CN114712309A - Ginsenoside docetaxel liposome, preparation method and application thereof - Google Patents

Abstract

The invention discloses a compound ginsenoside docetaxel liposome, a preparation method and application thereof. The invention provides a compound ginsenoside docetaxel liposome, which comprises the following components in percentage by mass: 8-18 parts of phospholipid, 1-1.5 parts of ginsenoside, 1 part of docetaxel and 20-35 parts of freeze-drying protective agent; the ginsenoside docetaxel plastid does not contain one or more of cholesterol, soybean oil and sodium oleate. The ginsenoside docetaxel liposome disclosed by the invention has better active targeting property of Glut 1; the drug effect is improved by more than 2 times compared with the conventional ginsenoside docetaxel liposome; the toxicity is reduced by more than 1.5 times compared with the conventional ginsenoside docetaxel liposome and is reduced by more than 4-6 times compared with the conventional cholesterol docetaxel liposome; the risk of accumulating toxicity in vivo is low.

Description

A kind of ginsenoside docetaxel liposome, its preparation method and application

technical field

The invention relates to a compound ginsenoside docetaxel liposome, a preparation method and application thereof; further discloses a compound ginsenoside docetaxel liposome with high efficiency and low toxicity for injection, its preparation method and application.

Background technique

Liposome is a kind of directional drug delivery system, which belongs to a special dosage form of targeted drug delivery system. It can encapsulate drugs in nanometer-sized particles, which are similar to bilayers in biological membrane structures. Microvesicles enter the human body and are mainly phagocytosed by the reticuloendothelial system, and change the distribution of the encapsulated drug in the body, so that the drug is mainly accumulated in the target tissue, thereby improving the therapeutic index of the drug, reducing the therapeutic dose of the drug and reducing the drug toxicity.

The present invention is a technical innovation based on Chinese invention application patents such as CN201610693884.2, CN201811447245.3 and CN201811447243.4. The above three patent applications all disclose the technical advantages of liposomes with ginsenosides as membrane materials, such as stable quality and remarkable drug efficacy after encapsulating chemotherapy drugs such as paclitaxel.

CN201610693884.2 discloses a blank liposome with ginsenoside Rg5 and its derivatives as membrane materials and its application. In addition to phospholipids, saponins, drugs, and freeze-drying protective agents, the prescription can further include cholesterol, antioxidants , soybean oil and/or other excipients such as sodium oleate.

CN201811447245.3 discloses a blank liposome with ginsenoside Rh5H and its derivatives as membrane materials and its application. On the basis of CN201610693884.2, this patent further solves the problem of hemolysis of ginsenoside. Similarly, in addition to phospholipids, saponins, drugs, and lyoprotectants, the prescription may further include cholesterol, antioxidants, soybean oil and/or sodium oleate and other adjuvants.

CN201811447243.4 discloses a blank liposome using ginsenoside Rg3 and its derivatives as membrane materials and its application. This patent solves the problem of the solubility of ginsenosides in chloroform by using Rg3, Rh2 and other saponins through ultrafine powder and other technical means, thereby solving the problem that ginsenosides such as Rg3 and Rh2 must be formed into films in chloroform, and the prepared quality meets the standard Rg3 liposomes.

There are still some deficiencies in the above-mentioned prior art. For example, in some schemes, the required pressure for the homogenization step of liposome production is large, the speed of filter membrane sterilization and filtration is slow, the retention rate is high, and the product yield is obviously poor; it is necessary to add 2-6 times the amount of soybean oil. However, due to the addition of soybean oil, it is not conducive to the freeze-drying of the preparation and affects the long-term storage of the drug.

The core of compound preparations is the synergistic interaction of drugs in the body, and can significantly improve the clinical treatment effect of drugs. The reasonable ratio range of each functional component of the compound preparation is the core of the compound preparation, especially the changes in drug synergy, in vivo pharmacokinetics, in vivo tissue distribution, and drug efficacy caused by the changes in the functional components of the compound liposome are all fresh. Someone involved. Therefore, for the compound ginsenoside docetaxel liposome for injection (hereinafter referred to as "Ginposome-DTX" or "Ginposome-CTX"), the main drug "docetaxel" has been determined, how to choose the most suitable "synergistic" The active drug and excipient "ginsenoside" and the key excipient "phospholipid" and their related ratios are prepared to achieve the best effect with reasonable compatibility, small particle size, stable quality, drug efficacy and toxicity, so that the drug and key excipients of the present invention have a specific ratio. The composition is innovative and unique, and is of great significance.

In the screening of prescriptions, among many factors such as drugs, phospholipids, saponins, lyophilized protective agents and preparation processes, any change in any one factor will have a fatal impact on the quality, efficacy and safety of the product. For example, increasing the mass ratio of saponins to paclitaxel can increase the quality stability and synergistic anti-tumor effect of compound liposomes, and increase the targeted distribution of tumor tissues, but it will also increase the cumulative toxicity of saponins in the human body and cause uncontrollable effects. Organ damage; the appropriate mass ratio of saponin to paclitaxel has a very important relationship with the stability, active targeting, pharmacodynamics and safety of this type of liposome. At the same time, the selection of different lyoprotectants plays a crucial role in the preservation of the lipid bilayer structure during the lyophilization of liposomes and the recovery of the properties of the liposomes after reconstitution of the lyophilized drug. For example, in the selection of lyoprotectants, different lyoprotectants have different effects on the lyophilization curve, especially in the co-dissolution point of the compound liposome, whether it collapses, whether the liposome changes significantly after reconstitution, once The freeze-drying temperature and time setting, the total freeze-drying time and many other aspects have important influences.

The safety and effectiveness of a drug are two basic attributes of a drug, and both are indispensable. The approval and use of a drug are based on the risk-benefit ratio between the two, especially for improved new drugs, whose core is to improve effectiveness. and security.

In toxicology research, formulation research plays a crucial role, especially the effects of formulation ratio and preparation process selection on acute toxicity, long-term toxicity and cumulative toxicity of various functional organs will directly determine the compound formula Whether liposomes meet the new drug application requirements.

It can be seen that the optimum ratio range of the phospholipids, ginsenosides, docetaxel and lyoprotectant carbohydrate components in the above-mentioned liposome prescription membrane components is good for the good pharmaceutical stability, in vivo distribution, in vivo distribution, and composition of the formed compound liposomes. Properties such as pharmacodynamics and toxicology play an important role. However, for this optimal ratio, the prior art does not provide any of the above-mentioned components and ratios, as well as the derivation relationship between the process and the pharmacological activity, pharmacokinetics and toxicology. Due to the large number of variables involved, its screening must pass a lot of experiments and creative labor.

Therefore, how to choose the best compound drug compatibility and how to formulate the best preparation process in order to produce a compound ginsenoside for injection with better efficacy, lower toxicity, quality and other indicators that can meet the drug requirements Sitaxel liposomes require a lot of research work and technical breakthroughs in order to meet the drug application requirements.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a (compound) ginsenoside docetaxel liposome, its preparation method and application in view of the deficiencies of the existing docetaxel liposome; Small diameter, high drug encapsulation rate, good in vivo compatibility, good in vivo drug release, better efficacy, lower toxicity, and reasonable compatibility; and it has a better preparation process, and the preparation conditions are easy to achieve, which is conducive to industrialization; The optimization of the combination of preparation process and product performance.

The present invention solves the above technical problems through the following technical solutions.

The present invention provides a (compound) ginsenoside docetaxel liposome (referred to as "Ginposome-DTX" or "Ginposome-CTX"), which comprises the following components by mass fraction: 8-18 parts of phospholipids, 1- 1.5 parts of ginsenoside, 1 part of docetaxel, and 15-35 parts of a freeze-drying protective agent; the ginsenoside docetaxel plastid does not contain one or more of cholesterol, soybean oil and sodium oleate.

In a certain scheme of the present invention, the ginsenoside docetaxel liposome is composed of the following components by mass fraction: 8-18 parts of phospholipids, 1-1.5 parts of ginsenoside, 1 part of docetaxel And 15-35 parts of lyoprotectant.

In a certain scheme of the present invention, the phospholipid is one or more of egg yolk lecithin, soybean lecithin, hydrogenated phospholipid and cephalin, or contains 0.01-10% distearoylphosphatidylethanolamine-methyl One or more of egg yolk lecithin, soybean lecithin, hydrogenated phospholipid and cephalin of oxypolyethylene glycol 2000 (mPEG2000-DSPE), preferably egg yolk lecithin and/or soybean lecithin, more preferably egg yolk lecithin.

In a certain aspect of the present invention, the mass ratio of the docetaxel to the phospholipid may be 1:8-12, such as 1:10; for example, the docetaxel and egg yolk lecithin The mass ratio is 1:10.

In a certain scheme of the present invention, the ginsenoside can be 20(S)-ginsenoside Rg3, ginsenoside pseudo-Rg3, 20(S)-ginsenoside Rh2, pseudo-ginsenoside GQ, ginsenoside Rg5, ginsenoside One or more of Rk1 and ginsenoside Rp1, preferably 20(S)-ginsenoside Rg3 and/or 20(S)-ginsenoside Rh2, more preferably 20(S)-ginsenoside Rg3.

In a certain scheme of the present invention, the mass ratio of the docetaxel to the ginsenoside may be 1:1 or 1:1.5.

In a certain scheme of the present invention, the mass ratio of the docetaxel to the 20(S)-ginsenoside Rg3 is 1:1; or, the docetaxel and the 20 The mass ratio of (S)-ginsenoside Rg3 can be 1:1.5; or, the mass ratio of the docetaxel to the 20(S)-ginsenoside Rh2 can be 1:1; or, the described The mass ratio of the docetaxel to the 20(S)-ginsenoside Rh2 is 1:1.5.

In a certain aspect of the present invention, the lyoprotectant may be one or more of trehalose, glucose, sucrose, lactose and galactose, preferably glucose.

In a certain scheme of the present invention, the mass ratio of the lyoprotectant to the docetaxel can be 20-35:1; for example 25-35:1, another example 25:1, 30:1 1 or 35:1. For example, the mass ratio of the glucose to the docetaxel is 25:1.

In a certain scheme of the present invention, the ginsenoside docetaxel liposome has a particle size D90≤150nm, and an encapsulation efficiency≥98%.

In a certain aspect of the present invention, the HPLC purity of the ginsenoside is ≥99%.

In a certain aspect of the present invention, the ginsenoside docetaxel liposome comprises the following components by mass fraction: 10 parts phospholipids, 1 part or 1.5 parts ginsenoside, 1 part docetaxel and 25 parts Freeze-drying protectant.

In a certain aspect of the present invention, the ginsenoside docetaxel liposome may include the following components in mass fraction:

10 parts of phospholipids, 1 part of 20(S)-ginsenoside Rg3, 1 part of docetaxel and 25 parts of lyoprotectant;

Or, 10 parts of phospholipids, 1.5 parts of 20(S)-ginsenoside Rh2, 1 part of docetaxel and 25 parts of lyoprotectant;

Or, 10 parts of phospholipids, 1 part of 20(S)-ginsenoside Rg3, 1 part of docetaxel and 30 parts of lyoprotectant;

Or, 10 parts of phospholipids, 1.5 parts of 20(S)-ginsenoside Rh2, 1 part of docetaxel and 30 parts of lyoprotectant.

In a certain aspect of the present invention, the ginsenoside docetaxel liposome may include the following components in mass fraction:

10 parts egg yolk lecithin, 1 part 20(S)-ginsenoside Rg3, 1 part docetaxel and 25 parts glucose;

Or, 10 parts egg yolk lecithin, 1.5 parts 20(S)-ginsenoside Rh2, 1 part docetaxel and 25 parts glucose.

Or, 10 parts egg yolk lecithin, 1 part 20(S)-ginsenoside Rg3, 1 part docetaxel and 30 parts glucose.

Or, 10 parts egg yolk lecithin, 1.5 parts 20(S)-ginsenoside Rh2, 1 part docetaxel and 30 parts glucose.

The present invention also provides a blank liposome, which comprises the following components by mass fraction: 8-18 parts of phospholipids, 1-2 parts of ginsenosides and 20-35 parts of lyophilized protective agent; the liposome does not contain One or more of cholesterol, soybean oil, and sodium oleate.

In a certain aspect of the present invention, the blank liposome can be loaded with a pharmaceutical active substance, preferably, the pharmaceutical active substance can be a taxane derivative; for example, paclitaxel and/or docetaxel; and such as docetaxel.

In a certain aspect of the present invention, in the blank liposome, the phospholipid is one or more of egg yolk lecithin, soybean phospholipid, hydrogenated phospholipid and cephalin, or contains 0.01-10% diphospholipid One or more of egg yolk lecithin, soybean lecithin, hydrogenated phospholipid and cephalin of stearoylphosphatidylethanolamine-methoxy polyethylene glycol 2000.

In a certain aspect of the present invention, in the blank liposome, the phospholipid can be 10 parts.

In a certain scheme of the present invention, in the blank liposome, the ginsenoside can be 20(S)-ginsenoside Rg3, ginsenoside pseudo-Rg3, 20(S)-ginsenoside Rh2, pseudo-ginsenoside One or more of GQ, ginsenoside Rg5, ginsenoside Rk1 and ginsenoside Rp1, preferably 20(S)-ginsenoside Rg3 and/or 20(S)-ginsenoside Rh2, more preferably 20(S)-ginsenoside Saponin Rg3.

In a certain scheme of the present invention, in the blank liposome, the mass ratio of the ginsenosides may be 1 part, 1.5 parts or 2 parts.

In a certain aspect of the present invention, in the blank liposome, the lyoprotectant may be one or more of trehalose, glucose, sucrose, lactose and galactose, preferably glucose.

In a certain aspect of the present invention, in the blank liposome, the lyophilized protective agent may be 25-35 parts, for example, 25 parts, 30 parts or 35 parts.

In a certain aspect of the present invention, the blank liposome may include the following components in mass fractions: 9 parts of egg yolk lecithin, 1.8 parts of Rg3 and 20-35 parts of lyoprotectant;

Or, 10 parts soybean lecithin, 2 parts Rh2 and 20-35 parts lyoprotectant;

Or, 10 parts egg yolk lecithin: 1.5 parts pseudo Rg3 and 20-35 parts lyoprotectant;

Or, 10 parts egg yolk lecithin: 1.5 parts pseudo-GQ and 20-35 parts lyoprotectant;

Or, 10 parts egg yolk lecithin: 1.5 parts Rk1 and 20-35 parts lyoprotectant;

Or, 10 parts egg yolk lecithin: 1.5 parts Rp1 and 20-35 parts lyoprotectant.

The present invention also provides a preparation method of ginsenoside docetaxel liposome, which comprises the following steps;

Step 1. Concentrate the solution A1 of docetaxel, ginsenoside, phospholipid and organic solvent to form a film;

Step 2. After the membrane obtained in Step 1 is kept in water for hydration, it is evenly mixed with the lyophilized protective agent solution to obtain a liposome solution A2;

Step 3, it is scheme 1 or scheme 2;

Scheme 1 (high pressure homogenization method) includes the following steps:

The liposome solution A2 obtained in step 2 is subjected to high pressure homogenization, and the particle diameter D90 is controlled to be less than 100 nm to obtain a liposome solution A3a.

Scheme 2 (high-speed shearing + extrusion method) includes the following steps:

After shearing the liposome solution A2 obtained in step 2, it is extruded through a 150nm aperture extrusion plate, and the particle diameter D90 is controlled to be less than 100nm to obtain a liposome solution A3b;

Wherein, the definitions of docetaxel, ginsenoside, phospholipid and lyoprotectant solution are the same as those described in the above (compound) ginsenoside docetaxel liposome.

In a certain scheme of the present invention, in the step 1, the organic solvent may be one or more of methanol, ethanol, chloroform, and dichloromethane, preferably methanol and/or ethanol and chloroform and/or Or a mixed solvent of dichloromethane; for example, a mixed solvent of ethanol:chloroform=1:1 (volume ratio). The amount of the organic solvent may not be specifically limited, as long as it can dissolve docetaxel, ginsenoside and phospholipid. For example, the mass-volume ratio of docetaxel to the organic solvent is 1 g/60-120 ml, for example, 1 g/80 mL.

In a certain scheme of the present invention, in the step 1, the solution A1 is preferably obtained by heating and dissolving docetaxel, the ginsenoside, the phospholipid, etc. in an organic solvent; For example, the ginsenoside and the phospholipid are added to the solution of docetaxel and the organic solvent, and heated to dissolve; the heating can be heated to 35-65°C in a water bath, such as 55°C .

In a certain scheme of the present invention, in the step 1, the concentration can be reduced pressure concentration; the reduced pressure concentration can be vacuum=-0.08mpa～-0.1mpa, for example -0.089～-0.1 MPa; the concentration can be completed until the solvent is completely volatilized; the total concentration time is preferably less than 4 hours.

In a certain solution of the present invention, in the step 1, the concentration can be carried out in a rotary flask, and the rotation speed can be 40-60 rp/min, for example, 50 rp/min.

In a certain scheme of the present invention, in the step 2, the water can be water for injection.

In a certain scheme of the present invention, in the step 2, the concentration of the lyophilized protective agent solution may be 0.20-0.35 mg/ml, for example, 0.25 mg/mL.

In a certain aspect of the present invention, in the step 2, the temperature of the hydration may be 35-65°C, preferably 40-45°C.

In a certain scheme of the present invention, in the step 2, the hydration is carried out in a rotary flask, and the rotational speed is 40-60 rp/min, for example, 50 rp/min.

In a certain scheme of the present invention, in the step 2, the hydration may be as long as the solution is uniform, for example, 2-4 hours.

In a certain scheme of the present invention, in the step 2, the docetaxel: lyoprotectant solution = 1 g: 100 mL.

In a certain aspect of the present invention, in the step 2, the volume of the lyophilized protective agent solution is the same as the volume of the water.

In a certain scheme of the present invention, in the scheme 1 of step 3, the high-pressure homogenization is to use 0-10° C. frozen water in a homogenizer to cool and cut a cycle; preferably, ensure that the liposome solution is The temperature is 5-10℃.

In a certain solution of the present invention, in the solution 1 of the step 3, the pressure of the high-pressure homogenization is between 800-1400 bar, for example, 1200 bar.

In a certain solution of the present invention, in the solution 1 of step 3, the number of times of the high-pressure homogenization may be 3-4 times, for example, 4 times.

In a certain scheme of the present invention, in scheme 2 of step 3, the shearing can be performed at room temperature.

In a certain solution of the present invention, in the solution 2 of the step 3, the rotation speed of the shearing is 1500-2200 rp/min; for example, 2000 rp/min.

In a certain scheme of the present invention, in the scheme 2 of the step 3, the shearing time is 5-10 minutes; for example, 5 minutes.

In a certain scheme of the present invention, in the scheme 2 of the step 3, the temperature of the extrusion is 35-45°C, for example, 40°C.

In a certain solution of the present invention, in the solution 2 of the step 3, the pore diameter of the extrusion plate is 150 nm.

In a certain solution of the present invention, in the solution 2 of step 3, the extrusion pressure is 600-800 psi; for example, 800 psi.

In a certain solution of the present invention, in the solution 2 of the step 3, the number of times of the extrusion may be 3-4 times, for example, 4 times.

The present invention also provides a preparation method of ginsenoside docetaxel liposome for injection, which comprises the following steps;

Step 1-1, step 1-3 in the above-mentioned preparation method of ginsenoside docetaxel liposome, obtain liposome solution A3a or A3b;

Step 2-1, sterilizing and filtering the liposome solution A3a or A3b to obtain the liposome solution A4;

Step 3-1, freeze-drying the liposome solution A4 to obtain ginsenoside docetaxel liposomes for injection.

In the described preparation method, the conditions and operations of the described sterilization filtration and freeze-drying can be conventional conditions and operations in this type of technology in the field; in the present invention, preferably as follows:

In a certain scheme of the present invention, in the step 2-1, a 0.22 μm filter membrane can be used for the sterile filtration.

In a certain scheme of the present invention, in the step 3-1, the freeze-drying can be performed in a vial, and the vial can be a conventional vial in the field, such as a 30ml or 50mL vial.

In a certain scheme of the present invention, in the step 3-1, the freeze-drying may sequentially include: pre-freezing, primary drying, and secondary drying; specifically, may include the following steps:

Step a. The liposome solution A4 quantitatively packed in the vial is in the freeze-drying box, wherein the freeze-dried shelf temperature is uniformly reduced to -10 ± 1 °C, incubated for 1 hour, and then the shelf temperature is raised to -13±1℃, keep warm for 1 hour, and then continue to reduce the shelf temperature to -55±1℃, when the temperature of the product reaches -45±1℃, start timing and continue to keep warm for 3 hours;

Step b. After the heat preservation in step a is completed, the temperature of the condenser is quickly lowered to below -50±1°C, the vacuum is evacuated to below 10pa, the temperature of the shelf (about 1.5 hours) is raised to -25±1°C, and the Keep the temperature for 18 hours, and then quickly raise the temperature of the shelf to -15±1℃, keep the temperature until the ice crystals of the product disappear completely, and then continue to keep the temperature for 4 hours;

Step c, after the insulation in step b is completed, the temperature of the shelf is quickly (within 1 hour) raised to 15±1°C, kept for 3 hours, then the temperature of the shelf is raised to 30±1°C, and the temperature of the product is raised to 25°C. When the temperature is ±1°C, the temperature is kept for 12 hours.

In a certain scheme of the present invention, after step 3-1, post-treatment may be further included, and the conditions and operations of the post-treatment may be conventional conditions and operations in such processes in the art; for example, the post-treatment It includes the following steps: after the heat preservation described in step c is completed, fully press the plug and take out the box; and then roll the cover.

The present invention also provides a compound ginsenoside docetaxel liposome for injection, which is prepared by the above-mentioned preparation method of ginsenoside docetaxel liposome for injection.

In a certain scheme of the present invention, the ginsenoside docetaxel liposome for injection has a particle size D90≤150 nm, and an encapsulation efficiency≥98%. In a certain aspect of the present invention, the purity of the ginsenoside is ≥99%.

The present invention also provides the above-mentioned ginsenoside docetaxel liposome, or the above-mentioned blank liposome, or the above-mentioned compound ginsenoside docetaxel liposome for injection. Use in medicines for the treatment and/or prevention of cancer.

The cancer can be breast cancer, ovarian cancer, lung cancer, gastric cancer, head and neck cancer and esophageal cancer.

The term "particle size D90" refers to the particle size at which the cumulative particle size distribution percentage of a sample reaches 90%. Its physical meaning is that particles with a particle size smaller than it account for 90%.

The prescription abbreviations of the present invention are explained as follows:

On the basis of not violating common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive improvement effect of the present invention is that: the compound ginsenoside docetaxel liposome provided by the present invention has a targeting effect on tumor cells, an anti-multidrug resistance effect, a synergistic effect and a drug synergistic effect. Taking the compound ginsenoside Rg3 docetaxel liposome for injection as an example in the embodiment, the drug effect is significantly better than the technical scheme that is not within the scope of protection of the present invention; The liposomes play a better role in "drugs, excipients, membrane materials, target heads", etc., and play a good drug synergy. specifically:

(1) The drug efficacy is significantly improved. Especially the DTX-Rg3(1.0)/Lp group and DTX-Rh2(1.5)/Lp group had the best efficacy, and the tumor inhibition rate (68%) of the dose (10mg/kg) both reached or exceeded the non-prescription of the present invention The tumor inhibition rate of the high dose (20mg/kg) in the liposome group was increased by 2 times;

(2) Glut1 targeting was significantly improved. In the Glut1 targeting experiment of tumor-bearing mice, the Glut1 targeting properties of the ginsenoside liposomes were more than 4 times higher than that of ordinary cholesterol liposomes, while the ordinary non-preferred ginsenosides The targeting of Glut1 of the liposomes is less than 2 times higher than that of ordinary cholesterol liposomes.

(3) Toxic and side effects are significantly reduced. The liposomes prepared according to the prescription of the present invention, the acute toxicity of DTX-Rg3(1.0)/Lp (LD50≥150mg/kg) and DTX-Rh2(1.5)/Lp (LD50≥150mg/kg) were higher than those of Taxotere group (LD50 About 25mg/kg) is more than 6 times lower than that of ordinary cholesterol docetaxel liposome (LD50 is between 25 and 50mg/kg), 3-4 times lower than that of DTX-C-Rg3(1.8)/Lp and DTX-C-Rh2(2.0)/Lp and other non-prescription ginsenoside liposomes of the present invention (LD50=about 50-100mg/kg) decreased about 1.5-2 times. In the long-term toxicity experiment of SD rats, there was animal death in the Taxotere group, indicating that the Taxotere group has a greater toxic effect. The toxicity of the ginsenoside Rg3 docetaxel liposome or the ginsenoside Rh2 docetaxel liposome of the present invention is more than 4-6 times lower than that of Taxotere.

Detailed ways

The present invention is further described below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods that do not specify specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product description.

Experimental drugs and equipment

Experimental drugs: 20(S)-ginsenoside Rg3 (abbreviation: Rg3), ginsenoside pseudo-Rg3 (abbreviation: pseudo-Rg3), ginsenoside Rp1 (abbreviation: Rp1), ginsenoside pseudo-GQ (abbreviation: pseudo-GQ), ginsenoside Rk1 (abbreviation: Rk1), ginsenoside Rg5 (abbreviation: Rg5), 20(S)-ginsenoside Rh2 (abbreviation: Rh2), ginsenoside Rk2 (abbreviation: Rk2), 20(S)-ginsenoside Rg2 (abbreviation: Rk2) Rg2), 20(S)-ginsenoside Rh1 (abbreviation: Rh1), 20(S)-protopanaxadiol (abbreviation: PPD), 20(S)-protopanaxatriol (abbreviation: PPT), etc. are routine in the field Commercially available, such as Shanghai Bensu Pharmaceutical Technology Co., Ltd., Suzhou Xinghai Jinsen Biomedical Co., Ltd., Shanghai Yuanye Biotechnology Co., Ltd., etc.

Docetaxel injection: Taxotere, manufacturer: British Aventis Company, purchased from Shanghai Lei's Pharmacy.

The molecular structural formula of ginsenosides of the present invention is as follows:

Test instrument: the instrument used in the following embodiment is the own instrument and equipment of Shanghai Bensu Pharmaceutical Technology Co., Ltd. and Fudan University School of Pharmacy, and its equipment model and source information are as follows:

Agilent liquid chromatography: a set of Agilent 1100, Aotai 3300ELSD, Agilent Technologies (China) Co., Ltd.;

Rotary evaporator: ZX98-1 5L, Shanghai Luyi Industry and Trade Co., Ltd.;

Ultrasonic cleaning machine (SB3200DT, Ningbo Xinzhi Biotechnology Co., Ltd.);

Nitrogen blowing instrument (HGC-12A, Tianjin Hengao Technology Development Co., Ltd.);

Probe ultrasound instrument (JYD-650, Shanghai Zhixin Instrument Co., Ltd., China);

High pressure homogenizer (B15, AVESTIN, Canada);

Micro-extruder (Mini-extruder, Avanti Polar Lipids Inc);

Laser particle size analyzer (Nano ZS, Malvern, UK);

Malvern Nanosizer ZS90 (Malvern, UK);

Microplate reader (Thermo Scientific, Waltham, MA, USA);

Microplate reader (Infinitie 200, Tecan Trading Co., Ltd, Switzerland);

Flow cytometer (BD Biosciences, USA);

Flow cytometer (CytoFlex S, Beckman Coulter, Inc., USA);

Inverted fluorescence microscope (Leica, DMI 4000D, Germany);

Fluorescence microscopy (Zeiss LSM 710, Oberkochen, Germany);

Laser confocal microscope (Leica, DMI 4000D, Germany);

Confocal intravital microscopy (IVM);

Upright two-photon microscope (DM5500 Q; Nikon);

Small animal in vivo imaging system (in vivo imaging system, IVIS) (PerkinElmer, USA);

Biomacromolecule interaction instrument BiaCore T 200 instrument (GE, USA);

Clean workbench (SW-CJ-1FD, Suzhou Antai Air Technology Co., Ltd.);

20L Rotary Evaporator: R5002K, Shanghai Xiafeng Industrial Co., Ltd.;

Freeze dryer: FD-1D-80, Shanghai Bilang Instrument Manufacturing Co., Ltd.;

Freeze dryer: PDFD GLZ-1B, Shanghai Pudong Freeze Drying Equipment Co., Ltd.;

Electronic balance: CPA2250 (accuracy 0.00001g), Sartorius (Shanghai) Trading Co., Ltd.;

Electronic balance: JY3003 (accuracy 0.001g), Shanghai Sunny Hengping Scientific Instrument Co., Ltd.;

Photoelectric microscope (XDS-1B, Chongqing Photoelectric Instrument Co., Ltd.);

Cell incubator (CCL-170B-8, ESCO Singapore).

Animals and Cell Lines

Animals: BALB/c nude mice, 3-4 weeks old, produced by Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Tumor cell line:

Breast cancer in situ tumor 4T1 cell line, provided by School of Pharmacy, Fudan University

Breast cancer MCF-7 cell line, provided by School of Pharmacy, Fudan University;

Triple-negative breast cancer MDA-MB-231 cell line, provided by School of Pharmacy, Fudan University;

Example 1 Preparation of compound ginsenoside Rg3 docetaxel liposome for injection

1. Prescription: egg yolk lecithin 10g, ginsenoside Rg3 1g, docetaxel 1g, glucose 25g, absolute ethanol 40ml, chloroform 40ml, water for injection 200ml.

2. Film formation: prepare a mixed solvent of absolute ethanol and chloroform (1:1) in the prescribed amount for use.

Add the prescription amount of docetaxel to the mixed solvent to dissolve for later use, then add the prescription amount of ginsenoside Rg3 and egg yolk lecithin into the mixed solvent, heat to dissolve, transfer into a 1L rotary steamer, concentrate under reduced pressure, and the water bath temperature is 55 ° C , rotating speed 50 rev/min, vacuum degree -0.089～-0.1MPa, rotary steam until all the solvent is completely volatilized.

3. Hydration: Preparation of glucose solution: Add 25g of anhydrous glucose to 100ml of water for injection, stir and dissolve, and then prepare a 0.25mg/ml aqueous glucose solution, and heat it in a 40°C water bath for later use.

Add 100ml of water for injection into the rotary evaporation bottle after film formation, the water bath temperature is 40-45°C, the rotation speed is 50 rev/min, hydrated and completely dissolved, and the time is about 2h.

Then add 100 ml of aqueous glucose solution, stir evenly, and set aside.

4. High-pressure homogenization: The hydrated solution is transferred to a homogenizer. The homogenizer uses 0-10°C chilled water to cool and circulate, and the homogenization pressure is set to 1200bar, and the cycle is homogenized 3--4 times until D90 is less than 100nm.

5. Sterilization filtration: The homogenized solution is sterilized and filtered through a 0.22 μm filter membrane.

6. Packing: Pack the solution after sterilization and filtration into 30 or 50ml vials according to the set amount of 8-10ml.

7. Pre-freezing: After the product is put into the box, the temperature of the shelf drops to about -10°C at a constant speed, and the temperature of the shelf is kept for 1 hour. Then the temperature of the shelf is raised to about -55°C, and the temperature of the shelf is kept for 1 hour. After the heat preservation, the temperature of the shelf is continued. When the temperature of the product reaches -45°C, start the timer and continue to keep the temperature for about 3 hours.

8. Primary drying: quickly reduce the temperature of the condenser to below -50°C, evacuate to below 10pa, raise the temperature of the shelf (about 1.5 hours) to -25±1°C, start timing and keep warm for about 18 hours, and then After quickly raising the shelf temperature to -15±1°C, keep the product until the ice crystals completely disappear, and then continue to keep the temperature for about 4 hours;

9. Secondary drying: The temperature of the shelf rises rapidly (within 1 hour) to about 15°C, and the temperature is kept for about 3 hours. Then the temperature of the shelf is raised to about 25°C. When the temperature of the product rises to 25°C, the temperature is kept for about 12 hours. . After the heat preservation is over, check the vacuum degree, end the entire freeze-drying process, fully press the plug, and take out the box.

10. Capping and packaging: Capping and packaging the above-mentioned liposomes to obtain compound ginsenoside Rg3 docetaxel liposomes for injection (Prescription 1).

Example 2 Preparation of Compound Ginsenoside Rg3 Docetaxel Liposomes for Injection

The prescription amount of the ginsenoside Rg3 in Example 1 was increased to 1.5 g, and the others were the same as in Example 1, and the compound ginsenoside Rg3 docetaxel liposome for injection was prepared.

Example 3 Preparation of Compound Ginsenoside Rg3 Docetaxel Liposomes for Injection

1. Prescription: egg yolk lecithin 10g, ginsenoside Rg3 1.5g, docetaxel 1g, glucose 25g, absolute ethanol 40ml, chloroform 40ml, water for injection 200ml.

2. Film formation: the same as the film formation method of Example 1.

3. Hydration: the same as the hydration method of Example 1.

4. High-speed shearing and extrusion: the above-mentioned liposome solution was rapidly sheared at 2000 rp/min for 5 min at room temperature.

The temperature of the liposome solution was controlled at 35-45 °C, an extrusion device was connected, an extrusion plate with a 150 nm aperture was installed, and the extrusion was performed under a pressure of 800 psi.

5. Subsequent steps are the same as those in Example 1.

Example 4 Preparation of Compound Ginsenoside Rh2 Docetaxel Liposomes for Injection

The ginsenoside Rg3 in Example 1 was changed to ginsenoside Rh2 1.0 g, and the others were the same as in Example 1, to prepare the compound ginsenoside Rh2 docetaxel liposome for injection.

Example 5 Preparation of Compound Ginsenoside Rh2 Docetaxel Liposomes for Injection

The ginsenoside Rg3 in Example 1 was changed to ginsenoside Rh2 1.5 g, and the others were the same as in Example 1, to prepare the compound ginsenoside Rh2 docetaxel liposome for injection.

Example 6 Preparation of Compound Ginsenoside Pseudo-Rg3 Docetaxel Liposomes for Injection

The ginsenoside Rg3 in Example 1 was changed to 1.5 g of ginsenoside pseudo-Rg3, and the others were the same as in Example 1, and the compound ginsenoside pseudo-Rg3 docetaxel liposome for injection was prepared.

Example 7 Preparation of compound ginsenoside Rg5 docetaxel liposome for injection

The ginsenoside Rg3 in Example 1 was changed to 1.5 g of ginsenoside Rg5, and the others were the same as in Example 1, to prepare the compound ginsenoside Rg5 docetaxel liposome for injection.

Example 8 Preparation of Compound Ginsenoside Rk1 Docetaxel Liposomes for Injection

The ginsenoside Rg3 in Example 1 was changed to 1.5 g of ginsenoside Rk1, and the others were the same as those in Example 1, and the compound ginsenoside Rk1 docetaxel liposome for injection was prepared.

Example 9 Preparation of compound ginsenoside Rp1 docetaxel liposome for injection

The ginsenoside Rg3 in Example 1 was changed to 1.5 g of ginsenoside Rp1, and the others were the same as in Example 1, and the compound ginsenoside Rp1 docetaxel liposome for injection was prepared.

Example 10 Preparation of Compound Ginsenoside Pseudo-GQ Docetaxel Liposomes for Injection

The ginsenoside Rg3 in Example 1 was changed to 1.5 g of ginsenoside pseudo-GQ, and the others were the same as in Example 1, and the compound ginsenoside pseudo-GQ docetaxel liposome for injection was prepared.

Effect Example 1

(a) prescription according to the following table, and by the same method of embodiment 1 (do not need freeze-drying step), to the kind comparison result of ginsenosides as following table:

The above-mentioned series of experiments prove that, without adding other auxiliary materials such as soybean oil or cholesterol, the ginsenosides in the present invention are: Rg3, pseudo-Rg3, Rh2, pseudo-GQ, Rg5, Rk1 and Rp1, etc. When 7, Ginposome-DTX or CTX has a better preparation process, and the preparation conditions are easy to realize, which is beneficial to industrialization. When ginsenosides are Rk2, Rg2, Rh1, PPD and PPT, other auxiliary materials need to be added, and the preparation conditions are relatively harsh.

(b) The types and proportions of phospholipids were compared:

Remarks: Egg yolk lecithin (EPC), soybean lecithin (SPC), cephalin (PE), sphingomyelin (SM), hydrogenated phospholipid (HSPC), phosphatidylserine (PS), dipalmitoyl phosphatidylglycerol (DPPG), Dioleoyl lecithin (DOPC), distearoyl phosphatidyl choline (DSPC), 1-palmitoyl-2-oleoyl phosphatidyl ethanolamine (POPE), dimyristoyl phosphatidyl choline (DMPC), Polyethylene glycol 2000-distearate phosphatidylethanolamine (mPEG-DSPE), polyethylene glycol 2000-dioleoyl phosphatidylethanolamine (mPEG-DOPE).

According to the same method as in Example 1 (no lyophilization step is required), the comparison results of the phospholipid species and ratio are as follows:

The above experiments have proved that docetaxel can be well packaged without adding other auxiliary materials such as soybean oil or cholesterol, and the phospholipids that can be easily realized by the preparation process are egg yolk lecithin, soybean lecithin, hydrogenated phospholipid, cephalin and mixed phospholipids (above-mentioned. 0.01-10% mPEG-DSPE in 4 phospholipids). Other phospholipids and Rg3 can also encapsulate docetaxel well, but other excipients such as soybean oil need to be added, or the homogenization pressure and times need to be increased. The above experiments also show that: in terms of film-forming properties, when the phospholipid in the present invention: docetaxel=8-18:1, the effect is better, preferably 8-12:1.

(c) by the same method of embodiment 1 (do not need freeze-drying step), the optimal ratio of ginsenosides has been compared:

The above experiments prove that, without adding other auxiliary materials such as soybean oil or cholesterol, the effect is better when the ratio of saponin:docetaxel=1-3:1 in the present invention. However, due to the research results of the toxicokinetics of the application example 4 of the present invention, the present invention only selects the ratio range of saponin: docetaxel=1-1.5:1.

(d) by the same method of embodiment 1, the freeze-drying protective agent was compared:

In the selection of freeze-drying protective agents, different freeze-drying protective agents have a significant impact on the encapsulation efficiency and particle size distribution of liposomes after product reconstitution, and also have a great impact on the economy of the freeze-drying curve. Through the above experiments, without adding other auxiliary materials such as soybean oil, the freeze-drying protective agent in the present invention is one or more of glucose, trehalose, sucrose, lactose and galactose, docetaxel and freeze-dried The ratio of protective agent is: lyophilized protective agent/docetaxel = 25-35 times the amount, for example, when 15 times the amount of glucose, the reconstituted particle size of Ginposome-DTX is 200nm, which is between qualified and unqualified, 25 Or 35 times the amount, it has a good match with docetaxel liposome. Due to comprehensive consideration of the empirical data of formulations, the ratio of docetaxel and lyophilized protective agent determined in the present invention is: lyophilized protective agent/docetaxel=25-35 times the amount.

Application Example 1: Cellular Uptake Experiment of Glut1

1) Experimental purpose: To prove the Glut1 targeting mechanism by adding glucose inhibitors, etc.; to verify the types and proportions of ginsenosides, phospholipids and proportions of the present invention through Glut1 targeting.

2) Experimental method: In order to compare the uptake of 4T1 to each experimental group and explore the uptake mechanism of the compound preparation, 4T1 cells were inoculated in a 12-well plate at a cell density of 2×10 ⁵ . For peridin and experimental group + quercetin group, the medium was replaced with 20 mM glucose solution, phloridzin solution and quercetin solution after 12 hours, respectively. These three solutes should be dissolved in glucose-free medium. After 1 hour of incubation, the drugs of each experimental group (the concentration of UV fluorescent color developer is 100ng/ml) were added. After 4 hours of incubation, digested and washed with fresh PBS solution. Analysis was performed by flow cytometry.

3) Preparation method of experimental group: prepared according to the method of Example 1 of the present invention (docetaxel is changed to coumarin, no freeze-drying step is required)

Using the results of the toxicity test in Example 4, the following experiments did not carry out the experiment of the prescription of ginsenoside/docetaxel exceeding 2.0.

test group:

Prescription content short name Egg yolk lecithin: cholesterol: fluorescent probe = 9:1.8:1 C6-C/Lp Egg yolk lecithin: Rg3: fluorescent probe: cholesterol = 9:1.8:1:2.25 C6-C-Rg3/Lp Egg yolk lecithin: Rg3: fluorescent probe = 9:1.8:1 C6-Rg3/Lp Soybean phospholipid: Rh2: fluorescent probe: cholesterol = 10:2:1:2.25 C6-C-Rh2/Lp Soybean phospholipid: Rh2: fluorescent probe = 10:2:1 C6-Rh2/Lp Soybean phospholipid: Rg5: Fluorescent probe: soybean oil = 4:3:1:2 C6-Rg5/Lp Egg yolk lecithin: pseudo-Rg3: fluorescent probe = 10:1.5:1 C6-pseudo-Rg3(1.5)/Lp Egg yolk lecithin:pseudo-GQ:fluorescent probe = 10:1.5:1 C6-pseudo GQ(1.5)/Lp Egg yolk lecithin: Rk1: fluorescent probe = 10:1.5:1 C6-Rk1(1.5)/Lp Egg yolk lecithin: Rp1: fluorescent probe = 10:1.5:1 C6-Rp1(1.5)/Lp

The experimental results are as follows:

Experimental results:

1) Under the same prescription, after adding cholesterol, its Glut1 targeting is greatly reduced.

2) The targeting performance of the prescription of the present invention is more than 4 times higher than that of cholesterol liposomes, and the targeting performance of non-preferred ginsenoside liposomes is about 2 times higher than that of cholesterol liposomes.

Application Example 2: In vivo pharmacodynamics study of breast cancer (MCF-7)

1) Test method: The tumor cell line (MCF-7) was injected into mice subcutaneously to establish a subcutaneous tumor model. When the tumor volume reached 100 mm ³ (7d after inoculation), the mice were randomly divided into groups (n=8 in each group) for treatment, and each group was injected with blank solvent (5% glucose, Blank), DTX-C/Lp group, DTX group through tail vein -Rg3(0.5)/Lp group, DTX-Rg3(1.0)/Lp group, DTX-Rg3(1.5)/Lp group, DTX-Rg3(2.0)/Lp group, DTX-Rh2(0.5)/Lp group, DTX -Rh2(1.0)/Lp group, DTX-Rh2(1.5)/Lp group, DTX-Rh2(2.0)/Lp group, DTX-C-Rg3(1.8)/Lp group (yolk lecithin: Rg3: cholesterol: more Sitaxel=9:1.8:2.25:1), DTX-C-Rh2(2.0)/Lp group (soybean phospholipid:Rh2:cholesterol:docetaxel=10:1:2.25:1), the dose is according to docetaxel Three groups (20mg, 10mg, 5mg) of high, middle and low levels of hexai meter were administered once every 7 days until the 28th day. The length, width and body weight of the tumor were measured at the same time of administration. The formula for calculating tumor volume (V) is: V=(W ² ×L)/2. Length (L) is the longest diameter of the solid tumor and width (W) is the shortest diameter perpendicular to the length. At the end of the experiment on day 28, all animals were sacrificed, and tumors were removed for imaging and histological examination.

Remarks: Docetaxel+Rg3=20mg/kg+30mg/kg, indicating the drug concentration, the same below.

Due to the toxicological results of application example 4, the study of DTX-Rg3 (above 2.0)/Lp group was not carried out in this example.

2) The test results are as follows:

in conclusion:

1) DTX-Rg3(1.0)/Lp group and DTX-Rh2(1.5)/Lp group had the best drug effect, and the tumor disappeared completely on the 21st day. The tumor inhibition rate of the middle dose (docetaxel = 10mg/kg) in the second group was comparable to the high dose group (docetaxel) of the DTX-C-Rg3(1.8)/Lp group and DTX-C-Rh2(2.0)/Lp group The tumor inhibition rate was basically the same with hexidine = 20 mg/kg). That is, the tumor-inhibiting effect was about 2 times higher than that of the non-invention group.

2) Animal death occurred in the DTX-C/Lp group, indicating that the toxic effect was large.

3) There is no linear relationship between the efficacy and the ratio of ginsenosides. According to this prescription, when the ratio of ginsenoside Rg3/docetaxel is 1.0 to 1.5, the efficacy is the best, preferably 1.0; ginsenoside Rh2/docetaxel When the ratio of hexidine is between 1.0 and 2.0, the efficacy is the best, preferably 1.5.

Application Example 3: Acute toxicity (LD50) study (SD rat)

1) Experimental method: 160-260 g rats, 6-9 weeks old, 6 rats in each group, administration method: slow intravenous push (about 1 mL/min), administration frequency: 3 times/day.

The doses of the test product docetaxel in this experiment were set at 25, 50, 100 and 150 mg/kg/day, and the Rg3 content of the test product was 25, 50, 100 and 150 mg/kg/day, respectively; the Rh2 content was 37.5, 75 mg/kg/day, respectively. , 150 and 225 mg/kg/day. At the same time, the vehicle control group (5% glucose injection), the commercially available positive control group (Taxotere group), the Rg3 liposome group and the DTX-C-Rg3/Lp group were set up. / day, at least 4h between each dose.

2) Experimental grouping: a total of 13 groups, 5% glucose group, DTX-C/Lp, DTX-C-Rg3(1.8)/Lp group, DTX-C-Rh2(2.0)/Lp group and pharmacodynamics experimental grouping Consistent, other group names are shown in the table below.

3) Preparation method of experimental group: according to the prescription requirements, prepare according to the method of Example 1.

3) The experimental results are as follows:

Remarks: Due to the toxic generation results of Example 4, the study of DTX-Rg3 (2.0 or above)/Lp group was not carried out in this example.

The above experiments show that the technical solution of the present invention has excellent formulations and Glut1 targeting, so that the related preparations have the best detoxification effect, compared with the cholesterol liposome group (DTX-C/Lp) and docetaxel injection. (Taxotere), the toxicity was generally reduced by about 4-6 times; compared with the DTX-C-Rg3(1.8)/Lp group and the DTX-C-Rh2(2.0)/Lp group, the toxicity was generally reduced by 1.5-2 times.

Application Example 4: Toxicokinetic (TK) Study

1. Experimental purpose: To study the cumulative toxicity of each experimental group.

2. Experimental method: 8 groups were set up in the TK group in this experiment, which were the vehicle control group (5% glucose injection), the commercially available positive control group Taxotere (10 mg/kg), the test sample 1 (DTX-Rg3 (1.0 )/Lp group: 10mg/kg and 20mg/kg), test article 2 (DTX-Rh2(1.5)/Lp group: 10mg/kg and 20mg/kg), test article 3 (DTX-Rh2(2.0)/ Lp group, 10 mg/kg and 20 mg/kg). There were 10 SD rats in each group, half male and half male, 80 rats in total. Intravenous injection, once a week, continuous administration for four weeks, the vehicle control group collected whole blood before D1 and D29 administration and 1 hr after administration, respectively. 3 and the marketed positive control group were collected whole blood before D1 and D29 administration and 3min, 15min, 30min, 1hr, 3hr, 6hr, 24hr after administration, and the whole blood was collected in a test tube containing EDTA-K2 anticoagulant , placed on crushed ice, and centrifuged to collect plasma for analysis.

The concentration of docetaxel, 20(S)-Rg3, 20(S)-Rh2 in plasma was detected by LC-MS/MS method. The lower limit of quantification of Rh2 analysis method was 50.000ng/mL. The non-compartmental model of WinNonlin software was used to analyze the plasma concentration data, and to calculate the parameters, to study the toxicokinetic characteristics of the test product, and to provide reference for clinical trials.

3. Experimental results, under the conditions of this test:

1) After administration of D1 and D29, the exposure of ginsenosides Rg3 and Rh2 in plasma (calculated by AUC(0-t) after administration of test article 1, test article 2 and test article 3 in male and female SD rats by intravenous injection ) increases with the increase of the administered dose, and the increase is higher than that of the dose.

2) After D1 administration, the exposure of ginsenosides Rg3 and Rh2 in plasma (calculated as Cmax) of ginsenosides Rg3 and Rh2 after intravenous injection of test article 1, test article 2 and test article 3 in male and female SD rats is proportional to the administered dose .

3) After administration of D1 and D29, the exposure of docetaxel in plasma (in terms of AUC(0-t) and Cmax) after intravenous injection of test article 1 in male and female SD rats increased with the administration dose And the increase, the increase rate is higher than the increase of the dose.

4) After administration of D1 and D29, the exposures of ginsenosides Rg3 and Rh2 in plasma (calculated by Cmax) of ginsenosides Rg3 and Rh2 after intravenous injection of test article 2 in male and female SD rats were proportional to the administration dose.

5) Under the conditions of this experiment, after continuous administration, the ginsenosides Rg3 and Rh2 in the plasma of male and female SD rats after intravenous injection of test article 1 and test article 2 basically had no tendency to accumulate; There was basically no accumulation tendency of docetaxel in plasma after test article 1, test article 2 and test article 3; the exposure of docetaxel and ginsenoside Rh2 in plasma after intravenous injection of test article 3 in male and female SD rats There was a slight accumulation of docetaxel in the plasma of male and female SD rats administered intravenously to the commercially available control group without accumulation of docetaxel exposure.

Conclusion: There is no cumulative toxicity risk in the DTX-Rg3(1.0)/Lp group.

There was no cumulative toxicity risk in the DTX-Rh2(1.5)/Lp group.

There was a slight risk of cumulative toxicity in the DTX-Rh2(2.0)/Lp group.

Application Example 5: Long-term toxicity study in SD rats

1) Experimental method: Rats 160-260 g, 6-9 weeks old, 6 rats in each group, administration method: slow intravenous push (about 1 mL/min), administration frequency: 1 time/week (D1, D8, D15 , D22, D29).

2) Experimental grouping: 5% glucose group (vehicle control group), Taxotere group (positive control group), Taxotere+Rg3/Lp group, DTX-Rg3/Lp group, DTX-Rh2/Lp group were set up, a total of 5 groups.

The doses of docetaxel tested in this experiment were set at 5, 10 and 20 mg/kg, the mass ratio of docetaxel and Rg3 in the test product was 1:1.0, and the mass ratio of docetaxel and Rh2 was 1:1. 1.5, so it contains 5, 10 and 20 mg/kg of Rg3, and 7.5, 15, and 30 mg/kg of Rh2, respectively. (prepared according to the preparation method of Example 1)

The dose settings for each group were as follows:

3) The experimental results are as follows:

Result 1: The results of the long-term toxicity test in the Taxotere group (exam results on the 30th day)

Dosage 5mg/kg 10mg/kg 20mg/kg animal death rate no death no death 33.3% Changes in the average body weight of animals +15% +2% -18% liver tissue damage no damage Moderate severe kidney tissue damage no damage Moderate severe Spleen tissue damage no damage Moderate severe heart tissue damage no damage Moderate severe

Result 2: Long-term toxicity test results of Taxotere group + Rg3/Lp group (test results on the 30th day)

Result 3: Long-term toxicity test results of DTX-Rg3/Lp group (examination results on the 30th day)

Result 4: Long-term toxicity test results of DTX-Rh2/Lp group (test results on the 30th day)

The above long-term toxicity test results in rats show that:

1) In Taxotere group, the rats died, the body weight was severely reduced, and the organs were severely damaged, indicating high toxicity.

2) In the DTX-Rg3/Lp group and the DTX-Rh2/Lp group, the rats did not die, the body weight almost did not decrease, and the organs were mainly slightly damaged, indicating that the long-term toxicity was greatly reduced.

Application Example 6: In vivo study of compound ginsenoside docetaxel liposome for injection on triple negative breast cancer (MDA-MB-231)

Animals: BALB/c nude mice, 3-4 weeks old, produced by Shanghai Institute of Materia Medica.

Tumor cell line: triple negative breast cancer MDA-MB-231 cell line

Provided by Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Transplantation tumor model: the above cell lines were inoculated into the right armpit of nude mice subcutaneously, and the inoculation amount of cells was 5×10 ⁶ / mouse. After the transplanted tumor was formed, it was passed down in nude mice for 1 generation before use.

Experimental methods: The tumor cell lines were injected into mice subcutaneously to establish a subcutaneous tumor model. When the tumor volume reached 100 mm ³ (7d after inoculation), the mice were randomly divided into 4 groups (n=8 in each group) for treatment, and each group was injected with blank solvent (5% glucose), Taxotere group, DTX-Rg3/ Lp group (20mg/kg docetaxel, 20mg/kg ginsenoside Rg3), DTX-Rh2/Lp group (20mg/kg docetaxel, 30mg/kg ginsenoside Rh2), every 7 days One dose, continued until day 28, was administered while tumor length, width and body weight were recorded. The formula for calculating tumor volume (V) is: V=(W2×L)/2. Length (L) is the longest diameter of the solid tumor and width (W) is the shortest diameter perpendicular to the length. At the end of the experiment on day 28, all animals were sacrificed, and tumors were removed for imaging and histological examination.

Triple-negative breast cancer MDA-MB-231: According to the in vivo pharmacodynamics experimental method, the in vivo pharmacodynamics data for triple-negative breast cancer MDA-MB-231 are as follows.

The results showed that: for triple-negative breast cancer MDA-MB-231 tumor-bearing mice, at the same dose, the Taxotere group had a general tumor inhibitory effect (the tumor inhibition rate was 68%), and the DTX-Rg3/Lp group and DTX-Rh2/Lp group had the most effect. Good, the tumors had completely disappeared on the 28th day. The experimental results show that: the compound preparation has significant efficacy and significant drug synergy.

Claims (12)

1. The ginsenoside docetaxel liposome comprises the following components in percentage by mass: 8-18 parts of phospholipid, 1-1.5 parts of ginsenoside, 1 part of docetaxel and 20-35 parts of freeze-drying protective agent; the ginsenoside docetaxel plastid does not contain one or more of cholesterol, soybean oil and sodium oleate.

2. A ginsenoside docetaxel liposome of claim 1, which consists of the following components in percentage by mass: 8-18 parts of phospholipid, 1-1.5 parts of ginsenoside, 1 part of docetaxel and 20-35 parts of freeze-drying protective agent.

3. A ginsenoside docetaxel liposome of claim 1 or 2, wherein,

the phospholipid is one or more of egg yolk lecithin, soybean phospholipid, hydrogenated phospholipid and cephalin, or one or more of egg yolk lecithin, soybean phospholipid, hydrogenated phospholipid and cephalin containing 0.01-10% of distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000;

and/or the mass ratio of the docetaxel to the phospholipid is 1 (8-12); e.g., 1: 10;

and/or the ginsenoside is one or more of 20(S) -ginsenoside Rg3, ginsenoside pseudo Rg3, 20(S) -ginsenoside Rh2, pseudo ginsenoside GQ, ginsenoside Rg5, ginsenoside Rk1 and ginsenoside Rp 1;

and/or the mass ratio of the docetaxel to the ginsenoside is 1:1 or 1: 1.5;

and/or the freeze-drying protective agent is one or more of trehalose, glucose, sucrose, lactose and galactose;

and/or the mass ratio of the lyoprotectant to the docetaxel is (25-35): 1; e.g., 30: 1;

and/or the particle size D90 of the ginsenoside docetaxel plastid is not more than 150 nm.

4. A ginsenoside docetaxel liposome of claim 1, wherein the liposome is a liposome comprising a liposome-containing liposome,

the composition comprises the following components in percentage by mass: 10 parts of phospholipid, 1 part of ginsenoside Rg3, 1 part of docetaxel and 25 parts of freeze-drying protective agent;

or, the composition comprises the following components in percentage by mass: 10 parts of phospholipid, 1.5 parts of ginsenoside Rh2, 1 part of docetaxel and 25 parts of freeze-drying protective agent;

or, the composition comprises the following components in percentage by mass: 10 parts of phospholipid, 1 part of ginsenoside Rg3, 1 part of docetaxel and 30 parts of freeze-drying protective agent;

or, the composition comprises the following components in percentage by mass: 10 parts of phospholipid, 1.5 parts of ginsenoside Rh2, 1 part of docetaxel and 30 parts of freeze-drying protective agent.

5. A blank liposome is characterized by comprising the following components in percentage by mass: 8-18 parts of phospholipid, 1-2 parts of ginsenoside and 20-35 parts of freeze-drying protective agent; the blank liposomes do not contain one or more of cholesterol, soybean oil, and sodium oleate.

6. The blank liposome of claim 5, wherein the blank liposome is loaded with a pharmaceutically active substance, wherein the pharmaceutically active substance can be a taxane derivative; such as paclitaxel and/or docetaxel;

and/or the phospholipid is one or more of egg yolk lecithin, soybean phospholipid, hydrogenated phospholipid and cephalin, or one or more of egg yolk lecithin, soybean phospholipid, hydrogenated phospholipid and cephalin containing 0.01-10% of distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000;

and/or 10 parts of phospholipid;

and/or the ginsenoside is one or more of 20(S) -ginsenoside Rg3, ginsenoside pseudo Rg3, 20(S) -ginsenoside Rh2, pseudo ginsenoside GQ, ginsenoside Rg5, ginsenoside Rk1 and ginsenoside Rp 1;

and/or the mass ratio of the ginsenoside is 1 part, 1.5 parts or 2 parts;

and/or the freeze-drying protective agent is one or more of trehalose, glucose, sucrose, lactose and galactose;

and/or 25-35 parts of a freeze-drying protective agent.

7. A method of preparing a ginsenoside docetaxel liposome of any one of claims 1-4, comprising the steps of:

step 1, concentrating a solution A1 of docetaxel, ginsenoside, phospholipid and an organic solvent to form a film;

step 2, after the membrane obtained in the step 1 is subjected to heat preservation hydration in water, uniformly mixing with a freeze-drying protective agent solution to obtain a liposome solution A2;

step 3, which is scheme 1 or scheme 2;

scheme 1 comprises the following steps:

homogenizing the liposome solution A2 obtained in step 2 under high pressure, and controlling the particle diameter D90 to be less than 100nm to obtain liposome solution A3 a;

scheme 2 includes the following steps:

and (3) shearing the liposome solution A2 obtained in the step (2), extruding the sheared liposome solution A2 through an extrusion plate with the aperture of 150nm, and controlling the particle size D90 to be smaller than 100nm to obtain a liposome solution A3 b.

8. A method of preparing ginsenoside docetaxel liposomes of claim 7 wherein,

in the step 1, the organic solvent is one or more of methanol, ethanol, chloroform and dichloromethane, preferably a mixed solvent of methanol and/or ethanol and chloroform and/or dichloromethane; for example, ethanol: the volume ratio of chloroform is 1:1, a mixed solvent;

and/or the mass volume ratio of the docetaxel to the organic solvent is 1mg/80 mL;

and/or, in the step 1, the solution A1 is obtained by heating and dissolving docetaxel, ginsenoside and phospholipid in an organic solvent; for example, the ginsenoside and the phospholipid are added into a solution of docetaxel and the organic solvent, and the mixture is heated and dissolved to obtain the compound; the heating may be carried out in a water bath to 35-65 deg.C, for example 55 deg.C;

and/or, in the step 1, the concentration is reduced pressure concentration; the reduced pressure concentration may be-0.08 MPa to-0.1 MPa, for example-0.089 to-0.1 MPa, under vacuum; concentrating until the solvent is completely volatilized; the total concentration time is preferably less than 4 hours;

and/or, in the step 1, the concentration is carried out in a rotary evaporation bottle at a rotating speed of 40-60 rp/min, such as 50 rp/min;

and/or, in the step 2, the water is water for injection;

and/or, in the step 2, the concentration of the lyoprotectant solution is 0.20-0.35mg/mL, such as 0.25 mg/mL;

and/or, in the step 2, the hydration temperature is 35-65 ℃, preferably 40-45 ℃;

and/or in the step 2, the hydration is carried out in a rotary evaporation bottle at a rotating speed of 40-60 rp/min, such as 50 rp/min;

and/or, in the step 2, the hydration is performed in the form of uniform solution, for example, 2-4 hours;

and/or, in the step 2, the ratio of docetaxel: lyophilized protectant solution ═ 1 g: 100 mL;

and/or, in the step 2, the volume of the lyoprotectant solution is the same as the volume of the water;

and/or in the scheme 1 of the step 3, the high-pressure homogenization is a cooling and cutting cycle in a homogenizer by using 0-10 ℃ chilled water; preferably, the temperature of the liposome solution is ensured to be 5-10 ℃;

and/or, in the scheme 1 of the step 3, the pressure of the high-pressure homogenization is between 800 and 1400 bar; for example 1200 bar;

and/or, in the scheme 1 of the step 3, the times of high-pressure homogenization are 3-4 times;

and/or, in case of scheme 2 of step 3, said shearing is carried out at room temperature;

and/or, in the scheme 2 of the step 3, the rotation speed of the shearing is 1500-2200 rp/min, such as 2000 rp/min;

and/or in the scheme 2 of the step 3, the shearing time is 5-10 min;

and/or, in case of scheme 2 of step 3, the temperature of the extrusion is 35-45 ℃, for example 40 ℃;

and/or, in the scheme 2 of the step 3, the pore diameter of the extruded plate is 150 nm;

and/or, in the scheme 2 of the step 3, the extrusion pressure is 600-800 psi;

and/or, in the scheme 2 of the step 3, the number of times of extrusion can be 3-4 times.

9. A method for preparing ginsenoside docetaxel liposome for injection comprises the following steps;

a liposome solution A3a or A3b obtained in the method for preparing ginsenoside docetaxel liposome of claim 7 or 8 of step 1-1;

step 2-1, sterilizing and filtering the liposome solution A3a or A3b to obtain a liposome solution A4;

and 3-1, freeze-drying the liposome solution A4 to obtain the ginsenoside docetaxel liposome for injection.

10. The method for preparing ginsenoside docetaxel liposome for injection in accordance with claim 9, wherein,

in the step 2-1, a 0.22 mu m filter membrane is adopted for sterilization filtration;

and/or, in the step 3-1, the freeze drying sequentially comprises: pre-freezing, primary drying and secondary drying; specifically, the method can comprise the following steps: step a, quantitatively placing liposome solution A4 which is subpackaged in penicillin bottles into a freeze drying box, wherein the temperature of a freeze-dried shelf is reduced to minus 10 +/-1 ℃ at a constant speed, preserving heat for 1 hour, then increasing the temperature of the shelf to minus 13 +/-1 ℃, preserving heat for 1 hour, then continuously reducing the temperature of the shelf to minus 55 +/-1 ℃, and starting timing and continuously preserving heat for 3 hours after the temperature of a product reaches minus 45 +/-1 ℃;

b, after the heat preservation in the step a is finished, quickly reducing the temperature of the condenser to below minus 50 +/-1 ℃, vacuumizing to below 10pa, raising the temperature of the shelf to minus 25 +/-1 ℃ for about 1.5 hours, starting timing and preserving heat for 18 hours, quickly raising the temperature of the shelf to minus 15 +/-1 ℃, preserving heat until the ice crystals of the product completely disappear, and continuing preserving heat for 4 hours;

step c, after the heat preservation in the step b is finished, rapidly raising the temperature of the shelf to 15 +/-1 ℃ within 1 hour, preserving the heat for 3 hours, then raising the temperature of the shelf to 30 +/-1 ℃, and finishing the heat preservation for 12 hours when the temperature of the product is raised to 25 +/-1 ℃;

and/or, after the step 3-1, further comprising post-treatment, wherein the post-treatment comprises the following steps: c, after the heat preservation in the step c is finished, fully pressing the plug, and discharging the box; and (4) rolling a cover.

11. A compound ginsenoside docetaxel liposome for injection, which is prepared by the preparation method of ginsenoside docetaxel liposome for injection as claimed in claim 9 or 10.

12. Use of a ginsenoside docetaxel liposome of any one of claims 1-4, or a blank liposome of claim 5, or a co-formulated ginsenoside docetaxel liposome for injection of claim 11 in the preparation of a medicament for the treatment and/or prevention of cancer; wherein the cancer may be one or more of breast cancer, ovarian cancer, lung cancer, gastric cancer, head and neck cancer and esophageal cancer.