WO1997021446A1 - Composition pharmaceutique pour la protection des cellules souches hematopoietiques, et applications - Google Patents

Composition pharmaceutique pour la protection des cellules souches hematopoietiques, et applications Download PDF

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WO1997021446A1
WO1997021446A1 PCT/CN1996/000106 CN9600106W WO9721446A1 WO 1997021446 A1 WO1997021446 A1 WO 1997021446A1 CN 9600106 W CN9600106 W CN 9600106W WO 9721446 A1 WO9721446 A1 WO 9721446A1
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cells
pharmaceutical composition
hematopoietic
tsp
composition according
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PCT/CN1996/000106
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French (fr)
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Zhongchao Han
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Shanghai Beite Biotechnology Co. Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors

Definitions

  • the present invention relates to a pharmaceutical composition for protecting hematopoietic precursor cells and its application. More specifically, it relates to a thrombin-sensitized protein (Thrombospondin (Referred to as TSP) or its active fragment or their active mutant protein as an active ingredient for a pharmaceutical composition for protecting hematopoietic precursor cells and its various blood types caused by the treatment of antitumor drugs-induced hemocytopenia and megakaryocytes. Microplate hyperplasia and its application in expanding hematopoietic stem cells and progenitor cells.
  • the most commonly used method of treating tumors is chemotherapy, that is, using some anti-tumor drugs to kill abnormally proliferating tumor cells. Because anti-tumor drugs kill tumor cells, they also have effects on normal cells, especially bone marrow hematopoietic cells.
  • the killing effect causes blood cells to decrease, while white blood cells decrease the body's ability to resist external infections, red blood cells decrease can cause anemia, and thrombocytopenia can cause the body to bleed.
  • the main methods currently used to treat blood cell reduction caused by chemotherapy include blood transfusion and blood transfusion components, the use of factors that promote blood cell proliferation, such as G-CSF, GM-CSF, EPO, and TPO.
  • hematopoietic stem cells can differentiate into progenitor cells of granulocytes, red blood cells or megakaryocytes under certain conditions, and further proliferate to produce mature white blood cells, red blood cells or megakaryocytes. Each giant cell can produce thousands of Functional platelets.
  • hematopoietic stem cells are regulated by many factors, including interleukins 3, 6, 11, 1 and 13 and SCF, GM-CSF, TPO, MPO, and EPO. They can be used alone or in combination to stimulate -1, Growth and differentiation of one or more series of hematopoietic cells.
  • hematopoietic stem cells and progenitor cells are mainly present in the bone marrow, and neonatal umbilical cord blood and embryonic liver are also abundant.
  • human peripheral blood is low, bone marrow, umbilical cord blood, embryonic liver, and peripheral blood are all abundant. It can be used as a source of hematopoietic stem cells, but the above sources are very limited.
  • the absolute number of hematopoietic stem and frustum cells that can be collected from bone marrow, umbilical cord blood and peripheral blood is low, which cannot meet the needs of transplantation.
  • TSP is one of the main components of platelet oc granules. It is also synthesized in endothelial cells, smooth muscle cells, fibroblasts and some tumor cells. TSP plays an important role in cell-matrix, cell-to-cell interaction, and can promote Many cells, including hematopoietic stem cells, adhere to the matrix, and TSP also has an antiangiogenic effect. TSP is a glycoprotein composed of homotrimers, with a molecule of £ 450Kd. It contains several functional regions, the most noticeable of which is the region that can bind to heparin. These regions can promote the binding of TSP to the sulfuric acid on the cell surface.
  • Heparin glycoproteins have the function of regulating cells.
  • TSP Frazier WA, Curr. Opion Cell Biol. 3: 792-799. 1 99 1: Taraboletti G. et al. J. Cell Biol., 1 1 1: 765- 772,! 996) .
  • the object of the present invention is to provide a pharmaceutical composition for protecting hematopoietic precursor cells, and the use of the pharmaceutical composition for the treatment of anti-tumor drugs i-hemocytopenia and various thrombocytosis caused by megakaryocytes and the present invention.
  • Method for the expansion of hematopoietic precursor cells The pharmaceutical composition for protecting hematopoietic precursor cells according to the present invention uses thrombin sensitizing protein (TSP) and / or its active fragment and / or their active mutant protein as an active ingredient.
  • TSP thrombin sensitizing protein
  • thrombin sensitizing proteins and their active fragments or their active mutant proteins may be natural, genetically recombinant, or a mixture thereof.
  • active mutein refers to an analog of TSP, one or more of which is naturally occurring
  • TSP or its active fragment are replaced or deleted by different amino acid residues, or one or more amino acid residues are added to the sequence of natural TSP, and compared with the active fragment of natural TSP, there is no The activity of the resulting product is significantly changed.
  • active muteins can be prepared using known artificial synthesis and / or site-directed mutagenesis techniques or any other known technique suitable for this purpose. '''
  • Any such active mutein preferably has an amino acid sequence sufficiently identical to the amino acid sequence of TSP so that it has substantially similar activity to TSP or its active fragment.
  • any such active mutant protein has at least 40% identity or homology with TSP. More preferably at least 50%, at least 60%, at least 70%, at least 80% or most preferably at least 90%-homology or homology,
  • Muteins of TSP or its active fragments that can be used in the present invention, or nucleic acids encoded thereby, include a limited series of substantially corresponding substitution peptides or polynucleotide sequences, which can be made by those skilled in the art. It is routinely obtained by appropriate experimental methods in accordance with the instructions and guidance given herein. According to the present invention, preferred changes in active muteins are those known "conservative" substitutions, TSPs or polypeptides or their activity
  • a conservative amino acid substitution of a fragment can include a group of synonymous amino acids that have sufficiently similar physicochemical properties such that substitutions between amino acids in the group will retain the biological function of the molecule [Grantham, Science, Vol, 1 85, pp.
  • the active fragment of the thrombin sensitizing protein include a fragment consisting of the first to fourth amino acid residues at the N-terminus of the thrombin sensitizing protein, referred to as TSP M 74 for short.
  • hematopoietic precursor cells refer to hematopoietic stem cells and targeted progenitor cells
  • progenitor cells including HPP-CFC (high proliferative potential colony forming cells), CFU-Mix or CFU-GEMM (mixed colony forming units), CFU-MK (megakaryocyte colony forming units), CFU-GM (granulocytes) Colony forming units), BFU-E (erythrocyte colony forming units), etc., whose sources include human umbilical cord blood, bone marrow, peripheral blood and embryonic liver.
  • HPP-CFC high proliferative potential colony forming cells
  • CFU-Mix or CFU-GEMM mixed colony forming units
  • CFU-MK megakaryocyte colony forming units
  • CFU-GM granulocytes Colony forming units
  • BFU-E erythrocyte colony forming units
  • the present invention is based on the following observations: TSP and fragments containing hepatic crest-binding regions were observed
  • TSP, _ 17 pairs of hematopoietic stem cells (HPP-CFC and CFU-GEMM) and megakaryocytes ⁇ cells (CFUMK) Growth has an inhibitory effect. This inhibitory effect is reversible. After hematopoietic precursor cells treated with TSP or its active fragments are washed, they can proliferate and differentiate in new cell culture fluids. In addition, the inhibitory effect of TSP and its active fragments can be completely neutralized by heparin. In addition, a fragment consisting of 559th to 669th amino acid residues at the N-terminus without a heparin binding site is used.
  • TSP 55 ⁇ ) - 6 «Performed the same test and found that it has no inhibitory effect on the growth of megakaryocyte progenitor cells (CFU-MK) and no protective effect on hematopoietic precursor cells.
  • CFU-MK megakaryocyte progenitor cells
  • TSP has several functional regions, especially The region that can bind to heparin can promote the binding of TSP to heparin sulfate glycoprotein on the cell surface, which has the function of regulating cells, and the inhibitory effect of TSP and TSP t- 174 on the growth of hematopoietic stem cells and megakaryocyte progenitor cells can be controlled Heparin is completely neutralized, that is, heparin has a neutralizing effect. It can be inferred that the heparin binding region in the thrombin-sensitized protein is an essential functional region of the active protein and active polypeptide of the present invention.
  • TSP and its active fragments have a reversible effect on hematopoietic precursor cells
  • the inventors envisage using TSP or its active fragments as a pharmaceutical composition for protecting hematopoietic precursor cells to pretreat the hematopoietic precursor cells so that The treated cells were temporarily inhibited due to their proliferation, and their sensitivity to subsequent antitumor drugs was reduced, so they were not killed by antitumor drugs.
  • the results of a series of experiments designed to fully confirm ⁇ TSP and its activity Fragments can reduce the sensitivity of hematopoietic stem and limulus cells to anti-tumor drugs and protect them from being killed by anti-tumor drugs.
  • TSP is injected into a mouse and then an anti-tumor drug is injected.
  • the experimental results showed that the mice that had been pre-injected with TSP had faster recovery of hematopoietic function than the control mice.
  • These in vivo results are consistent with the above in vitro results, suggesting that TSP does have a protective effect on bone marrow hematopoietic precursor cells and can prevent or alleviate bone marrow failure and hemocytopenia induced by chemotherapy or radiation therapy.
  • the pharmaceutical composition for protecting hematopoietic precursor cells of the present invention can be used alone or in combination with other drugs that can promote blood cell growth to produce an additive or f
  • the drugs that promote blood cell growth include interleukin 3 (IL3), interleukin 6 (IL6), interleukin 1 1 (IL 1 1), and interleukin 13 (IL 1 3), Granulocyte Colony Stimulating Factor (G-CSF), Granulocyte Colony Stimulating Factor (GM-CSF), Stem Cell Factor (SCF), Thrombopoietin (c-Mpl Ligand), Megakaryocyte Gene ( MPO) and mucopolysaccharides.
  • IL3 interleukin 3
  • IL6 interleukin 6
  • IL 1 1 1 interleukin 1 1
  • IL 13 interleukin 13
  • G-CSF Granulocyte Colony Stimulating Factor
  • GM-CSF Granulocyte Colony Stimulating Factor
  • TSP is injected into the mouse, then the antitumor drug is injected, and finally the cell growth factor G-CSF is injected.
  • the experimental results show that the combined use of TSP and growth factors can accelerate the recovery of hematopoietic function after chemotherapy, compared with the use of TSP or growth factors alone.
  • TSP alone or in combination with the above-mentioned hematopoietic cell growth factors can expand hematopoietic stem cells and progenitor cells, especially megakaryocytes and their progenitor cells, in vitro.
  • the basic method is: incubate bone marrow or umbilical cord blood cells and the donor serum as a nutrient source with TSP alone or with growth factors for a period of time, or incubate with growth factors, and then add TSP and then incubate for a certain period of time to promote early stem cells Differentiate and proliferate to progenitor cells, and at the same time prevent juvenile cells from entering the G 2 / M phase to divide and proliferate, so that the ratio of hematopoietic stem cells and callus cells is greatly increased, and hematopoietic stem cells and progenitor cells are obtained.
  • Growth factor can be targeted to expand different hematopoietic stem cells and progenitor cells as well as
  • CD 34+ cells are generally recognized as hematopoietic progenitor cells because CD34 antigen is expressed only in hematopoietic progenitor cells such as CFU-GEMM, CFU-MK, CFU-GM, and BFU-E.
  • CD 34+ cells isolated from human umbilical cord blood are used as a research object, TSP and CD34 + cells are incubated for 3 days, and then an anti-tumor drug is added for 24 hours.
  • TSP has a significant protective effect on human cord blood CD 34+ cells, indicating that the effect of TSP on mice is also applicable to dry humans.
  • the pharmaceutical composition for protecting hematopoietic precursor cells of the present invention can be used for the treatment of tumors and other diseases by chemotherapy or radiotherapy-induced reduction of blood cells, especially thrombocytopenia, and can also promote the development of large-dose chemotherapy to promote tumors. cure.
  • the pharmaceutical composition for protecting hematopoietic precursor cells of the present invention can also be used as an agent for expanding hematopoietic stem cells in vitro, and a large number of hematopoietic stem cells and progenitor cells can be expanded from human umbilical cord blood, bone marrow or embryonic liver cells for use by the donor himself. Or other people use it for clinical treatments such as blood cell or thrombocytopenia, various bone marrow transplantation indications, infusion or transplantation, or cryopreservation to establish a cell bank.
  • the pharmaceutical composition for protecting hematopoietic precursor cells of the present invention is a megakaryocyte growth inhibitor. Although this inhibitory effect is reversible, continuous administration can inhibit the generation of megakaryocytes and platelets, so it can be used to treat megakaryocytes. Various thrombocytosis caused by cytosis.
  • the method for clinically treating hemacytopenia induced by chemotherapy or radiotherapy of the present invention includes using a sufficient amount of the pharmaceutical composition of the present invention to a patient receiving an antitumor drug.
  • the method for clinically treating thrombocytosis of the present invention includes administering to a patient a sufficient amount of the pharmaceutical composition of the present invention.
  • the aforementioned precursor cell pharmaceutical composition of the present invention contains the thrombin sensitizing protein and / or its active fragment and / or their active mutant protein as an active ingredient and a pharmaceutically acceptable carrier.
  • the active protein or active polypeptide can be in free form or in the form of an acid addition salt, which can be used alone (such as when used to expand hematopoietic precursor cells in vitro), but is usually dissolved in physiological saline or a buffer solution (such as PBS buffer) or mixed with other pharmaceutically acceptable carriers to prepare a pharmaceutical composition.
  • compositions may be solid, liquid or semi-fluid, and fillers, diluents, stabilizers, pH regulators, osmotic pressure regulators and excipients may be added to the pharmaceutical composition as needed.
  • the active protein or active polypeptide can be prepared into various dosage forms by, for example, freeze-drying.
  • the administration method may be a commonly used administration method similar to an agent and depends on the treatment conditions, and for example, it may be intramuscular injection, intravenous drip, subcutaneous injection, or oral administration.
  • the amount of active ingredient administered depends on the route of administration, the disease being treated, and the weight and condition of the patient. In any case, the dosage used is determined by the physician who understands the condition of the recipient.
  • the pharmaceutical composition of the present invention usually contains 10 ⁇ g to 10 mg of active ingredient per dosage unit. However, it is clear that in some compositions, the amount of active ingredient may be greater or less than the above limit,
  • 74 is a gene recombinant protein expressed by E. coli, provided by Biotechnology General Ltd. of Israel. The present invention will be specifically described below through implementation, but the present invention is not limited to these embodiments.
  • TSP and TSP have inhibitory effects, and this effect can be completely neutralized by small molecule heparin.
  • Table 1 Effects of thrombin sensitizing protein (TSP) and its fragment TSP, 174 alone or in combination with small molecules although heparin on the growth of different mouse hematopoietic progenitor cells
  • TSP and TSP, 174 have an inhibitory effect on megakaryocyte production. Although this inhibitory effect is reversible, continuous administration can inhibit megakaryocytes and platelets. Generation, so TSP and TSP,-
  • the cells in the experimental group were added with TSP, and the control group was added with PBS. After the incubation, the cells were washed with the culture solution, and a part was cultured by the plasma clot method and the methyl cellulose method, and the progenitor cells were analyzed. content. Incubate another part of the cells with 5-fluorouracil (0.30 ⁇ ⁇ / ⁇ 1) for 24 hours, then wash and culture to analyze the cell content. See Table 2 for specific results.
  • TSP thrombin sensitizing protein
  • the number of various progenitor cells in the table is the total number.
  • CFU-GEMM CFU-GM and BFU-E were analyzed by methyl cellulose culture method, and CFU-MK was analyzed by plasma clot method.
  • mice Twelve 8-week-old Balb / c mice were selected and divided into two groups of 6 mice each. TSP was injected intraperitoneally in a group of mice at a dose of 5 micrograms each, once every hour, and two consecutive injections were performed. The other group of mice was used as a control, and an equal volume of PBS was injected intraperitoneally. 20 hours after the second injection, yes All mice were injected with 5-air uracil (5-FU) once at a dose of 150 mg / kg body weight. On the 8th day after 5-air uracil injection, about 0.4 ml of blood was taken from the orbital vein for all mice. After analysis, the femur was taken, bone marrow cells were collected, and the hematopoietic progenitor cell content was cultured (Han et al., CRAcad Sci. Paris 3 13: 553, 1991).
  • 5-air uracil 5-air uracil
  • Table 3 is the average data of three experiments. It can be seen that the CFU-GEMM, CFU-MK, CFU-GM, the number of single megakaryocytes, and the number of platelets and leukocytes in peripheral blood of mouse bone marrow pretreated by TSP were higher than those in the control group, indicating that TSP has protection Hematopoietic cells are not killed by the anti-tumor drug 5-fluorouracil and accelerate the recovery of hematopoietic function. Table 3.In vivo protective effect of TSP on bone marrow hematopoietic precursor cells and peripheral blood cells in 5-FU treated mice
  • TSP + 5-FU group 462 ⁇ 24 * ⁇ 4 soil ⁇ 12 ⁇ 2 * 16 ⁇ 2 * 65 ⁇ 5 * 5 ⁇ 1 ⁇ 980 ⁇ 20 ⁇
  • HPP-CFC CFU-MK and megakaryocytes were analyzed by plasma clot culture.
  • CFU-GEMM and CFU-GM were analyzed by methyl cellulose culture method.
  • Peripheral blood leukocyte and platelet counts were measured using an automatic blood cell counter (Coulter type).
  • mice Twenty Balb / c mice aged 8 weeks were divided into 4 groups of 5 mice each. Except for G-CSF, according to the same injection dose and time as in Example 3, 1) after intraperitoneal injection of TSP in mice of TSP group, 5-FU was injected once again; 2) at the same time as in TSP group, G-CSF group mice were injected intraperitoneally twice with PBS, then once with 5-FU, and after 24 hours with 5-FU, they were injected with G-CSF once a day at 5 micrograms per mouse for 5 days; 3) For the TSP and G-CSF group mice, as in the TSP group, two TSP injections followed by 5-FU once, and 24 hours after 5-FU injection, the same as G-CSF group, G-CSF injection 5 days; 4) In the same manner as in the G-CSF group, in the abdominal cavity of the 5-FU group, the rats were injected with PBS twice and then with 5-FU once.
  • TSP group 460 ⁇ 2 22 ⁇ 3 * 6 ⁇ 2 ⁇ 7 + 2 * 40 ⁇ 5 * 4 ⁇ 1 ⁇ 320 ⁇ 3 ⁇
  • G-CSF group 201 ⁇ 24 12 ⁇ 2 30 ⁇ 3 * 9 ⁇ 2 38 ⁇ 3 7 ⁇ 2 ⁇ 760 140
  • CD34 + cells were divided into two equal amounts, each tube 2--104 cells, at a concentration of 10 4 cells / 500 [mu Rise. TSP was added to one of the tubes so that the final concentration was 5 ⁇ ⁇ / ⁇ 1, and an equal volume of PBS was added to the other tube as a control. Place cells with TSP at 37 'C, containing 5%. Incubate in a 0 2 incubator for 3 days.
  • intravenous drip Before or early in patients receiving anti-tumor treatment (chemotherapy or radiotherapy), intravenous drip. Intramuscular or subcutaneous injection or oral route, the patient is administered an effective dose of the pharmaceutical composition of the present invention, so that normal hematopoietic stem cells and Thallium cells respond in advance, making them less sensitive to anti-tumor treatment and therefore protected. As a result, the phenomenon of blood cell reduction induced by anti-tumor therapy can be avoided or significantly reduced.
  • the dosage of the pharmaceutical composition of the present invention should be determined by the attending doctor based on the patient's weight, the results of blood sample analysis, and the dosage of anti-tumor therapy. If the pharmaceutical composition of the present invention is used in combination with an effective amount of a blood cell growth factor such as G-CSF and TPO, it is possible to more effectively and promptly prevent and treat hemocytopenia induced by antitumor therapy.
  • a blood cell growth factor such as G-CSF and TPO
  • An effective dose of the pharmaceutical composition of the present invention is administered to a patient with thrombocytosis by intravenous drip, intramuscular or subcutaneous injection, or oral route. Once or several times a day, or once a few days.
  • the length of medication should refer to the platelet number and function of the patient after the medication. Usually, the patient's platelet count and function are normal or close to normal as a course of treatment. Generally, intermittent treatment is required for 2 to 3 courses.

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Description

用于保护造血前体细胞的药物组合物及其应用 本发明涉及一种用于保护造血前体细胞的药物组合物及其应用， 更具体地 说， 涉及一种以凝血酶致敏蛋白（Thrombospondin, 简称 TSP )或其活性片段或它们 的活性突变蛋白为有效成分的用于保护造血前体细胞的药物组合物及其在治疗 抗肿瘤药物诱发的血细胞减少症和巨核细胞增多导致的各种血小扳增多症以及 在扩增造血干细胞、 祖细胞中的应用。
现在最常用的治疗肿瘤的方法是化学疗法， 即， 使用一些抗肿瘤药物将异常 增殖的肿瘤细胞杀死.， 由于抗肿瘤药物在杀死肿瘤细胞的同时， 对正常细胞特别 是骨髓造血细胞也有杀伤作用， 造成血细胞减少， 而白细胞减少会使机体抵御外 来感染的能力降低， 红细胞减少会造成贫血， 血小板减少则可导致机体出血。 目 前治疗由化学疗法导致的血细胞减少的主要方法包括输血和输血液成份、 使用能 促使血细胞增殖的因子如 G-CSF , GM-CSF , EPO和 TPO等
已知造血多能干细胞在一定的条件下能向粒单细胞、 红细胞或巨核细胞的祖 细胞分化， 进一步增殖产生成熟的白细胞、 红细胞或巨核细胞， 每个巨^细胞又 能产生数千个有功能的血小板。
造血细胞的增殖和分化受许多因子的调节，这些因子包括白细胞介素 3 、 6 、 1 1 、 1 3和 SCF , GM-CSF 、 TPO 、 MPO以及 EPO ,它们单独使用或合用能刺 激一 1、系列或多个系列造血细胞的生长和分化。 在正常情况下， 造血干细胞和祖 细胞主要存在于骨髓中， 新生儿脐带血和胚胎肝脏中含量也较丰富， 人末梢血中 d虽较低,. 骨髓、 脐带血、 胚胎肝脏以及末梢血都可用作造血干细胞的来源， 但 上述来源十分有限， 而且， 骨髓， 脐带血和末梢血中可采集的造血干沮细胞绝对 数较低， 不能满足移植的需要。
已知 TSP是血小板 oc颗粒的主要成份之一， 在内皮细胞、 平滑肌细胞， 成纤 维细胞和一些肿瘤细胞中也有合成 TSP在细胞与基质、 细胞与细胞的相互作用 中起重要作用， 还能促使包括造血干细胞在内的许多细胞粘附到基质上， 此外， TS P还有抗血管形成作用。 TSP是由同源三聚体构成的糖蛋白， 分子 l£ 450Kd ， 含有几个功能区域， 其中最令人注意的是能与肝素结合的区域， 这些区域能促使 TS P结合在细胞表面的硫酸肝素糖聚蛋白（proteoglycans )上， 有调节细胞的功能。 关于 TSP的详细情况,可参见 Frazier WA, Curr. Opion Cell Biol . 3 : 792-799. 1 99 1 : Taraboletti G. et al. J. Cell Biol.、 1 1 1 : 765- 772 , ! 996)。
本发明的目的在于提供一种用于保护造血前体细胞的药物组合物及使用该药 物组合物治疗抗肿瘤药物 i秀发的血细胞减少症和巨核细胞增多导致的各种血小 板增多症以及 ί本外扩增造血前体细胞的方法 确认本 本发明所述的用于保护造血前体细胞的药物组合物是以凝血酶致敏蛋白 (TSP) 和 /或其活性片段和 /或它们的活性突变蛋白作为有效成分的。
上述凝血酶致敏蛋白及其活性片段或它们的活性突变蛋白可以是天然的、 基 因重组的或它们的混合物。
此处所用的术语 "活性突变蛋白" 是指 TSP 的类似物， 其中一个或多个天然
TSP 或其活性片段的氨基酸残基被不同的氨基酸残基所替换或缺失， 或者一个或 多个氨基酸残基被加入到天然 TSP的顺序中， 而且， 与天然的 TSP的活性片段相 比， 没有显著改变所得到的产物的活性。 这些活性突变蛋白可以用已知的人工合 成和 /或定点诱变技术或任何其它已知的适合用于该目的的技术来制备」
任何一种这样的活性突变蛋白最好具有与 TSP的氨基酸顺序足够一样的氨基 酸顺序， 从而使其具有与 TSP或其活性片段实质上相似的活性。
在较佳的具体例子中， 任何这样的活性突变蛋白与 TSP 至少具有 40%的一致 性或同源性。 更佳的具有至少 50%、 至少 60%、 至少 70%、 至少 80%或最佳的具 有至少 90%—致性或同源性，
可在本发明中使用的 TSP 或其活性片段的突变蛋白， 或者为其编码的核酸， 包括一系列有限的实质上对应的置换肽或多聚核苷酸顺序， 可以由本领域的一般 熟练技术人员， 依据此处给出的说明和指导， 通过适当的实验方法常规地获得. 根据本发明， 较佳的活性突变蛋白的改变是那些已知的 "保守的" 置换， TSP 或多肽或它们的活性片段的保守的氨基酸置换可以包括一组同义氨基酸， 这些氨 基酸具有足够相似的物化性能使得组中氨基酸之间的置换会保留该分子的生物 学功能 〔 Grantham, Science, Vol, 1 85, pp. 862 - 864 ( 1974)〕 ， 已经清楚， 在上 述限定的顺序中也可以插入或缺失一些氨基酸而不改变其功能， 尤其是如果插人 或缺失只包括少数氨基酸例如小于 30个， 较佳的小于 1 0 个， 并且不去除或替换 对功能构象至关重要的氨基酸如 Cys 残基时 〔 Anfmsen, " Principles That Govern The Folding of Protein Chains " , Science, Vol, 1 8 1 , pp. 223 - 230 ( 1973) ] 。 这样的缺失和 /或插人而形成的蛋白质和活性突变蛋白归于本发明的范 围。
凝血酶致敏蛋白的活性片段的具体例子包括由凝血酶致敏蛋白 N 末端的第 1 至第 Π4个氨基酸残基组成的片段， 简称 TSP M 74 。
上述造血前体细胞是指造血多能干细胞（stem cell)和各系列定向祖细胞
(progenitor cells),包括 HPP-CFC (高增殖潜能集落形成细胞)、 CFU-Mix或 CFU- GEMM (混合集落形成单位)、 CFU-MK (巨核细胞集落形成单位)、 CFU-GM (粒单 细胞集落形成单位)、 BFU-E (红细胞集落形成单位)等， 其来源包括人脐带血、 骨 髓、 末梢血和胚肝.
本发明是以下述发现为基础的： 观察到 TSP 和其含肝柰结合区域的片段
TSP , _ 1 7 对造血多能干细胞（HPP-CFC 和 CFU-GEMM )和巨核细胞袓细胞 (CFUMK)生长有抑制作用， 这种抑制作用是可逆性的， 用 TSP或其活性片段作用 过的造血前体细胞经洗涤后， 可在新的细胞培养液中增殖分化。 另外， TSP及其 活性片段的抑制作用能完全被肝素中和， 此外， 使用不含肝素结合位点的、 由 N 末端的第 559至第 669个氨基酸残基组成的片段 TSP55<) - 6(«进行 Γ同样的试验， 发现其对巨核细胞祖细胞 (CFU - MK)的生长没有抑制作用， 也没有保护造血前 体细胞的作用， 由于如上所述， TSP具有几个功能区域， 尤其是能与肝素结合的 区域能促使 TSP结合在细胞表面的硫酸肝素糖聚蛋白上， 有调节细胞的功能， 且 TSP 和 TSP t ― 174对造血多能干细胞和巨核细胞祖细胞生长的抑制作用可被肝素 完全中和， 即, 肝素具有中和作用。 由此可推知， 凝血酶致敏蛋白中的肝素结合 区域是本发明的活性蛋白和活性多肽的必不可少的功能区域。
利用 TSP 及其活性片段对造血前体细胞有可逆性作用这一待点， 本发明者设 想以 TSP 或其活性片段作为用于保护造血前体细胞的药物组合物预处理造血前 体细胞， 使处理后的细胞因其增殖一时性受抑， 对随后加人的抗肿瘤药物的敏感 性降低，从而不被抗肿瘤药物杀死.由此设计的一系列实验的结果完全证实 Γ TSP 和其活性片段能降低造血干、 袓细胞对抗肿瘤药物的敏感性， 保护它们免受抗肿 瘤药物的杀伤.
在本发明的一个实施例中， 将 TSP先注射入小鼠体内， 然后再注射抗肿瘤药 物。 实验结果发现， 预先注射过 TSP的小鼠， 其造血功能的恢复较对照组小鼠快。 这些体内结果与上述体外结果相符合， 表明 TSP确实对骨髓造血前体细胞有保护 作用， 可预防或缓解化疗或放疗诱发的骨髓衰竭和血细胞减少症。
在治疗由抗肿瘤药物诱发的血细胞减少症时， 本发明的用于保护造血前体细 胞的药物组合物除可单独使用外， 还可以与其它能促进血细胞生长的药物合用， 产生相加或 f办同作用 所述促进血细胞生长的药物也即造血细胞生长因子的例子 包括白细胞介素 3(IL3)、 白细胞介素 6(IL6)、 白细胞介素 1 1 (IL 1 1 )、 白细胞介素 13(IL 1 3), 粒细胞集落刺激因子 (G-CSF)、 粒单细胞集落刺激因子 (GM-CSF)、 干 细胞因子（SCF)、 血小板生成素 (c-Mpl Ligand)、 巨核细胞生成素 (MPO)以及粘多 糖等。
在本发明的另一实施例中， 先注射 TSP到小鼠体内， 然后注射抗肿瘤药物， 最后再注射细胞生长因子 G-CSF。 实验结果表明， 合用 TSP和生长因子， 其加 速化疗后造血功能恢复的作用优于 TSP或生长因子的单独使用。
将 TSP单独地或与上述造血细胞生长因子联合使用， 能在体外扩增造血干细 胞和祖细胞， 特别是巨核细胞及其祖细胞。 其基本方法是： 将骨髓或脐带血细胞 和作为营养源的供体血清与 TSP单独地或加生长因子共孵育若干时间， 或先经生 长因子孵育， 后加人 TSP再孵育若干时间， 促使早期干细胞向祖细胞分化繁殖， 同时阻止沮细胞进入 G 2 /M期进行分裂增殖， 从而使造血干细胞和袓细胞的比 ί列 和绝对 it大大增高， 茯得大壁的造血干细胞和祖细胞。 通过使用不同的造血细胞 生长因子， 可定向扩增不同的造血干细胞和祖细胞以及巨核细胞和血小板 .
在造血细胞中， CD 34+细胞通常被公认为造血祖细胞， 因为 CD34抗原只在 CFU-GEMM 、 CFU-MK 、 CFU-GM和 BFU-E等造血祖细胞中表达。 在本发明 的又一实施 ί列中， 采用从人脐带血分离出来的 CD 34+细胞作为研究对象， 将 TSP 与 CD34+细胞共孵育 3天， 然后再加人抗肿瘤药物孵宵 24小时。 与对照组相比， TSP对人脐带血 CD 34+细胞具有明显的保护作用， 表明 TSP对小鼠的作用同样 适用干人体，
本发明的用于保护造血前体细胞的药物组合物可用于治疗肿瘤和其它疾病的 化学治疗或放射治疗诱发的血细胞减少， 特别是血小板减少， 还可促使大剂量化 学疗法的开展， 促进肿瘤的治愈。 本发明的用于保护造血前体细胞的药物组合物 还可用作体外扩增造血干细胞试剂， 从人脐带血、 骨髓或胚肝细胞中大量扩增造 血干细胞和祖细胞， 用于供者本人或其他人在患血细胞或血小板减少症， 各种骨 髓移植适应症时输注或移植等临床治疗使用， 或冷冻保存， 建立细胞库 ί寺用。
本发明的用于保护造血前体细胞的药物组合物是一种巨核细胞生长抑制剂， 虽然这种抑制作用是可逆的， 但持续给药可抑制巨核细胞和血小板的生成， 因此 可用于治疗巨核细胞增多导致的各种血小板增多症。
本发明的临床治疗由化疗或放疗诱发的血细胞减少症的方法包括给接受抗肿 瘤药物患者使用足够量的本发明的药物组合物。
本发明的临床治疗血小板增多症的方法包括给患者使用足够蛩的本发明的药 物组合物。
上述本发明的前体细胞药物组合物含作为有效成分的凝血酶致敏蛋白和 /或其 活性片段和 /或它们的活性突变蛋白以及药学上可接受的载体。活性蛋白或活性多 肽可以是游离的形式， 也可以是酸加成盐的形式， 可以单独使用 （如在用于体外 扩增造血前体细胞时） ， 但通常溶解在生理盐水或缓冲溶液（如 PBS缓冲液） 中 或与药学上可接受的其他载体混合， 制成药物组合物。 这些药物组合物可以是固 体的， 也可以是液体的或半流体的， 可视需要在药物组合物中加入填料、 稀释剂， 稳定剂、 ρΗ调节剂、 渗透压调节剂和赋形剂等。 活性蛋白或活性多肽可通过例 如冷冻干燥法等制成各种剂量形式。 给药方法可以是常用的类似药剂的给药方法 并视治疗条件而定， 例如， 可以是肌肉注射、 静脉滴注、 皮下注射或口服等。 所 投与的活性成分量视用药途径、 所治疗的疾病和患者的体重和病情而定， 在任何 情况下， 所用剂量均由 Γ解接受治疗者情况的内科医生来定。
本发明的药物组合物通常每个剂量单位中含有 1 0 μ g - 1 0mg活性成分。 但 是， 很显然， 在一些组合物中， 活性成分的量可以大于或小于上述限定直，
本发明实施例中使用的 TSP系按 Dubernard和 Legrand的方法 (J. Lab. & Clin. Med. . 1 1 8 :446 - 457. 1 99 1 )分离得到。 TSP , - | 74为用大肠扞菌（E. Col i )表达的基 因重组蛋白质， 由以色列 B iotechnology General Ltd. 提供。 下面通过实施冽进一歩具体说明本发明， 但本发明并不局限于这些实施例。 实施例 1
取 Balb/c小鼠的骨髓细胞， 用血浆凝块法 (Han等, Br. 丄 Haematol. 81: - 5, 1992和 J. Lab. & Clin. Med., 123:610 - 6, 1994),将细胞 (2 - 105/培养皿)加人含 2- 巯基乙醇 10%、 氯化钙 10%、 牛血清白蛋白 10%、 猪血清 5%、 牛血浆 10%的 α - 培养液中，于 37 C在 C02孵箱中培养，分析 TSP单独或与小分子. 肝素 (Fraxiparin) 合用对不同的造血祖细胞生长的影响， 结果见表 1 。 从表 I 可以看出， TSP和 TSP,.17 ^ CFU-MK , CFU - GEMM以及 HPP-CFC有抑制作用， 这种作用可被 小分子肝素完全中和。 表 1. 凝血酶致敏蛋白 (TSP)和其片断 TSP, _ 174单独或与小分子虽肝素合用 对不同的小鼠造血祖细胞体外生长的影响
Figure imgf000007_0001
结果以每 105个种植骨髓单个核细胞所获集落的、 均数 ± ^准误¾示， 数据来自二 以上的独立实验.
*表示经 t 检验， 与对照组比较， p < 0.0 5 . 由于 TSP和 TSP, 174对巨核细胞生成有抑制作用， 虽然这种抑制作用是可逆 的， 但持续给药可抑制巨核细胞和血小板的生成， 因此， TSP和 TSP, - |74可用 来治疗血小板增多症。 实施例 2
取 Balb/c小鼠骨髓单个核细胞， 将部分细胞立即作血浆凝块法和甲基纤维素 法（即， 将细胞（1 0%)加人含甲基纤维素 20%、 胎牛血淸 20% , 生长因子 1 0%的 IMDM培养液中， 于 37 'C在 C02孵箱中培养)培养， 分析祖细胞含量， 以每毫升 1 06个细胞的浓度将细胞加人含 1 0%胎牛血清、 1 %再障猪血清 (作为生长刺激因 子的来源)、 1 %牛血清白蛋白和 1 0·4Μ 2-巯基乙醇的培养液中， 在 37 'C、 含 5% 02的培养箱中第一次孵育 3天。 实验组细胞加 TSP , 对照组加等 ί本积磷酸盐 缓冲液 (PBS). 孵育结束后， 将细胞用培养液洗涤， 取一部分作血浆凝块法和甲 基纤维素法培养， 分析祖细胞含量。 将另一部分细胞与 5-氟尿嘧啶 (0.30μ§/π 1)共 孵育 24小时， 然后洗涤， 培养分析袓细胞含量， 具体结果见表 2
. 凝血酶致敏蛋白 (TSP)对骨髓造血干细胞的保护作用
Figure imgf000008_0001
表中各种祖细胞数为总数. 其中 CFU - GEMM . CFU - GM和 BFU - E采用甲 基纤维素培养方法分析， CFU - MK则采用血浆凝块法分析.
* 表示与 PBS再孵育一天组比较， p<0.01 . 从表 2可以看出， TSP对造血细胞的抑制作用是可逆性的， TSP孵育后的细 胞经洗 ί条后仍可继续生长， 经 TSP预处理的细胞对 5-氟尿嘧啶的敏感性减低。 这 些结果表明， TSP能保护造血干细胞不受化疗药物损伤， 因此， 可用于肿瘤化疗 期间的骨髓保护。 实施 ί列 3
取 8周龄 Balb/c小鼠 12只， 分成二组， 每组 6只。 在一组小鼠腹腔注射 TSP , 剂量为每只 5微克， 每小时一次， 连续注射 2次； 将另一组小鼠作为对照， 腹腔 注射等体积 PBS , 注射次数和时间与实验组的相同。 第二次注射后 20小时， 对 所有小鼠注射一次 5-氣尿嘧啶 (5-FU), 剂量为 150毫克 /公斤体重. 5-氣尿嘧啶注 射后第 8天， 对所有小鼠从眼眶静脉取血约 0.4毫升， 作血象分析， 然后取其股 骨， 采集骨髓细胞， 培养分折造血祖细胞含量 (Han 等， C.R.Acad Sci. Paris 3 13 :553, 1991 )。
表 3是三次实验的平均数据。 从中可以看出， 经 TSP预处理的小鼠骨髓中的 CFU-GEMM , CFU-MK , CFU-GM、 单个巨核细胞数和末梢血中的血小板、 白 细胞数均比对照组高， 说明 TSP 有保护造血细胞不被抗肿瘤药物 5-氟尿嘧啶杀 伤、 加速造血功能恢复的作用。 表 3. TSP对 5-FU处理小鼠骨髓造血前体细胞和末梢血细胞的体内保护作用
HPP-CFC CFU-GEMM CFU-GM CFU-MK 巨核细胞 白细胞 血小板
/股骨 103/股骨 109/L - I O'VL 对照组 10 ± 2 5 ± 2 10 ± 2 4.5 ± 1 30 ± 4 7 + "> 1050 ± 30
5-FU组 203 ± 16 9 ± 2 4 ± 1 9.6 ± 1 46 ± 4 3 ± 1 750 ± 25
TSP+5-FU组 462 ± 24* 】4 土 ；： Δ 12 ± 2* 16 ± 2* 65 ± 5* 5 ± 1 Δ 980 ± 20 Δ 表中数据均以平均值 ±标准误表示，
HPP-CFC CFU-MK和巨核细胞是用血浆凝块培养法分析.
CFU - GEMM和 CFU - GM是用甲基纤维素培养法分析。
末梢血白细胞和血小板数是用自动血细胞计数仪 (Coulter 型)测定.
*和 Δ分别表示与 5 - FU组比较， 经 [检验， p<0.01和 p<0.05 . 实施例 4
取 8周龄 Balb/c小鼠 20只， 分成 4组， 每组 5只。 除 G- CSF夕卜， 按与实施例 3相同的注射剂量和时间， 1 ) 在 TSP组小鼠腹腔注射二次 TSP后， 再注射一次 5-FU ; 2)在与 TSP组相同的时间， 先在 G-CSF组小鼠腹腔注射二次 PBS , 再注 射一次 5-FU , 注射 5-FU 24小时后， 再注射 G-CSF , 每天一次， 每次每鼠 5微 克， 共注射 5天； 3) 对 TSP和 G-CSF组小鼠， 与 TSP组同样， 先注射二次 TSP , 再注射一次 5-FU , 在注射 5-FU 24小时后， 与 G-CSF组同样， 注射 G-CSF 5天； 4) 与 G-CSF组同样， 在 5-FU组小鼠腹腔， 先注射二次 PBS， 再注射一次 5-FU 在 5-FU注射后第 6天和第 8天， 按与实施例 3相同的方法， 对所有小鼠采集末 梢血， 作血象分析； 采集股骨， 取骨髓细胞作造血前体细胞分折. 表 4为一次实 验的数据， 从中可以看出， TSP与 G-CSF合用， 有相加或协同作用， 能明显加 速化疗后造血功能的恢复。 表 4. TSP和 G-CSF体内合用对 5-FU处理小鼠造血系统的影响
HPP-CFC CFU-GEMM CFU-GM CFU-MK 巨 f 细胞 白细胞 iia小板
/股骨 103/股骨 109/L 109/L 第 6天
5-FU组 320±16 12±3 2±1 3土 ί 21±3 2±i 220±25
TSP组 460±2 22±3* 6±2 Δ 7+2* 40±5* 4±1 Δ 320±3 Δ
G-CSF组 280±30 8±2 7±2 Δ 2±1 23±4 5±2 Δ 25O±40
TSP加 G-CSF ill 450±40* 21±4* 8±2* 6±2* 46±6* 7±2* 460±30*
¾ 8天
5-FU ϊ\\ 194±22 10±2 8±2 10±2 40±5 3±1 720±46
TSP ί\ 436±30* 18±2* 19±3* 17土 3* 64±4 Δ 5±2 990±3 Λ
G-CSF组 201±24 12±2 30±3* 9±2 38±3 7±2 Δ 760140
TSP力 fi G-CSF m 440±2 22+2* 22±4* 21±3* 56±5 Δ 9±3 1100±40* 表中敉据均以 T均 ftL ±标准误表示， 测定方法与实施例 3相同。 实施例 5
将按免疫磁珠法（Xi等， Sr. J Haematol, 90:921-922, 1995)分离得到的 CD34+ 细胞分成等量二管， 每管 2 - 104细胞， 细胞浓度为 104/500微升。 在其中一管内 加 TSP , 使其最终浓度为 5μ§/πι1， 在另一管内加等体积 PBS作为对照。 将细胞 与 TSP在 37 'C、 含 5%。02的培养箱中孵育 3天。 孵育结束后， 用培养液洗涤细 胞， 再加入 5-氟尿嘧啶 (0.3 μ g/ml)， 共孵育 24小时。 然后洗涤， 用甲基纤维素 法和血浆凝块法培养分析祖细胞含量， 具体结果见表 5 表 5. 凝血酶致敏蛋白（TSP)对人脐带血 CD34+细胞的保护作用
CFU-GEMM CFU-GM CFU-MK
PBS+5-FU组 242 ± 12 450 ± 22 335 ± 12
TSP+5-FU组 427 土 18* 640 ± 17* 742 土 24* 本实验共做三次， 表中结果为总的细胞数. 其中， CFU-GEMM和 CFU-GM 用甲 基纤维素法分折， CFU-MK是用血浆凝块法分析。
* 表示 1 j PBS+5 - FU组比较， p<0.05. 实施例 6
将 10ύ脐血单个核细胞与 1 毫升含有 5 %体积的 PBS或 TSP(5 μ g)或 TSP(1 M g)的培养液共孵育 48小时， 然后加入 5 μ g鬼臼乙叉甙 (FTP)再 ^育 24小时。 将细胞用培养液洗涤 2次后重新种植到 24孔培养板中 ( 5 1 04个细胞 /0.25毫升 / 孔）， 用甲基纤维素培养体系 ( Xi等, Br. J. Haematol, 93 : 490, 1 996)培养 14天 然后计算各种造血祖细胞的集落数， 结果见表 6。 表 6 TSP和 TSP 1 - 1 74对化疗药物处理过的脐血干细胞存活率的影响
Figure imgf000011_0001
本实验共做三次， ¾中结果为总的细胞数. 表中数据均以平均 ii ±标准误¾示.
* ¾/T; L-j PBS + PBS组比较， p < 0.05 . 实施 ί列 7
预防和治疗抗肿瘤治疗诱发的血细胞减少症的方法
在患者接受抗肿瘤治疗 (化疗或放疗)前或早期， 经静脉滴注. 肌肉或皮下注射 或口服途径， 给患者使用有效剂量的本发明的药物组合物， 以使患者体内正常的 造血干细胞和袓细胞预先反应， 从而对抗肿瘤治疗不敏感并因此得到保护。 由此 使抗肿瘤治疗诱发的血细胞减少现象得以避免或明显减轻. 本发明药物组合物的 使用剂量应由主治医生根据患者体重， 血样分析结果和抗肿瘤治疗的使用剂量等 而定。 如将本发明的药物组合物与有效量的血细胞生长因子如 G - CSF和 ΤΡΟ 等合用， 则可更有效并及时地预防和治疗抗肿瘤治疗诱发的血细胞减少症。 实施例 8
临床治疗各种血小板增多症的方法
将有效剂量的本发明的药物组合物经静脉滴注、 肌肉或皮下注射或口服途 径给血小板增多症患者使用。 每日一次或数次， 或数日一次。 用药时间的长短应 参考用药后患者血小板数量和功能情况， 通常以用到患者血小板数和功能正常或 接近正常为一疗程， 一般需间歇治疗 2 - 3疗程。 上述具体实施例的描述揭示了本发明的一般性质， 从而使本领域的技术人员 应用目前的知识， 为 Γ各种应用， 能容易地修饰和 /或改动这些具 ί本实施 ί列， 代之 以其它形式的实施 ί列而不背离本发明的基本构思和内涵， 这些改动和修饰因此应 属于这些公开例子的等价事 ί列的范围， 应理解， 此处所采用的措辞和术 i吾是为丁 便于描述而不起限制作用。

Claims

权 利 要 求 书
1 . 用于保护造血前体细胞的药物组合物， 其特征在于， 含有作为有效成 分的凝血酶致敏蛋白和 /或其活性片段和 /或它们的突变蛋白以及药学上可接受 的载体
2. 如权利要求 1 所述的药物组合物， 其特征在于， 所述活性片段至少含 凝血酶致敏蛋白的 N末端的肝素结合区域。
3. 如权利要求 2 所述的药物组合物， 其特征在于， 所述活性片段至少含 凝血酶致敏蛋白 N-末端第 1至第 174个氨基酸残基。
4. 如权利要求 3 所述的药物组合物， 其特征在于， 所述活性片段由凝血 酶致敏蛋白 N-末端第 1至第 174个氨基酸残基组成，
5. 如权利要求 1 所述的药物组合物， 其特征在于， 所述突变蛋白为与天 然凝血酶致敏蛋白具有 80 %以上的同源性的多肽。
6. 如权利要求 1所述的药物组合物，其特征在于，所述突变蛋白为与 TSP t - 174具有 80 %以上的同源性的多肽。
7. 权利要求 1 所述的药物组合物在体外扩增造血前体细胞中的应用， 其 特征在于， 将药物组合物与造血前体细胞共孵育。
8. 如权利要求 7所述的的应用， 其特征在于， 将药物组合物和造血细胞 生长因子与造血细胞共孵育
9. 如权利要求 7 所述的应用， 其特征在于， 所述造血前体细胞为造血多 能干细胞和 /或各系列定向祖细胞.
1 0. 如权利要求 9所述的应用， 其特征在于， 所述造血多能干细胞和 /或各 系列定向祖细胞包括高增殖潜能集落形成细胞、 混合集落形成单位、 巨核细胞 集落形成单位、 粒单细胞集落形成单位和红细胞集落形成单位。
1 1 . 如权利要求 7所述的应用， 其特征在于， 所述造血前体细胞为来自人 脐带血或骨髓、 末梢血和胚肝的造血前体细胞。
12. 如权利要求 8所述的应用， 其特征在于， 所述造血细胞生长因子为选 自白细胞介素 3、 白细胞介素 6、 白细胞介素 U、 白细胞介素！ 3、 粒单细胞 集落刺激因子、 粒细胞集落刺激因子、 干细胞因子、 血小板 生成素、 巨核细 胞生成素以及粘多糖中的一种或多种，
1 3. 临床治疗由化疗或放疗诱发的血细胞减少症的方法， 其特征在于， 给 接受抗肿瘤药物的患者使用足够量的权利要求 1所述的药物组合物。
14. 临床治疗血小板增多症的方法， 其特征在于， 给患者使用足够量的权 利要求 1所述的药物组合物。
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