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WO2018133109A1 - 一种地榆皂苷ⅱ脂质体及其制备方法 - Google Patents

一种地榆皂苷ⅱ脂质体及其制备方法 Download PDF

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WO2018133109A1
WO2018133109A1 PCT/CN2017/072229 CN2017072229W WO2018133109A1 WO 2018133109 A1 WO2018133109 A1 WO 2018133109A1 CN 2017072229 W CN2017072229 W CN 2017072229W WO 2018133109 A1 WO2018133109 A1 WO 2018133109A1
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saponin
liposome
carrier material
weight ratio
liposome according
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PCT/CN2017/072229
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English (en)
French (fr)
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杨世林
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四川英路维特医药科技有限公司
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Publication of WO2018133109A1 publication Critical patent/WO2018133109A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

Definitions

  • the invention relates to a saponin II liposome and a preparation method thereof, relating to the field of medicine.
  • Myelosuppression is a clinically common hematopoietic disease that can occur in radiation therapy and/or chemotherapy of various systemic neoplastic diseases, radiation damage caused by ionizing radiation, viral hepatitis, parvovirus infection or drugs (chloramphenicol). , benzene, sulfonamides, anti-epileptic drugs, sedatives, anti-thyroid drugs, anti-diabetes drugs, anti-malaria, sleeping pills and other factors. Myelosuppression can cause damage to the bone marrow microenvironment, hematopoietic stem cells, hematopoietic growth factors, etc., and the granulosa, red, and megakaryocyte systems are inhibited.
  • Diltiazem II is a mantle from the genus Rosaceae or A pharmacologically active compound is extracted from the roots of the longleaf mantle.
  • CN101119740A discloses the use of saponin II in the preparation of a medicament for increasing red blood cells and hemoglobin.
  • saponin II is not effective, and it is often difficult to obtain a better blood cell level and a bone marrow suppression treatment when used alone. Therefore, there is an urgent need to improve the efficacy of the saponin II and promote the clinical application of the drug.
  • the protective agent is selected from one or a mixture of two or more of glucose, sucrose, trehalose, fructose, mannitol or lactose.
  • the invention provides a preparation method of the liposome, comprising the following steps:
  • step b removing the organic solvent in the mixed solution of step a, and then adding a protective agent and water to the concentration of saponin II is 0.2 mg / mL;
  • the organic solvent is ethanol
  • the homogenization condition is: homogenization 4 times under a pressure of 1000 bar
  • the sterilization condition is: 0.22 ⁇ m microporous membrane filtration sterilization.
  • the invention provides the use of the liposomes in the manufacture of a medicament for the treatment and/or prevention of myelosuppression.
  • the present invention provides the liposome in the preparation of a drug for raising the number of blood cells and hemoglobin use.
  • the present invention provides a method of treating and/or preventing myelosuppression using the liposome.
  • the present invention provides a method of increasing the number of blood cells and hemoglobin using the liposome.
  • the reason that the effect of the saponin II on raising the blood cell level is poor is that the solubility is low and the gastrointestinal absorption rate is small, resulting in low bioavailability of the drug and limiting the exertion of its efficacy.
  • the mixed lipid of HSPC, DSPE-PEG 2000 and cholesterol is used as a carrier material, and the saponin II can be prepared into a liposome of better quality.
  • the saponin II liposome of the present invention can significantly increase the number of WBC, RBC, PLT, NEUT and HGB in the peripheral blood, and the drug effect is obviously superior to that of the saponin II drug, indicating that the present invention
  • the invention can improve the bioavailability of the main drug, and increase the blood cell number and prevent bone marrow suppression by preparing the saponin II into a liposome.
  • Figure 1 is a graph comparing the counts of bone marrow hematopoietic stem cells in mice of each experimental group.
  • the raw materials and equipment used in the specific embodiments of the present invention are known products and are obtained by purchasing commercially available products.
  • Hydrogenated Soy Lecithin (HSPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000), cholesterol, and stigmasterol were purchased from Hangzhou Dayang Chemical Co., Ltd.; lecithin was purchased from Chubby Corporation of Japan.
  • Glucose, sucrose, trehalose, fructose, mannitol, lactose, starch, and cellulose were purchased from Jiangsu Manshi Biotechnology Co., Ltd.
  • Preparation method Mixing saponin II with total lipid (composed of HSPC, DSPE-PEG 2000 and cholesterol), dissolving in ethanol, removing ethanol by rotary evaporation under reduced pressure, adding sugar and water to make liposome suspension
  • concentration of the saponin II was 0.2 mg/mL, and the pressure was high pressure 4 times under the pressure of 1000 bar, and the 0.22 ⁇ m microporous membrane was filtered and sterilized.
  • the drug content was determined by HPLC-ELSD, and the particle size results were measured by a Malvern particle size analyzer.
  • the PDI is tested using a particle size analyzer.
  • the mixed lipids of 0.5mg of saponin II and different carrier materials HSPC, DSPE-PEG 2000, cholesterol, lecithin and stigmasterol were mixed at a mass ratio of 1:20, dissolved in ethanol, and ethanol was removed by rotary evaporation under reduced pressure.
  • 5 mg of sucrose and water were used to make the concentration of saponin II in the liposome suspension 0.2 mg/mL, high pressure homogenization 4 times under a pressure of 1000 bar, and 0.22 ⁇ m microporous membrane filtration sterilization.
  • the entrapment efficiency, particle size distribution and dispersion index (PDI) of the saponin II in the liposome were measured. The results are shown in Table 1.
  • the quality of the saponin II liposome prepared by using the mixed lipid of HSPC, DSPE-PEG 2000 and cholesterol as the carrier material is better: the encapsulation efficiency is above 65%, the average particle size is below 220 nm, and the dispersion is The index (PDI) is less than 0.125; using the other two excipients, lecithin or stigmasterol, results in a significant decrease in the encapsulation efficiency and particle size uniformity of the drug, and the average particle size of the liposome is large.
  • the above results indicate that the mixed lipid of HSPC, DSPE-PEG 2000 and cholesterol is the most suitable carrier material for preparing the saponin II liposome.
  • HSPC DSPE-PEG 2000: cholesterol weight ratio of 5:1:1, the prepared saponin II liposome encapsulation efficiency, average particle size, dispersion index (PDI) and other indicators are the best.
  • the saponin II and total lipid were weighed according to the mass ratio shown in Table 2 (fixed saponin II mass 0.5 mg, total lipid) The mass is changed according to the ratio), dissolved in ethanol, and the ethanol is removed by rotary evaporation under reduced pressure. 5 mg of sucrose and water are added to make the concentration of saponin II in the liposome suspension 0.2 mg/mL, and the pressure is high pressure homogeneous under 1000 bar. The 0.22 ⁇ m microporous membrane was sterilized by filtration. The entrapment efficiency, average particle size and dispersion index (PDI) of the saponin II in the liposome were measured, and the results are shown in Table 2.
  • Table 2 The saponin II and total lipid
  • Test drug The saponin II liposome group (A, B, C, D, E, F) and the saponin II 10% DMSO-salt group prepared according to Example 1.
  • tool drugs cyclophosphamide.
  • mice All animals were adaptively fed for 1 week and were randomly divided into: blank group; model group; saponin II liposome group (A, B, C, D, E, F) prepared according to different prescriptions of Example 1, Formulated into 2.5mg ⁇ kg -1 suspension, prepared before use; saponin II group: saponin II powder, dissolved in 10% DMSO-physiological saline, formulated into 2.5mg ⁇ kg -1 suspension, Prepare before use.
  • the other groups of mice were intraperitoneally injected with cyclophosphamide physiological saline solution at a dose of 50 mg ⁇ kg -1 for 3 consecutive days, and the blank mice were injected with the same volume of normal saline in the tail vein.
  • the experimental groups were given the corresponding drugs by dose and tail vein from the first day of the experiment.
  • the blank group and the model group were injected with the same volume of normal saline in the tail vein for 7 consecutive days.
  • Peripheral blood test Peripheral blood leukocytes (WBC), neutrophils (NEUT) red blood cells (RBC), platelets (PLT), and hemoglobin (HGB) were counted in each experimental group by an automatic blood cell counter.
  • WBC Peripheral blood leukocytes
  • NUT neutrophils
  • RBC red blood cells
  • PHT platelets
  • HGB hemoglobin
  • Bone marrow hematopoietic stem cell count (based on bone marrow cell CD34 + antigen expression), the right femur bone marrow cells were washed out with PBS buffer containing bovine serum albumin at a concentration of 0.2%, and 10 6 cells were removed and centrifuged. The supernatant was added with 30 ⁇ L of normal mouse serum to block the non-specific binding site, and then 10 ⁇ L of FITC-labeled rat anti-mouse CD34 + antibody was added, 10 ⁇ L of the corresponding control antibody was added to the control tube, and the reaction was incubated at 4 ° C for 30 min in the dark.
  • the number of hematopoietic stem cells in the saponin II liposome group of the present invention was significantly increased (P ⁇ 0.05), and there was no significant difference in the saponin II group; Compared with the group II, the number of hematopoietic stem cells in the saponin II liposome group of the present invention was significantly increased (P ⁇ 0.05).

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Abstract

一种地榆皂苷Ⅱ脂质体、其制备方法及其在制备治疗和/或预防骨髓抑制和升高血细胞、血红蛋白数量的药物中的用途。所述地榆皂苷Ⅱ脂质体包含地榆皂苷Ⅱ1份、载体材料2～25份；其中，所述的载体材料由下述重量配比的组分组成：氢化大豆卵磷脂:二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000:胆固醇＝5:(1～4):(1～2)。

Description

一种地榆皂苷Ⅱ脂质体及其制备方法 技术领域
本发明涉及一种地榆皂苷Ⅱ脂质体及其制备方法，涉及医药领域。
背景技术
骨髓抑制是临床上常见的造血系统疾病,它可发生于各系统肿瘤性疾病的放射治疗及(或)化学治疗、电离辐射引起的放射损伤、病毒性肝炎、微小病毒感染或药物(氯霉素、苯、磺胺、抗癫痈药、镇静剂、抗甲状腺药、抗糖尿病药、抗疟疾、安眠药)等因素。骨髓抑制可引起骨髓微环境、造血干细胞、造血细胞生长因子等的损伤,粒、红、巨核细胞系统一系、二系或三系细胞受抑制。粒细胞缺乏会引起严重感染；红细胞明显减少会引起严重贫血；血小板明显下降引起严重出血,甚至导致死亡。目前，临床上对于骨髓抑制尚缺乏有效的治疗手段，亟需开发出药效较好的治疗药物。
地榆皂苷Ⅱ，化学名：3-O-α-L-阿拉伯糖基-19α-羟基乌索-12烯-28-羧酸(ziyu-glycosideⅡ)，是从蔷薇科地榆属植物地榆或长叶地榆的根中提取得到的具有药理活性的化合物。CN101119740A公开了地榆皂苷Ⅱ在制备升高红细胞和血红蛋白的药物中的用途。然而，实际使用中发现，地榆皂苷Ⅱ药效欠佳，单独使用时往往难以取得较好的升高血细胞水平、治疗骨髓抑制的效果。因此，亟需提高地榆皂苷Ⅱ的药效，促进该药物在临床上的应用。
发明内容
本发明的目的在于提供一种地榆皂苷Ⅱ脂质体及其制备方法。
本发明提供了一种地榆皂苷Ⅱ脂质体，它是包含下述重量配比的原辅料制备而成：地榆皂苷Ⅱ1份、载体材料2～25份；其中，所述的载体材料由下述重量配比的组分组成：氢化大豆卵磷脂:二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000:胆固醇＝5:(1～4):(1～2)。
进一步的，所述的载体材料由下述重量配比的组分组成：氢化大豆卵磷脂:二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000:胆固醇＝5:1:1。
进一步的，它是包含下述重量配比的原辅料制备而成：地榆皂苷Ⅱ1份、载体材料20份。
进一步的，它还包含保护剂1～10份。
进一步的，所述的保护剂选自葡萄糖、蔗糖、海藻糖、果糖、甘露醇或乳糖中一种或两种以上的混合物。
进一步的，它是由下述重量配比的原辅料制备而成：地榆皂苷Ⅱ1份、载体材料20份、蔗糖5份；其中，所述的载体材料由下述重量配比的组分组成：氢化大豆卵磷脂:二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000:胆固醇＝5:1:1。
本发明提供了一种所述脂质体的制备方法，包括如下步骤：
a、将地榆皂苷Ⅱ和载体材料溶解于有机溶剂，混合溶液备用；
b、除去a步骤混合溶液中的有机溶剂，再加入保护剂及水至地榆皂苷Ⅱ的浓度为0.2mg/mL；
c、均质，除菌，即得。
进一步的，所述的有机溶剂为乙醇；所述的均质条件为：1000bar压力下均质4次；所述的除菌条件为：0.22μm微孔滤膜过滤除菌。
本发明提供了所述脂质体在制备治疗和/或预防骨髓抑制的药物的用途。
本发明提供了所述脂质体在制备升高血细胞、血红蛋白数量的药物中的 用途。
本发明提供了一种使用所述脂质体治疗和/或预防骨髓抑制的方法。
本发明提供了一种使用所述脂质体升高血细胞、血红蛋白数量的方法。发明人在研究过程中发现，地榆皂苷II升高血细胞水平效果欠佳的原因在于其溶解度低、胃肠吸收率小，导致该药物的生物利用度较低，限制其药效的发挥。本发明以HSPC、DSPE-PEG 2000和胆固醇的混合脂质作为载体材料，可将地榆皂苷Ⅱ制备成质量较好的脂质体。药效实验中，与模型组比较，本发明地榆皂苷Ⅱ脂质体能显著升高外周血WBC、RBC、PLT、NEUT和HGB数量，且药效明显优于地榆皂苷Ⅱ原药，表明本发明将地榆皂苷Ⅱ制备成脂质体后能够提高主药的生物利用度，增强其升高血细胞数量、防治骨髓抑制的作用。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为各实验组小鼠骨髓造血干细胞计数比较图。
具体实施方式
本发明具体实施方式中使用的原料、设备均为已知产品，通过购买市售产品获得。
氢化大豆卵磷脂(HSPC)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000 (DSPE-PEG 2000)、胆固醇、豆甾醇，均购于杭州大阳化工有限公司；卵磷脂购于日本丘比株式会社。
葡萄糖，蔗糖，海藻糖，果糖，甘露醇，乳糖，淀粉，纤维素均购于江苏曼氏生物科技有限公司。
实施例1本发明地榆皂苷Ⅱ脂质体的制备
处方一(C)：地榆皂苷Ⅱ0.5mg、HSPC 1mg、DSPE-PEG 2000 20mg、胆固醇10mg、海藻糖5mg
处方二(A)：地榆皂苷Ⅱ1mg、HSPC 10mg、DSPE-PEG 2000 20mg、胆固醇20mg、葡萄糖5mg
处方三(D)：地榆皂苷Ⅱ5mg、HSPC 10mg、DSPE-PEG 2000 40mg、胆固醇20mg、果糖5mg
处方四(E)：地榆皂苷Ⅱ5mg、HSPC 5mg、DSPE-PEG 2000 20mg、胆固醇50mg、甘露糖5mg
处方五(F)：地榆皂苷Ⅱ10mg、HSPC 5mg、DSPE-PEG 2000 20mg、胆固醇30mg、乳糖5mg
处方六(B)：地榆皂苷Ⅱ10mg、HSPC 10mg、DSPE-PEG 2000 40mg、胆固醇50mg)、蔗糖5mg
制备方法：将地榆皂苷Ⅱ与总脂质(由HSPC、DSPE-PEG 2000和胆固醇组成)混合，以乙醇溶解，减压旋转蒸发除去乙醇，加入糖和水，使脂质体混悬液中地榆皂苷Ⅱ的浓度为0.2mg/mL，1000bar压力下高压均质4次，0.22μm微孔滤膜过滤除菌，即得。
以下通过实验例证明本发明的有益效果。
药物含量采用HPLC-ELSD进行测定，马尔文粒径测定仪测得粒径结果， PDI采用粒度测定仪进行检测。
实验例1采用不同载体材料制备地榆皂苷Ⅱ脂质体的质量评价
本实验设置7个实验组。分别将0.5mg地榆皂苷Ⅱ与不同载体材料HSPC、DSPE-PEG 2000、胆固醇、卵磷脂、豆甾醇的混合脂质按照质量比1:20混合，以乙醇溶解，减压旋转蒸发除去乙醇，加入5mg蔗糖和水，使脂质体混悬液中地榆皂苷Ⅱ的浓度为0.2mg/mL，1000bar压力下高压均质4次，0.22μm微孔滤膜过滤除菌。测定脂质体中地榆皂苷Ⅱ包封率、粒径分布及分散指数(PDI)，结果见表1。
表1 地榆皂苷Ⅱ脂质体的质量评价
Figure PCTCN2017072229-appb-000001
实验结果：以HSPC、DSPE-PEG 2000和胆固醇的混合脂质作为载体材料，制备得到的地榆皂苷Ⅱ脂质体质量较好：包封率达到65％以上，平均粒径在220nm以下，分散指数(PDI)小于0.125；采用另两种辅料卵磷脂或豆甾醇，则导致药物包封率及粒径均匀度的明显降低，而且脂质体的平均粒径较大。上述结果表明，HSPC、DSPE-PEG 2000和胆固醇的混合脂质是制备地榆皂苷Ⅱ脂质体最适宜的载体材料。
另外，HSPC:DSPE-PEG 2000:胆固醇重量配比为5:1:1时，制备得到的地榆皂苷Ⅱ脂质体包封率、平均粒径、分散指数(PDI)等指标最佳。
实验例2载体材料用量对地榆皂苷Ⅱ脂质体质量的影响
本实验设置6个实验组。分别按照如表2所示的质量比例称取地榆皂苷Ⅱ与总脂质(HSPC:DSPE-PEG 2000:胆固醇＝5:1:1)(固定地榆皂苷Ⅱ质量为0.5mg，总脂质质量随比例变化)，以乙醇溶解，减压旋转蒸发除去乙醇，加入5mg蔗糖和水，使脂质体混悬液中地榆皂苷Ⅱ的浓度为0.2mg/mL，1000bar压力下高压均质4次，0.22μm微孔滤膜过滤除菌。测定脂质体中地榆皂苷Ⅱ包封率、平均粒径及分散指数(PDI)，结果见表2。
表2 地榆皂苷Ⅱ脂质体的质量评价
Figure PCTCN2017072229-appb-000002
实验结果：载体材料用量为地榆皂苷Ⅱ2-25倍时均能得到质量较好的脂质体：包封率不低于78％，平均粒径在210nm以下，PDI低于0.106；其中，地榆皂苷Ⅱ：载体材料重量配比为1：20时，地榆皂苷Ⅱ脂质体质量最佳。
实验例3本发明地榆皂苷Ⅱ脂质体的药效实验
1、实验材料、试剂、仪器
1.1、受试药物：根据实施例1制备的地榆皂苷Ⅱ脂质体组(A、B、C、D、E、F)、地榆皂苷Ⅱ10％DMSO-生理盐水组。
1.2、工具药物：环磷酰胺。
1.3、实验动物KM-小鼠：18.5-22.5g。
1.4、实验仪器：全自动血球分析仪；BS-600L电子天平：规格：600g/0.1g，上海友声衡器有限公司。
2、统计方法
用SPSS 17.0软件进行统计分析。数据以均数±标准差(
Figure PCTCN2017072229-appb-000003
)表示，组间采用单因素方差分析，方差齐者组间进行LSD检验，方差不齐者进行Tamhane’s T2检验。
3、实验方法
3.1、实验动物分组及模型制备
所有动物适应性喂养1周后按体重随机分为：空白组；模型组；根据实施例1不同处方制备的地榆皂苷Ⅱ脂质体组(A、B、C、D、E、F)，配制成2.5mg·kg-1混悬液，临用前配制；地榆皂苷Ⅱ组：地榆皂苷Ⅱ粉末，用10％DMSO-生理盐水溶解，配制成2.5mg·kg-1混悬液，临用前配制。实验第1天，除空白组外，其余各组小鼠按50mg·kg-1剂量腹腔注射环磷酰胺生理盐水溶液，连续3天，空白组小鼠尾静脉注射等体积生理盐水。
3.2、给药
各实验组自实验第1天开始按剂量、尾静脉注射给予相应药物，空白组和模型组小鼠尾静脉注射等体积生理盐水，连续7天。
3.3、标本采集
实验第8天，各实验组小鼠眼眶取血，用装有EDTA抗凝剂的0.5mlEP管收集待测。
3.4、检测指标及方法
(1)外周血象检测：采用全自动血球计数仪对各实验组小鼠外周血白细胞(WBC)、中性粒细胞(NEUT)红细胞(RBC)、血小板(PLT)、血红蛋白(HGB)进行计数。
(2)骨髓造血干细胞计数(以骨髓细胞CD34+抗原表达量计)用含牛血清白蛋白浓度为0.2％的PBS缓冲液冲出小鼠右侧股骨骨髓细胞，取出106个细胞离心，弃上清，加入30μL正常小鼠血清以封闭非特异结合位点，再加 入10μL FITC标记的大鼠抗小鼠CD34+抗体，对照管加入10μL相应对照抗体，4℃避光反应30min。加入2mL红细胞裂解液，作用5min，洗细胞2次，加入终浓度为3μg/mL的PI染液，采用流式细胞仪检测骨髓细胞CD34+抗原表达。
4、实验结果
4.1、外周血主要血细胞计数比较
表3 各实验组小鼠外周血血细胞数量
Figure PCTCN2017072229-appb-000004
注：与模型组比较，*P<0.05，**P<0.01；注：与地榆皂苷Ⅱ组比较，△P<0.05，△△P<0.01。
由表3可知，与模型组比较，本发明地榆皂苷Ⅱ脂质体组(A、B、C、D、E、F)小鼠外周血WBC、RBC、PLT数量均有显著升高(P<0.05)，地榆皂苷Ⅱ组无显著性差异；与地榆皂苷Ⅱ组比较，本发明地榆皂苷Ⅱ脂质体组(A、B、 C、D、E、F)小鼠外周血WBC、RBC、PLT数量均有显著升高(P<0.05)。
表4 各实验组小鼠外周血血细胞数量
Figure PCTCN2017072229-appb-000005
注：与模型组比较，*P<0.05，**P<0.01；与地榆皂苷Ⅱ组比较，△P<0.05，△△P<0.01。
由表4可知，与模型组比较，本发明地榆皂苷Ⅱ脂质体组小鼠外周血NEUT和HGB数量均有显著升高(P<0.05)，地榆皂苷Ⅱ组无显著性差异；与地榆皂苷Ⅱ组比较，本发明地榆皂苷Ⅱ脂质体A、B、C、D、E、F组小鼠外周血NEUT和HGB数量均有显著升高(P<0.05)。
4.2、骨髓造血干细胞计数比较
从图1可知，与模型组比较，本发明地榆皂苷Ⅱ脂质体组小鼠造血干细胞数量均有显著升高(P<0.05)，地榆皂苷Ⅱ组无显著性差异；与地榆皂苷Ⅱ组比较，本发明地榆皂苷Ⅱ脂质体组小鼠造血干细胞数量均有显著升高(P<0.05)。
以上实验结果表明，根据本发明制剂处方将地榆皂苷Ⅱ制备成脂质体后，可显著提高药物升高血细胞水平的作用，能有效防治骨髓抑制。

Claims (12)

  1. 一种地榆皂苷Ⅱ脂质体，其特征是：它是包含下述重量配比的原辅料制备而成：地榆皂苷Ⅱ 1份、载体材料2～25份；其中，所述的载体材料由下述重量配比的组分组成：氢化大豆卵磷脂:二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000:胆固醇＝5:(1～4):(1～2)。
  2. 如权利要求1所述的脂质体，其特征是：所述的载体材料由下述重量配比的组分组成：氢化大豆卵磷脂:二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000:胆固醇＝5:1:1。
  3. 如权利要求1或2所述的脂质体，其特征是：它是包含下述重量配比的原辅料制备而成：地榆皂苷Ⅱ 1份、载体材料20份。
  4. 如权利要求1～3任意一项所述的脂质体，其特征是：它还包含保护剂1～10份。
  5. 如权利要求4所述的脂质体，其特征是：所述的保护剂选自葡萄糖、蔗糖、海藻糖、果糖、甘露醇或乳糖中一种或两种以上的混合物。
  6. 如权利要求1～5任意一项所述的脂质体，其特征是：它是由下述重量配比的原辅料制备而成：地榆皂苷Ⅱ 1份、载体材料20份、蔗糖5份；其中，所述的载体材料由下述重量配比的组分组成：氢化大豆卵磷脂:二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000:胆固醇＝5:1:1。
  7. 一种权利要求1～6任意一项所述脂质体的制备方法，其特征是：包括如下步骤：
    a、将地榆皂苷Ⅱ和载体材料溶解于有机溶剂，混合溶液备用；
    b、除去a步骤混合溶液中的有机溶剂，再加入保护剂及水至地榆皂苷Ⅱ的浓度为0.2mg/mL；
    c、均质，除菌，即得。
  8. 如权利要求7所述的制备方法，其特征是：所述的有机溶剂为乙醇；所述的均质条件为：1000bar压力下均质4次；所述的除菌条件为：0.22μm微孔滤膜过滤除菌。
  9. 权利要求1～6任意一项所述脂质体在制备治疗和/或预防骨髓抑制的药物的用途。
  10. 权利要求1～6任意一项所述脂质体在制备升高血细胞、血红蛋白数量的药物中的用途。
  11. 一种治疗和/或预防骨髓抑制的方法，其特征是：使用权利要求1～6任意一项所述脂质体。
  12. 一种升高血细胞、血红蛋白数量的方法，其特征是：使用权利要求1～6任意一项所述脂质体。
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CN1593436A (zh) * 2003-09-08 2005-03-16 成都地奥制药集团有限公司 乌索烷型三萜皂苷在制备升高白细胞和/或血小板药物中的应用
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