WO2015024512A1 - 阿可拉定在制备用于治疗flt-3有关疾病的药物中的用途 - Google Patents

阿可拉定在制备用于治疗flt-3有关疾病的药物中的用途 Download PDF

Info

Publication number
WO2015024512A1
WO2015024512A1 PCT/CN2014/084781 CN2014084781W WO2015024512A1 WO 2015024512 A1 WO2015024512 A1 WO 2015024512A1 CN 2014084781 W CN2014084781 W CN 2014084781W WO 2015024512 A1 WO2015024512 A1 WO 2015024512A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
flt
proportion
cell
purchased
Prior art date
Application number
PCT/CN2014/084781
Other languages
English (en)
French (fr)
Inventor
孟坤
王骏
王静
张波
王宗慧
Original Assignee
北京珅奥基医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京珅奥基医药科技有限公司 filed Critical 北京珅奥基医药科技有限公司
Priority to CN201480039760.1A priority Critical patent/CN105579039A/zh
Publication of WO2015024512A1 publication Critical patent/WO2015024512A1/zh

Links

Classifications

    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention relates to the use of acrotadine for the preparation of a medicament for the treatment of a disease associated with FLT-3, and belongs to the field of medicine.
  • FLT-3 is a family member of the type III receptor tyrosine kinase and is a signaling molecule. FLT-3 is expressed in various tissues such as liver, spleen, lymph, brain, placenta and gonads, and is also expressed in normal myeloid and lymphoid cell precursors, and is expressed in many hematopoietic malignancies. Its signal transduction pathway is associated with many tumor conduction pathways. Therefore, FLT-3 has become an ideal anti-tumor drug target. Studies have shown that it is expressed in 70-90% of acute myeloid leukemia and acute lymphoblastic leukemia cells.
  • FLT-3 As a signaling molecule, FLT-3 is susceptible to mutations, including: internal tandem replication of the membrane domain, kinase domain mutations and near-membrane domain point mutations, triggering the sustained activation of autoreceptors, and continuing to activate a series of downstream Signaling pathways lead to abnormal proliferation of leukemia cells. And FLT3-ITD mutation is one of the important reasons leading to poor prognosis, high recurrence rate and low survival rate of acute leukemia.
  • Acrolatine also known as icariin, is a new effective monomer obtained by enzymatic conversion of the main active ingredient icariin extracted from Chinese medicinal herbs, and its structural formula is as shown in the following formula (I). :
  • a method for preparing the compound is disclosed in CN101302548B.
  • the method uses icariin as a raw material, and is hydrolyzed by ⁇ -glucosidase, and the precipitate obtained by centrifugation of the hydrolyzate is dissolved in acetone, and the supernatant is obtained by centrifugal filtration. The supernatant obtained by centrifugation was again recrystallized with water to obtain pure extract of icariin.
  • the present inventors have surprisingly found that acrolatine is capable of treating diseases associated with FLT-3.
  • One aspect of the invention provides the use of acrotadine for the manufacture of a medicament for the treatment of a FLT-3 related disorder.
  • the FLT-3 related diseases include acute myeloid leukemia, acute lymphocytic leukemia or autoimmune system diseases.
  • the autoimmune system disease comprises lupus erythematosus.
  • the drug is an oral preparation or an injection.
  • the injection is an intramuscular injection or an intravenous injection.
  • the beneficial effects of the present invention are:
  • the acrolatine of the present invention has achieved very good effects in treating diseases related to FLT-3, especially for acute myeloid leukemia, acute lymphocytic leukemia or autoimmune diseases, Akola Both have shown good pharmaceutical effects. Therefore, the present invention provides a broad prospect for the further application of acaradine.
  • acrolatin is derived from a single monomer of Chinese herbal medicine Epimedium, the raw materials of acrolatin are easily available, with little side effects and high safety.
  • Figure 1 shows the inhibition rate of acrolatine on FLT-3 kinase
  • Figure 2 is a graph showing the inhibition curve of acrolatine on proliferation of acute myeloid leukemia cells MV-4-11 overexpressing FLT-3;
  • Figure 3 is a graph showing the apoptosis effect of acrolatine on MV-4-11 of acute myeloid leukemia cells overexpressing FLT-3, and Figures 3A, 3B, 3C, 3D, 3E and 3F are shown at 0 ⁇ , 10 ⁇ , 15 ⁇ , respectively. Apoptosis detected by flow cytometry after exposure to atropine at a concentration of 20 ⁇ , 25 ⁇ , 30 ⁇ for 24 hours;
  • Figure 4 is a graph showing the effect of acolidine on the cell cycle of MV-4-11.
  • Figure 4 ⁇ , Figure 4 ⁇ , Figure 4C, Figure 4D, Figure 4E and Figure 4F show that MV-4-11 cells are at 0 ⁇ , 0.625 ⁇ , Flow cytometry was used to detect cell cycle distribution after exposure to 1.25 ⁇ , 2.5 ⁇ , 5 ⁇ , ⁇ concentrations of acrolatine for 48 hours;
  • Figure 5 is a graph showing the inhibition curve of alecabadine on subcutaneous allograft tumors of MV-4-11 cells in NOD/SCID mice.
  • 5A is where the tumor volume reached about 300mm 3, the start of administration, A may affect tumor growth cephradine;
  • FIG. 5B is a showing tumor volume reached about 215mm 3, the start of administration, given A pull on tumor growth influences;
  • Figure 6 is a graph showing the inhibition of acyclodine on FLT-3 overexpressing acute lymphoblastic leukemia cell line RS4;
  • FLT-3 related disease refers to diseases related to or involving FLT-3 activity, such as FLT-3 overexpression and abnormal activation, and disorders associated with these diseases.
  • FLT-3 overexpressing refers to 1. expressing FLT-3 in cells that are generally not expected to express FLT-3; 2. causing unwanted cell proliferation, thereby increasing FLT-3 expression; 3. FLT- The cell expression level of 3 exceeded the normal level.
  • a disease abnormally activated by FLT-3 refers to a disease caused by an abnormally high amount of FLT-3 or a mutation in FLT-3.
  • acute myeloid leukemia refers to the accumulation of immature myeloid cells in the bone marrow, resulting in bone marrow hematopoiesis being inhibited by the disease.
  • acute lymphocytic leukemia refers to a hematological malignancy caused by the infinite proliferation of undifferentiated or poorly differentiated lymphocytes in hematopoietic tissue.
  • autoimmune system disease refers to a disease caused by the body's immune response to its own antigen resulting in damage to its own tissues. These include, but are not limited to, organ-specific autoimmune diseases and systemic autoimmune diseases.
  • organ-specific autoimmune system diseases includes, but is not limited to, chronic lymphatic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, chronic ulcerative colitis, pernicious anemia, semi-chronic atrophic gastritis, pulmonary hemorrhage Nephritis syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, etc.
  • systemic autoimmune system disorders includes, but is not limited to, systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed mustard tissue disease, autoimmune Hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, etc.
  • the acute myeloid leukemia cell MV-4-11 of the present invention was purchased from the American Type Culture Collection (ATCC) commercial number CRL-9591, and the FLT-3 expression of this cell line was positive.
  • FLT-3 kinase was purchased from Carna Bioscience Ltd., model number 08-154, batch number 07CBS-2350.
  • the acute lymphoblastic leukemia cell RS4; 11 of the present invention was purchased from ATCC, commercial number CRL-1873, and the FLT-3 expression of this cell line was positive.
  • mice were non-obese diabetic/severe combined immunodeficiency (Nod/SCID) mice, 6 weeks, weighing 18.0-22.0 g, male, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., feeding and sterile independent air supply.
  • Cage which is a sterile feed specially formulated for mice, free to drink pure water.
  • Acrolatine was prepared by the method of Example 1.
  • a method for preparing epimedium is disclosed in the patent publication CN 101302548.
  • icariin is used as a raw material, and hydrolysis is carried out by ⁇ -glucosidase, and the precipitate obtained by centrifugation of the hydrolyzate is dissolved in acetone, and the supernatant is obtained by centrifugal filtration. The supernatant obtained by centrifugation was again recrystallized with water to obtain pure cumin.
  • the icariin in the present invention is purchased from Shaanxi Jiahe Plant Chemical Co., Ltd., and the purity is 90%.
  • polyoxyethylene lauryl ether (0.0015% Bnj-35) (purchased from Shenzhen Shidejia Technology Co., Ltd., trade name: Bridger-35, product number BRIJ-35), 10mM M g C12 and 2 mM dithiothreitol (DTT) (purchased from Solvay Technology Co., Ltd., trade name dithiothreitol, commercial number D8220-1).
  • 0.0015% (m/v) purchased from Anheji Chemical Co., Ltd., trade name 4-hydroxyethylpiperazineethanesulfonic acid, commercial product number A010752; >, polyoxyethylene lauryl ether (0.0015% Brij-35), 0.2%
  • the acrolatine was dissolved in 100% DMSO to a 10 mM solution, and the 10 mM acorramine solution was diluted to the corresponding concentration with 10% DMSO according to the concentration of the arazidine solution to be tested.
  • the reaction wells of the enzyme-free control group, the reaction wells of the control group and the reaction wells of the drug group were separately set.
  • 5 L of 10% dimethyl sulfoxide (DMSO), 1 (L 1 -fold kinase buffer solution) was added to the reaction well of the enzyme-free control group;
  • 5 L of 10% dimethyl sulfoxide (DMSO) was added to the control well.
  • DMSO dimethyl sulfoxide
  • DMSO 10% dimethyl sulfoxide
  • inhibition rate (control group conversion rate - drug group conversion rate) I (control group conversion rate - no enzyme live control conversion rate) * 100%
  • the axeidine-induced FLT-3 kinase inhibition curve shows that alcondamine has an inhibitory effect on FLT-3 kinase with an IC50 of 28 nM. Because FLT-3 is expressed in both acute myeloid leukemia and acute lymphocytic leukemia, it is believed that acrolatine has a therapeutic effect on both diseases.
  • a medium suspension of 8.0*104 cells/ml was prepared using a medium, and each well was added to a 96-well plate. After 24 hours of culture, different concentrations of acrolatine were added to each well, with 4 parallel wells per concentration. After 72 hours of incubation, 20 ⁇ l of a 50 mg/ml sputum-free serum-free medium solution was added to each well. After 4 hours of culture, the medium was discarded, 200 W of DMSO was added to each well, and the mixture was gently shaken for 10 minutes. After the formazan was sufficiently dissolved, the cell absorbance (OD value) was measured at a wavelength of 570 nm using a microplate reader.
  • the final concentration of acrolatine was set to 169.66 ⁇ , 84.83 ⁇ , 42.41 ⁇ , 21.21 ⁇ , 10.6 ⁇ , 5.3 ⁇ , 2.65 ⁇ , 1.33 ⁇ , 0.66 ⁇ , and the growth of MV-4-11 cells in each exposure concentration was calculated.
  • the MTT assay showed that MV-4-11 cells were exposed in 169.66 ⁇ , 84.83 ⁇ , 42.41 ⁇ , 21.21 ⁇ , 10.6 ⁇ , 5.3 ⁇ , 2.65 ⁇ , 1.33 ⁇ , 0.66 ⁇ acortadine.
  • the cell growth inhibition rates were 88.12%, 84.17%, 87.14%, 76.22%, 55.44%, 32.72%, 30.20%, 13.27%, 5.56%, and IC50 was 7.48 ⁇ (see Figure 2).
  • PS Phosphatidylserine
  • Annexm V apoptosis detection agent
  • PI propidium iodide
  • the proportion of live B3 in the ⁇ acoladine group was 88.2%, the ratio of early apoptotic cells ( ⁇ 4 region) was 8.2%, and the proportion of middle and late apoptotic cells ( ⁇ 2 region) was 3.3%, dead cells (B1)
  • the ratio of the region is 0.2% (see Figure 3 ⁇ ); the ratio of living cells ( ⁇ 3 region) in the ⁇ acoladine group is 72.9%, and the proportion of early apoptotic cells ( ⁇ 4 region) is 17.3%, middle and late apoptotic cells ( ⁇ 2 region)
  • the ratio is 9.5%, the proportion of dead cells (B1 region) is 0.2% (see Figure 3 ⁇ ); the ratio of living cells ( ⁇ 3 region) in the 15 ⁇ acapradine group is 59.7%, early apoptotic cells ( ⁇ 4 region)
  • the ratio was 23.6%, the proportion of apoptotic cells in the middle and late stages ( ⁇ 2 region) was 16.4%, and the proportion of dead cells (B1 region) was
  • ⁇ can enter the intracellular and nucleic acid binding.
  • the fluorescence of the dye is proportional to the DNA content in the nucleus. Therefore, the ratio of the amount of DNA in the cell can be measured by flow cytometry.
  • the percentage of cells in the G0/G1 phase ie, the ratio of cells in the quiescent phase and the pre-DNA synthesis phase
  • the double fluorescence amount represents the G2/M phase (S ⁇ : DNA).
  • S ⁇ DNA
  • the flowmeter was named Cytomics FC500 (purchased from Beckman Coulter, USA), ribonuclease (RNaseA was purchased from Sigma, USA), propidium iodide (PI, purchased from Sigma, USA).
  • MV-4-11 cells were at 0 ⁇ , 0.625 ⁇ , 1.25 ⁇ , 2.5 ⁇ , 5 ⁇ and
  • the cells were collected, centrifuged and the medium removed, washed twice with phosphate buffered saline (PBS), resuspended in 1 ml of 70% ethanol, fixed overnight at -20 ° C, and removed by centrifugation.
  • PBS phosphate buffered saline
  • EXPERIMENTAL RESULTS Since MV-4-11 cells had a higher apoptotic rate of exposure to aculeidine above ⁇ for 24 hours, see Example 5, so reduce the drug concentration and choose ⁇ , 0.625 ⁇ , 1.25 ⁇ , 2.5 ⁇ , 5 ⁇ . ⁇ abradine, MV-4-11 cells were exposed to the above concentrations of acrolatine for 48 hours, flow cytometry to detect cell cycle distribution, the results showed that with the increase of drug concentration, the proportion of apoptotic cells Apo And the ratio of 0 () / 0 phase 1 cells increased (see Figure 4).
  • the cultured MV-4-11 cells were collected, centrifuged and the medium was removed, and a 4*10 7 /ml cell suspension was prepared with an appropriate amount of physiological saline, mixed with Matrigel at a ratio of 1:1, and inoculated into non-obese diabetes.
  • / Severe combined immunodeficiency NOD/SCID mice were subcutaneously in the right forelimb, 0.2 ml (8 * 10 6 /pc) per mouse. The blank group was given with corn oil.
  • Matrigel (trade name Matrigel was purchased from BD Biocoat, USA).
  • MV-4-11 cells were inoculated subcutaneously in NOD/SCID mice, and the tumors reached about 300 mm 3 in about 2 weeks.
  • the cells were divided into groups and administered continuously for 18 days.
  • the doses were 12.5 mg/kg and 25 mg/d, respectively.
  • the inhibition rates of kg and 50 mg/kg acrolatine tumor growth were 20.4%, 37.4%, and 47.2% (PO.001), respectively, as shown in Figure 5A.
  • L00% ; Vc is the control tumor volume, Vt is the tumor volume of the test group.
  • Tumor volume l / 2 * a * ba is the long diameter of the tumor block, b is the short diameter of the tumor block :).
  • the inhibition rates of tumor growth at 12.5 mg/kg, 25 mg/kg, and 50 mg/kg were 44.6% (PO.05), 43.7%, and 50.4% (PO.05), respectively, as shown in Figure 5B.
  • acrolatine has a good inhibition rate for tumors caused by MV-4-11 cells, especially when the tumor volume is small, and the inhibition rate of the drug is higher.
  • RPMI 1640 medium purchased from Gibco Life Technologies, USA
  • fetal bovine serum purchased from Gibco Life Technologies, USA
  • C0 2 cell culture medium was purchased from Heraeus, Germany
  • tetrazolium blue purchased from Sigma, USA
  • DMSO purchased from Sigma, USA
  • Cell culture method Human acute lymphoblastic leukemia RS4; 11 cells cultured in RPMI 1640 medium containing 10% fetal bovine serum, placed in 37 ° C, 5% CO 2 cell incubator, passed through the next day, change the liquid .
  • the cells After culturing the cultured RS4; 11 cells, the cells were mixed with a medium of 8.0*10 4 cells/ml, and added to a 96-well plate at a per ⁇ hole. After 24 hours of culture, different concentrations of aconradin were added to each well, with 4 parallel wells per concentration. After 72 hours of culture, 20 ⁇ l of a 50 mg/ml MTT serum-free medium solution was added to each well. After 4 hours of culture, the medium was discarded, 20 ( ⁇ 1 DMSO was added per well, and the mixture was gently shaken for 10 minutes. After the formazan was fully dissolved, the cell absorbance (OD value) was measured at a wavelength of 570 nm using a microplate reader.
  • OD value cell absorbance
  • the final concentrations of tartar were 88.2215 ⁇ , 44.11075 ⁇ , 22.05538 ⁇ , 11.02769 ⁇ , 5.513844 ⁇ , 2.756922 ⁇ , 1.378461 ⁇ , 0.68923 ⁇ , and the RS4 in each exposure concentration was calculated; 11 cell growth inhibition rate, by Origin 5.0 software The cell up inhibition rate curve was fitted and the half effect inhibitory concentration IC 5Q was obtained .
  • the results of MTT assay showed that RS4; 11 cells were exposed for 72 hours in the concentration of 88.2215 ⁇ , 44.11075 ⁇ , 22.05538 ⁇ , 11.02769 ⁇ , 5.513844 ⁇ , 2.569922 ⁇ , 1.378461 ⁇ , 0.68923 ⁇ acortadine, cells.
  • the growth inhibition rates were 97.3637%, 98.00679%, 91.9099%, 77.55591%, 72.00618%, 26.09831%, 3.821215%, 1.618376%, IC 5 , respectively . It is 3.82009 ⁇ .

Landscapes

  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Hematology (AREA)
  • Oncology (AREA)

Abstract

本发明提供阿可拉定在制备用于治疗FLT-3有关疾病的药物中的用途，特别是对于急性随性白血病、急性淋巴白血病或者自身免疫***疾病。

Description

阿可拉定在制备用于治疗 FLT-3有关疾病的药物中的用途 技术领域
本发明涉及阿可拉定在制备用于治疗 FLT-3有关疾病的药物中的用途， 属于 医药领域。
FLT-3属于 III型受体酪氨酸激酶的家族成员， 是一种信号分子。 FLT-3在肝 脏、 脾脏、 淋巴、 脑、 胎盘和生殖腺等各种组织中均有表达， 同时在正常的髓 系和淋巴系细胞前体中也有表达， 在许多造血***恶性病中都有表达， 其信号 传导途径与众多肿瘤传导途径有关联， 因此， FLT-3成为一个理想抗肿瘤的药物 作用靶点。 研究表明， 在 70-90%的急性髓细胞白血病和急性淋巴细胞白血病细 胞中均有表达。
在大多数急性髓细胞白血病患者中均有 FLT-3的过度表达和异常激活。
FLT-3作为一种信号分子， 易发生突变， 突变包括： 近膜结构域内部串联复制、 激酶结构域突变和近膜结构域点突变， 引发自身受体的持续活化， 并持续激活 一系列下游信号传导途径， 导致白血病细胞的异常增殖。 并且 FLT3-ITD突变是 导致急性白血病预后差、 疾病复发率高和生存率低的重要原因之一。
阿可拉定， 又名淫羊藿素， 是从中药淫羊藿中提取分离得到的主要活性成分 淫羊藿苷经酶转化得到的新的有效单体， 其结构式如下式 （I) 所示：
Figure imgf000002_0001
该化合物的制备方法公开于 CN101302548B。 该方法以淫羊藿苷为原料， 以 β-葡萄糖苷酶进行水解，水解产物离心得到的沉淀用丙酮溶解， 离心过滤得到上 清液。 再将离心得到的上清液用水进行重结晶， 得到淫羊藿素纯品。
在申请号为 200910180879.1的中国专利申请中公开了淫羊藿素在制备预防 或治疗***受体 ER-a36表达为阳性的慢性粒细胞性白血病的药物中的应用， 其中涉及到的疾病有乳腺癌、 肺癌、 ***癌、 ***和子宫内膜癌。 但是在现有技术中， 并没有针对淫羊藿素对 ER-a36以外的其他靶点的进一 歩研究。 发明内容
本发明人^ t奇地发现， 阿可拉定能够治疗与 FLT-3有关的疾病。
本发明的目的是提供阿可拉定在制备用于治疗 FLT-3有关疾病的药物中的 用途。
本发明一方面提供了阿可拉定在制备用于治疗 FLT-3有关疾病的药物中的 用途。
优选地， 所述的 FLT-3有关疾病包括急性髓性白血病、 急性淋巴细胞白血病 或自身免疫***疾病。
优选地， 所述的自身免疫***疾病包括红斑狼疮。
优选地， 所述的药物为口服制剂或注射剂。
优选地， 所述的注射剂为肌肉注射剂或静脉注射剂。
本发明的有益效果在于： 本发明的阿可拉定在治疗 FLT-3的相关疾病中取得 了非常好的效果， 特别是对于急性髓性白血病、 急性淋巴白血病或者自身免疫 ***疾病， 阿可拉定都表现出了良好的药学效果。 因此， 本发明为阿可拉定的 进一歩应用提供了广阔的前景。 同时， 由于阿可拉定是源自中药淫羊藿中的一 个单体， 所以阿可拉定的原料易得， 毒副作用小， 安全性高。 附图说明
图 1表示阿可拉定对 FLT-3激酶的抑制率；
图 2表示阿可拉定对过表达 FLT-3的急性髓性白血病细胞 MV-4-11细胞增殖 抑制曲线图；
图 3表示阿可拉定对过表达 FLT-3的急性髓性白血病细胞 MV-4-11的凋亡作 用图， 图 3A、 3B、 3C、 3D、 3E和 3F分别表示在 0μΜ、 10μΜ、 15μΜ、 20μΜ、 25μΜ、 30μΜ浓度的阿可拉定中暴露 24小时， 通过流式细胞仪检测到的细胞凋 亡情况；
图 4表示阿可拉定对 MV-4-11细胞周期的影响图， 图 4Α、 图 4Β、 图 4C、 图 4D、图 4E和图 4F分别表示 MV-4-11细胞在 0μΜ、0.625μΜ、 1.25μΜ、 2.5μΜ、 5μΜ、 ΙΟμΜ浓度的阿可拉定中暴露 48小时后， 流式细胞仪检测细胞周期分布 情况； 图 5表示阿可拉定对 MV-4-11细胞 NOD/SCID小鼠皮下异体抑制瘤的抑制 曲线。 其中图 5A是肿瘤体积达到 300mm3左右时， 开始给药， 阿可拉定对肿瘤 生长的影响； 图 5B表示是肿瘤体积达到 215mm3左右时， 开始给药， 阿可拉定 对肿瘤生长的影响；
图 6表示阿可拉定对 FLT-3过表达的急性淋巴白血病细胞 RS4;11细胞增殖 的抑制曲线图。
以下实施例仅用于对本发明进行示例性说明， 不用于限制本发明， 在本发 明保护范围内所做的修改、 改变、 变型等都在本发明的保护范围内。
术语" FLT-3有关疾病"指的是有关或者涉及 FLT-3活性，如 FLT-3过度表达和 异常激活的疾病， 以及伴有这些疾病的病症。
术语" FLT-3"过度表达指的是 1.在通常不希望表达 FLT-3的细胞中表达 FLT-3 ; 2.引起不希望的细胞增殖， 从而增加 FLT-3的表达； 3.FLT-3的细胞表达 水平超过正常水平。
术语" FLT-3异常激活的疾病"指的是由于 FLT-3反常高量或者 FLT-3中突变 引起的疾病。
术语"急性髓性白血病"指的是不成熟髓细胞在骨髓里聚集，造成的骨髓造血 受到抑制疾病。
术语"急性淋巴细胞白血病 "指的是由于未分化或分化很差的淋巴细胞在造 血组织无限增殖所致的恶性血液病。
术语"自身免疫***疾病"指的是机体对自身抗原发生免疫反应而导致自身 组织损害所引起的疾病。 包括但不限于器官特异性自身免疫***疾病和***性 自身免疫疾病。
术语"器官特异性自身免疫***疾病"包括但不限于慢性淋巴性甲状腺炎、甲 状腺功能亢进、 胰岛素依赖性糖尿病、 重症肌无力、 慢性溃疡性结肠炎、 恶性 贫血性半慢性萎缩性胃炎、 肺出血肾炎综合征、 寻常天皰疮、 类天皰疮、 原发 性胆汁性肝硬变、 多发性脑脊髓硬化症、 急性特发性多神经炎等。
术语"***性自身免疫***疾病"包括但不限于***性红斑狼疮、类风湿性关 节炎、 ***性脉管炎、 硬皮病、 天疱疮、 皮肌炎、 混合芥蒂组织病、 自身免疫 性溶血性贫血、 甲状腺自身免疫病、 溃疡性结肠炎等。 本发明中的急性髓性白血病细胞 MV-4-11购自美国菌种保藏中心 （ATCC) 商品号 CRL-9591,该细胞系的 FLT-3表达为阳性。
FLT-3激酶购自 Carna生物科学公司 （ Carna Bioscience Ltd. ) ， 产品型号为 08-154， 批号 07CBS-2350。
本发明中的急性淋巴白血病细胞 RS4; 11购自 ATCC, 商品号 CRL-1873 , 该细胞系的 FLT-3表达为阳性。
小鼠为非肥胖糖尿病 /重症联合免疫缺陷 （Nod/SCID) 小鼠， 6周， 体重 18.0-22.0g, 雄性， 购自北京维通利华实验动物技术有限公司， 饲养与无菌独立 送风笼 （IVC) ， 饲养专门为小鼠配置的消毒饲料， 自由饮用纯净水。
阿可拉定通过实施例 1方法制备。 实施例
实施例 1
阿可拉定的制备
阿可拉定又名淫羊藿素， 是从中药淫羊藿中提取分离得到。
在公开号为 CN 101302548的专利中公开了淫羊藿的制备方法。该方法以淫 羊藿苷为原料， 以 β-葡萄糖苷酶进行水解， 水解产物离心得到的沉淀用丙酮溶 解， 离心过滤得到上清液。 再将离心得到的上清液用水进行重结晶， 得到淫羊 藿纯品。 本发明中的淫羊藿苷购自陕西嘉禾植物化工有限责任公司公司， 纯度 90%。 实施例 2
阿可拉定对 FLT-3激酶的抑制作用
实验方法：
1. 配置 1倍激酶缓冲液
缓冲液含有 50mM的 4-羟乙基哌嗪乙磺酸（HEPES pH=7.5 )、 0.0015% (m/v) (购自安耐吉化学公司， 商品名 4-羟乙基哌嗪乙磺酸， 商品号 A010752;>、 聚氧乙 烯月桂醇醚（0.0015%Bnj-35 ) (购自深圳市时得佳科技有限公司， 商品名： 布里 杰 -35， 商品编号 BRIJ-35)、 10mM MgC12和 2mM二硫苏糖醇（DTT) (购自索莱 宝科技有限公司， 商品名二硫苏糖醇， 商品号 D8220-1) 。
2. 配置终止液
终止液含有 lOOmM的 4-羟乙基哌嗪乙磺酸（HEPES pH=7.5 )、0.0015%(m/v) (：购自安耐吉化学公司， 商品名 4-羟乙基哌嗪乙磺酸， 商品号 A010752;>、 聚氧乙 烯月桂醇醚 （0.0015%Brij-35 ) 、 0.2%的包被试剂 （coatingreagent #3购自 caliper 公司， 货号为 PN760050) 和 50mM的乙二胺四乙酸螯合剂 （EDTA) 。
3. 阿可拉定溶液的配置
将阿可拉定用 100%DMSO溶解为 10mM的溶液，再根据需要检测的阿可拉定 溶液的浓度， 将 10mM的阿可拉定溶液用 10%DMSO稀释成相应的浓度。
4. 激酶反应： 将 FLT-3激酶加入 1倍激酶缓冲液， 配制成 2.5倍酶溶液。 再将 荧光染料 （FAM) 标记的多肽和 ATP (购自 sigma公司， 商品名 ATP二钠水合物， 商品号 213-579-1；)加入 1倍激酶缓冲液， 配制成 2.5倍底物溶液。
在 384孔板上分别设置无酶活对照组反应孔， 对照组反应孔和药物组反应 孔。 无酶活对照组反应孔中加入 5 L 10%二甲基亚砜 （DMSO) ， 1( L 1倍激酶 缓冲溶液； 对照组反应孔中加入 5 L的 10%二甲基亚砜 （DMSO) ， ΙΟμΙ^ 2.5倍 酶溶液； 药物组反应孔中加入 5 L 10%二甲基亚砜 （DMSO) 溶解的阿可拉定， 1(^L 2.5倍酶溶液。室温孵育 10分钟， 再分别向各孔中加入 ΙΟμΙ^ 2.5倍底物溶液。
28°C孵育 1小时， 加入 25 L终止液终止反应， 通过药物筛选平台 （购自 Caliper 公司的 EZ reader2.0) 检测得到无酶活对照转化率， 对照组转化率和药物反应组 转化率， 转化率 =产物量 / (产物量 +底物量） *100%。
5. 抑制率的计算：
根据公式： 抑制率 = (对照组转化率-药物组转化率） I (对照组转化率 -无酶 活对照转化率） *100%
通过图 1，阿可拉定对 FLT-3激酶抑制曲线可以看出阿可拉定对 FLT-3激酶具 有抑制作用， 其 IC50值为 28nM。 因为 FLT-3在急性髓性白血病和急性淋巴白血 病中都有表达， 所以认为阿可拉定对于这两种疾病都有治疗作用。 实施例 3
通过 MTT细胞增殖实验 （比色法检测药效） 测定阿可拉定对急性髓性白血 病细胞 MV-4-11细胞的抑制作用
1. 实验材料： RPMI IMDM培养基 （购自美国 Gibco生命技术公司） ， 胎牛 血清 （购自美国 Gibco生命技术公司） ， C02细胞培养基购自德国贺立氏仪器公 司， 四氮唑蓝 （MTT, 购自美国 Sigma公司） ， DMSO (购自美国 Sigma公司） 。
2. 细胞培养方法：人急性髓性白血病 MV-4-11细胞培养于含 10%胎牛血清的 RPMI IMDM培养基中， 置于 37°C， 5%C02细胞培养箱中， 隔天传代、 换液。 3. MTT细胞增殖实验 （比色法检测药效） 方法
将培养的 MV-4-11细胞离心后， 用培养基配成 8.0*104细胞 /ml细胞悬液， 按 每孔 ΙΟΟμΙ加到 96孔板中。 培养 24小时后， 每孔加入 ΙΟΟμΙ不同浓度的阿可拉定， 每个浓度 4个平行孔。 培养 72小时后， 每孔加入 20μ1 的 50mg/ml的 ΜΤΤ无血清培 养基溶液。 培养 4小时后， 弃培养基， 每孔加入 200W DMSO， 轻微震荡 10分钟， 甲臜充分溶解后， 用酶标仪在 570nm波长下测定细胞光吸收值 （OD值） 。 设定 阿可拉定的终浓度为 169.66μΜ， 84.83μΜ、 42.41μΜ、 21.21μΜ、 10.6μΜ、 5.3μΜ、 2.65μΜ、 1.33μΜ、 0.66μΜ， 计算各暴露浓度中的 MV-4-11细胞生长抑制率， 通 过 Origin5.0软件拟合细胞上涨抑制率曲线， 并得出半效抑制浓度 IC50。
4. 实验结果
通过图 2可见， 由 MTT实验结果显示， MV-4-11细胞在 169.66μΜ， 84.83μΜ、 42.41μΜ、 21.21μΜ、 10.6μΜ、 5.3μΜ、 2.65μΜ、 1.33μΜ、 0.66μΜ阿可拉定中暴 露 72小时， 细胞生长抑制率分别为 88.12%、 84.17%、 87.14%、 76.22%、 55.44%、 32.72%、 30.20%、 13.27%、 5.56%, IC50为 7.48μΜ (见图 2) 。 实施例 4
阿可拉定诱导细胞凋亡作用
实验原理： 磷脂酰丝氨酸 （PS ) 分布在细胞膜脂质双层的内侧， 而在细胞 凋亡早期， 细胞膜中的 PS由脂膜内侧翻向夕卜侧， Annexm V (细胞凋亡检测剂） 是 Ca2+依赖性磷脂结合蛋白， 与 PS有高度亲和力， 故可用 Annexm V-FITC标 记早期凋亡细胞； 碘化丙啶（PI)不能透过完整的细胞膜， 但能透过凋亡中晚期 的细胞和死细胞的细胞膜， 而使细胞核染红， 故可用 PI标记中晚期凋亡和死亡 的细胞。
实验材料： 细胞凋亡试剂盒 （购自美国 Biolegend公司） ， Annexin-V (购 自美国 Biolegend公司） 。
实验方法： 将 MV-4-11细胞在 0μΜ、 10μΜ、 15μΜ、 20μΜ、 25μΜ和 30μΜ 的阿可拉定中暴露 24小时后， 收集细胞， 离心并去除培养基， 用磷酸盐缓冲液 (PBS)清洗两次，加入 ΙΟΟμΙ结合缓冲液和 2μ1细胞凋亡检测剂（Annexin-V)， 室温避光 30min， 再加入 4μ1碘化丙啶 （ΡΙ) ， 避光反应 5min， 加入 400μ1 结 合缓冲液 （Binding Buffer) ， 用流式仪检测凋亡细胞百分比。
实验结果： MV-4-11细胞在 0μΜ、 10μΜ、 15μΜ、 20μΜ、 25μΜ和 30μΜ 的阿可拉定中暴露 24小时后， 可观察到： 随着药物浓度升高， 活细胞比例减少， 早期凋亡和中晚期凋亡的细胞比例增加， 并呈现浓度依赖性 （见图 3 ) 。
ΟμΜ阿可拉定组中活细胞B3区域:)比例为 88.2%,早期凋亡细胞（Β4区域） 比例为 8.2%， 中、 晚期凋亡细胞（Β2区域） 比例为 3.3%， 死亡细胞（B1区域） 比例为 0.2% (见图 3Α) ； ΙΟμΜ阿可拉定组中活细胞 (Β3区域)比例为 72.9%， 早期凋亡细胞（Β4区域） 比例为 17.3%， 中、 晚期凋亡细胞（Β2区域） 比例为 9.5%, 死亡细胞 （B1区域） 比例为 0.2% (见图 3Β ) ； 15μΜ阿可拉定组中活 细胞 （Β3区域） 比例为 59.7%, 早期凋亡细胞 （Β4区域） 比例为 23.6%， 中、 晚期凋亡细胞（Β2区域） 比例为 16.4%， 死亡细胞（B1区域） 比例为 0.3% (见 图 3C)； 20μΜ阿可拉定组中活细胞 (Β3区域：)比例为 39.0%， 早期凋亡细胞（Β4 区域） 比例为 34.0%， 中、 晚期凋亡细胞 （Β2区域） 比例为 26.8%， 死亡细胞 (B1区域） 比例为 0.2% (见图 3D) ； 25μΜ阿可拉定组中活细胞 (Β3区域)比 例为 13.8%， 早期凋亡细胞 （Β4区域） 比例为 46.6%， 中、 晚期凋亡细胞 （Β2 区域） 比例为 39.5%， 死亡细胞 （B1区域） 比例为 0.0% (见图 3Ε) ； 30μΜ阿 可拉定组中活细胞 (Β3区域)比例为 10.3%， 早期凋亡细胞 （Β4区域） 比例为 53.1%, 中、 晚期凋亡细胞 （Β2区域） 比例为 36.5%， 死亡细胞 （B1区域） 比 例为 0.0% (见图 3F ) 。 实施例 5
阿可拉定对细胞周期的影响
实验原理： 细胞经乙醇固定后， 细胞膜的通透性增加， DNA特异荧光染料
ΡΙ可以进入胞内与核酸结合， 在 RNase消化 RNA后， 染料的荧光量与核内的 DNA含量呈比例，因此可通过流式测定 PI荧光量而反映细胞 DNA含量的比例。 根据细胞有丝***的特征， 可通过软件分析单倍荧光量代表 G0/G1期 （即： 静 止期和 DNA合成前期的细胞比例）的细胞比例，双倍荧光量代表 G2/M期（S卩： DNA合成完到有丝***前和细胞***开始到结束的细胞） 的细胞比例， 两者之 间为 DNA合成期即 S期。
实验材料： 流式仪商品名称为 Cytomics FC500 (购自美国 Beckman Coulter 公司） ， 核糖核酸酶 （RNaseA购自美国 Sigma公司） ， 碘化丙啶 （PI, 购自美 国 Sigma公司） 。
实验方法： 将 MV-4-11细胞在 0μΜ、 0.625μΜ、 1.25μΜ、 2.5μΜ、 5μΜ和
10μΜ的阿可拉定中暴露 48小时后， 收集细胞， 离心并去除培养基， 用磷酸盐 缓冲液 （PBS) 清洗两次， 加入 lml 70%乙醇重悬， -20°C固定过夜， 离心去除 乙醇后， 用磷酸盐缓冲液 （PBS ) 清洗两次， 加入 200μ1磷酸盐缓冲液 （PBS ) 重悬，加入核糖核酸酶 (RnaseA)10( g/ml， 37 °C孵育 30分钟，加入碘化丙啶（PI) 5(^g/ml， 室温避光 30分钟， 使用流式仪检测细胞周期变化。
实验结果： 由于 MV-4-11细胞在 ΙΟμΜ以上的阿可拉定暴露 24小时凋亡比 例较高， 见实施例 5， 故降低药物浓度， 选择 ΟμΜ、 0.625μΜ、 1.25μΜ、 2.5μΜ、 5μΜ、 ΙΟμΜ 阿可拉定， MV-4-11细胞在以上浓度的阿可拉定中暴露 48小时后， 流式细胞仪检测细胞周期分布情况， 结果表明随药物浓度升高， 凋亡细胞 Apo 比例和 0()/01期细胞比例增加 （见图 4 ) 。
ΟμΜ阿可拉定组中， G0/G1期细胞比例为 64.82%， G2/M期细胞比例为 12.25%, S期细胞比例为 22.93%， 凋亡细胞比例为 0% (见图 4Α) ； 0.625μΜ 阿可拉定组中， G0/G1期细胞比例为 65.35%， G2/M期细胞比例为 13.49%， S 期细胞比例为 21.16%,凋亡细胞比例为 0% (见图 4Β )； 1.25μΜ阿可拉定组中， G0/G1期细胞比例为 66.64%， G2/M期细胞比例为 11.92%， S期细胞比例为 21.44%, 凋亡细胞比例为 0% (见图 4C) ； 2.5μΜ阿可拉定组中， G0/G1期细 胞比例为 67.30%， G2/M期细胞比例为 10.59%， S期细胞比例为 22.11%， 凋亡 细胞比例为 5.11%(见图40); 5.(^1 阿可拉定组中，00/01期细胞比例为72.07%， G2/M期细胞比例为 8.89%, S期细胞比例为 19.04%,凋亡细胞比例为 9.82% (见 图 4Ε) ； ΙΟ.ΟμΜ阿可拉定组中， G0/G1期细胞比例为 84.64%， G2/M期细胞比 例为 4.9%， S期细胞比例为 10.46%， 凋亡细胞比例为 11.46% (见图 4F ) 。 实施例 6
动物实验
动物实验模型的建立
收集培养的 MV-4-11细胞， 离心并去除培养基， 用适量生理盐水配置成 4* 107/ml细胞悬液， 与基质胶 （Matrigel ) 以 1 : 1比例混合， 接种于非肥胖糖尿 病 /重症联合免疫缺陷 NOD/SCID小鼠右前肢腋部皮下， 每只小鼠 0.2ml ( 8* 106/ 只） 。 空白组以给以玉米油。
实验材料： 基质胶 （商品名称 Matrigel购自美国 BD Biocoat公司） 。
在 NOD/SCID小鼠皮下接种 MV-4-11细胞， 2周左右， 瘤体达到 300mm3 左右， 分组并开始给药， 连续给药 18天， 给药量分别为 12.5mg/kg、 25mg/kg、 50mg/kg阿可拉定肿瘤生长的抑制率分别为 20.4%、 37.4%、 47.2% (PO.001 ) ， 具体见图 5A。
Figure imgf000009_0001
l00%; Vc是对照组肿瘤体积， Vt是受试组肿瘤体积。 肿瘤体积 =l/2*a*b a为瘤块的长径， b为瘤块的短径：)。 重复实验在瘤体积达到 215mm3左右， 分组并开始给药， 给药量分别为
12.5mg/kg、 25mg/kg、 50mg/kg阿可拉定对肿瘤生长的抑制率分别为 44.6% (PO.05 ) 、 43.7%和 50.4% (PO.05 ) ，具体见图 5B。
因此可见， 阿可拉定对于 MV-4-11细胞引起的肿瘤具有良好的抑制率， 特 别是在肿瘤体积较小时， 药物的抑制率更高。 实施例 7
通过 MTT细胞增殖实验（比色法检测药效）测定阿可拉定对急性淋巴白血 病 RS4; 11细胞的抑制作用
1. 实验材料： RPMI 1640培养基 （购自美国 Gibco生命技术公司） ， 胎牛 血清（购自美国 Gibco生命技术公司） ， C02细胞培养基购自德国贺立氏仪器公 司， 四氮唑蓝（MTT,购自美国 Sigma公司） ， DMSO (购自美国 Sigma公司） 。
2. 细胞培养方法： 人急性淋巴白血病 RS4; 11细胞培养于含 10%胎牛血清 的 RPMI 1640培养基中， 置于 37°C， 5%C02细胞培养箱中， 隔天传代、 换液。
3. MTT细胞增殖实验 （比色法检测药效） 方法
将培养的 RS4; 11细胞离心后， 用培养基配成 8.0*104细胞 /ml细胞悬液， 按每孔 ΙΟΟμΙ加到 96孔板中。 培养 24小时后， 每孔加入 ΙΟΟμΙ不同浓度的阿可 拉定，每个浓度 4个平行孔。培养 72小时后，每孔加入 20μ1 的 50mg/ml的 MTT 无血清培养基溶液。 培养 4小时后， 弃培养基， 每孔加入 20(^1 DMSO， 轻微震 荡 10分钟， 甲臜充分溶解后，用酶标仪在 570nm波长下测定细胞光吸收值（OD 值） 。 设定阿可拉定的终浓度为 88.2215μΜ， 44.11075μΜ、 22.05538μΜ、 11.02769μΜ、 5.513844μΜ、 2.756922μΜ、 1.378461μΜ、 0.68923μΜ, 计算各暴 露浓度中的 RS4; 11细胞生长抑制率， 通过 Origin5.0软件拟合细胞上涨抑制率 曲线， 并得出半效抑制浓度 IC5Q。
4. 实验结果
通过图 6可见， 由 MTT实验结果显示， RS4; 11细胞在 88.2215μΜ， 44.11075μΜ、22.05538μΜ、 11.02769μΜ、 5.513844μΜ、2.756922μΜ、 1.378461μΜ、 0.68923μΜ浓度阿可拉定中暴露 72小时， 细胞生长抑制率分别为 97.3637%、 98.00679%、 91.9099%、 77.55591%、 72.00618%、 26.09831%、 3.821215%、 1.618376%, IC5。为 3.82009μΜ。

Claims

权 利 要 求 书
1. 阿可拉定在制备用于治疗 FLT-3有关疾病的药物中的用途。
2. 根据权利要求 1所述的用途， 其特征在于， 所述的 FLT-3有关疾病包括急 性髓性白血病、 急性淋巴细胞白血病或自身免疫***疾病。
3. 根据权利要求 2所述的用途， 其特征在于， 所述的自身免疫***疾病包括 红斑狼疮。
4. 根据权利要求 1所述的用途， 其特征在于， 所述的药物为口服制剂或注射 剂。
5. 根据权利要求 2所述的用途， 其特征在于， 所述的注射剂为肌肉注射剂 或静脉注射剂。
PCT/CN2014/084781 2013-08-23 2014-08-20 阿可拉定在制备用于治疗flt-3有关疾病的药物中的用途 WO2015024512A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201480039760.1A CN105579039A (zh) 2013-08-23 2014-08-20 阿可拉定在制备用于治疗flt-3有关疾病的药物中的用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201310370640.7 2013-08-23
CN201310370640 2013-08-23

Publications (1)

Publication Number Publication Date
WO2015024512A1 true WO2015024512A1 (zh) 2015-02-26

Family

ID=52483084

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2014/084781 WO2015024512A1 (zh) 2013-08-23 2014-08-20 阿可拉定在制备用于治疗flt-3有关疾病的药物中的用途

Country Status (2)

Country Link
CN (1) CN105579039A (zh)
WO (1) WO2015024512A1 (zh)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002254842B2 (en) * 2002-04-09 2008-05-29 Guangzhou Chen Li Ji Pharmaceutical Factory An anti-rheumatism medicament and method to prepare thereof
US20080214844A1 (en) * 2007-01-23 2008-09-04 Nan Zhang Icaritin and desmethylicaritin as anti-cancer agents
CN102038673B (zh) * 2009-10-20 2012-07-25 北京珅奥基医药科技有限公司 羟基苯并吡喃酮类化合物在制备治疗白血病药物中的应用
CN101843629B (zh) * 2010-06-11 2012-03-14 首都医科大学宣武医院 淫羊藿苷和含有淫羊藿苷的淫羊藿黄酮的新用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SANG-HUN KANG ET AL.: "Icariside II induces apoptosis in U937 acute myeloid leukemia cells: role of inactivation of STAT3-related signaling", PLOS ONE, vol. 7, no. 4, 6 April 2012 (2012-04-06) *

Also Published As

Publication number Publication date
CN105579039A (zh) 2016-05-11

Similar Documents

Publication Publication Date Title
Rajendran et al. Suppression of signal transducer and activator of transcription 3 activation by butein inhibits growth of human hepatocellular carcinoma in vivo
US10420814B2 (en) Composition for treating cancer stem cells
JP2014530247A (ja) 乳がんの治療に使用されるフィトカンナビノイド
US20070207989A1 (en) Diterpene derivatives for the treatment of cardiovascular, cancer and inflammatory diseases
Xia et al. Vorinostat upregulates MICA via the PI3K/Akt pathway to enhance the ability of natural killer cells to kill tumor cells
Alamdari et al. Melatonin as a promising modulator of aging related neurodegenerative disorders: role of microRNAs
Kim et al. Sequential treatment of HPV E6 and E7-expressing TC-1 cells with bortezomib and celecoxib promotes apoptosis through p-p38 MAPK-mediated downregulation of cyclin D1 and CDK2
Li et al. Proapoptotic and Growth-inhibitory Effects of Plumbagin on Human Gastric Cancer Cells Via Suppression of Signal Transducer and Activator of Transcription 3 and Protein Kinase B.
Tan et al. Erchen Plus Huiyanzhuyu Decoction Inhibits the Growth of Laryngeal Carcinoma in a Mouse Model of Phlegm‐Coagulation‐Blood‐Stasis Syndrome via the STAT3/Cyclin D1 Pathway
Zheng et al. Gentiana scabra restrains hepatic pro-inflammatory macrophages to ameliorate non-alcoholic fatty liver disease
JP7026263B2 (ja) 抗腫瘍の漢方薬組成物及びその揮発性油エキス
WO2015024512A1 (zh) 阿可拉定在制备用于治疗flt-3有关疾病的药物中的用途
EP2942056B1 (en) Novel uses of licochalcone a
CN115177618A (zh) Flt3抑制剂在制备治疗急性髓系白血病药物中的应用
CA3065332A1 (en) Garcinol compositions for therapeutic management of endoplasmic reticulum stress
Yang et al. Fucoidan enhances cisplatin-induced effects on SCC-25 human oral cancer cells by inhibiting the PI3K/AKT pathway
KR101603279B1 (ko) 프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 nfat5의 활성 관련 질환의 예방 또는 치료용 약학적 조성물
KR102235218B1 (ko) 감마-테르피넨을 포함하는 자궁경부암 예방 또는 치료용 조성물
Jiang et al. Daily chemical evodiamine from Chinese prickly ash attenuates osteoclast differentiation through RANKL induced NFAT pathways
Ren et al. An integrated in vitro/in silico approach to assess the anti-androgenic potency of isobavachin
Yong-Jiea et al. STAT3 Inhibition by Centipede Scolopendra Extract in Liver Cancer HepG2 Cells and Orthotopic Mouse Models of Hepatocellular Carcinoma
WO1999004777A1 (fr) Carcinostatiques
TW201444561A (zh) 可治療癌症之藥學組合物
KR101620022B1 (ko) 닥나무로부터 추출된 폴리페놀을 유효성분으로 함유하는 제1형 당뇨병 예방 및 치료용 약학적 조성물
WO2015113511A1 (zh) 串联表达的siRNA及其在治疗慢性淋巴细胞白血病中的应用

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201480039760.1

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14838158

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205N DATED 02/05/2016)

122 Ep: pct application non-entry in european phase

Ref document number: 14838158

Country of ref document: EP

Kind code of ref document: A1