WO2018133109A1

WO2018133109A1 - Ziyuglycoside ii liposome and preparation method therefor

Info

Publication number: WO2018133109A1
Application number: PCT/CN2017/072229
Authority: WO
Inventors: 杨世林
Original assignee: 四川英路维特医药科技有限公司
Priority date: 2017-01-23
Filing date: 2017-01-23
Publication date: 2018-07-26

Abstract

A ziyuglycoside II liposome, a preparation method therefor and a use thereof in preparing medicines for treating and/or preventing myelosuppression and medicines for increasing the count of blood cells and hemoglobin. The ziyuglycoside II liposome comprises 1 part of ziyuglycoside II and 2-25 parts of a carrier material, wherein the carrier material consists of the following components in weight proportion: hydrogenated soybean phospholipid:Poly(ethylene glycol)-distearoylphosphatidylethanolamine-polyethyleneglycol 2000:cholesterol=5:(1-4):(1-2).

Description

Ligustrum saponin II liposome and preparation method thereof

Technical field

The invention relates to a saponin II liposome and a preparation method thereof, relating to the field of medicine.

Background technique

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. Lack of granulocytes can cause serious infections; a marked reduction in red blood cells can cause severe anemia; a marked drop in platelets causes severe bleeding and even death. At present, there is still no effective treatment for bone marrow suppression in the clinic, and it is urgent to develop a therapeutic drug with better efficacy.

Diltiazem II, chemical name: 3-O-α-L-arabino-19-carboxylic acid (ziyu-glycoside 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. However, in actual use, it has been found that 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.

Summary of the invention

It is an object of the present invention to provide a saponin II liposome and a process for the preparation thereof.

The present invention provides a saponin II liposome prepared by the following raw materials in a weight ratio: saponin II 1 part, carrier material 2-25 parts; wherein the carrier material is composed of Component composition of the following weight ratio: hydrogenated soybean lecithin: distearoylphosphatidylethanolamine-polyethylene glycol 2000: cholesterol = 5: (1 to 4): (1 to 2).

Further, the carrier material consists of the following weight ratio components: hydrogenated soy lecithin: distearoylphosphatidylethanolamine-polyethylene glycol 2000: cholesterol = 5:1:1.

Further, it is prepared by including a raw material of the following weight ratio: 1 part of saponin II and 20 parts of a carrier material.

Further, it further contains 1 to 10 parts of a protective agent.

Further, the protective agent is selected from one or a mixture of two or more of glucose, sucrose, trehalose, fructose, mannitol or lactose.

Further, it is prepared from the following weight ratio raw materials: saponin II 1 part, carrier material 20 parts, sucrose 5 parts; wherein the carrier material is composed of the following weight ratio components : Hydrogenated soybean lecithin: distearoylphosphatidylethanolamine-polyethylene glycol 2000: cholesterol = 5:1:1.

The invention provides a preparation method of the liposome, comprising the following steps:

a, dissolving the saponin II and the carrier material in an organic solvent, and mixing the solution for use;

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;

c, homogenization, sterilization, that is.

Further, the organic solvent is ethanol; the homogenization condition is: homogenization 4 times under a pressure of 1000 bar; the sterilization condition is: 0.22 μm 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. During the research, the inventors found that 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. In the present invention, 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. In the pharmacodynamic experiment, compared with the model group, 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.

It is apparent that various other modifications, substitutions and changes can be made in the form of the above-described embodiments of the present invention.

The above content of the present invention will be further described in detail below by way of specific embodiments in the form of embodiments. However, the scope of the above-mentioned subject matter of the present invention should not be construed as being limited to the following examples. Any technique implemented based on the above description of the present invention is within the scope of the present invention.

DRAWINGS

Figure 1 is a graph comparing the counts of bone marrow hematopoietic stem cells in mice of each experimental group.

detailed description

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.

Example 1 Preparation of the saponin II liposome of the present invention

Prescription one (C): saponin II 0.5 mg, HSPC 1 mg, DSPE-PEG 2000 20 mg, cholesterol 10 mg, trehalose 5 mg

Prescription 2 (A): saponin II 1 mg, HSPC 10 mg, DSPE-PEG 2000 20 mg, cholesterol 20 mg, glucose 5 mg

Prescription III (D): saponin II 5 mg, HSPC 10 mg, DSPE-PEG 2000 40 mg, cholesterol 20 mg, fructose 5 mg

Prescription IV (E): saponin II 5 mg, HSPC 5 mg, DSPE-PEG 2000 20 mg, cholesterol 50 mg, mannose 5 mg

Prescription 5 (F): saponin II 10 mg, HSPC 5 mg, DSPE-PEG 2000 20 mg, cholesterol 30 mg, lactose 5 mg

Prescription 6 (B): saponin II 10 mg, HSPC 10 mg, DSPE-PEG 2000 40 mg, cholesterol 50 mg), sucrose 5 mg

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 The 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 μm microporous membrane was filtered and sterilized.

The beneficial effects of the present invention are exemplified below by experiments.

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.

Experimental Example 1 Quality Evaluation of Preparation of Ligustrazine II Liposomes Using Different Carrier Materials

Seven experimental groups were set up in this experiment. 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 μm 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.

Table 1 Quality evaluation of saponin II liposome

Experimental results: 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.

In addition, 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.

Experimental Example 2 Effect of the Amount of Carrier Materials on the Quality of Ligustrazine II Liposomes

Six experimental groups were set up in this experiment. The saponin II and total lipid (HSPC: DSPE-PEG 2000: cholesterol = 5:1:1) 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 μm 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 Quality evaluation of saponin II liposome

Experimental results: when the amount of carrier material is 2-25 times of saponin II, the liposome with better quality can be obtained: the encapsulation efficiency is not less than 78%, the average particle diameter is below 210 nm, and the PDI is less than 0.106; When the weight ratio of the saponin II: carrier material is 1:20, the quality of the saponin II liposome is the best.

Experimental Example 3 Experimental study on the saponin II liposome of the present invention

1. Experimental materials, reagents and instruments

1.1. 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.

1.2, tool drugs: cyclophosphamide.

1.3, experimental animal KM-mouse: 18.5-22.5g.

1.4, experimental equipment: automatic blood cell analyzer; BS-600L electronic balance: specifications: 600g / 0.1g, Shanghai Yousheng Weighing Apparatus Co., Ltd.

2, statistical methods

Statistical analysis was performed using SPSS 17.0 software. Data in mean ± standard deviation (

), one-way analysis of variance was used between groups, LSD test was performed between groups with variance, and Tamhane's T2 test was performed for those with irregular variance.

3. Experimental methods

3.1, experimental animal grouping and model preparation

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. 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.

3.2, administration

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.

3.3, specimen collection

On the 8th day of the experiment, blood was taken from the eye of each experimental group and collected with a 0.5 ml EP tube containing EDTA anticoagulant.

3.4. Test indicators and methods

(1) 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.

(2) 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 μL of normal mouse serum to block the non-specific binding site, and then 10 μL of FITC-labeled rat anti-mouse CD34 ⁺ antibody was added, 10 μL 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. 2 mL of red blood cell lysate was added for 5 min, the cells were washed twice, and PI staining solution with a final concentration of 3 μg/mL was added, and the expression of CD34 ⁺ antigen in bone marrow cells was detected by flow cytometry.

4. Experimental results

4.1. Comparison of major blood cell counts in peripheral blood

Table 3 Number of peripheral blood cells in each experimental group

Note: Compared with the model group, *P<0.05, **P<0.01; Note: Compared with the saponin II group, △P<0.05, △△P<0.01.

As can be seen from Table 3, compared with the model group, the number of WBC, RBC, and PLT in the peripheral blood of the saponin II liposome group (A, B, C, D, E, F) of the present invention was significantly increased (P <0.05), there was no significant difference in the saponin II group; compared with the saponin II group, the saponin II liposome group of the present invention (A, B, The number of WBC, RBC and PLT in peripheral blood of mice with C, D, E and F) were significantly increased (P<0.05).

Table 4 Number of peripheral blood cells in each experimental group

Note: Compared with the model group, *P<0.05, **P<0.01; compared with the saponin II group, △P<0.05, △△P<0.01.

As can be seen from Table 4, compared with the model group, the NEUT and HGB numbers in the peripheral blood of the saponin II liposome group of the present invention were significantly increased (P<0.05), and there was no significant difference in the saponin II group; Compared with the group of saponins II, the levels of NEUT and HGB in the peripheral blood of the mice of the saponins II, B, C, D, E and F groups of the present invention were significantly increased (P<0.05).

4.2, comparison of bone marrow hematopoietic stem cell count

As can be seen from Fig. 1, compared with the model group, 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).

The above experimental results show that the preparation of the saponin II into a liposome according to the formulation of the present invention can significantly improve the blood cell level of the drug, and can effectively prevent bone marrow suppression.

Claims

A saponin II liposome, which is prepared by containing a raw material of the following weight ratio: 1 part of saponin II, 2-25 parts of carrier material; wherein the carrier material It consists of the following weight ratio components: hydrogenated soy lecithin: distearoylphosphatidylethanolamine-polyethylene glycol 2000: cholesterol = 5: (1 to 4): (1 to 2).
The liposome of claim 1 wherein said carrier material consists of the following weight ratio components: hydrogenated soy lecithin: distearoylphosphatidylethanolamine-polyethylene glycol 2000: Cholesterol = 5:1:1.
The liposome according to claim 1 or 2, which is prepared by containing a raw material of the following weight ratio: 1 part of saponin II and 20 parts of a carrier material.
The liposome according to any one of claims 1 to 3, which further comprises 1 to 10 parts of a protective agent.
The liposome according to claim 4, wherein the protective agent is one or more selected from the group consisting of glucose, sucrose, trehalose, fructose, mannitol or lactose.
The liposome according to any one of claims 1 to 5, which is prepared from the following raw materials by weight ratio: 1 part of saponin II, 20 parts of carrier material, and 5 parts of sucrose. Wherein the carrier material consists of the following weight ratio components: hydrogenated soy lecithin: distearoylphosphatidylethanolamine-polyethylene glycol 2000: cholesterol = 5:1:1.
A method for preparing a liposome according to any one of claims 1 to 6, comprising the steps of:

a, dissolving the saponin II and the carrier material in an organic solvent, and mixing the solution for use;

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;

c, homogenization, sterilization, that is.
The preparation method according to claim 7, wherein the organic solvent is ethanol; the homogenization condition is: homogenization 4 times under a pressure of 1000 bar; and the sterilization condition is 0.22 μm micropores. The filter membrane is sterilized by filtration.
Use of a liposome according to any one of claims 1 to 6 for the manufacture of a medicament for the treatment and/or prevention of myelosuppression.
Use of the liposome according to any one of claims 1 to 6 for the preparation of a medicament for increasing the number of blood cells and hemoglobin.
A method for treating and/or preventing myelosuppression, which comprises using the liposome according to any one of claims 1 to 6.
A method for increasing the number of blood cells and hemoglobin, which comprises using the liposome according to any one of claims 1 to 6.