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WO2018133113A1 - 一种地榆苷元脂质体及其制备方法、用途 - Google Patents

一种地榆苷元脂质体及其制备方法、用途 Download PDF

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WO2018133113A1
WO2018133113A1 PCT/CN2017/072233 CN2017072233W WO2018133113A1 WO 2018133113 A1 WO2018133113 A1 WO 2018133113A1 CN 2017072233 W CN2017072233 W CN 2017072233W WO 2018133113 A1 WO2018133113 A1 WO 2018133113A1
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liposome
carrier material
mantle
aglycone
weight ratio
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PCT/CN2017/072233
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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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Priority to PCT/CN2017/072233 priority Critical patent/WO2018133113A1/zh
Publication of WO2018133113A1 publication Critical patent/WO2018133113A1/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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • 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
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • 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
    • 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
    • A61P35/00—Antineoplastic agents
    • 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 mantle aglycone liposome, a preparation method thereof and use thereof, and belongs 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.
  • the mantle aglycone is one of the roots extracted from the roots of Sanguisorba officinalis L. or S. officinalis L. var. longifolia (Bertol.) Yu et Li.
  • the active ingredient is an aglycon of saponin I and saponin II, chemical name: 3 ⁇ , 19 ⁇ -hydroxy-Uso-12--28-carboxylic acid, and its structural formula is as follows:
  • CN101119740A discloses the use of saponin II in the preparation of a medicament for increasing red blood cells and hemoglobin.
  • saponin II in the preparation of a medicament for increasing red blood cells and hemoglobin.
  • the geniposide has poor efficacy, and when used alone, the effect of increasing blood cell level and treating bone marrow suppression is poor, which greatly limits the clinical application of the geniposide.
  • An object of the present invention is to provide a guanosine aglycon liposome, a preparation method thereof and use thereof.
  • HSPC hydrogenated soybean lecithin
  • DSPE-PEG 2000 distearoylphosphatidylethanolamine-polyethylene glycol 2000.
  • the protective agent is selected from one or a mixture of two or more of glucose, sucrose, trehalose, fructose, mannitol or lactose.
  • the invention also provides a preparation method of the above liposome, comprising the following steps:
  • step b removing the organic solvent in the mixed solution of step a, and adding a protective agent and water;
  • the organic solvent described in the step a is ethanol; in the step b, after adding the protective agent and water, the concentration of the geniposide is 0.2 mg/mL; in the step c, the homogenous condition is: 1000 bar The mixture was homogenized 4 times under pressure; the sterilization condition was: 0.22 ⁇ m microporous membrane filtration sterilization.
  • the present invention also provides the use of the above liposome for the preparation of a medicament for the treatment and/or prevention of myelosuppression.
  • the present invention also provides the use of the above liposome for the preparation of a medicament for increasing the number of one or more of peripheral blood leukocytes, neutrophils, red blood cells, platelets, hemoglobin or bone marrow hematopoietic stem cells.
  • the invention also provides a method of treating and/or preventing myelosuppression, in particular using the aforementioned solid liposomes.
  • the present invention also provides a method for increasing the amount of one or more of peripheral blood leukocytes, neutrophils, red blood cells, platelets, hemoglobin, bone marrow hematopoietic stem cells, in particular, using the aforementioned liposomes.
  • the inventors found that the reason that the effect of the cellar aglycone on the elevated blood cell level is poor is that its solubility is low and the gastrointestinal absorption rate is small, which leads to low bioavailability of the drug and limits its efficacy.
  • the scorpion aglycone liposome prepared by the invention can significantly increase the number of peripheral blood leukocytes, neutrophils, red blood cells, platelets, hemoglobin and bone marrow hematopoietic stem cells, and the pharmacological effect is obviously superior to the scorpion aglycone original drug, indicating
  • the preparation of the diterpene aglycone into a liposome can improve the bioavailability of the main drug, enhance the blood cell number and prevent bone marrow suppression.
  • 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.
  • Prescription seven (G): 1 mg of mantle aglycon and 20 mg of carrier material (HSPC: DSPE-PEG 2000: cholesterol 5:1:1, ie, HSPC 14 mg, DSPE-PEG 2000 3 mg, cholesterol 3 mg), and sucrose 5 mg.
  • aglycone ie, saponin
  • total lipid composed of HSPC, PEG 2000-DSPE and cholesterol
  • sugar corresponding glucose, sucrose
  • trehalose fructose
  • mannitol mannitol or lactose
  • appropriate amount of water so that the concentration of the aglycone in the liposome suspension is 0.2 mg / mL
  • high pressure homogenization 4 times under 1000 bar pressure, 0.22 ⁇ m microporous membrane Filter and sterilize, that is.
  • the drug content was determined by HPLC-ELSD, the particle size was measured by a Malvern particle size analyzer, and the PDI was detected by a particle size analyzer.
  • the mixed lipids of 1mg of scutellarin 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, and 5 mg was added.
  • Sucrose and water were used to make the concentration of the aglycone in the liposome suspension 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 encapsulation efficiency, particle size distribution and dispersion index (PDI) of the indole aglycone in the liposome were measured. The results are shown in Table 1.
  • the quality of the mantle aglycone 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 70%, and the average particle diameter is below 215 nm.
  • the dispersion index (PDI) is less than 0.127, the effect is significantly better than other excipients and proportioning group (P ⁇ 0.05); using the other two excipients lecithin or stigmasterol, the drug encapsulation rate and particle size uniformity are significantly reduced. And the average particle size of the liposome is large.
  • HSPC DSPE-PEG 2000: cholesterol weight ratio of 5:1:1, the prepared mantle aglycons liposome encapsulation efficiency, average particle size, dispersion index (PDI) and other indicators are the best.
  • the bacteria were sterilized by filtration through a 0.22 ⁇ m microporous membrane.
  • the encapsulation efficiency, average particle size and dispersion index (PDI) of the indole aglycone in the liposome were measured. The results are shown in Table 2.
  • test drug was prepared with different excipients to prepare the aglycone liposome solution group (A, B, C, D, E, F, G) and the mantle aglycone 10% DMSO-saline group.
  • tool drugs cyclophosphamide.
  • All animals were fed ad libitum for 1 week and were randomly divided into: blank group; model group; different prescriptions of mantle aglycone liposome group (A, B, C, D, E, F, G). 2.5mg ⁇ kg -1 suspension, prepared before use; mantle aglycone group: mantle aglycone powder, dissolved in 10% DMSO-physiological saline, formulated into 2.5mg ⁇ kg -1 suspension, use Pre-formulation.
  • mice On the first day of the experiment, except for the blank group, 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.
  • Each experimental group was given the corresponding drug by dose and tail vein from the first day of the experiment, and the blank group and The model group mice were injected with an equal 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 scorpion aglycone liposome group of the present invention was significantly increased (P ⁇ 0.05), and there was no significant difference in the scutellarin group;
  • the number of hematopoietic stem cells in the scorpion aglycone liposome group of the present invention was significantly increased (P ⁇ 0.05).
  • the mantle aglycone liposome prepared by the invention effectively solves the problem of low solubility of the mantle aglycone, improves the bioavailability of the mantle aglycone, thereby improving the therapeutic effect of raising blood cells, and effectively preventing and preventing bone marrow suppression. It is of great significance for the clinical application of geniposide.

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Abstract

一种地榆苷元脂质体、其制备方法及其在制备治疗和/或预防骨髓抑制和升高外周血细胞的药物中的用途。所述地榆苷元脂质体包含地榆苷元1份、载体材料2～40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

一种地榆苷元脂质体及其制备方法、用途 技术领域
本发明涉及一种地榆苷元脂质体及其制备方法、用途，属于医药领域。
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，尤其是放化疗引起的骨髓抑制，尚缺乏有效的治疗手段，亟需开发出药效较好的治疗药物。
地榆苷元是从蔷薇科地榆属植物地榆(Sanguisorba officinalis L.)或长叶地榆[S.officinalis L.var.longifolia(Bertol.)Yu et Li]的根中提取得到的一种活性成分，是地榆皂苷I和地榆皂苷II的苷元，化学名：3β，19α-羟基乌索-12烯-28-羧酸，其结构式如下所示：
Figure PCTCN2017072233-appb-000001
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。实际使用中发现，地榆苷元药效欠佳，单独使用时，升高血细胞水平、治疗骨髓抑制的效果不佳，大大限制了地榆苷元在临床上的应用。
目前尚未见以地榆皂苷元为活性成分制备脂质体，用于治疗和/或预防骨髓抑制的公开报道。
发明内容
本发明的目的在于提供一种地榆苷元脂质体及其制备方法、用途。
本发明提供了一种地榆苷元脂质体，它是包含下述重量配比的原辅料制备而成：地榆苷元1份、载体材料2～40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)。
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。
HSPC：氢化大豆卵磷脂；
DSPE-PEG 2000：二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000。
其中，它是由下述重量配比的原辅料制备而成的制剂：地榆苷元1份、载体材料20份。
其中，它还包含保护剂1～5份。
其中，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。
其中，它是由下述重量配比的原辅料制备而成：地榆苷元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。
本发明还提供了上述脂质体的制备方法，包括如下步骤：
a、将地榆苷元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，再加入保护剂和水；
c、均质，除菌，即得。
其中，步骤a中所述的有机溶剂为乙醇；步骤b中，加入保护剂和水后，使地榆苷元的浓度为0.2mg/mL；步骤c中，所述均质的条件为：1000bar压力下均质4次；所述除菌的条件为：0.22μm微孔滤膜过滤除菌。
本发明还提供了上述脂质体在制备治疗和/或预防骨髓抑制的药物的用途。
本发明还提供了上述脂质体在制备升高外周血白细胞、中性粒细胞、红细胞、血小板、血红蛋白或骨髓造血干细胞中一种或几种的数量的药物中的用途。
本发明还提供了一种治疗和/或预防骨髓抑制的方法，具体是采用前述的固体脂质体进行治疗。
本发明还提供了一种升高外周血白细胞、中性粒细胞、红细胞、血小板、血红蛋白、骨髓造血干细胞中一种或几种的数量的方法，具体是采用前述的脂质体进行治疗。
发明人在研究过程中发现，地榆苷元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。本发明制备的地榆苷元脂质体，能显著提高外周血白细胞、中性粒细胞、红细胞、血小板、血红蛋白和骨髓造血干细胞的数量，且药效明显优于地榆苷元原药，表明本发明将地榆苷元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各实验组小鼠CD34-/Sca-1+的变化情况
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。
实施例1本发明脂质体的制备
处方一(A)：地榆苷元1mg、HSPC 1mg、DSPE-PEG 2000 10mg、胆固醇10mg、葡萄糖1mg；
处方二(B)：地榆苷元1mg、HSPC 5mg、DSPE-PEG 2000 30mg、胆固醇3mg、蔗糖3mg；
处方三(C)：地榆苷元2mg、HSPC 5mg、DSPE-PEG 2000 20mg、胆固醇10mg、海藻糖3mg；
处方四(D)：地榆苷元5mg、HSPC 10mg、DSPE-PEG 2000 30mg、胆固醇30mg、果糖5mg；
处方五(E)：地榆苷元7mg、HSPC 10mg、DSPE-PEG 2000 40mg、胆固醇50mg、甘露糖5mg；
处方六(F)：地榆苷元10mg、HSPC 10mg、DSPE-PEG 2000 40mg、胆固醇50mg、乳糖5mg。
处方七(G)：地榆苷元1mg、载体材料20mg(HSPC:DSPE-PEG 2000:胆固醇＝5:1:1即HSPC 14mg、DSPE-PEG 2000 3mg、胆固醇3mg)、蔗糖5mg。
制备方法：
将地榆苷元(即地榆皂苷元)与总脂质(由HSPC、PEG 2000-DSPE和胆固醇组成)混合，以乙醇溶解，减压旋转蒸发除去乙醇，加入糖类(相应的葡萄糖，蔗糖，海藻糖，果糖，甘露醇或乳糖)和适量水，使脂质体混悬液中地榆苷元的浓度为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苷元脂质体的质量评价
本实验设置7个实验组。
分别将1mg地榆苷元与不同载体材料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苷元的浓度为0.2mg/mL，1000bar压力下高压均质4次，0.22μm微孔滤膜过滤除菌。测定脂质体中地榆苷元包封率、粒径分布及分散指数(PDI)，结果见表1。
表1 地榆苷元脂质体的质量评价
Figure PCTCN2017072233-appb-000002
注：与HSPC:DSPE-PEG 2000:胆固醇＝5:1:1组比较，*P<0.05。
由表1可见，以HSPC、DSPE-PEG 2000和胆固醇的混合脂质作为载体材料，制备得到的地榆苷元脂质体质量较好：包封率达到70％以上，平均粒径在215nm以下，分散指数(PDI)小于0.127，效果显著优于其他辅料及配比组(P<0.05)；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。
结果表明，本发明采用HSPC、DSPE-PEG 2000和胆固醇的混合脂质可以制备得到包封率高的地榆苷元脂质体，而采用其他常规辅料则无法制备得到包封率高的地榆苷元脂质体。
另外，HSPC:DSPE-PEG 2000:胆固醇重量配比为5:1:1时，制备得到的地榆苷元脂质体包封率、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苷元脂质体质量的影响
本实验设置7个实验组。
分别按照如表2所示的质量比例称取地榆苷元与总脂质(HSPC:DSPE-PEG2000:胆固醇＝5:1:1)(固定地榆苷元质量为1mg，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苷元的浓度为0.2mg/mL，1000bar压力下高压均质4次，0.22μm微孔滤膜过滤除菌。测定脂质体中地榆苷元包封率、平均粒径及分散指数(PDI)，结果见表2。
表2 地榆苷元脂质体的质量评价
Figure PCTCN2017072233-appb-000003
注：与1:20组比较，*P<0.05。
实验结果：载体材料用量为地榆苷元2-40倍时均能得到质量较好的脂质体：包封率不低于75％，平均粒径在210nm以下，PDI低于0.108；其中，地榆苷元：载体材料重量配比为1：20时，地榆皂苷元脂质体质量最佳，包封率显著优于其他载体材料重量配比组(P<0.05)。
实验例3本发明地榆苷元脂质体的药效实验
1、实验材料、试剂、仪器
1.1、受试药物不同辅料制地榆苷元脂质体溶液组(A、B、C、D、E、F、G)、地榆苷元10％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 PCTCN2017072233-appb-000004
)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；不同处方制备的地榆苷元脂质体组(A、B、C、D、E、F、G)，配制成2.5mg·kg-1混悬液，临用前配制；地榆苷元组：地榆苷元粉末，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,见表3-4。
表3 各实验组小鼠外周血血细胞数量
Figure PCTCN2017072233-appb-000005
注：与模型组比较，*P<0.05，**P<0.01；注：与地榆苷元组比较，△P<0.05，△△P<0.01。
由表3可知，与模型组比较，地榆苷元组无显著性差异，本发明地榆苷元脂质体组(A、B、C、D、E、F、G)小鼠外周血WBC、RBC、PLT数量均有显著升高(P<0.05)；与地榆苷元组比较，本发明地榆苷元脂质体组(A、B、C、D、E、F、G)小鼠外周血WBC、RBC、PLT数量均有显著升高(P<0.05)，其中地榆苷元脂质体G组小鼠外周血WBC、RBC、PLT数量升高最显著。
表4 各实验组小鼠外周血血细胞数量
Figure PCTCN2017072233-appb-000006
注：与模型组比较，*P<0.05，**P<0.01；与地榆苷元组比较，△P<0.05，△△P<0.01。
由表4可知，与模型组比较，地榆苷元组无显著性差异，本发明地榆苷元脂质体组小鼠外周血NEUT和HGB数量均有显著升高(P<0.05)；与地榆苷元组比较，A、B、C、D、E、F、G组小鼠外周血NEUT和HGB数量均有显著升高(P<0.05)，其中地榆苷元脂质体G组小鼠外周血NEUT和HGB数量升高最显著。
4.2、骨髓造血干细胞计数比较
见图1。
由图1可知，与模型组比较，本发明地榆苷元脂质体组小鼠造血干细胞数量均有显著升高(P<0.05)，地榆苷元组无显著性差异；与地榆苷元组比较，本发明地榆苷元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，本发明地榆苷元脂质体可以有效升高血细胞，药效明显优于直接用地榆苷元原药。
综上，本发明制备的地榆苷元脂质体有效解决了地榆苷元溶解度低的问题，提高了地榆苷元生物利用度，进而提高其升高血细胞的疗效，能有效防治骨髓抑制，对地榆苷元的临床应用具有十分重要的意义。

Claims (12)

  1. 一种地榆苷元脂质体，其特征是：它是包含下述重量配比的原辅料制备而成：地榆苷元1份、载体材料2～40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. 根据权利要求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. 根据权利要求1或2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的制剂：地榆苷元1份、载体材料20份。
  4. 根据权利要求1-3任意一项所述的脂质体，其特征是：它还包含保护剂1～5份。
  5. 根据权利要求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. 根据权利要求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：地榆苷元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、将地榆苷元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，再加入保护剂和水；
    c、均质，除菌，即得。
  8. 根据权利要求7所述的制备方法，其特征是：步骤a中所述的有机溶剂为乙醇；步骤b中，加入保护剂和水后，使地榆苷元的浓度为0.2mg/mL；步骤c中，所述均质的条件为：1000bar压力下均质4次；所述除菌的条件为：0.22μm微孔滤膜过滤除菌。
  9. 权利要求1-6任意一项所述的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任意一项所述的脂质体在制备升高外周血白细胞、中性粒细胞、红细胞、血小板、血红蛋白或骨髓造血干细胞中一种或几种的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，其特征在于：采用权利要求1-6任意一项所述的脂质体进行治疗。
  12. 一种升高外周血白细胞、中性粒细胞、红细胞、血小板、血红蛋白、骨髓造血干细胞中一种或几种的数量的方法，其特征在于：采用权利要求1-6任意一项所述的脂质体进行治疗。
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