WO2018133105A1

WO2018133105A1 - ZIYUGLYCOGENIN-HYDROXYPROPYL β-CYCLODEXTRIN INCLUSION COMPLEX, PREPARATION METHOD THEREFOR AND USE THEREOF

Info

Publication number: WO2018133105A1
Application number: PCT/CN2017/072225
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-hydroxypropyl β-cyclodextrin inclusion complex and a preparation method therefor and a use thereof. The ziyuglycogenin-hydroxypropyl β-cyclodextrin inclusion complex is a formulation prepared from the following raw materials in weight proportion: 0.5-10 parts of ziyuglycogenin and 1-20 parts of hydroxypropyl β-cyclodextrin, and a pharmaceutically accepted solvent.

Description

Saponin hydroxypropyl β-cyclodextrin inclusion compound, preparation method and use thereof

Technical field

The invention relates to a saponin hydroxypropyl β-cyclodextrin inclusion compound, 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 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:

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

Summary of the invention

The technical proposal of the present invention provides a saponin hydroxypropyl β-cyclodextrin inclusion compound, a preparation method thereof and use thereof.

The present invention provides a saponin hydroxypropyl β-cyclodextrin inclusion compound prepared from the following stoichiometric starting materials plus a pharmaceutically acceptable solvent:

0.5-10 parts of saponin and 1-20 parts of hydroxypropyl β-cyclodextrin.

Hydroxypropyl β-cyclodextrin: H-β-CD.

Among them, it is prepared from the following weight ratio raw materials plus a pharmaceutically acceptable solvent:

1 part of saponin, 1-30 parts of hydroxypropyl β-cyclodextrin.

Wherein: it is prepared from the following weight ratio of raw materials plus a pharmaceutically acceptable solvent:

2.5 parts of saponin and 10 parts of hydroxypropyl β-cyclodextrin;

Or 1 part of saponin, 10 parts of hydroxypropyl β-cyclodextrin.

Wherein the pharmaceutically acceptable solvent is at least one of absolute ethanol, n-butanol and diethyl ether.

The present invention also provides a method of preparing the above clathrate, comprising the steps of:

a according to the ratio of the saponin, adding a pharmaceutically acceptable solvent, dissolved, to obtain a solution 1;

b. According to the ratio of taking hydroxypropyl β-cyclodextrin, adding a pharmaceutically acceptable solvent, dissolved, to obtain a solution 2;

c. While stirring the solution 2, the solution 1 is added thereto, and after all the addition, stirring, evaporation and grinding are carried out to obtain a clathrate.

Wherein in step a and / or b, the pharmaceutically acceptable solvent is at least one of absolute ethanol, n-butanol and diethyl ether;

In the step c, the stirring time was 4 hours, and the evaporation to dryness was 60 °C.

The invention also provides the use of the above clathrate 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 clathrate 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 present invention also provides a saponin preparation which is prepared by using a saponin hydroxypropyl β-cyclodextrin inclusion compound as an active ingredient, adding a pharmaceutically acceptable auxiliary or auxiliary component. preparation.

Wherein, the preparation is a powder, a tablet, a pill, a granule, a powder injection, an injection or a capsule.

The present invention also provides a method of treating and/or preventing myelosuppression, in particular, using the aforementioned clathrate.

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 clathrate.

The saponin hydroxypropyl β-cyclodextrin inclusion compound prepared by the invention can significantly improve the solubility and dissolution of the drug, and significantly increase the peripheral blood leukocytes, neutrophils, red blood cells, platelets, hemoglobin and bone marrow hematopoietic stem cells. The quantity, with obvious therapeutic and/or prevention of myelosuppression, is of great significance for the clinical application of saponin.

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 Comparison of bone marrow hematopoietic stem cell counts 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 the inclusion complex of the present invention

Weigh the raw materials according to the following ratio:

There is 0.5 g of saponin and 1.5 g of hydroxypropyl β-cyclodextrin.

The preparation method is as follows:

The saponin was taken according to the ratio, and 50 ml of anhydrous ethanol was added thereto to ultrasonically dissolve to obtain a solution 1. Further, H-β-CD was taken according to the ratio, and 15 ml of absolute ethanol was added to ultrasonicate 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 clathrate.

Example 2 Preparation of the inclusion complex of the present invention

Weigh the raw materials according to the following ratio:

1.5 g of saponin and 5 g of hydroxypropyl β-cyclodextrin.

The preparation method is as follows:

The saponin was taken according to the ratio, and 150 ml of absolute ethanol was added to dissolve to dissolve to obtain a solution 1. Another H-β-CD was taken according to the ratio, and 50 ml of absolute ethanol was added to ultrasonicate 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 clathrate.

Example 3 Preparation of the inclusion complex of the present invention

Weigh the raw materials according to the following ratio:

2 g of saponin and 10 g of hydroxypropyl β-cyclodextrin.

The preparation method is as follows:

The saponin was taken according to the ratio, and 250 ml of absolute ethanol was added to dissolve to dissolve to obtain a solution 1. Further, H-β-CD was taken according to the ratio, and 100 ml of anhydrous ethanol was added thereto to ultrasonically 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 clathrate.

Example 4 Preparation of the inclusion complex of the present invention

Weigh the raw materials according to the following ratio:

There were 4.5 g of saponin and 45 g of hydroxypropyl β-cyclodextrin.

The preparation method is as follows:

The saponin was taken according to the ratio, and 450 ml of anhydrous ethanol was added to dissolve to dissolve to obtain a solution 1. Further, H-β-CD was taken according to the ratio, and 450 ml of absolute ethanol was added to ultrasonicate 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 clathrate.

Example 5 Preparation of the inclusion complex of the present invention

Weigh the raw materials according to the following ratio:

There were 6.5 g of saponin and 16.5 g of hydroxypropyl β-cyclodextrin.

The preparation method is as follows:

The saponin was taken according to the ratio, and 650 ml of absolute ethanol was added to ultrasonicate to dissolve to obtain a solution 1. Further, H-β-CD was taken according to the ratio, and 165 ml of absolute ethanol was added to ultrasonicate 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 clathrate.

Example 6 Preparation of the inclusion complex of the present invention

Weigh the raw materials according to the following ratio:

Mantle saponin 8.5 g, hydroxypropyl β-cyclodextrin 35 g.

The preparation method is as follows:

The saponin was taken according to the ratio, and 850 ml of absolute ethanol was added to dissolve to dissolve to obtain a solution 1. Further, H-β-CD was taken according to the ratio, and 350 ml of absolute ethanol was added thereto to ultrasonically 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 clathrate.

Example 7 Preparation of the inclusion complex of the present invention

Weigh the raw materials according to the following ratio:

10 g of saponin and 45 g of hydroxypropyl β-cyclodextrin.

The preparation method is as follows:

The saponin was taken according to the ratio, and 1000 ml of absolute ethanol was added to ultrasonicate to dissolve to obtain a solution 1. Further, H-β-CD was taken according to the ratio, and 450 ml of absolute ethanol was added to ultrasonicate to dissolve to obtain a solution 2. Stir the solution 2 on a constant temperature magnetic stirrer while slowly adding the solution 1 to the solution 2 with a dropper. After all of Solution 1 was 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 clathrate.

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

Experimental Example 1 Quality Evaluation of Preparation of Mantle Saponin Inclusion Complex Using Different Inclusion Materials

In this study, based on the properties of the saponin, β-cyclodextrin, methyl β-cyclodextrin, and hydroxypropyl β-cyclodextrin were selected as the carrier materials to prepare the saponin inclusion complex.

The following materials were weighed and weighed: 5 g of saponin and 15 g of clathrate.

The preparation method is as follows:

The saponin was weighed and added with 100 times the amount of absolute ethanol to dissolve to obtain a solution 1. The inclusion material was further taken, and 10 times of anhydrous ethanol was added thereto to ultrasonically 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 clathrate.

The inclusion ratio (inclusion rate (%) = mass of saponin in the inclusion compound / mass of saponin administration × 100%) was measured, and the results are shown in Table 1.

Table 1 Quality evaluation of preparation of saponin inclusion complexes with different inclusion materials

Note: *P<0.05 compared to the β-cyclodextrin group.

It can be seen from Table 1 that the saponin hydroxypropyl β-cyclodextrin inclusion compound prepared by using the hydroxypropyl β-cyclodextrin of the present invention has high inclusion ratio and good inclusion effect. Inclusion packages prepared using other inclusion materials have poor inclusion rates.

The results showed that the quality of the saponin hydroxypropyl β-cyclodextrin inclusion compound prepared only using the hydroxypropyl β-cyclodextrin of the present invention was the best.

Experimental Example 2: Quality Evaluation of Preparation of Diosgenin Hydroxypropyl β-Cyclodextrin Inclusion Complex Using Different Amounts of Hydroxypropyl β-Cyclodextrin Carrier Material

Different proportions of saponin and hydroxypropyl β-cyclodextrin were weighed and tested according to the following method: a certain amount of saponin was added, and 100 times of absolute ethanol was added to dissolve to obtain solution 1 . Further, hydroxypropyl β-cyclodextrin was taken according to the ratio, and 10 times the amount of absolute ethanol was added thereto to ultrasonically 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, and evaporated to dryness. The dried product was finely ground to obtain a clathrate, and the inclusion ratio was measured. The results are shown in Table 2.

Table 2 Inclusion ratio of saponin hydroxypropyl β-cyclodextrin inclusion compound prepared by different amounts of hydroxypropyl β-cyclodextrin carrier materials

Note: *P<0.05 compared to 1:0.5 group.

It can be seen from Table 2 that the saponin hydroxypropyl β-cyclodextrin inclusion complex can be obtained only in the proportion of the saponin: hydroxypropyl β-cyclodextrin of the present invention; wherein, the saponin is used Element: Hydroxypropyl β-cyclodextrin is in the range of 1:1 to 1:20, and the inclusion ratio is over 90%, which is significantly different from that of hydroxypropyl β-cyclodextrin 1:0.5 (P< 0.05), indicating hydroxypropyl β-cyclodextrin to saponin pack The effect is good.

Experimental Example 3 Pharmacodynamic study of the saponin hydroxypropyl β-cyclodextrin inclusion compound of the present invention

Experimental material

1.1 Test drug: saponin-H-β-CD clathrate (prepared according to Examples 1 to 7), saponin, cyclophosphamide.

1.2 Experimental animals: KM-mouse: 18.5-22.5 g.

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

1.4 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.

2. Experimental methods

2.1 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-H-β-CD inclusion complex group, prepared according to Examples 1-7, formulated into 2.5 mg·kg ^{- 1} suspension, set to sample groups 1 to 7, respectively, saponin group; dissolved in 10% DMSO-physiological saline, formulated into a 2.5mg·kg ^-1 suspension. 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.

2.2 administration

The experimental groups were given the corresponding drugs according to the dose and administration mode 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 physiological saline for 7 consecutive days.

2.3 specimen collection

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

2.4 Detection indicators and methods

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.

Bone marrow hematopoietic stem cell count (based on bone marrow cell CD34+ antigen expression), the right femur bone marrow cells were pulverized with PBS buffer containing 0.2% bovine serum albumin, 106 cells were removed, the supernatant was discarded, and 30 μL was added. Normal mouse serum was blocked with non-specific binding sites, 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 protected from light for 30 min at 4 °C. 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.

3. Experimental results

3.1 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 saponin group, ^△ P<0.05, ^△△ P<0.01.

As can be seen from Table 3, there was no significant difference in the saponin group compared with the model group, and the number of WBC, RBC, and PLT in the peripheral blood of the saponin-H-β-CD inclusion complex group was significantly increased ( P<0.05); Compared with the saponin group, the number of WBC, RBC and PLT in the peripheral blood of the saponin-H-β-CD inclusion complex group was significantly increased (P<0.05). Among them, the sample 3 group had a very significant increase (P<0.01).

Table 4 Number of peripheral blood cells in each experimental group

As can be seen from Table 4, there was no significant difference in the saponin group compared with the model group. The number of NEUT and HGB in the peripheral blood of the saponin-H-β-CD inclusion complex group was significantly increased (P< 0.05); Compared with the saponin group, the NEUT and HGB levels in the peripheral blood of the saponin-H-β-CD inclusion complex group were significantly increased (P<0.05). Among them, the sample 3 group had a very significant increase (P<0.01).

3.2 Comparison of bone marrow hematopoietic stem cell counts

see picture 1.

As can be seen from Fig. 1, compared with the model group, the number of hematopoietic stem cells in the saponin hydroxypropyl β-cyclodextrin inclusion compound group of the present invention was significantly increased (P<0.05), and the saponin group was absent. Significant difference; compared with the saponin group, the number of hematopoietic stem cells in the saponin hydroxypropyl β-cyclodextrin inclusion compound group of the present invention was significantly increased (P<0.05).

The above experimental results show that the saponin hydroxypropyl β-cyclodextrin inclusion compound of the present invention can effectively increase blood cells, and the direct use of the saponin is ineffective.

In summary, the saponin inclusion complex prepared by the invention effectively solves the problem of low solubility of the saponin, improves the bioavailability of the saponin, can effectively raise blood cells, and effectively prevent and treat bone marrow suppression. The clinical application of sapogenin is of great significance.

Claims

A saponin hydroxypropyl β-cyclodextrin inclusion compound, which is prepared from the following weight ratio raw materials together with a pharmaceutically acceptable solvent:

0.5-10 parts of saponin and 1-20 parts of hydroxypropyl β-cyclodextrin.
The clathrate according to claim 1, which is prepared from the following stoichiometric starting materials plus a pharmaceutically acceptable solvent:

1 part of saponin, 1-30 parts of hydroxypropyl β-cyclodextrin.
The clathrate according to claim 1 or 2, which is prepared from the following stoichiometric starting materials together with a pharmaceutically acceptable solvent:

2.5 parts of saponin and 10 parts of hydroxypropyl β-cyclodextrin;

Or 1 part of saponin, 10 parts of hydroxypropyl β-cyclodextrin.
The clathrate according to any one of claims 1 to 3, wherein the pharmaceutically acceptable solvent is at least one of absolute ethanol, n-butanol and diethyl ether.
A method of preparing a clathrate according to any one of claims 1 to 4, comprising the steps of:

a according to the ratio of the saponin, adding a pharmaceutically acceptable solvent, dissolved, to obtain a solution 1;

b. According to the ratio of taking hydroxypropyl β-cyclodextrin, adding a pharmaceutically acceptable solvent, dissolved, to obtain a solution 2;

c. While stirring the solution 2, the solution 1 is added thereto, and after all the addition, stirring, evaporation and grinding are carried out to obtain a clathrate.
The method of claim 5 wherein:

In step a and / or b, the pharmaceutically acceptable solvent is at least one of absolute ethanol, n-butanol and diethyl ether;

In the step c, the stirring time was 4 hours, and the evaporation to dryness was 60 °C.
Use of the clathrate of any of claims 1-4 for the manufacture of a medicament for the treatment and/or prevention of myelosuppression.
The use according to claim 7, wherein the drug is a drug for treating and/or preventing a bone marrow suppression caused by a chemical substance.
The use of the inclusion complex according to any one of claims 1 to 4 for the preparation of a medicament for increasing the amount of one or more of peripheral blood leukocytes, neutrophils, red blood cells, platelets, hemoglobin, bone marrow hematopoietic stem cells.
A saponin preparation, which is prepared by adding a saponin hydroxypropyl β-cyclodextrin inclusion compound as an active ingredient, adding a pharmaceutically acceptable auxiliary or auxiliary component. .
A method for the treatment and/or prevention of myelosuppression, characterized in that the composition according to any one of claims 1 to 4 is used for treatment.
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 package according to any one of claims 1-4 The compound is treated.