WO2018133106A1

WO2018133106A1 - Ziyuglycogenin solid dispersion, preparation method therefor and use thereof

Info

Publication number: WO2018133106A1
Application number: PCT/CN2017/072226
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 ziyuglycogenin solid dispersion, a preparation method therefor and a use thereof. The ziyuglycogenin solid dispersion is a formulation prepared from the following raw and auxiliary materials in weight proportion: 1 part of ziyuglycogenin and 2-30 parts of polyvinylpyrrolidone.

Description

A saponin solid dispersion and preparation method and use thereof

Technical field

The invention relates to a saponin solid dispersion, a preparation method thereof and a 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 saponin is 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 was found that the saponin 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 saponin.

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

Summary of the invention

The object of the present invention is to provide a saponin solid dispersion, a preparation method thereof and use thereof.

The present invention provides a solid dispersion of saponin, which is prepared from a raw material of the following weight ratio: 1 part of saponin and 2 to 30 parts of povidone.

Among them, it is a preparation prepared from the following raw materials by weight ratio: 1 part of saponin and 2-20 parts of povidone.

Further, it is a preparation prepared from the following raw materials by weight ratio: 1 part of saponin and 8 parts of povidone.

Further, it is a preparation prepared from the following raw materials by weight ratio: 1 part of saponin and 5 parts of povidone.

Wherein the povidone is PVP K30.

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

a. Dissolving the saponin and povidone in an organic solvent to obtain a saponin solution, Povidone solution, spare;

b, adding the saponin solution to the povidone solution, and mixing uniformly;

c. The organic solvent in the mixture obtained in the step b is removed and pulverized.

Wherein, the organic solvent described in the step a is anhydrous ethanol.

The invention also provides the use of a solid dispersion as described above for the manufacture of a medicament for the treatment and/or prevention of myelosuppression.

Wherein the drug is a drug for treating and/or preventing bone marrow suppression caused by a chemical substance.

The present invention also provides the use of the above solid dispersion for the preparation of a medicament for increasing the number of one or more of peripheral blood leukocytes, neutrophils, red blood cells, platelets, hemoglobin, bone marrow hematopoietic stem cells.

The invention also provides a method of treating and/or preventing myelosuppression, in particular using the solid dispersion described above.

Among them, the method is a method of treating and/or preventing bone marrow suppression caused by a chemical substance.

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 solid dispersion.

The present invention provides a saponin solid dispersion and a preparation method thereof. During the research, the inventors found that the effect of saponin increasing blood cell level is poor because of its low solubility and low gastrointestinal absorption rate, resulting in low bioavailability of the drug and limiting its efficacy. The solid dispersion of saponin prepared by the invention can significantly improve the solubility and dissolution of the drug, and significantly increase the number of peripheral blood leukocytes, neutrophils, red blood cells, platelets, hemoglobin and bone marrow hematopoietic stem cells, and the effect is significantly better than the ground. The original drug of saponin has obvious effects of treating and/or preventing myelosuppression.

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

1 each experimental group of mice CD34 ^- / Sca-1 ⁺ changes in

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 Solid Dispersion of the Invention

1 g of saponin was taken and dissolved by adding 100 mL of absolute ethanol to obtain a solution 1.

Another 5 g of PVP K30 was added and ultrasonically added to 20 mL of absolute ethanol to dissolve to obtain a solution 2.

The solution 2 was stirred on a thermostatic magnetic stirrer while the solution 1 was slowly added to the solution 2 with a dropper. After the solution 1 was all added to the solution 2, the mixture was stirred on a thermostatic magnetic stirrer for four hours. The mixture was poured into an evaporating dish, placed in a 60 ° C water bath, evaporated to dryness, and the dried product was finely ground to obtain a solid dispersion.

Example 2 Preparation of Solid Dispersion of the Invention

5 g of saponin was taken and 500 mL of absolute ethanol was added to dissolve to dissolve to obtain a solution 1.

Another 40 g of PVP K30 was added and ultrasonically added to 160 mL of absolute ethanol to dissolve to obtain a solution 2.

Example 3 Preparation of Solid Dispersion of the Invention

10 g of mantle saponin was taken and ultrasonically added to 1000 mL of absolute ethanol to dissolve to obtain a solution 1.

Another 200 g of PVP K30 was added, and 800 mL of absolute ethanol was added to ultrasonicate to dissolve to obtain a solution 2.

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

Dissolution method: Take one tablet of each batch, according to the dissolution method ("Chinese Pharmacopoeia" Appendix XC second method), with pH 6.8PBS 1 000mL as the dissolution medium, the rotation speed is 100r·min ^-1 , temperature ( 37±0.5)°C, sample 5mL at 0, 5, 15, 20, 25min, filter with 0.45μm microporous membrane, place the filtrate in a 100mL volumetric flask, dilute to the mark with pH 6.8PBS, shake well, and UV- Visible spectrophotometry (Chinese Pharmacopoeia Appendix IVA) measured absorbance at 345nm wavelength; after 25min sampling, the whole solution was transferred to a beaker, heated and stirred at 100 ° C for 2 ~ 3min, cooled to 37 ° C, according to the above method After sampling and dilution, the absorbance measured by the eluate after treatment at 100 ° C was the denominator, and the absorbance measured at each time point was taken as a molecule, and the cumulative dissolution rate was calculated.

Experimental Example 1 Quality Evaluation of Preparation of Diosgenin Solid Dispersion Using Different Carrier Materials

Five experimental groups were set up in this experiment.

1.0 g of saponin was taken separately, and 100 ml of anhydrous ethanol was added thereto to dissolve to obtain a solution 1. Another 8.0g of different polymer materials PVP K30, poloxamer 188 (F68), polyethylene glycol (PEG) 8000, polyethylene glycol (PEG) 6000, polyethylene glycol (PEG) 4000, add 40ml The anhydrous ethanol was sonicated to dissolve to obtain a solution 2. Solution 2 was heated to melt on a thermostatic magnetic stirrer while solution 1 was slowly added to solution 2 with a dropper. After solution 1 was all added to solution 2, the mixture was stirred on a thermostatic magnetic stirrer for four hours. The mixture was poured into an evaporating dish, placed in a 60 ° C water bath, evaporated to dryness, and the dried product was finely ground to obtain a solid dispersion. Determining the solubility of solid dispersions and The dissolution rate is shown in Table 1.

Table 1 Results of determination of solubility and dissolution of saponin solid dispersion

Note: Compared with PVP K30, *P<0.05.

The solubility of the original drug of the saponin was measured in the early stage of the experiment, which was only 0.021 mg/ml. It can be seen from Table 1 that when the solid dispersion of saponin is prepared by using the povidone of the present invention, the solubility and dissolution of the drug are the highest, which is significantly better than other carrier materials (P<0.05); if other carrier materials, such as Pollock, are used, With sam, polyethylene glycol, etc., the dissolution rate of the solid dispersion is greatly reduced, and the solubility of the drug is not as obvious as that of PVP K30.

The above results show that the quality of the saponin solid dispersion prepared by using povidone as the carrier material is the best.

Experimental Example 2 Effect of the Amount of Carrier Materials on the Quality of Diosgenin Solid Dispersion

Six experimental groups were set up in this experiment.

1.0 g of saponin was taken separately, and 100 ml of anhydrous ethanol was added thereto to dissolve to obtain a solution 1. Further, PVP K30 (the mass of the fixed saponin was changed to 1 part, and the mass of the PVP K30 as a function of the ratio) was weighed according to the mass ratio shown in Table 2, and 40 ml of absolute ethanol was added thereto to ultrasonically dissolve to obtain a solution 2. Solution 2 was heated to melt on a thermostatic magnetic stirrer while solution 1 was slowly added to solution 2 with a dropper. After solution 1 was all added to solution 2, the mixture was stirred on a thermostatic magnetic stirrer for four hours. The mixture was poured into an evaporating dish, placed in a 60 ° C water bath, evaporated to dryness, and the dried product was finely ground to obtain a solid dispersion. The solubility and dissolution of the solid dispersion were measured, and the results are shown in Table 2.

Table 2 Determination of Solubility and Dissolution of Diquat Saponin Solid Dispersion

Note: *P<0.05 compared to 1:8 group.

The experimental results: when the amount of PVP K30 is 2-30 times of saponins, the solid dispersion with good quality can be obtained, the dissolution rate is above 65%; when the dosage of PVP K30 is 2-20 times of saponin, The quality of the preparation was further optimized, the solubility of the drug was increased to 0.48 mg/mL or more, and the dissolution rate was not less than 70%. Among them, the drug solubility increased to 0.599 under the condition that the mass ratio of saponin: PVP K30 was 1:8. Mg/mL, the dissolution rate is as high as 93%, the prepared solid dispersion has the highest solubility and dissolution, which is significantly better than other ratios (P<0.05).

Experimental Example 3 Pharmacodynamic study of the saponin solid dispersion of the present invention

1. Experimental materials, reagents and instruments

1.1. The test drug saponin solid dispersion group (prepared according to Example 2), saponin 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

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

It is indicated that one-way analysis of variance is used between groups, LSD test is performed between groups with variance, and Tamhane's T2 test is 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; saponin solid dispersion group, powder must be formulated into 0.5mg·kg ^-1 , 5mg·kg ^-1 , 10mg·kg ^-1 suspension, prepared before use; saponin group, formulated into 10mg·kg ^-1 suspension, prepared 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 gavage from the first day of the experiment. The mice in the blank group and the model group were intragastrically administered with the same volume of normal saline 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 using 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

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; compared with the saponin group, ^△ P<0.05, ^△△ P<0.01.

As can be seen from Table 3, compared with the model group, there was no significant difference in the saponin group, and the numbers of WBC, RBC and PLT in the peripheral blood of the mice in the solid dispersion of saponin were significantly increased (P<0.05). Compared with the saponin group, the number of WBC, RBC and PLT in the peripheral blood of the mice in the solid dispersion of saponin was significantly increased (P<0.05).

Table 4 Number of peripheral blood cells in each experimental group

As can be seen from Table 4, compared with the model group, there was no significant difference in the saponin group, and the number of NEUT and HGB in the peripheral blood of the saponin solid dispersion group was significantly increased (P<0.05); Compared with the sapogenin group, the number of NEUT and HGB in the peripheral blood of the saponin solid dispersion group was significantly increased (P<0.05).

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 saponin solid dispersion group of the present invention was significantly increased (P<0.05), and there was no significant difference in the saponin group; Compared with the tuple group, the number of hematopoietic stem cells in the saponin solid dispersion group of the present invention was significantly increased (P<0.05).

The above experimental results show that the solid dispersion of the saponin of the present invention can effectively raise blood cells, and the drug effect is obviously superior to that of the direct use of saponin.

In summary, the solid dispersion of saponin prepared by the invention effectively solves the problem of low solubility of the saponin, improves the bioavailability of the saponin, thereby improving the therapeutic effect of raising blood cells, and effectively preventing and treating bone marrow suppression. It is of great significance for the clinical application of saponin.

Claims

A solid dispersion of saponin, characterized in that it is prepared from the following raw materials by weight ratio: 1 part of saponin and 2-30 parts of povidone.
The solid dispersion according to claim 1, which is prepared from the following raw materials of a weight ratio: 1 part of saponin and 2 to 20 parts of povidone.
The solid dispersion according to claim 1 or 2, which is a preparation prepared from the following raw materials by weight ratio: 1 part of saponin and 8 parts of povidone.
The solid dispersion according to claim 1 or 2, which is a preparation prepared from the following raw materials by weight ratio: 1 part of saponin and 5 parts of povidone.
The solid dispersion according to any one of claims 1 to 4, wherein the povidone is PVP K30.
A method for preparing a solid dispersion according to any one of claims 1 to 5, comprising the steps of:

a, the saponin and povidone are dissolved in an organic solvent to obtain a saponin solution, a povidone solution, and set aside;

b, adding the saponin solution to the povidone solution, and mixing uniformly;

c. The organic solvent in the mixture obtained in the step b is removed and pulverized.
The preparation method according to claim 6, wherein the organic solvent in the step a is anhydrous ethanol.
Use of the solid dispersion according to any one of claims 1 to 5 for the preparation of a medicament for the treatment and/or prevention of myelosuppression.
The use according to claim 8, wherein the drug is a drug for treating and/or preventing a bone marrow suppression caused by a chemical substance.
The use of the solid dispersion according to any one of claims 1 to 5 for the preparation of a medicament for increasing the number of one or more of peripheral blood leukocytes, neutrophils, red blood cells, platelets, hemoglobin, bone marrow hematopoietic stem cells.
A method for treating and/or preventing myelosuppression, which comprises treating the solid dispersion according to any one of claims 1 to 5.
The method according to claim 11, wherein the method is a method of treating and/or preventing chemical substance-induced myelosuppression.
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 solid according to any one of claims 1 to 5. The dispersion is treated.