WO2018133112A1

WO2018133112A1 - Ziyuglycogenin injection, preparation method therefor and use thereof

Info

Publication number: WO2018133112A1
Application number: PCT/CN2017/072232
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 injection, a preparation method therefor and use thereof. The ziyuglycogenin injection comprises a formulation prepared from the following raw materials: 0.5-10 parts of ziyuglycogenin and 0.001-60 parts of solubilizer.

Description

Mantle aglycone injection, preparation method and use thereof

Technical field

The invention relates to a diterpene aglycone injection, 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, especially myelosuppression caused by radiotherapy and chemotherapy. Therefore, finding an effective drug to alleviate bone marrow suppression has become an urgent problem to be solved.

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:

However, to date, there has been no public report on the preparation of an injection using the indole aglycone as an active ingredient for the treatment and/or prevention of myelosuppression.

Summary of the invention

In order to solve the above problems, the present invention provides a diterpene aglycone injection, which is characterized in that it is a preparation comprising the following raw materials:

Mantle aglycon and solubilizer.

Further, it is a preparation prepared from a raw material having the following weight ratio:

0.5-10 parts of mantle aglycone and 0.001-60 parts of solubilizer.

Further, the weight ratio of the mantle aglycone to the solubilizer is 1:0.005-300.

Further, the solubilizing agent is Tween 80, polyoxyethylene castor oil EL or poloxamer 188; and the pH adjusting agent is sodium hydroxide.

Further, the concentration of the indole aglycon in the injection was 0.2 mg/mL.

Further, the injection is an injection, a powder injection, a water injection or a large infusion.

Further, the following steps are included:

The genistein is weighed and dispersed in water for injection according to the ratio, and the corresponding ratio of solubilizer and pH adjuster is added, the pH is adjusted to 10, stirred and dissolved, and then water for injection is added, filtered, and sterilized to obtain the injection of the present invention.

The invention also provides the use of the injection for the preparation of a medicament for the treatment and/or prevention of myelosuppression.

Further, the medicament is a medicament for treating and/or preventing bone marrow suppression caused by a chemical substance.

Further, the drug is a drug that raises blood cells and/or bone marrow hematopoietic stem cells.

Further, the drug is a drug that increases peripheral blood leukocytes, neutrophils, red blood cells, platelets, and/or hemoglobin.

The present invention also provides a method of treating and/or preventing myelosuppression by using the aforementioned injection The agent is treated and/or prevented.

Previous studies by the inventors have found that mantle aglycones have problems such as low solubility and low oral absorption rate, which results in low oral bioavailability of the component, which limits the effect of the component on blood cell growth.

The inventors have studied the dosage form and its formulation, and the mantle aglycone injection prepared by the invention solves the problem of low solubility of the mantle aglycone, improves the bioavailability of the mantle aglycone, and further improves the therapeutic effect of the blood cell. It is of great significance for the development of new dosage forms of geniposide and better clinical application.

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 comparison of bone marrow hematopoietic stem cell counts in mice of each experimental group.

detailed description

Example 1 Preparation of Injectable Agent of the Present Invention

Weigh the raw materials according to the following ratio:

1.5 g of mantle aglycone, 10 g of Tween 80, and an appropriate amount of pH adjuster.

The preparation method is as follows:

The aglycone was dispersed in 10 mL of water for injection, Tween 80 and sodium hydroxide were added, the pH was adjusted to 10, stirred and dissolved, and then water for injection was added to 7500 mL to make the concentration of the aglycone in the solution 0.2 mg/mL. The injection of the present invention is obtained by filtration and sterilization with a 0.22 um microporous membrane.

Example 2 Preparation of Injectable Agent of the Present Invention

Weigh the raw materials according to the following ratio:

2.5 g of mantle aglycone, 60 g of polyoxyethylene castor oil, and an appropriate amount of pH adjuster.

The preparation method is as follows:

The mantle aglycone was dispersed in 10 mL of water for injection, polyoxyethylene castor oil EL and sodium hydroxide were added, the pH was adjusted to 10, stirred and dissolved, and then water for injection was added to 12500 mL to make the concentration of the aglycone in the solution 0.2 mg. /mL, filtered and sterilized with a 0.22 um microporous membrane to obtain the injection of the present invention.

Example 3 Preparation of the Injection of the Invention

Weigh the raw materials according to the following ratio:

10 g of mantle aglycone, 188 60 g of poloxamer, and an appropriate amount of pH adjuster.

The preparation method is as follows:

The mantle aglycone was dispersed in 10 mL of water for injection, poloxamer 188, sodium hydroxide was added, the pH was adjusted to 10, stirred and dissolved, and then water for injection was added to 50,000 mL to make the concentration of the aglycone in the solution 0.2 mg/ The mL was filtered and sterilized with a 0.22 um microporous membrane to obtain the injection of the present invention.

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

Experimental Example 1 Quality Evaluation of Mantle Aglycone Injection Prepared with Different Excipients

According to the nature of mantle aglycones, Tween 80, polyoxyethylene castor oil EL, poloxamer 188, propylene glycol and vitamin C were used as excipients to prepare scutellarin injection.

The preparation method is as follows: 0.2 g of mantle aglycone is dispersed in 100 mL of water for injection, and different auxiliary materials are added (Tween 80, polyoxyethylene castor oil EL, poloxamer 188, propylene glycol, vitamin C), sodium hydroxide. Adjust the pH to 10, stir and dissolve, then add water for injection to 1000 mL to make the concentration of the aglycone in the solution 0.2mg/mL, filter and sterilize with 0.22um microporous membrane to obtain the injection of the present invention. Table 1.

The pyrogen is determined according to the Chinese Pharmacopoeia (2010 edition) pyrogen inspection method.

According to the Chinese Pharmacopoeia (2010 edition), the clarification degree can be checked by the light inspection method under the foreign matter inspection law.

The content of the aglycone was determined by HPLC. Chromatographic conditions: Agilent extend-C18 Column (250 x 4.6 mm, 5 μm); mobile phase: acetonitrile-0.5% phosphoric acid (70:30). Flow rate: 0.8 mL·min-1, column temperature: 40 ° C, detection wavelength: 205 nm, injection amount: 10 μL.

Table 1 Quality evaluation of mantle aglycone injection prepared with different excipients

Note: Compared with Tween 80 group, *P<0.05.

EXPERIMENTAL RESULTS: The mantle aglycone injection prepared by using Tween 80, polyoxyethylene castor oil EL and poloxamer 188 of the invention was qualified for heat source examination, and the degree of clarity was checked. The content of mantle aglycone showed high content. It is indicated that the mantle aglycone injection prepared by using the excipient Tween 80, polyoxyethylene castor oil EL and poloxamer 188 of the invention meets the requirements of the preparation, and retains the aglycone component to the greatest extent. The other two kinds of excipients, propylene glycol and vitamin C, were prepared by geniposide injection, which was qualified by heat source, and the clarity of the test was unqualified. The content of genistein was low.

Experimental Example 2 Quality Evaluation of Mantle Aglycone Injection Prepared with Different Amounts of Excipients

The preparation method is as follows: 0.2 g of mantle aglycone is dispersed in 100 mL of water for injection, and different amounts of solubilizing agent (Tween 80) and sodium hydroxide are added respectively, the pH is adjusted to 10, stirred and dissolved, and then water for injection is added to 1000 mL. The concentration of the indole aglycon in the solution was 0.2 mg/mL, and the mixture was filtered and sterilized by a 0.22 um microporous membrane to obtain the injection of the present invention. The results are shown in Table 2.

Table 2 Quality evaluation of mantle aglycone injection prepared with different amounts of solubilizer

Note: *P<0.05 compared to the 1.0 mg group.

Experimental results: Only in the present invention, the aglycone 0.2g and the solubilizer dosage of 1.0mg-60g ratio prepared by the geniposide injection heat source qualified, the degree of clarity meets the requirements, the content is high, the preparation is the best, and the preparation can meet the requirements. A mantle aglycone injection.

Experimental Example 3 Pharmacodynamic study of the indole aglycone injection of the present invention

1 experimental material

1.1 Test drug The present invention is a geniposide injection group A (prepared according to Example 1), group B (prepared according to Example 2), group C (prepared according to Example 3), mantle aglycone 10% DMSO-physiology Saline group, cyclophosphamide.

1.2 Experimental animal 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 (

), 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.

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; mantle aglycone injection group A, B, C, 2.5 mg·kg ^-1 suspension, prepared before use; Indole aglycone group: Mantle aglycone powder, dissolved in 10% DMSO-physiological saline, formulated into a 2.5 mg·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.

2.2 administration

Each experimental group was given the corresponding drug by dose and administration 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.

2.3 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.

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 and Table 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.

The results showed that compared with the model group, the number of hematopoietic stem cells in the aglycone injection group A, the mantle aglycone injection group B, and the mantle aglycone injection group C were significantly increased (P<0.05). There was no significant difference in the tuple group. Compared with the mantle aglycone group, the number of WBC, RBC and PLT in the peripheral blood of the mice in the group A, the aglycone injection group B and the mantle aglycone injection group C were all different. Significantly elevated (P <0.05).

Table 4 Number of peripheral blood cells in each experimental group

The results showed that compared with the model group, the number of NEUT and HGB in the peripheral blood of the mice treated with the aglycone injection group A, the mantle aglycone injection group B and the mantle aglycone injection group C were significantly increased (P<0.05). There was no significant difference in the mantle aglycon group; compared with the mantle aglycon group, the number of NEUT and HGB in peripheral blood of the mice in the group of the mantle aglycone injection group A, the mantle aglycone injection group B, and the mantle aglycone injection group C group There was a significant increase (P<0.05)

3.2, 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 indole aglycone injection group of the present invention was significantly increased (P<0.05), and there was no significant difference in the mantle aglycon group; In comparison, the number of hematopoietic stem cells in the inoculum aglycone injection group of the present invention was significantly increased (P<0.05).

The above experimental results show that the indole aglycone injection of the present invention can effectively raise blood cells, and the drug effect is obviously superior to that of the direct use of the geniposide.

In summary, the mantle aglycone injection prepared by the invention solves the problem of low solubility of the mantle aglycone, improves the bioavailability of the mantle aglycone, and further improves the therapeutic effect of the blood cell, thereby improving the therapeutic effect of the cell aglycone. Good clinical application is of great significance.

Claims

A mantle aglycone injection, which is prepared by preparing a raw material having the following weight ratio:

0.5-10 parts of mantle aglycone and 0.001-60 parts of solubilizer.
The injection according to claim 1, wherein the weight ratio of the mantle aglycone to the solubilizer is 1:0.005 to 300.
The injection according to claim 1 or 2, wherein the solubilizing agent is Tween 80, polyoxyethylene castor oil EL or poloxamer 188; and the pH adjusting agent is sodium hydroxide.
The injection according to any one of claims 1 to 3, wherein the concentration of the indole aglycon in the injection is 0.2 mg/mL.
The injection according to any one of claims 1 to 4, wherein the injection is an injection, a powder injection, a water injection or a large infusion.
A method of preparing an injection according to any one of claims 1 to 5, comprising the steps of:

The genistein is weighed and dispersed in water for injection according to the ratio, and the corresponding ratio of solubilizer and pH adjuster is added, the pH is adjusted to 10, stirred and dissolved, and then water for injection is added, filtered, and sterilized to obtain the injection of the present invention.
Use of the injection according to any one of claims 1 to 6 for the preparation 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.
Use according to claim 7 or 8, characterized in that the medicament is a medicament for raising blood cells and/or bone marrow hematopoietic stem cells.
The use according to claim 9, characterized in that the drug is a drug for raising peripheral blood leukocytes, neutrophils, red blood cells, platelets and/or hemoglobin.
A method for treating and/or preventing myelosuppression, which is characterized in that it is treated and/or prevented by the injection according to any one of claims 1 to 6.