α-SMA和PDGFRβ的mRNA水平(A)，随着处理时间的延长，对细胞增殖(B)有明显的抑制作用(*，P＜0.05；**，P＜0.01)。
动物肝纤维化的SYK靶向治疗
大鼠肝纤维化/肝癌模型：选用雄性Wistar大鼠(体重100-120g)，每组8只，腹腔注射二乙基亚硝胺(DEN)，50mg/kg，每周1次；对照组腹腔注射等体积PBS，连续18周。SYK抑制剂GS-9973采用灌胃方式给药，2.5mg/kg，每日给药1次，自第13周开始，连续给药至第17周，共5周。
小鼠肝纤维化模型：选用雄性C57BL/6小鼠(8周龄)，每组8只，每只腹腔注射10％CCl4共0.2ml(橄榄油稀释)，每周2次；对照组腹腔注射等体积橄榄油，连续18周。SYK抑制剂GS-9973采用灌胃方式给药，5mg/kg，每日给药1次，自第13周开始，连续给药至第18周，共6周。
SYK抑制剂GS-9973最后一次给药后一周，处死动物。下腔静脉取血，用于血清学生化肝功能测定，包括ALP、ALT、AST、TBIL等；少量动物肝脏保存RNA及DNA标本；部分肝中叶组织用4％甲醛固定后制备石蜡切片；光镜下肝脏组织病理学观察，HE、Sirius Red染色、免疫组化等方法检测各大鼠(或小鼠)肝脏病理组织学改变、肝星状细胞激活状态、肝纤维化指标等(图4和5)。
图4：在四氯化碳(CCl4)诱导的小鼠肝纤维化模型中，SYK小分子化合物GS-9973可显著抑制肝脏胶原生成(Sirius Red染色)，以及肝星状细胞的激活指标α-SMA蛋白的表达(**，P＜0.01；***，P＜0.001)；
图5：在二乙基亚硝胺(DEN)诱导的大鼠肝纤维化/肝癌模型中，SYK靶向治疗可抑制大鼠肝胶原生成、肝星状细胞激活状态，并抑制肝纤维化基础上肝癌的形成(##比较直径小于8mm的新生肝癌，P＜0.01)。
可以预见，通过使用核酸干扰技术，调降SYK基因表达量或抑制SYK基因表达，或使其转录的DNA无法翻译，对肝纤维化或肝硬化同样具有治疗作用。
其他SYK抑制剂，同样对肝纤维化或肝硬化具有治疗作用。
因此，SYK可以作为肝纤维化或肝硬化的治疗靶点。

Claims (9)

  1. 脾酪氨酸激酶作为肝纤维化/肝硬化治疗靶点的应用。
  2. 根据权利要求1所述的应用，其特征在于：通过干扰脾酪氨酸激酶基因表达或者抑制脾酪氨酸激酶的活性，实现肝纤维化/肝硬化的治疗。
  3. 调降脾酪氨酸激酶基因表达的化合物或核酸在制备治疗或改善肝纤维化/肝硬化药物中的应用。
  4. 根据权利要求3所述的应用，其特征在于：调降脾酪氨酸激酶基因表达的核酸为SYK-siRNA、以SYK为靶标的miRNA或反义核苷酸。
  5. 脾酪氨酸激酶抑制剂在制备治疗或改善肝纤维化/肝硬化药物中的应用。
  6. 根据权利要求5所述的应用，其特征在于：脾酪氨酸激酶抑制剂选自Entospletinib(GS-9973)、Fostamatinib(R788)、R406、PRT062607。
  7. 一种治疗肝纤维化/肝硬化的方法，包括对患者给予治疗量的脾酪氨酸激酶基因表达调降剂和脾酪氨酸激酶活性抑制剂中的至少一种。
  8. 根据权利要求7所述的方法，其特征在于：脾酪氨酸激酶基因表达调降剂选自SYK-siRNA、以SYK为靶标的miRNA或反义核苷酸。
  9. 根据权利要求7所述的方法，其特征在于：脾酪氨酸激酶活性抑制剂选自Entospletinib(GS-9973)、Fostamatinib(R788)、R406、PRT062607。
PCT/CN2016/094618 2015-09-24 2016-08-11 Syk作为肝纤维化/肝硬化治疗靶点的应用 WO2017050052A1 (zh)

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