WO2020249132A1 - 一种葡聚糖在制备药物的应用 - Google Patents

一种葡聚糖在制备药物的应用 Download PDF

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WO2020249132A1
WO2020249132A1 PCT/CN2020/096223 CN2020096223W WO2020249132A1 WO 2020249132 A1 WO2020249132 A1 WO 2020249132A1 CN 2020096223 W CN2020096223 W CN 2020096223W WO 2020249132 A1 WO2020249132 A1 WO 2020249132A1
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glucan
preparation
cancer
tumor
pharmaceutical composition
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French (fr)
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杨金波
于广利
宋巧玲
吴丽娟
赵俊
吕友晶
赵晨阳
管华诗
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Qingdao Marine Biomedical Research Institute Co Ltd
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Qingdao Marine Biomedical Research Institute Co Ltd
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Priority to AU2020291757A priority patent/AU2020291757B2/en
Priority to JP2021574203A priority patent/JP7358516B2/ja
Priority to CN202080043041.2A priority patent/CN114026129B/zh
Priority to US17/618,311 priority patent/US12357654B2/en
Priority to EP20822937.7A priority patent/EP3985031B1/en
Application filed by Qingdao Marine Biomedical Research Institute Co Ltd filed Critical Qingdao Marine Biomedical Research Institute Co Ltd
Priority to KR1020227001244A priority patent/KR102837755B1/ko
Publication of WO2020249132A1 publication Critical patent/WO2020249132A1/zh
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Priority to JP2023166009A priority patent/JP7668318B2/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • 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
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A61K36/03—Phaeophycota or phaeophyta (brown algae), e.g. Fucus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Definitions

  • the present invention relates to the field of marine medicine, in particular to an application of ⁇ -1,3/1,6-glucan, which is used to prepare pharmaceutical compositions or The preparation can be used to enhance the anti-tumor effect of immunotherapy, radiotherapy, or chemotherapy, and treat leukopenia and/or thrombocytopenia.
  • Beta-glucan is a long-chain polysaccharide composed of glucose from the cell walls of fungi, yeast, certain bacteria and plants.
  • the main chain of these polymers contains linear ⁇ -D-(1,3) glucosyl units, which are substituted by side chains connected by ⁇ -D-(1,6) glucosyl units at O-6.
  • the molecular weight is And the distribution can vary.
  • ⁇ -glucan is considered to be pathogen-associated molecular patterns (PAMPs) that regulate the host immune response by triggering innate immune cells such as neutrophils, macrophages and granulocytes.
  • PAMPs pathogen-associated molecular patterns
  • ⁇ -1,3-glucans on the market mostly come from terrestrial organisms such as barley, oats, edible fungi (Lentinus edodes, Grifola frondosa, Schizophyllum), yeast, etc. Due to the different sources of raw materials, the ⁇ -1, The molecular weight, connection method and branching degree of 3-glucan are very different, and the quality is difficult to control.
  • the ⁇ -glucan for injection medicine is mainly derived from mushrooms, which is a kind of ⁇ -1,6-branched ⁇ -1,3-glucan has poor water solubility due to its molecular weight of 400-800kDa.
  • Cancer immunotherapy which stimulates the immune system exogenously to treat cancer, has become a promising cancer treatment strategy.
  • inhibitors of immune checkpoints such as cytotoxic T lymphocyte antigen 4 (CTLA4), programmed cell death receptor 1 (PD-1) and its ligand (PD-L1), etc., by blocking immunosuppressive signals and enhancing autonomy Anti-tumor response has achieved great success in a variety of cancers.
  • CTL4 cytotoxic T lymphocyte antigen 4
  • PD-1 programmed cell death receptor 1
  • PD-L1 its ligand
  • Cancer immunotherapy targeting PD-1 has achieved great success by regulating the immune environment to induce a more effective anti-tumor response. However, only some patients will benefit from single-agent PD-1 blockade. Chemoradiation is the most commonly used method of cancer treatment. However, due to the high toxicity of radiotherapy and chemotherapy, cancer patients will have various side effects during radiotherapy and chemotherapy. The most common one may be low white blood cells, leading to infections that endanger the lives of patients. .
  • the purpose of the present invention is a use of ⁇ -1,3/1,6-glucan, which includes anti-tumor, increase white blood cell and resist platelet reduction, said ⁇ -1,3/1,6 -Glucan has the characteristics of good water solubility and high safety.
  • the first aspect of the present invention provides an application of ⁇ -1,3/1,6-glucan, characterized in that the ⁇ -1,3/1,6-glucan is from Antarctica For brown algae, the ⁇ -1,3/1,6-glucan is used to prepare a pharmaceutical composition or preparation, and the pharmaceutical composition or preparation is used to treat leukopenia and/or thrombocytopenia.
  • the H1 signal of the ⁇ -1,3/1,6-glucan is located in the 4.40-4.64 ppm region in 1H-NMR, and the C1 signal is located in the 102.4-102.67 ppm region in 13C-NMR. .
  • the Antarctic brown algae is sea velvet, sea bamboo shoot, or Leissonella, Durvillaea Antarctica.
  • the ⁇ -1,3/1,6-glucan is a ⁇ -glucan represented by formula (I) and/or formula (II),
  • n is an integer selected from 1-20 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 Or 20), R is H and/or not more than 4 glucose residues (such as 1, 2, 3 or 4 glucose residues).
  • R in the structure of formula (I) or formula (II) is one or more of the structure of formula (III) or formula (IV) or formula (V) or formula (VI), among them
  • the molecular weight of the ⁇ -1,3/1,6-glucan is 1-50kDa; preferably, it is 2-30kDa; more preferably, it is 2-10kDa; most preferably , Is 4-7kDa.
  • the specific rotation of the ⁇ -1,3/1,6-glucan is not less than -15.0°; preferably, -15°-25°; more preferably, -15 ⁇ -21°.
  • the ultraviolet full-wavelength scanning pattern of the ⁇ -1,3/1,6-glucan has no obvious absorption in the wavelength range of 300 to 900 nm; more preferably, it is in the wavelength range of 230 to 900 nm No obvious absorption.
  • the ultraviolet full-wavelength scanning pattern of the ⁇ -1,3/1,6-glucan has no absorption peak in the wavelength range of 260-280 nm.
  • the side chain length of the ⁇ -1,3/1,6-glucan ⁇ 5.
  • the ⁇ -1,3/1,6-glucan can be prepared by the following steps:
  • step (4) is purified as follows: dissolve the crude polysaccharide in step (3) with distilled water, use distilled water and aqueous sodium chloride as mobile phases, separate and purify by anion exchange resin, and detect by sulfuric acid phenol method. The water-eluted fractions are collected, concentrated under reduced pressure, and freeze-dried to obtain the ⁇ -1,3/1,6-glucan.
  • the anion resin separation and purification is strong anion ion resin separation and purification.
  • the anion resin separation and purification is: firstly, the separation and purification by strong anion resin and then by weak anion resin; or the separation and purification by weak anion ion resin and then the separation and purification by strong anion ion resin.
  • the strong anionic resin is an anionic resin containing quaternary ammonium groups.
  • the weak anionic resin is an anionic resin containing diethylaminoethyl.
  • the present invention provides an application of ⁇ -1,3/1,6-glucan, characterized in that the white blood cells are lymphocytes.
  • the lymphocytes are B cells and/or T cells.
  • the application of the ⁇ -1,3/1,6-glucan is characterized in that the pharmaceutical composition or preparation also has an anti-tumor effect.
  • the application of the ⁇ -1,3/1,6-glucan is characterized in that the pharmaceutical composition or preparation can also be used in combination with immune checkpoint drugs.
  • the application of the ⁇ -1,3/1,6-glucan is characterized in that the immune checkpoint drug is selected from the group consisting of programmed death 1 protein (PD-1) antagonist Antagonist, or PD-L1 antagonist, or cytotoxic T lymphocyte antigen (CTLA-4) antagonist, or lymphocyte activation gene-3 (LAG-3) antagonist, or T cell immunoglobulin-3 (TIM- 3) Antagonist, or T cell immunoglobulin and ITIM domain protein (TIGIT) antagonist.
  • PD-1 protein PD-1 antagonist
  • CTL-3 lymphocyte activation gene-3
  • T cell immunoglobulin-3 TIM- 3 Antagonist
  • T cell immunoglobulin and ITIM domain protein (TIGIT) antagonist T cell immunoglobulin and ITIM domain protein
  • the application of the ⁇ -1,3/1,6-glucan is characterized in that the immune checkpoint drug is selected from anti-PD-1 antibody, anti-PD-L1 antibody .
  • the application of the ⁇ -1,3/1,6-glucan in the preparation of drugs for the treatment of white blood cells and thrombocytopenia is characterized in that the anti-PD-1 antibody or PD-
  • the L1 antibody is selected from Durvalumab, Atezolizumab, Nivolumab, BMS202, Spartalizumab, and Camrelizumab.
  • the combination of the pharmaceutical composition or preparation and the programmed death 1 protein (PD-1) or PD-L1 antagonist is administered simultaneously, sequentially or separately.
  • the application of the ⁇ -1,3/1,6-glucan is characterized in that the pharmaceutical composition or preparation can also be used in combination with at least one chemotherapeutic agent.
  • the application of the ⁇ -1,3/1,6-glucan is characterized in that the chemotherapeutic agent is selected from cytotoxic chemotherapeutic agents.
  • the application of the ⁇ -1,3/1,6-glucan is characterized in that the chemotherapeutic agent is selected from the group consisting of anthracyclines, 5-Fu, and alkaloids One of them.
  • the application of the ⁇ -1,3/1,6-glucan is characterized in that the chemotherapeutic agent is selected from one of cisplatin and carboplatin.
  • the application of the ⁇ -1,3/1,6-glucan, the combined application of the pharmaceutical composition or preparation and the chemotherapeutic agent is simultaneous, sequential or separate administration .
  • the application of the ⁇ -1,3/1,6-glucan is characterized in that the pharmaceutical composition or preparation can also be used in combination with radiotherapy.
  • the application of the ⁇ -1,3/1,6-glucan, the combined application of the pharmaceutical composition or preparation and radiotherapy is simultaneous, sequential or separate administration.
  • the application of the ⁇ -1,3/1,6-glucan is characterized in that the pharmaceutical composition or preparation is used to treat cancer in an individual.
  • the cancer is one or more of melanoma, colorectal cancer, lung cancer, kidney cancer, liver cancer, and breast cancer.
  • the pharmaceutical composition or preparation contains a safe and effective amount of ⁇ -1,3/1,6-glucan, and a pharmaceutically acceptable carrier or excipient.
  • Figure 1 shows the effect of ⁇ -1,3/1,6-glucan in combination with anti-PD-1 antibody on B16 tumor model.
  • Figure 2 shows the effect of ⁇ -1,3/1,6-glucan in combination with anti-PD-1 antibody on T lymphocytes in B16 syngeneic tumor model.
  • Figure 3 shows the effect of ⁇ -1,3/1,6-glucan in combination with anti-PD-1 antibody on platelets of B16 syngeneic tumor model.
  • Figure 4 shows the effect of ⁇ -1,3/1,6-glucan in combination with anti-PD-1 antibody on lymphocytes of B16 syngeneic tumor model.
  • Figures 5-7 show the anti-tumor effect of ⁇ -1,3/1,6-glucan combined with radiotherapy on the B16 tumor model.
  • Figures 8-15 show the effect of ⁇ -1,3/1,6-glucan combined with radiotherapy on leukocytes of B16 syngeneic tumor model.
  • Figure 16 shows the effect of ⁇ -1,3/1,6-glucan combined with radiotherapy on the peripheral blood of the B16 tumor model.
  • Figures 17 to 21 show the effect of ⁇ -1,3/1,6-glucan combined with chemotherapy on the B16 syngeneic tumor model.
  • PLT platelet count
  • NEUT neutrophils
  • LYMPH lymphocytes
  • MONO monocytes WBC means white blood cells.
  • strong anion resin and “strong anion exchange resin” can be used interchangeably and refer to anionic resins containing strong reactive groups such as quaternary amine groups.
  • weak anion resin and “weak anion exchange resin” can be used interchangeably to refer to anionic resins containing weaker reactive groups such as diethylaminoethyl.
  • the ⁇ -1,3/1,6-glucan of the present invention is derived from Antarctic brown algae from the ocean. It has the characteristics of small molecular weight, good water solubility, and high safety, and it can resist leukopenia and thrombocytopenia It has a good effect especially on the reduction of T lymphocytes and B lymphocytes caused by tumor treatment.
  • Example 1 The effect of combined use of ⁇ -1,3/1,6-glucan and anti-PD-1 antibody on B16 syngeneic tumor model
  • a cell suspension of 3 ⁇ 10 5 mouse melanoma cell lines B16 (gifted by PerkinElmer) was injected subcutaneously into C57BL/6J mice (female, 6-8 weeks old, purchased from Jinan Pengyue Experimental Animal Company). Approximately 4 days after the tumor was implanted, the B16 tumor grew to be palpable before the drug administration experiment was performed. Mice were divided into four groups. They were treated with solvent or ⁇ -1,3/1,6-glucan (4mg/kg, intravenous injection, twice a week) alone, or with anti-PD-1 antibody (anti-mouse PD -1 antibody was purchased from BioxCell, 200 ⁇ g per mouse, once a week, intravenous injection) combined treatment mice. Assess tumor volume and record tumor weight. After the mice were sacrificed, blood and tumor samples were collected for flow cytometric analysis.
  • Blood samples were collected by cardiac puncture, collected in EDTA-anticoagulation tubes, 50 ⁇ l whole blood was taken for blood analysis, 50 ⁇ l whole blood was taken for red blood cell lysis (erythrocyte lysis buffer, Meltenyi), the remaining whole blood was centrifuged at 3500rpm for 7 minutes, and plasma was taken Store at -80°C. Take the spleen and thymus to weigh and record. Take the mouse subcutaneous tumor, take approximately 0.2-0.5 g of tumor tissue, and obtain a single cell suspension from the tumor sample through the mouse tumor dissociation kit (Meltenyi). The lysed red blood cells and the dissociated tumor cell suspension are subjected to subsequent processing and flow cytometry
  • CD11b-PE, CD4- BV510, CD8-PerCP-Cy5.5, CD19-APC, CD3-FITC, CD206-PE-Cy7, Ly6C-APC incubate and stain for 30 minutes.
  • FACS Arial III BD Biosciences. The specific process is to delimit the cell population in the FSC/SSC quadrant, delimit the single cell population through FSC-H and FSC-A, delineate the corresponding immune cells through immune cell surface markers, and calculate the cell ratio or relative concentration.
  • ⁇ -1,3/1,6-glucan and anti-PD-1 antibodies can synergistically increase tumor infiltration of pro-inflammatory macrophages, reduce the percentage of immunosuppressive TAM, and increase acquired immune cells T and B The proportion of cells, thereby establishing a more pro-inflammatory and anti-tumor tumor microenvironment.
  • Example 2 The effect of ⁇ -1,3/1,6-glucan in combination with anti-PD-1 antibody on platelets in B16 homologous tumor model
  • the experimental method was the same as that in Example 1. On the 14th day, blood was taken from the heart after the animal was sacrificed, put into an EDTA-anticoagulation tube, and mixed, 50 ⁇ l was taken to detect the platelet concentration with a blood analyzer. As shown in Figure 3, PD-1 antibody alone can reduce platelet concentration, while ⁇ -1,3/1,6-glucan alone does not affect platelet concentration, but combined use with PD-1 antibody can reverse PD The platelet reduction side effect of -1 antibody.
  • Example 3 The effect of combined use of ⁇ -1,3/1,6-glucan and anti-PD-1 antibody on the number of lymphocytes in peripheral blood
  • the experimental method was the same as that in Example 1. On the 14th day, the animal was sacrificed and the blood was taken from the heart, put in an EDTA-anticoagulation tube, and mixed, 50 ⁇ l of whole blood was taken, and 5 ⁇ l of GFP microspheres were added to each sample for red blood cell lysis.
  • Figure 4 The experimental results show that the PD-1 antibody alone can increase the relative concentration of CD3, CD4 and CD8 in the blood, while the combination of 8mg/kg ⁇ -1,3/1,6-glucan and PD-1 antibody can Further increase the relative concentration of CD3, CD4, CD8 and CD19 in the blood, indicating that ⁇ -1,3/1,6-glucan can further enhance the immune activation effect of PD-1 antibody.
  • the cell suspension of 3 ⁇ 10 5 mouse melanoma cell lines B16 (gifted by PerkinElmer) was injected subcutaneously into C57BL/6J mice (female, 6-8 weeks old, purchased from Jinan Pengyue Experimental Animal Company) (Overwijk&Restifo, 2001). Approximately 5 days after tumor implantation, local radiotherapy (tumor injection area, 10Gy) was used. On the 6th and 14th days, administer solvent or ⁇ -1,3/1,6-glucan (injected into the tail vein, once a week), during which the tumor volume and the weight of the mouse were measured, and the animals were sacrificed on the 17th Afterwards, the tumor quality was tested.
  • radiotherapy can significantly inhibit the growth of tumor cells, and ⁇ -1,3/1,6-glucan can enhance the anti-tumor effect of radiotherapy.
  • the cell suspension of 3 ⁇ 10 5 mouse melanoma cell lines B16 (gifted by PerkinElmer) was injected subcutaneously into C57BL/6J mice (female, 6-8 weeks old, purchased from Jinan Pengyue Experimental Animal Company) (Overwijk&Restifo, 2001). Approximately 5 days after tumor implantation, local radiotherapy (tumor injection area, 10Gy) was used. On the 6th and 14th days, the solvent or ⁇ -1,3/1,6-glucan was administered (tail vein injection, once a week). On the 17th day, after the animals were sacrificed, the tumor quality was measured. After the animals were sacrificed, the blood was taken from the heart, put into an EDTA-anticoagulation tube, and after mixing, 50 ⁇ l of whole blood was taken, and the concentration of immune cells was detected by a blood analyzer.
  • radiotherapy reduces the concentration of immune cells in the animal body while anti-tumor, which is not good for the survival state of mice, and ⁇ -1,3/1,6-glucan can be used as an immunostimulant to stimulate Immune cells can increase the concentration of immune cells and effectively reduce the side effects of radiotherapy.
  • the cell suspension of 3x105 mouse melanoma cell line B16 (gifted by PerkinElmer) was injected subcutaneously into C57BL/6J mice (female, 6-8 weeks old, purchased from Jinan Pengyue Experimental Animal Company) (Overwijk & Restifo, 2001 ). About 2 days after tumor implantation, intraperitoneal injection of carboplatin (30mg/kg, twice a week) and tail vein injection of BG136 (4mg/kg) or lentinan LNT (2mg/kg), the tumor volume was measured during the administration, on the 15th day After the animals were sacrificed, the tumor quality was tested. After the animals were sacrificed, the blood was taken from the heart, put into an EDTA-anticoagulation tube, and after mixing, 50 ⁇ l of whole blood was taken, and the concentration of immune cells was detected by a blood analyzer.
  • BG136 can enhance the tumor suppressive effect of carboplatin, and can stimulate the immune response, reverse the immune suppression after carboplatin administration, and the reduction of platelets.
  • ⁇ -1,3/1,6-glucan Since immune cells play an important role in a variety of tumor cell types, the anti-tumor, whitening and platelet-increasing effects of ⁇ -1,3/1,6-glucan will be effective in a variety of tumor cells. Play a role (for example, lung cancer, kidney cancer, liver cancer, breast cancer, etc.)
  • ⁇ -1,3/1,6-glucan may inhibit tumor cell metastasis and occurrence.

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Abstract

一种葡聚糖在制备药物的应用。所述β-1,3/1,6-葡聚糖用于制备药物组合物或制剂，所述的药物组合物或制剂可以增强免疫治疗，放疗，或化疗的抗肿瘤效果，用于治疗白细胞和/或血小板减少。

Description

一种葡聚糖在制备药物的应用 技术领域
本发明涉及海洋药物领域，具体地涉及一种β-1,3/1,6-葡聚糖的应用，所述β-1,3/1,6-葡聚糖用于制备药物组合物或制剂，可以用于增强免疫治疗，放疗，或化疗的抗肿瘤效果，治疗白细胞和/或血小板减少。
背景技术
β-葡聚糖是由来自真菌，酵母，某些细菌和植物的细胞壁的葡萄糖构成的长链多糖。这些聚合物的主链包含线性β-D-(1,3)葡糖基单元，在O-6处被β-D-(1,6)葡糖基单元连接的侧链取代，其分子量大小和分布可以变化。
β-葡聚糖被认为是通过引发先天免疫细胞如中性粒细胞，巨噬细胞和粒细胞来调节宿主免疫反应的病原体相关分子模式(PAMPs)。目前，市场上β-1,3-葡聚糖多来自于大麦、燕麦、食用真菌(香菇，灰树花，裂褶菌)、酵母等陆生生物，由于原料来源不同，所得β-1,3-葡聚糖的分子量、连接方式及分支度等差别很大，质量很难控制，如注射药用的β-葡聚糖主要来源于香菇，是一种具有β-1,6-分支的β-1,3-葡聚糖，由于其分子量高达400-800kDa，水溶性较差。
通过外源性刺激免疫系统来治疗癌症的癌症免疫疗法，已经成为一种有前景的癌症治疗策略。例如，细胞毒性T淋巴细胞抗原4(CTLA4)，程序性细胞死亡受体1(PD-1)及其配体(PD-L1)等免疫检查点的抑制剂，通过阻碍免疫抑制信号并增强自主抗肿瘤反应，在多种癌症取得了巨大成功。
靶向PD-1的癌症免疫疗法通过调节免疫环境以引起更有效的抗肿瘤反应，取得了巨大成功，然而只有一部分患者会受益于单药PD-1阻断。放化疗是最常用的癌症治疗方法，但由于放化疗毒性大等特点，癌症患者在接受放化疗期间会出现各种不同程度的副作用，其中最常见的可能是导致白细胞低，导致感染危及患者生命。
发明内容
本发明的目的是一种β-1,3/1,6-葡聚糖的用途，所述用途包括抗肿瘤，升高白细胞和抵抗血小板降低，所述的β-1,3/1,6-葡聚糖具有水溶性好，安全性高的特点。
本发明的第一方面，提供了一种β-1,3/1,6-葡聚糖的应用，其特征在于，所述的β-1,3/1,6-葡聚糖来源于南极褐藻，所述β-1,3/1,6-葡聚糖用于制备药物组合物或制剂，所述的药物组合物或制剂用于治疗白细胞和/或血小板减少。
在另一优选例中，所述的β-1,3/1,6-葡聚糖在1H-NMR中H1信号位于4.40-4.64ppm区域，在13C-NMR中C1信号位于102.4-102.67ppm区域。
在另一优选例中，所述南极褐藻为海茸、海笋或雷松藻，南极公牛藻(Durvillaea Antarctica)。
在另一优选例中，所述的β-1,3/1,6-葡聚糖为如式(I)和/或式(II)所示的β-葡聚糖，
Figure PCTCN2020096223-appb-000001
其中，n为选自1-20的整数(如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)，R为H和/或不超过4个葡萄糖残基(如1、2、3或4个葡萄糖残基)。
在另一优选例中，所述式(I)或式(II)结构中的R为式(III)或式(Ⅳ)或式(V)或式(Ⅵ)结构的一种或几种，其中
式(III)：Glcβ1-；
式(Ⅳ)：Glcβ1-3Glcβ1-或者Glcβ1-6Glcβ1-；
式(V)：Glcβ1-3Glcβ1-3Glcβ1-或者Glcβ1-6Glcβ1-3Glcβ1-或者
Glcβ1-3Glcβ1-6Glcβ1-或者Glcβ1-6Glcβ1-6Glcβ1-；
式(Ⅵ)：
Glcβ1-3Glcβ1-3Glcβ1-3Glcβ1-或者
Glcβ1-6Glcβ1-3Glcβ1-3Glcβ1-或者
Glcβ1-3Glcβ1-6Glcβ1-3Glcβ1-或者
Glcβ1-3Glcβ1-3Glcβ1-6Glcβ1-或者
Glcβ1-6Glcβ1-6Glcβ1-3Glcβ1-或者
Glcβ1-6Glcβ1-3Glcβ1-6Glcβ1-或者
Glcβ1-3Glcβ1-6Glcβ1-6Glcβ1-或者
Glcβ1-6Glcβ1-6Glcβ1-6Glcβ-。
在另一优选例中，所述的β-1,3/1,6-葡聚糖分子量为1-50kDa；较佳地，为2～30kDa；更佳地，为2-10kDa；最佳地，为4-7kDa。
在另一优选例中，所述β-1,3/1,6-葡聚糖的比旋度不低于-15.0°；较佳地，-15°～25°；更佳地，-15～-21°。
在另一优选例中，所述β-1,3/1,6-葡聚糖的紫外全波长扫描图谱在300～900nm波长范围内无明显吸收；更优选地，在230～900nm波长范围内无明显吸收。
在另一优选例中，所述β-1,3/1,6-葡聚糖的紫外全波长扫描图谱在260～280nm波长范围内无吸收峰。
在另一优选例中，所述的β-1,3/1,6-葡聚糖侧链长度≤5.
在另一优选例中，所述的β-1,3/1,6-葡聚糖可由以下步骤制备：
(1)脱脂：将南极褐藻烘干、粉碎，有机溶剂浸泡、搅拌，得脱脂藻粉；
(2)水提：所述脱脂藻粉室温下用水搅拌提取得水提液；
(3)分级：将步骤(2)所得水提液离心，在离心获得的上清液中加入1～3mol/L氯化钙水溶液；搅拌后离心，取上清液进行透析或超滤脱盐，减压浓缩干燥获得粗多糖；
(4)纯化：将步骤(3)所述粗多糖用蒸馏水溶解，以蒸馏水和氯化钠水溶液为流动相，经过阴离子交换树脂分离纯化，收集水洗脱组分，减压浓缩、冻干，获得所述β-1,3/1,6-葡聚糖。
在另一优选例中，步骤(4)纯化为：将步骤(3)所述粗多糖用蒸馏水溶解，以蒸馏水和氯化钠水溶液为流动相，经过阴离子交换树脂分离纯化，硫酸苯酚法检测，收集水洗脱组分，减压浓缩、冻干，获得所述β-1,3/1,6-葡聚糖。
在另一优选例中，所述的阴离子树脂分离纯化为强阴离子子树脂分离纯化。
在另一优选例中，所述的阴离子树脂分离纯化为：先经强阴离子树脂分离纯化再经弱阴离子树脂分离纯化；或者经弱阴离子子树脂分离纯化再经强阴离子子树脂分离纯化。
在另一优选例中，所述的强阴离子树脂为含季铵基团的阴离子树脂。
在另一优选例中，所述的弱阴离子树脂为含二乙基氨基乙基的阴离子树脂。
在另一优选例中，本发明提供了一种β-1,3/1,6-葡聚糖的应用，其特征在于，所述的白细胞为淋巴细胞。
在另一优选例中，所述的淋巴细胞为B细胞和/或T细胞。
在另一优选例中，所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的药物 组合物或制剂还具有抗肿瘤的作用。
在另一优选例中，所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的药物组合物或制剂还可与免疫检查点药物联合应用。
在另一优选例中，所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的免疫检查点药物选自程序性死亡1蛋白(PD-1)拮抗剂，或PD-L1拮抗剂，或细胞毒性T淋巴细胞抗原(CTLA-4)拮抗剂，或淋巴细胞活化基因-3(LAG-3)拮抗剂，或T细胞免疫球蛋白-3(TIM-3)拮抗剂，或T细胞免疫球蛋白和ITIM结构域蛋白(TIGIT)拮抗剂。
在另一优选例中，所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的免疫检查点药物选自抗PD-1抗体,抗PD-L1抗体。
在另一优选例中，所述的β-1,3/1,6-葡聚糖在制备治疗白细胞和血小板减少药物中的应用，其特征在于，所述的抗PD-1抗体或PD-L1抗体选自度伐单抗(Durvalumab)，阿特朱单抗(Atezolizumab)，纳武利尤单抗(Nivolumab)，BMS202，斯巴达珠单抗(Spartalizumab)，卡瑞利珠单抗(Camrelizumab)。
在另一优选例中，所述的药物组合物或制剂与程序性死亡1蛋白(PD-1)或PD-L1的拮抗剂联合应用为同时地、依次地或单独地施用。
在另一优选例中，所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的药物组合物或制剂还可与至少一种化学治疗剂联合应用。
在另一优选例中，所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的化学治疗剂选自细胞毒类化学治疗剂。
在另一优选例中，所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的化学治疗剂选自蒽环类，5-Fu类，生物碱类中的一种。
在另一优选例中，所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的化学治疗剂选自顺铂，卡铂中的一种。
在另一优选例中，所述的β-1,3/1,6-葡聚糖的应用，所述的药物组合物或制剂与化学治疗剂联合应用为同时地、依次地或单独地施用。
在另一优选例中，所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的药物组合物或制剂还可与放射疗法联合应用。
在另一优选例中，所述的β-1,3/1,6-葡聚糖的应用，所述的药物组合物或制剂与放射疗法联合应用为同时地、依次地或单独地施用。
在另一优选例中，所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述药物组 合物或制剂用于治疗个体的癌症。
在另一优选例中，所述的癌症为黑色素瘤、结直肠癌、肺癌、肾癌、肝癌、乳腺癌中的一种或多种。
在另一优选例中，所述的药物组合物或制剂，含有安全有效量β-1,3/1,6-葡聚糖，以及药学上可接受的载体或赋形剂。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示β-1,3/1,6-葡聚糖与抗PD-1抗体联合使用对B16同系肿瘤模型的作用。
图2显示β-1,3/1,6-葡聚糖与抗PD-1抗体联合使用对B16同系肿瘤模型的T淋巴细胞的作用。
图3显示β-1,3/1,6-葡聚糖与抗PD-1抗体联合使用对B16同系肿瘤模型的血小板的作用。
图4显示β-1,3/1,6-葡聚糖与抗PD-1抗体联合使用对B16同系肿瘤模型的淋巴细胞的作用。
图5-7显示β-1,3/1,6-葡聚糖与放疗联合使用对B16同系肿瘤模型的抗肿瘤作用。
图8-15显示β-1,3/1,6-葡聚糖与放疗联合使用对B16同系肿瘤模型的白细胞的作用。
图16显示β-1,3/1,6-葡聚糖与放疗联合使用对B16同系肿瘤模型的外周血的作用。
图17至21显示β-1,3/1,6-葡聚糖与化疗联合使用对B16同系肿瘤模型的作用。
具体实施方式
术语
除非另有定义，否则在说明书和权利要求书中所使用的下述术语具有的含义为所属领域技术人员通常理解的涵义。除非另有说明，本文全文引用的所有专利、 专利申请、公开材料通过引用方式整体并入本文。本发明中PLT,血小板计数；NEUT，中性粒细胞；LYMPH,淋巴细胞；MONO单核细胞；WBC，白细胞。
在本文中，术语“强阴离子树脂”和“强阴离子交换树脂”可以互换使用，是指含有较强的反应基如季胺基团等的阴离子树脂。
在本文中，术语“弱阴离子树脂”和“弱阴离子交换树脂”可以互换使用，是指含有较弱的反应基如二乙基氨基乙基的阴离子树脂。
本发明的主要优点
(1)本发明所述的β-1,3/1,6-葡聚糖，来源于海洋的南极褐藻，具有分子量小，水溶性好，安全性高的特点，具有抵抗白细胞降低和血小板减少的作用，尤其是对肿瘤治疗导致的T淋巴细胞和B淋巴细胞减少具有良好效果。
下面结合具体实施例，进一步阐述本发明，结合附图阅读本发明的具体实施例后，本发明的其他优点和特点将变得更加清晰。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
实施例1.β-1,3/1,6-葡聚糖与抗PD-1抗体联合使用对B16同系肿瘤模型的作用
将3x10 5个小鼠黑素瘤细胞系B16(PerkinElmer馈赠)的细胞悬浮液皮下注射到C57BL/6J小鼠(雌性，6-8周龄，购买自济南朋悦实验动物公司)中。植瘤大约4天后，B16肿瘤长到可触摸到后进行给药实验。小鼠分为四组，分别用溶剂或β-1,3/1,6-葡聚糖(4mg/kg，静脉注射，一周两次)单独，或者与抗PD-1抗体(抗小鼠PD-1抗体购自BioxCell，每只小鼠200μg，每周一次，静脉注射)联合处理小鼠。评估肿瘤体积并记录肿瘤重量。处死小鼠后，收集血液和肿瘤样本用于流式细胞分析。
通过心脏穿刺采集血液样本，收集到EDTA-抗凝管中，取50μl全血进行血液分析，取50μl全血进行红细胞裂解(红细胞裂解缓冲液，Meltenyi)，剩余全血3500rpm离心7分钟，取血浆-80℃保存。取脾脏和胸腺称重记录。取小鼠皮下肿瘤，取大约0.2-0.5g肿瘤组织，通过小鼠肿瘤解离试剂盒(Meltenyi)获得来自肿瘤样本的单细胞悬液。将红细胞裂解后的血细胞以及解离的肿瘤细胞悬液进行后续处理，以及流式细胞检测
用封闭缓冲液(20％FBS，1：100CD16/CD32抗体和1：100大鼠IgG)封闭单细胞悬液20分钟，在4℃下用相应的免疫细胞表面蛋白抗体(CD11b-PE,CD4-BV510, CD8-PerCP-Cy5.5,CD19-APC,CD3-FITC,CD206-PE-Cy7,Ly6C-APC)孵育染色30分钟。通过FACS Arial III(BD Biosciences)进行免疫细胞群体比例的分析。具体过程为在FSC/SSC象限圈定细胞群体，通过FSC-H和FSC-A圈定单细胞群体，通过免疫细胞表面标志物圈定相应免疫细胞，计算细胞比例或相对浓度。
如图1A和1B所示，用溶剂，β-1,3/1,6-葡聚糖(4mg/kg，静脉注射，每周两次)，抗PD-1抗体(200μg/小鼠，静脉注射，每周一次)，或β-1,3/1,6-葡聚糖和抗PD-1抗体联合处理B16同系肿瘤小鼠(第4天开始给药，第4天溶剂组，β-1,3/1,6-葡聚糖和PD-1联合用药组以及单独用药组，尾静脉注射，第7天溶剂组和PD-1组给溶剂，β-1,3/1,6-葡聚糖和β-1,3/1,6-葡聚糖和PD-1联合用药组给β-1,3/1,6-葡聚糖，第10天给药同第4天，第13天给药同第7天)，给药期间评估肿瘤体积，第14天小鼠处死，评估肿瘤重量等指标。结果表明β-1,3/1,6-葡聚糖增强了B16同系肿瘤模型中抗PD-1抗体诱导的肿瘤消退，和抗PD-1抗体的组合比单一治疗更有效地抑制肿瘤生长。在治疗期间小鼠体重和死亡没有明显变化，表明该组合疗法没有引起严重的毒性(图1C)。通过流式细胞仪分析血液和肿瘤中免疫细胞亚群表明，联合治疗增加了小鼠血液中CD19+细胞的百分比并降低了CD11b+细胞的百分比(图1D)。联合治疗还上调了外周血中促炎的单核细胞来源的巨噬细胞(CD11b+Ly6Chi，图1E)。检测肿瘤浸润免疫细胞发现，肿瘤内部有更多的髓样细胞(CD11b+)浸润，尤其是促炎性巨噬细胞(CD11b+Ly6Chi)(图1F)，与单独的抗PD-1抗体处理相比，联合治疗后免疫抑制性的肿瘤相关巨噬细胞TAM(CD11b+CD206+)的百分比也降低(图1G)。此外，还检测到β-1,3/1,6-葡聚糖和PD-1抗体联合应用增强了血液和肿瘤中CD4和CD8T细胞的比例(图2)。表明β-1,3/1,6-葡聚糖和抗PD-1抗体可协同增加促炎性巨噬细胞的肿瘤浸润，并降低免疫抑制性TAM的百分比，增加获得性免疫细胞T和B细胞的比例，从而建立更具促炎性和抗肿瘤性的肿瘤微环境。
实施例2.β-1,3/1,6-葡聚糖与抗PD-1抗体联合使用对B16同系肿瘤模型中血小板的作用
实验方法同实施例1，在第14天，动物处死后心脏取血，放入EDTA-抗凝管中，混匀后取50μl用血液分析仪检测血小板浓度。如图3所示，PD-1抗体单独使用会降低血小板浓度，而β-1,3/1,6-葡聚糖单独用药并不影响血小板浓度，但是与PD-1抗体联合使用可以逆转PD-1抗体的血小板降低副作用。
实施例3.β-1,3/1,6-葡聚糖与抗PD-1抗体联合使用对外周血中的淋巴细胞数目的影响
实验方法同实施例1，在第14天，动物处死后心脏取血，放入EDTA-抗凝管中，混匀后取50μl全血，每个样品加入5μl GFP微球，进行红细胞裂解。
用封闭缓冲液(20％FBS，1：100CD16/CD32抗体和1：100大鼠IgG)封闭单细胞悬液20分钟，在4℃下用相应的免疫细胞表面蛋白抗体(CD4-BV510,CD8-PerCP-Cy5.5,CD19-APC,CD3-FITC)孵育染色30分钟。通过FACS Arial III(BD Biosciences)进行免疫细胞群体比例的分析。具体过程为在FSC/SSC象限圈定细胞群体，通过FSC-H和FSC-A圈定单细胞群体，通过免疫细胞表面标志物圈定相应免疫细胞，计算相对免疫细胞浓度。
图4实验结果表明，PD-1抗体单独用药可以增加血液中的CD3，CD4和CD8的相对浓度，而8mg/kgβ-1,3/1,6-葡聚糖和PD-1抗体联合用药可以进一步增加血液中CD3，CD4，CD8和CD19的相对浓度，说明β-1,3/1,6-葡聚糖可以进一步增强PD-1抗体的免疫激活效果。
实施例4.β-1,3/1,6-葡聚糖应用于放疗后的小鼠模型
将3x10 5个小鼠黑素瘤细胞系B16(PerkinElmer馈赠)的细胞悬浮液皮下注射到C57BL/6J小鼠(雌性，6-8周龄，购买自济南朋悦实验动物公司)中(Overwijk&Restifo,2001)。植瘤大约5天后，采用局部放疗(肿瘤注射区域，10Gy)。第6和第14天，给药溶剂或β-1,3/1,6-葡聚糖(尾静脉注射，1周1次)，期间检测肿瘤体积和小鼠体重，第17天，动物处死后，检测肿瘤质量。
如图5-7所示，放疗可以显著抑制肿瘤细胞生长，而β-1,3/1,6-葡聚糖可以增强放疗的抗肿瘤效果。
将3x10 5个小鼠黑素瘤细胞系B16(PerkinElmer馈赠)的细胞悬浮液皮下注射到C57BL/6J小鼠(雌性，6-8周龄，购买自济南朋悦实验动物公司)中(Overwijk&Restifo,2001)。植瘤大约5天后，采用局部放疗(肿瘤注射区域，10Gy)。第6和第14天，给药溶剂或β-1,3/1,6-葡聚糖(尾静脉注射，1周1次),第17天，动物处死后，检测肿瘤质量。动物处死后心脏取血，放入EDTA-抗凝管中，混匀后取50μl全血，通过血液分析仪检测免疫细胞浓度。
如图8-15，放疗在抗肿瘤的同时降低了动物体内的免疫细胞浓度，对小鼠的生存 状态不利，而β-1,3/1,6-葡聚糖可以作为免疫刺激剂，刺激免疫细胞，起到提高免疫细胞浓度的效果，有效降低放疗的副作用。
在第14天，动物处死后心脏取血，放入EDTA-抗凝管中，混匀后取50μl全血，每个样品加入5μl GFP微球，进行红细胞裂解。
用封闭缓冲液(20％FBS，1：100CD16/CD32抗体和1：100大鼠IgG)封闭单细胞悬液20分钟，在4℃下用相应的免疫细胞表面蛋白抗体(CD4-BV510,CD8-PerCP-Cy5.5,CD19-APC)孵育染色30分钟。通过FACS Arial III(BD Biosciences)进行免疫细胞群体比例的分析。具体过程为在FSC/SSC象限圈定细胞群体，通过FSC-H和FSC-A圈定单细胞群体，通过免疫细胞表面标志物圈定相应免疫细胞，计算相对免疫细胞浓度。
图16结果表明，放疗降低了CD4，CD8和CD19的细胞浓度，而8mg/kgβ-1,3/1,6-葡聚糖可以提高相应细胞的浓度，从而起到刺激免疫系统的作用。
实施例5.β-1,3/1,6-葡聚糖联合化疗用于小鼠肿瘤模型
β-1,3/1,6-葡聚糖联合化疗对白细胞和血小板的作用
将3x105个小鼠黑素瘤细胞系B16(PerkinElmer馈赠)的细胞悬浮液皮下注射到C57BL/6J小鼠(雌性，6-8周龄，购买自济南朋悦实验动物公司)中(Overwijk&Restifo,2001)。植瘤大约2天后，腹腔注射卡铂(30mg/kg，每周两次)和尾静脉注射BG136(4mg/kg)或者香菇多糖LNT(2mg/kg)，给药期间检测肿瘤体积，第15天，动物处死后，检测肿瘤质量。动物处死后心脏取血，放入EDTA-抗凝管中，混匀后取50μl全血，通过血液分析仪检测免疫细胞浓度。
如图17-21所示BG136可以增强卡铂的肿瘤抑制效果，并且可以刺激免疫反应，逆转卡铂用药后的免疫抑制，以及血小板的降低。
实施例6.
由于免疫细胞在多种肿瘤细胞类型中均发挥着重要的作用，因此，β-1,3/1,6-葡聚糖的抗肿瘤以及升白和升血小板作用在多种肿瘤细胞中均会发挥作用(例如，肺癌，肾癌，肝癌，乳腺癌等)
并且免疫细胞对于肿瘤转移和发生也发挥调节作用，因此，β-1,3/1,6-葡聚糖可能抑制肿瘤细胞的转移和发生。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种β-1,3/1,6-葡聚糖的应用，其特征在于，所述的β-1,3/1,6-葡聚糖来源于南极褐藻，所述β-1,3/1,6-葡聚糖用于制备药物组合物或制剂，所述的药物组合物或制剂用于治疗白细胞和/或血小板减少。
  2. 根据权利要求1所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的白细胞为淋巴细胞。
  3. 根据权利要求1所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的药物组合物或制剂还可与免疫检查点药物联合应用。
  4. 根据权利要求1所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的免疫检查点药物选自程序性死亡1蛋白(PD-1)拮抗剂，或PD-L1拮抗剂，或细胞毒性T淋巴细胞抗原(CTLA-4)拮抗剂，或淋巴细胞活化基因-3(LAG-3)拮抗剂，或T细胞免疫球蛋白-3(TIM-3)拮抗剂，或T细胞免疫球蛋白和ITIM结构域蛋白(TIGIT)拮抗剂。
  5. 根据权利要求3所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的免疫检查点药物选自抗PD-1抗体,抗PD-L1抗体。
  6. 根据权利要求1所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的药物组合物或制剂还可与至少一种化学治疗剂联合应用。
  7. 根据权利要求6所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的化学治疗剂选自细胞毒类化学治疗剂。
  8. 根据权利要求1所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述的药物组合物或制剂还可与放射疗法联合应用。
  9. 根据权利要求1-8所述的β-1,3/1,6-葡聚糖的应用，其特征在于，所述药物组合物或制剂用于治疗个体的癌症。
  10. 根据权利要求9所述的癌症，其特征在于，所述的癌症选自黑色素瘤、结直肠癌、肺癌、肾癌、肝癌、乳腺癌中的一种或多种。
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