WO2018133113A1 - Ziyuglycogenin liposome, preparation method therefor and use thereof - Google Patents

Abstract

A ziyuglycogenin liposome, a preparation method therefor and a use thereof in preparing drugs for treating and/or preventing myelosuppression and increasing peripheral blood cells. The ziyuglycogenin liposome comprises 1 part of ziyuglycogenin and 2-40 parts of carrier material, wherein the carrier material consists of the following components in proportion by weight: hydrogenated soybean lecithin:distearoyl phosphatidylethanolamine-polyethylene glycol 2000:cholesterol=5:(1-4):(1-2).

Description

Mantle aglycone liposome, preparation method thereof and use thereof Technical field

The invention relates to a mantle aglycone liposome, a preparation method thereof and use thereof, and belongs 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, clinically, there is no effective treatment for myelosuppression caused by bone marrow suppression, especially chemoradiotherapy, and it is urgent to develop a therapeutic drug with better efficacy.

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: 3β, 19α-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. In actual use, it has been found that 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.

There have been no reports of the preparation of liposomes using saponins as active ingredients for the treatment and/or prevention of myelosuppression.

Summary of the invention

An object of the present invention is to provide a guanosine aglycon liposome, a preparation method thereof and use thereof.

The present invention provides a mantle aglycon liposome prepared by adding a raw material of the following weight ratio: 1 part of mantle aglycon and 2-40 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).

Wherein, the carrier material is composed of the following weight ratio components: hydrogenated soybean lecithin: distearoylphosphatidylethanolamine-polyethylene glycol 2000: cholesterol = 5:1:1.

HSPC: hydrogenated soybean lecithin;

DSPE-PEG 2000: distearoylphosphatidylethanolamine-polyethylene glycol 2000.

Among them, it is a preparation prepared from the following raw materials by weight ratio: 1 part of mantle aglycon and 20 parts of carrier material.

Among them, it also contains 1 to 5 parts of a protective agent.

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

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

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

a, dissolving the mantle aglycon 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 adding a protective agent and water;

c, homogenization, sterilization, that is.

Wherein, 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 μm 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.

During the research, 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.

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

Fig.1 Changes of CD34 ^- /Sca-1 ⁺ 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.

Example 1 Preparation of Liposomes of the Invention

Prescription one (A): mantle aglycon 1mg, HSPC 1mg, DSPE-PEG 2000 10mg, cholesterol 10mg, glucose 1mg;

Prescription 2 (B): mantle aglycon 1mg, HSPC 5mg, DSPE-PEG 2000 30mg, cholesterol 3mg, sucrose 3mg;

Prescription III (C): mantle aglycon 2mg, HSPC 5mg, DSPE-PEG 2000 20mg, cholesterol 10mg, trehalose 3mg;

Prescription IV (D): mantle aglycon 5mg, HSPC 10mg, DSPE-PEG 2000 30mg, cholesterol 30mg, fructose 5mg;

Prescription 5 (E): mantle aglycon 7mg, HSPC 10mg, DSPE-PEG 2000 40mg, cholesterol 50mg, mannose 5mg;

Prescription six (F): mantle aglycon 10mg, HSPC 10mg, DSPE-PEG 2000 40mg, cholesterol 50mg, lactose 5mg.

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.

Preparation:

Mixing the aglycone (ie, saponin) with total lipid (composed of HSPC, PEG 2000-DSPE and cholesterol), dissolving in ethanol, removing ethanol by rotary evaporation under reduced pressure, adding sugar (corresponding glucose, sucrose) , trehalose, fructose, mannitol or lactose) and 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 μm microporous membrane Filter and sterilize, that is.

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

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.

Experimental Example 1 Quality Evaluation of Glycosides of Glycosides Prepared by Different Carrier Materials

Seven experimental groups were set up in this experiment.

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 μm 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.

Table 1 Quality evaluation of mantle aglycons liposomes

Note: Compared with HSPC: DSPE-PEG 2000: Cholesterol = 5: 1:1 group, *P < 0.05.

It can be seen from Table 1 that 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.

The results show that the present invention can prepare the mantle aglycone liposome with high encapsulation efficiency by using the mixed lipid of HSPC, DSPE-PEG 2000 and cholesterol, and the mantle with high encapsulation rate cannot be prepared by using other conventional excipients. Aglycone liposomes.

In addition, 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.

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

Seven experimental groups were set up in this experiment.

The mantle aglycon and total lipid (HSPC: DSPE-PEG2000: cholesterol = 5:1:1) were weighed according to the mass ratio shown in Table 2 (the mass of the fixed aglycone was 1 mg, and the total lipid mass was The ratio is changed), 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 the aglycone in the liposome suspension 0.2 mg/mL, and the pressure is high pressure 4 times under the pressure of 1000 bar. The bacteria were sterilized by filtration through a 0.22 μm 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.

Table 2 Quality evaluation of mantle aglycons liposomes

Note: *P<0.05 compared to 1:20 group.

Experimental results: when the amount of carrier material is 2-40 times of mantle aglycone, the liposome with better quality can be obtained: the encapsulation efficiency is not less than 75%, the average particle diameter is below 210 nm, and the PDI is lower than 0.108; Glycosides: When the weight ratio of carrier materials was 1:20, the quality of saponin liposomes was the best, and the encapsulation efficiency was significantly better than that of other carrier materials (P<0.05).

Experimental Example 3 Pharmacodynamic experiment of the indole aglycone liposome of the present invention

1. Experimental materials, reagents and instruments

1.1. The 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.

1.2, tool drugs: cyclophosphamide.

1.3. Experimental animal KM-mouse: 18.5-22.5 g.

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

)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。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 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. 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

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.

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 the main blood cell counts of peripheral blood, see Table 3-4.

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 mantle aglycon group, △P<0.05, △△P<0.01.

As can be seen from Table 3, there was no significant difference in the mantle aglycon group compared with the model group, and the peripheral blood WBC of the scorpion aglycone liposome group (A, B, C, D, E, F, G) of the present invention. The number of RBC and PLT were significantly increased (P<0.05). Compared with the mantle aglycon group, the scorpion aglycone liposome group (A, B, C, D, E, F, G) was small. The number of WBC, RBC and PLT in peripheral blood of rats was significantly increased (P<0.05). The number of WBC, RBC and PLT in the peripheral blood of mice in the G of the Glycosides was the most significant.

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 mantle aglycon group, ^△ P<0.05, ^△△ P<0.01.

As can be seen from Table 4, compared with the model group, there was no significant difference in the geniposide group, and the number of NEUT and HGB in the peripheral blood of the scorpion aglycone liposome group of the present invention was significantly increased (P<0.05); Compared with the mantle aglycone group, the number of NEUT and HGB in peripheral blood of mice in group A, B, C, D, E, F and G were significantly increased (P<0.05), among which group of geniposide liposome G group The increase in the number of NEUT and HGB in peripheral blood of mice was the most significant.

4.2, comparison of bone marrow hematopoietic stem cell count

see picture 1.

As can be seen from Fig. 1, compared with the model group, 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; Compared with the tuple group, the number of hematopoietic stem cells in the scorpion aglycone liposome group of the present invention was significantly increased (P<0.05).

The above experimental results show that the inoculum aglycone liposome of the present invention can effectively raise blood cells, and the pharmacological effect is obviously superior to that of the direct use of the gill aglycone.

In summary, 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.

Claims (12)

A glucoside aglycone liposome, which is prepared by the following raw materials comprising a weight ratio: 1 part of mantle aglycon and 2-40 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 according to 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 a preparation prepared from the following raw materials by weight ratio: 1 part of mantle aglycon and 20 parts of a carrier material. The liposome according to any one of claims 1 to 3, which further comprises 1 to 5 parts of a protective agent. The liposome according to claim 4, wherein the protective agent is selected from one or a mixture of two or more 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 mantle aglycone, 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, characterized in that it comprises the following steps: a, dissolving the mantle aglycon 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 adding a protective agent and water; c, homogenization, sterilization, that is. The preparation method according to claim 7, wherein the organic solvent in the step a is ethanol; in the step b, after adding the protective agent and water, the concentration of the aglycone is 0.2 mg/mL; In c, the homogenous conditions are: homogenization 4 times under a pressure of 1000 bar; the sterilization condition is: 0.22 μm microporous membrane filtration sterilization. Use of the liposome of any of claims 1-6 for the manufacture of a medicament for the treatment and/or prevention of myelosuppression. Use of the liposome of any of claims 1-6 for the preparation of a medicament for increasing the amount of one or more of peripheral blood leukocytes, neutrophils, red blood cells, platelets, hemoglobin or bone marrow hematopoietic stem cells. A method for treating and/or preventing myelosuppression, which comprises the treatment with a liposome according to any one of claims 1 to 6. A method for increasing the number of one or more of peripheral blood leukocytes, neutrophils, red blood cells, platelets, hemoglobin, bone marrow hematopoietic stem cells, characterized by using the lipid according to any one of claims 1 to The plastid is treated.

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