CN114712309B - A kind of ginsenoside docetaxel liposome, its preparation method and application - Google Patents

Abstract

The invention discloses a compound ginsenoside docetaxel liposome, its preparation method and application. The invention provides a compound ginsenoside docetaxel liposome, which includes the following mass fraction components: 8-18 parts of phospholipid, 1-1.5 parts of ginsenoside, 1 part of docetaxel and 20-35 parts Lyophilizing protective agent; the ginsenoside docetaxel plasmid does not contain one or more of cholesterol, soybean oil and sodium oleate. The ginsenoside docetaxel liposome described in the present invention has better active targeting of Glut1; the efficacy is more than 2 times higher than that of conventional ginsenoside docetaxel liposome; and the toxicity is higher than that of conventional ginseng The saponin docetaxel liposome reduces cholesterol by more than 1.5 times and is more than 4-6 times lower than conventional cholesterol docetaxel liposome; the risk of accumulated toxicity in the body is low.

Description

Ginsenoside docetaxel liposome, preparation method and application thereof

Technical Field

The present invention relates to a compound ginsenoside docetaxel liposome, a preparation method and application thereof; further discloses a highly effective and low-toxic compound ginsenoside docetaxel liposome for injection, a preparation method and application thereof.

Background Art

Liposome is a directional drug delivery system and a special dosage form of targeted drug delivery system. It can encapsulate drugs in nanometer-sized particles. These particles are similar to bilayer microvesicles in biological membrane structures. When they enter the human body, they are mainly engulfed by the reticuloendothelial system and change the distribution of the encapsulated drugs in the body, so that the drugs accumulate mainly in the targeted tissues, thereby improving the therapeutic index of the drugs, reducing the therapeutic dose of the drugs and reducing the toxicity of the drugs.

The present invention is a technical innovation based on the Chinese invention patent applications CN201610693884.2, CN201811447245.3 and CN201811447243.4. The above three patent applications all disclose the technical advantages of liposomes with ginsenoside as membrane material, such as stable quality and significant 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 lyophilization protectants, the prescription may further include other auxiliary materials such as cholesterol, antioxidants, soybean oil and/or sodium oleate.

CN201811447245.3 discloses a blank liposome with ginsenoside Rh5H and its derivatives as membrane materials and its application. Based on CN201610693884.2, this patent further solves the hemolytic problem of ginsenoside. Similarly, in addition to phospholipids, saponins, drugs, and freeze-drying protective agents, the prescription may further include other excipients such as cholesterol, antioxidants, soybean oil and/or sodium oleate.

CN201811447243.4 discloses a blank liposome with ginsenoside Rg3 and its derivatives as membrane materials and its application. The patent solves the solubility problem of ginsenosides in chloroform by using ultrafine powder and other technical means to make saponins such as Rg3 and Rh2, thereby solving the problem that ginsenosides such as Rg3 and Rh2 must form membranes in chloroform, and prepares Rg3 liposomes that meet quality standards.

The above-mentioned prior art still has some shortcomings. For example, in some schemes, the pressure required for the homogenization step of liposome production is relatively high, the speed of membrane sterilization filtration is slow, the interception rate is high, and the product yield is significantly poor; 2-6 times the amount of soybean oil needs to be added. However, the addition of soybean oil 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 vivo, and can significantly improve the clinical therapeutic 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 pharmacokinetic, in vivo tissue distribution, drug efficacy, etc. caused by changes in the functional components of the compound liposomes, which are rarely involved. Therefore, for the compound ginsenoside docetaxel liposomes for injection (hereinafter referred to as "Ginposome-DTX" or "Ginposome-CTX"), after the main drug "docetaxel" has been determined, how to select the most suitable "synergistic drug and excipient" ginsenoside and key excipient "phospholipid" and their related proportions, prepare a reasonable compatibility, small particle size, stable quality, drug efficacy and toxicity to achieve the best effect, so that the composition of the specific ratio of the drug and key excipients of the present invention is innovative and unique, which is of great significance.

In prescription screening, any change in any of the factors, including drugs, phospholipids, saponins, lyoprotectants and preparation processes, will have a fatal impact on the quality, efficacy and safety of the product. For example, increasing the mass ratio of saponin to paclitaxel can increase the quality stability and synergistic anti-tumor effect of the compound liposomes, increase the targeted distribution of tumor tissues, but it will also increase the cumulative toxicity of saponins in the human body and cause uncontrollable organ damage; the appropriate mass ratio of saponin to paclitaxel has a very important correlation with the stability, active targeting, pharmacodynamics and safety of this type of liposomes. At the same time, the selection of different lyoprotectants plays a vital role in the non-destruction of the lipid bilayer structure during the freeze-drying process of the liposomes and the restoration of the properties of the liposomes after the freeze-dried drugs are reconstituted. For example, in the selection of lyoprotectants, different lyoprotectants have different effects on the freeze-drying curve, especially in the co-solubility point of the compound liposomes, whether it collapses, whether the liposomes change significantly after reconstitution, the temperature and time setting of the first freeze-drying, and the total freeze-drying time.

The safety and effectiveness of drugs are two basic attributes of drugs, and neither is dispensable. The approval and use of drugs are based on the risk-benefit ratio between the two, especially for improved new drugs, the core of which is to improve effectiveness and safety.

In toxicological research, pharmaceutical research plays a vital role, especially the effects of the selection of prescription ratio and preparation process on acute toxicity, long-term toxicity and cumulative toxicity of various functional organs, which will directly determine whether the compound liposome meets the requirements for new drug application.

It can be seen that the optimal ratio range of phospholipids, ginsenosides, docetaxel and lyoprotectant sugar components in the above liposome formulation membrane components plays an important role in the good pharmaceutical stability, in vivo distribution, pharmacodynamics and toxicology of the composite liposomes. However, the prior art does not provide any derivation relationship between the above components and ratios and the process and pharmacological activity, pharmacokinetics and toxicology. Due to the many variables involved, its screening must be done through a lot of experiments and creative labor.

Therefore, how to choose an optimal compound drug combination and how to develop the best preparation process in order to produce an injectable compound ginsenoside docetaxel liposome with better efficacy, lower toxicity, quality and other indicators that meet the requirements of drugs in order to meet the drug registration requirements requires a lot of research work and technical breakthroughs.

Summary of the invention

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

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

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

In a certain embodiment of the present invention, the ginsenoside docetaxel liposomes are composed of the following components in mass fractions: 8-18 parts of phospholipids, 1-1.5 parts of ginsenosides, 1 part of docetaxel and 15-35 parts of lyoprotectants.

In a certain embodiment of the present invention, the phospholipid is one or more of egg yolk lecithin, soybean lecithin, hydrogenated lecithin and cephalin, or one or more of egg yolk lecithin, soybean lecithin, hydrogenated lecithin and cephalin containing 0.01-10% distearoyl phosphatidylethanolamine-methoxypolyethylene glycol 2000 (mPEG2000-DSPE), preferably egg yolk lecithin and/or soybean lecithin, more preferably egg yolk lecithin.

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

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

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

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

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

In a certain embodiment of the present invention, the mass ratio of the lyoprotectant to the docetaxel may be 20-35:1, such as 25-35:1, and also such as 25:1, 30:1 or 35:1. For example, the mass ratio of the glucose to the docetaxel is 25:1.

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

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

In a certain embodiment of the present invention, the ginsenoside docetaxel liposomes include the following components in mass fractions: 10 parts of phospholipids, 1 part or 1.5 parts of ginsenosides, 1 part of docetaxel and 25 parts of lyophilization protectants.

In a certain embodiment of the present invention, the ginsenoside docetaxel liposomes may include the following components in mass fractions:

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

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 lyophilization protectant.

In a certain embodiment of the present invention, the ginsenoside docetaxel liposomes may include the following components in mass fractions:

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

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

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

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

The present invention also provides a blank liposome, which comprises the following components in mass fractions: 8-18 parts of phospholipids, 1-2 parts of ginsenosides and 20-35 parts of freeze-drying protective agents; the liposome does not contain one or more of cholesterol, soybean oil and sodium oleate.

In a certain embodiment of the present invention, the blank liposomes may be loaded with a pharmaceutically active substance. Preferably, the pharmaceutically active substance may be a taxane derivative; for example, paclitaxel and/or docetaxel; for example, docetaxel.

In a certain embodiment of the present invention, in the blank liposomes, the phospholipids are one or more of egg yolk lecithin, soybean lecithin, hydrogenated lecithin and cephalin, or one or more of egg yolk lecithin, soybean lecithin, hydrogenated lecithin and cephalin containing 0.01-10% distearoyl phosphatidylethanolamine-methoxypolyethylene glycol 2000.

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

In a certain embodiment of the present invention, the ginsenoside in the blank liposomes may be one or more of 20(S)-ginsenoside Rg3, ginsenoside pseudo-Rg3, 20(S)-ginsenoside Rh2, pseudo-ginsenoside GQ, ginsenoside Rg5, ginsenoside Rk1 and ginsenoside Rp1, preferably 20(S)-ginsenoside Rg3 and/or 20(S)-ginsenoside Rh2, and more preferably 20(S)-ginsenoside Rg3.

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

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

In a certain embodiment of the present invention, the lyoprotectant may be present in 25-35 parts of the blank liposome, for example, 25 parts, 30 parts or 35 parts.

In a certain embodiment of the present invention, the blank liposome may include the following components by mass fraction: 9 parts of egg yolk lecithin, 1.8 parts of Rg3 and 20-35 parts of lyophilization protective agent;

Or, 10 parts of soybean lecithin, 2 parts of Rh2 and 20-35 parts of lyophilizing protectant;

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

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

Or, 10 parts of egg yolk lecithin: 1.5 parts of Rk1 and 20-35 parts of lyophilization protectant;

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

The present invention also provides a method for preparing ginsenoside docetaxel liposomes, which comprises the following steps:

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

Step 2, the membrane obtained in step 1 is kept warm in water for hydration, and then mixed evenly with a freeze-dried protective agent solution to obtain a liposome solution A2;

Step 3, which 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 to control the particle size D90 to be less than 100 nm to obtain a liposome solution A3a.

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

The liposome solution A2 obtained in step 2 is sheared and then extruded through a 150 nm aperture extrusion plate to control the particle size D90 to be less than 100 nm, thereby obtaining a liposome solution A3b;

Wherein, definitions of docetaxel, ginsenoside, phospholipid and lyophilization protective agent solution are the same as those described in the above-mentioned (compound) ginsenoside docetaxel liposome.

In a certain embodiment of the present invention, in step 1, the organic solvent may be one or more of methanol, ethanol, chloroform, and dichloromethane, preferably a mixed solvent of methanol and/or ethanol with chloroform and/or 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, ginsenosides, and phospholipids. For example, the mass volume ratio of docetaxel to the organic solvent is 1g/60-120ml, for example, 1g/80mL.

In a certain embodiment 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 a solution of docetaxel and the organic solvent, and heated to dissolve; the heating can be heating in a water bath to 35-65°C, for example, 55°C.

In a certain embodiment of the present invention, in the step 1, the concentration may be reduced pressure concentration; the reduced pressure concentration may be vacuum = -0.08 MPa to -0.1 MPa, for example -0.089 to -0.1 MPa; the concentration may be performed until all the solvents are completely evaporated; the total concentration time is preferably less than 4 hours.

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

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

In one embodiment of the present invention, in step 2, the concentration of the lyoprotectant solution may be 0.20-0.35 mg/ml, for example 0.25 mg/mL.

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

In a certain embodiment of the present invention, in the step 2, the hydration is carried out in a rotary evaporator at a rotation speed of 40 to 60 rp/min, for example, 50 rp/min.

In a certain embodiment of the present invention, in the step 2, the hydration may be performed until the solution is uniform, for example, for 2-4 hours.

In a certain embodiment of the present invention, in the step 2, the docetaxel: lyophilized protective agent solution = 1 g: 100 mL.

In a certain embodiment of the present invention, in the step 2, the volume of the lyophilization protectant solution is the same as the volume of the water.

In a certain scheme of the present invention, in scheme 1 of step 3, the high-pressure homogenization is to use a 0-10°C chilled water cooling cycle in a homogenizer; preferably, the temperature of the liposome solution is ensured to be 5-10°C.

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

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

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

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

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

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

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

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

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

The present invention also provides a method for preparing ginsenoside docetaxel liposomes for injection, which comprises the following steps:

Step 1-1, step 1-3 in the method for preparing ginsenoside docetaxel liposomes as described above, obtaining a liposome solution A3a or A3b;

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

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

In the preparation method, the sterilization filtration and freeze-drying conditions and operations may be conventional conditions and operations in this type of process in the art; the following are preferred in the present invention:

In one embodiment of the present invention, in step 2-1, the sterilization filtration may use a 0.22 μm filter membrane.

In one embodiment of the present invention, in step 3-1, the freeze-drying can be carried out in a vial, and the vial can be a conventional vial in the art, such as a 30 ml or 50 ml vial.

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

Step a, quantitatively packing the liposome solution A4 in the vial into a freeze drying box, wherein the freeze drying shelf temperature is uniformly reduced to -10±1°C, kept warm for 1 hour, then the shelf temperature is raised to -13±1°C, kept warm for 1 hour, and then the shelf temperature is further reduced to -55±1°C, and when the product temperature reaches -45±1°C, start timing and continue to keep warm for 3 hours;

Step b, after the heat preservation in step a is completed, the condenser temperature is quickly reduced to below -50±1°C, and the condenser is evacuated to below 10Pa. After the shelf temperature is raised to -25±1°C (about 1.5 hours), the heat preservation timer is started for 18 hours, and then the shelf temperature is quickly raised to -15±1°C. After the ice crystals of the product completely disappear, the heat preservation is continued for another 4 hours;

Step c. When the insulation in step b is completed, quickly (within 1 hour) raise the shelf temperature to 15±1°C, keep it warm for 3 hours, then raise the shelf temperature to 30±1°C, and when the product temperature rises to 25±1°C, end the insulation for 12 hours.

In a certain embodiment of the present invention, step 3-1 may further include post-processing, and the conditions and operations of the post-processing may be conventional conditions and operations in this type of process in the art; for example, the post-processing includes the following steps: after the insulation described in step c is completed, the whole plug is pressed and taken out of the box; and the cover is rolled.

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

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

The present invention also provides a use of the above-mentioned ginsenoside docetaxel liposome, or the above-mentioned blank liposome, or the above-mentioned compound ginsenoside docetaxel liposome for injection in the preparation of a drug for treating and/or preventing cancer.

The cancer may be breast cancer, ovarian cancer, lung cancer, stomach cancer, head and neck cancer, and esophageal cancer.

The term "particle size D90" refers to the particle size corresponding to when the cumulative particle size distribution percentage of a sample reaches 90%. Its physical meaning is that 90% of the particles have a particle size smaller than it.

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

Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

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

The positive and progressive effects of the present invention are: the compound ginsenoside docetaxel liposomes provided by the present invention have a targeting effect on tumor cells, an anti-multidrug resistance effect, an efficacious and toxic reduction effect, and a drug synergistic effect. Taking the compound ginsenoside Rg3 docetaxel liposomes for injection in the embodiment as an example, the drug efficacy is significantly better than the technical solutions that are not within the scope of the present invention; it shows that Rg3 plays a better role as a "drug, excipient, membrane material, target head" and other multiple roles in the compound ginsenoside Rg3 docetaxel liposomes for injection, and plays a good drug synergistic effect. Specifically:

(1) The efficacy was significantly improved. In particular, the DTX-Rg3 (1.0) / Lp group and the DTX-Rh2 (1.5) / Lp group had the best efficacy, and the tumor inhibition rate (68%) of the dose (10 mg/kg) reached or exceeded the tumor inhibition rate of the high dose (20 mg/kg) of the liposome group not prescribed by the present invention, so the tumor inhibition effect was improved by 2 times;

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

(3) Significantly reduced toxic and side effects. The acute toxicity of DTX-Rg3(1.0)/Lp (LD50≥150mg/kg) and DTX-Rh2(1.5)/Lp (LD50≥150mg/kg) prepared according to the prescription of the present invention was reduced by more than 6 times compared with the Taxotere group (LD50 about 25mg/kg), 3-4 times compared with ordinary cholesterol docetaxel liposomes (LD50 between 25 and 50mg/kg), and 1.5-2 times compared with non-prescription ginsenoside liposomes of the present invention such as DTX-C-Rg3(1.8)/Lp and DTX-C-Rh2(2.0)/Lp (LD50=about 50-100mg/kg). In the long-term toxicity experiment on SD rats, animal deaths occurred in the Taxotere group, indicating that the toxic effect of the Taxotere group was greater. The toxicity of the ginsenoside Rg3 docetaxel liposome or the ginsenoside Rh2 docetaxel liposome of the present invention is reduced by more than 4-6 times compared with the toxicity of Taxotere.

DETAILED DESCRIPTION

The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

Experimental drugs and equipment

Experimental drugs: 20(S)-ginsenoside Rg3 (abbreviated as: Rg3), ginsenoside pseudo-Rg3 (abbreviated as: pseudo-Rg3), ginsenoside Rp1 (abbreviated as: Rp1), ginsenoside pseudo-GQ (abbreviated as: pseudo-GQ), ginsenoside Rk1 (abbreviated as: Rk1), ginsenoside Rg5 (abbreviated as: Rg5), 20(S)-ginsenoside Rh2 (abbreviated as: Rh2), ginsenoside Rk2 (abbreviated as: Rk2), 20(S)-ginsenoside Rg2 (abbreviated as: Rg2), 20(S)-ginsenoside Rh1 (abbreviated as: Rh1), 20(S)-protopanaxadiol (abbreviated as: PPD), 20(S)-protopanaxatriol (abbreviated as PPT), etc. are conventionally commercially available in the art, for example, Shanghai Bensu Pharmaceutical Technology Co., Ltd., Suzhou Xinghai Jinsen Biotechnology Co., Ltd., Shanghai Yuanye Biotechnology Co., Ltd., etc.

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

The molecular structural formula of ginsenoside described in the present invention is as follows:

Test instruments: The instruments used in the following examples are owned by Shanghai Bensu Pharmaceutical Technology Co., Ltd. and the School of Pharmacy of Fudan University. The equipment models and source information are as follows:

Agilent liquid chromatography: one 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);

Mini-extruder (Avanti Polar Lipids Inc);

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

Malvern Nanosizer ZS90 (Malvern Company, 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 observation (Zeiss LSM 710, Oberkochen, Germany);

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

Confocal intravital microscopy (IVM);

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

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

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

Clean bench (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 Sunyu Hengping Scientific Instrument Co., Ltd.;

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

Cell culture 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 lines:

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

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

The triple-negative breast cancer MDA-MB-231 cell line was provided by the School of Pharmacy, Fudan University;

Example 1 Preparation of Compound Ginsenoside Rg3 Docetaxel Liposomes for Injection

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

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

Add the prescribed amount of docetaxel into the mixed solvent and dissolve for later use. Then add the prescribed amount of ginsenoside Rg3 and egg yolk lecithin into the mixed solvent, heat to dissolve, transfer into a 1L rotary evaporator, and concentrate under reduced pressure at a water bath temperature of 55°C, a rotation speed of 50 rpm, a vacuum degree of -0.089 to -0.1 MPa, and rotary evaporation until the solvent is completely evaporated.

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

Add 100 ml of water for injection into the rotary evaporator after film formation, set the water bath temperature to 40-45°C, rotate at 50 rpm, hydrate and completely dissolve for about 2 hours.

Then add 100 ml of glucose aqueous solution, stir well 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 the cycle. The homogenization pressure is set to 1200 bar. The cycle is repeated 3-4 times until D90 is less than 100 nm.

5. Sterile filtration: Sterile filter the homogenized solution through a 0.22 μm filter membrane.

6. Packaging: Dispense the sterile filtered solution into 30 or 50 ml vials according to the set amount of 8 to 10 ml.

7. Pre-freezing: After the product enters the box, the shelf temperature drops to about -10℃ at a uniform rate and keeps warm for 1 hour. Then the shelf temperature is raised to about -55℃ and kept warm for 1 hour. After the insulation is completed, the shelf temperature is further dropped to about -55℃. After the product temperature reaches -45℃, start timing and continue to keep warm for about 3 hours.

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

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

10. Capping and packaging: Cap and package the above liposomes to obtain the compound ginsenoside Rg3 docetaxel liposomes for injection (prescription 1).

Example 2 Preparation of Compound Ginsenoside Rg3 Docetaxel Liposomes for Injection

The amount of ginsenoside Rg3 in Example 1 was increased to 1.5 g, and the other conditions were the same as in Example 1 to prepare compound ginsenoside Rg3 docetaxel liposomes for injection.

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, anhydrous ethanol 40ml, chloroform 40ml, water for injection 200ml.

2. Film formation: Same film formation method as in Example 1.

3. Hydration: Same as the hydration method in Example 1.

4. High-speed shearing and extrusion: The above 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, and the extrusion device was connected, an extrusion plate with a pore size of 150 nm was installed, and extrusion was performed at a pressure of 800 psi.

5. The 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 replaced with 1.0 g of ginsenoside Rh2, and the other ingredients were the same as in Example 1 to prepare compound ginsenoside Rh2 docetaxel liposomes for injection.

Example 5 Preparation of Compound Ginsenoside Rh2 Docetaxel Liposomes for Injection

The ginsenoside Rg3 in Example 1 was replaced with 1.5 g of ginsenoside Rh2, and the other ingredients were the same as in Example 1 to prepare compound ginsenoside Rh2 docetaxel liposomes 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 other ingredients were the same as in Example 1 to prepare compound ginsenoside pseudo-Rg3 docetaxel liposomes for injection.

Example 7 Preparation of Compound Ginsenoside Rg5 Docetaxel Liposomes for Injection

The ginsenoside Rg3 in Example 1 was replaced with 1.5 g of ginsenoside Rg5, and the other ingredients were the same as in Example 1 to prepare compound ginsenoside Rg5 docetaxel liposomes for injection.

Example 8 Preparation of Compound Ginsenoside Rk1 Docetaxel Liposomes for Injection

The ginsenoside Rg3 in Example 1 was replaced with 1.5 g of ginsenoside Rk1, and the other conditions were the same as in Example 1 to prepare compound ginsenoside Rk1 docetaxel liposomes for injection.

Example 9 Preparation of Compound Ginsenoside Rp1 Docetaxel Liposomes for Injection

The ginsenoside Rg3 in Example 1 was replaced with 1.5 g of ginsenoside Rp1, and the other ingredients were the same as in Example 1 to prepare compound ginsenoside Rp1 docetaxel liposomes for injection.

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 other ingredients were the same as in Example 1 to prepare compound ginsenoside pseudo-GQ docetaxel liposomes for injection.

Effect Example 1

(a) According to the prescription in the following table, and in the same manner as in Example 1 (without the freeze-drying step), the comparison results of the types of ginsenosides are as follows:

The above series of experiments prove that without adding other excipients such as soybean oil or cholesterol, when the ginsenosides in the present invention are: Rg3, pseudo Rg3, Rh2, pseudo GQ, Rg5, Rk1 and Rp1, Ginposome-DTX or CTX has a good preparation process, the preparation conditions are easy to achieve, and it is conducive to industrialization. When the ginsenosides are Rk2, Rg2, Rh1, PPD and PPT, other excipients need to be added, and the preparation conditions are relatively harsh.

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

Notes: Egg yolk phosphatidylcholine (EPC), soybean phospholipids (SPC), cephalin (PE), sphingomyelin (SM), hydrogenated phospholipids (HSPC), phosphatidylserine (PS), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl phosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), dimyristoylphosphatidylcholine (DMPC), polyethylene glycol 2000-distearoylphosphatidylethanolamine (mPEG-DSPE), polyethylene glycol 2000-dioleoylphosphatidylethanolamine (mPEG-DOPE).

According to the same method as Example 1 (freeze-drying step is not required), the comparison results of phospholipid types and proportions are as follows:

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

(c) The optimal ratios of ginsenosides were compared using the same method as in Example 1 (without freeze-drying step):

The above experiments prove that without adding other excipients such as soybean oil or cholesterol, the saponin:docetaxel ratio of the present invention is 1-3:1, and the effect is better. However, due to the toxicokinetics research results of Example 4 of the present invention, the present invention only selects the saponin:docetaxel ratio of 1-1.5:1.

(d) The freeze-dried protective agents were compared in the same manner as in Example 1:

In the selection of lyoprotectants, different lyoprotectants have a significant effect on the encapsulation rate and particle size distribution of the liposomes after the product is reconstituted, and also have a great impact on the economy of the freeze-drying curve. Through the above experiments, without adding other excipients such as soybean oil, the lyoprotectant in the present invention is one or more of glucose, trehalose, sucrose, lactose and galactose, and the ratio of docetaxel to lyoprotectant is: lyoprotectant/docetaxel = 25-35 times the amount, for example, when the amount is 15 times the amount of glucose, the reconstituted particle size of Ginposome-DTX is 200nm, which is between qualified and unqualified, and when it is 25 or 35 times the amount, it has a good match with docetaxel liposomes. Taking into account the empirical data of pharmaceutics, the ratio of docetaxel to lyoprotectant determined by the present invention is: lyoprotectant/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 and phospholipids of the present invention by Glut1 targeting.

2) Experimental methods: In order to compare the uptake of 4T1 in each experimental group and explore the uptake mechanism of the compound preparation, 4T1 cells were seeded in 12-well plates at a cell density of 2×10 ^5. For the experimental group + glucose, experimental group + phlorizin and experimental group + quercetin groups, the culture medium was replaced with 20mM glucose solution, phlorizin solution and quercetin solution after 12 hours. These three solutes should be dissolved in the glucose-free culture medium. After incubation for 1 hour, the drugs of each experimental group (the concentration of the ultraviolet fluorescent color developer is 100ng/ml) were added. After incubation for 4 hours, digestion, washing with fresh PBS solution and analysis by flow cytometry were performed.

3) Preparation method of the experimental group: prepared according to the method of Example 1 of the present invention (docetaxel was replaced with coumarin, and the freeze-drying step was not required)

Based on the results of the toxicological test in Example 4, the following experiment did not conduct an experiment with a prescription of ginsenoside/docetaxel exceeding 2.0.

Experimental Group:

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

The experimental results are as follows:

Experimental conclusion:

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

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

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

1) Experimental method: The tumor cell line (MCF-7) was injected subcutaneously into mice to establish a subcutaneous tumor model. When the tumor volume reached 100 mm ³ (7 days after inoculation), the mice were randomly divided into groups (n=8 per group) for treatment. Each group was injected with blank solvent (5% glucose, Blank), DTX-C/Lp group, DTX-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, DT X-Rh2(2.0)/Lp group, DTX-C-Rg3(1.8)/Lp group (egg yolk lecithin: Rg3: cholesterol: docetaxel = 9:1.8:2.25:1), DTX-C-Rh2(2.0)/Lp group (soybean lecithin: Rh2: cholesterol: docetaxel = 10:1:2.25:1), three groups of docetaxel (20mg, 10mg, 5mg), were given once every 7 days until the 28th day. The length and width of the tumor were measured and the body weight was recorded at the same time. The formula for calculating tumor volume (V) is: V = (W ² × L)/2. The length (L) is the longest diameter of the solid tumor, and the width (W) is the shortest diameter perpendicular to the length. At the end of the experiment on the 28th day, all animals were killed and the tumors were removed for imaging and histological examinations.

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

Due to the toxicological results of Example 4, the present example did not conduct research on the DTX-Rg3 (2.0 or more)/Lp group.

2) The test results are as follows:

in conclusion:

1) The drug efficacy of the DTX-Rg3 (1.0) / Lp group and the DTX-Rh2 (1.5) / Lp group was the best, and the tumor completely disappeared on the 21st day. The tumor inhibition rate of the medium dose (docetaxel = 10 mg/kg) of the two groups was basically consistent with the tumor inhibition rate of the high dose group (docetaxel = 20 mg/kg) of the DTX-C-Rg3 (1.8) / Lp group and the DTX-C-Rh2 (2.0) / Lp group. That is, the tumor inhibition effect was improved by more than about 2 times compared with the non-invention group.

2) Animal deaths occurred in the DTX-C/Lp group, indicating a strong toxic effect.

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

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

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

The dose of docetaxel in this study was set at 25, 50, 100 and 150 mg/kg/day, and the doses of Rg3 in the test sample were 25, 50, 100 and 150 mg/kg/day, respectively; the doses of Rh2 were 37.5, 75, 150 and 225 mg/kg/day, respectively. At the same time, a vehicle control group (5% glucose injection), a commercially available positive control group (Taxotere group), an Rg3 liposome group and a DTX-C-Rg3/Lp group were set up, and the drug was slowly injected intravenously (about 1 mL/min) 3 times/day, with at least 4 hours between each administration.

2) Experimental groups: There were 13 groups in total, including 5% glucose group, DTX-C/Lp group, DTX-C-Rg3(1.8)/Lp group, DTX-C-Rh2(2.0)/Lp group, which were consistent with the pharmacodynamic experimental groups. The names of other groups are shown in the table below.

3) Preparation method of the experimental group: According to the prescription requirements, the preparation was carried out according to the method of Example 1.

3) The experimental results are shown in the following table:

Note: Due to the toxicological results of Example 4, the study of the 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 pharmacology and Glut1 targeting, so that the toxicity reduction effect of the relevant preparations is the best. Compared with the cholesterol liposome group (DTX-C/Lp) and docetaxel injection (Taxotere), the toxicity is 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 is 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 methods: There were 8 groups in total in this experiment, including vehicle control group (5% glucose injection), commercially available positive control group Taxotere group (10 mg/kg), test product 1 (DTX-Rg3 (1.0)/Lp group: 10 mg/kg and 20 mg/kg), test product 2 (DTX-Rh2 (1.5)/Lp group: 10 mg/kg and 20 mg/kg), and test product 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 female, for a total of 80 rats. The drug was administered intravenously once a week for four consecutive weeks. Whole blood was collected from the vehicle control group before administration on D1 and D29 and 1 hr after administration. Whole blood was collected from test sample 1, test sample 2, test sample 3 and commercially available positive control group before administration on D1 and D29 and 3 min, 15 min, 30 min, 1 hr, 3 hr, 6 hr, 24 hr after administration. The whole blood was collected into a test tube containing EDTA-K2 anticoagulant, placed on crushed ice, and centrifuged to collect plasma for analysis and detection.

The concentrations of docetaxel, 20(S)-Rg3 and 20(S)-Rh2 in plasma were detected by LC-MS/MS. The lower limit of quantification of docetaxel analysis method was 25.000ng/mL, and the lower limit of quantification of Rg3 and Rh2 analysis method was 50.000ng/mL. The non-compartmental model of WinNonlin software was used to analyze the plasma concentration data and calculate the parameters to study the toxicokinetics characteristics of the test product and provide a reference for clinical trials.

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

1) After administration on D1 and D29, the exposure of ginsenosides Rg3 and Rh2 in the plasma of male and female SD rats after intravenous injection of test sample 1, test sample 2 and test sample 3 (measured in AUC(0-t)) increased with the increase of the administration dose, and the increase rate was higher than the increase of the dose.

2) After administration on D1, the exposure of ginsenosides Rg3 and Rh2 in the plasma of male and female SD rats after intravenous injection of test sample 1, test sample 2 and test sample 3 (measured in Cmax) was directly proportional to the administered dose.

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

4) After administration of D1 and D29, the exposure of ginsenosides Rg3 and Rh2 in the plasma of male and female SD rats after intravenous injection of test sample 2 (measured in Cmax) was directly proportional to the administered dose.

5) Under the conditions of this experiment, after continuous administration, there was basically no accumulation tendency of ginsenosides Rg3 and Rh2 in the plasma of male and female SD rats after intravenous injection of test samples 1 and 2; there was basically no accumulation tendency of docetaxel in the plasma of male and female SD rats after intravenous injection of test samples 1, 2 and 3; there was a slight accumulation of docetaxel and ginsenoside Rh2 in the plasma of male and female SD rats after intravenous injection of test sample 3; there was no accumulation of docetaxel in the plasma of male and female SD rats given intravenous injection of the commercially available control group.

Conclusion: There was 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 weighing 160-260 g, aged 6-9 weeks, 6 rats in each group, administration method: slow intravenous push (about 1 mL/min), administration frequency: once/week (D1, D8, D15, D22, D29).

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

The docetaxel dosage of the test sample in this experiment was set at 5, 10 and 20 mg/kg. The mass ratio of docetaxel to Rg3 in the test sample was 1:1.0, and the mass ratio of docetaxel to Rh2 was 1:1.5. Therefore, the Rg3 content was 5, 10 and 20 mg/kg, and the Rh2 content was 7.5, 15 and 30 mg/kg, respectively. (Prepared according to the preparation method of Example 1)

The dosage for each group is set as follows:

3) The experimental results are as follows:

Result 1: Taxotere group long-term toxicity test results (30-day test results)

Dosage 5mg/kg 10mg/kg 20mg/kg Animal mortality rate No deaths No deaths 33.3% Average weight changes of animals +15% +2% -18% Liver tissue damage No damage Moderate Severe Kidney tissue damage No damage Moderate Severe Splenic tissue damage No damage Moderate Severe Heart tissue damage No damage Moderate Severe

Result 2: Taxotere group + Rg3/Lp group long-term toxicity test results (30-day test results)

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

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

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

1) In the Taxotere group, rats died, had severe weight loss, and had severe damage to various organs, indicating high toxicity.

2) In both the DTX-Rg3/Lp and DTX-Rh2/Lp groups, no rats died, body weight was almost not reduced, and various organs were mainly slightly damaged, indicating that long-term toxicity was greatly reduced.

Application Example 6: In vivo study of compound ginsenoside docetaxel liposome 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.

Transplant tumor model: The above cell lines were inoculated subcutaneously in the right axilla of nude mice at a cell inoculation amount of 5×10 ⁶ /mouse. After the transplant tumor was formed, the cells were propagated once in the nude mice before use.

Experimental method: Tumor cell lines were injected subcutaneously into mice to establish a subcutaneous tumor model. When the tumor volume reached 100 mm ³ (7 days after inoculation), the mice were randomly divided into 4 groups (n = 8 per group) for treatment. Each group was injected with blank solvent (5% glucose), Taxotere group, DTX-Rg3/Lp group (20 mg/kg docetaxel, 20 mg/kg ginsenoside Rg3), and DTX-Rh2/Lp group (20 mg/kg docetaxel, 30 mg/kg ginsenoside Rh2) through the tail vein. The drug was given once every 7 days until the 28th day. The length and width of the tumor were measured and the body weight was recorded at the same time of drug administration. 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 the 28th day, all animals were killed and the tumors were removed for imaging and histological examinations.

Triple-negative breast cancer MDA-MB-231: Based on the in vivo pharmacodynamics experimental method, the in vivo pharmacodynamics research data of 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 inhibition effect (tumor inhibition rate of 68%), while the DTX-Rg3/Lp group and the DTX-Rh2/Lp group had the best effect, and the tumors had completely disappeared on the 28th day. The experimental results showed that the compound preparation had significant efficacy and significant drug synergy.

Claims (17)

1. A ginsenoside docetaxel liposome, which is composed of the following mass fraction components: 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 plasmid does not contain cholesterol, soybean oil and sodium oleate; 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 lecithin, hydrogenated phospholipid and cephalin containing 0.01-10% distearoyl phosphatidylethanolamine-methoxypolyethylene glycol 2000; the ginsenoside is 20 One or more of (S)-ginsenoside Rg3, ginsenoside pseudo-Rg3, 20(S)-ginsenoside Rh2, pseudo-ginsenoside GQ, ginsenoside Rg5, ginsenoside Rk1 and ginsenoside Rp1; the frozen The dry protective agent is one or more of trehalose, glucose, sucrose, lactose and galactose; the mass ratio of docetaxel to the phospholipid is 1:(8-12); the freeze-drying protection The mass ratio of the agent to docetaxel is (25-35):1. 2. Ginsenoside docetaxel liposome as claimed in claim 1, characterized in that, The mass ratio of docetaxel to the ginsenoside is 1:1 or 1:1.5; And/or, the particle size D90 of the ginsenoside docetaxel liposome is ≤150 nm. 3. Ginsenoside docetaxel liposome as claimed in claim 2, characterized in that, The mass ratio of docetaxel to the phospholipid is 1:10; And/or, the mass ratio of the lyophilized protective agent and docetaxel is 30:1. 4. Ginsenoside docetaxel liposome as claimed in claim 1, characterized in that, It consists of components with the following mass fractions: 10 parts of phospholipids, 1 part of ginsenoside Rg3, 1 part of docetaxel and 25 parts of freeze-dried protective agent; Or, it consists of components with the following mass fractions: 10 parts of phospholipid, 1.5 parts of ginsenoside Rh2, 1 part of docetaxel and 25 parts of freeze-dried protective agent; Or, it consists of components with the following mass fractions: 10 parts of phospholipid, 1 part of ginsenoside Rg3, 1 part of docetaxel and 30 parts of freeze-dried protective agent; Or, it consists of components with the following mass fractions: 10 parts of phospholipid, 1.5 parts of ginsenoside Rh2, 1 part of docetaxel and 30 parts of freeze-dried protective agent. 5. A blank liposome, characterized in that it is composed of the following mass fraction components: 8-18 parts of phospholipids, 1-1.5 parts of ginsenosides and 20-35 parts of freeze-dried protective agent; the blank liposome It does not contain cholesterol, soybean oil and sodium oleate; the phospholipid is one or more of egg yolk lecithin, soybean lecithin, hydrogenated phospholipid and cephalin; the ginsenoside is 20(S)-ginsenoside Rg3 , one or more of ginsenoside pseudo-Rg3, 20(S)-ginsenoside Rh2, pseudo-ginsenoside GQ, ginsenoside Rg5, ginsenoside Rk1 and ginsenoside Rp1; the freeze-drying protective agent is trehalose, One or more of glucose, sucrose, lactose and galactose. 6. Blank liposome as claimed in claim 5, characterized in that, The phospholipid is 10 parts. 7. A method for preparing ginsenoside docetaxel liposomes according to any one of claims 1-4, which includes the following steps: Step 1. Concentrate the solution A1 of docetaxel, ginsenoside, phospholipid and organic solvent to form a film; the organic solvent is one or more of methanol, ethanol, chloroform and methylene chloride; Step 2. After the membrane obtained in step 1 is kept warm and hydrated in water, it is evenly mixed with the freeze-dried protective agent solution to obtain liposome solution A2; Step 3. It is option 1 or option 2; Option 1 includes the following steps: Perform high-pressure homogenization of the liposome solution A2 obtained in step 2, and control the particle size D90 to be less than 100 nm to obtain liposome solution A3a; Option 2 includes the following steps: After shearing the liposome solution A2 obtained in step 2, it is extruded through an extrusion plate with a pore size of 150 nm, and the particle size D90 is controlled to be less than 100 nm to obtain liposome solution A3b. 8. The preparation method of ginsenoside docetaxel liposome as claimed in claim 7, characterized in that, The mass-to-volume ratio of docetaxel to the organic solvent is 1 mg/80 mL; And/or, in the step 1, the solution A1 is obtained by heating and dissolving docetaxel, the ginsenoside, and the phospholipid in an organic solvent; And/or, in the step 1, the concentration is concentration under reduced pressure; And/or, in the step 1, the concentration is carried out in a rotary evaporator, and the rotation speed is = 40 to 60 rpm; 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; And/or, in the step 2, the hydration temperature is 35-65°C; And/or, in the step 2, the hydration is carried out in a rotary evaporator bottle, and the rotation speed is 40 to 60 rpm; And/or, in the step 2, the hydration can be as long as the solution is uniform; And/or, in the step 2, the docetaxel: freeze-drying protectant solution = 1g: 100mL; 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 solution 1 of step 3, the high-pressure homogenization is to use a 0-10°C refrigerated water cooling cycle in a homogenizer; And/or, in the solution 1 of step 3, the pressure of the high-pressure homogenization is between 800-1400 bar; And/or, in the solution 1 of step 3, the number of times of high-pressure homogenization is 3-4 times; And/or, in the solution 2 of step 3, the shearing is performed at room temperature; And/or, in the solution 2 of step 3, the shearing speed is 1500 to 2200 rpm; And/or, in the solution 2 of step 3, the shearing time is 5 to 10 minutes; And/or, in the solution 2 of step 3, the extrusion temperature is 35-45°C; And/or, in the solution 2 of step 3, the pore diameter of the extrusion plate is 150nm; And/or, in the solution 2 of step 3, the extrusion pressure is 600 to 800 psi; And/or, in the solution 2 of step 3, the number of extrusions is 3-4 times. 9. The preparation method of ginsenoside docetaxel liposomes as claimed in claim 7, characterized in that, in the step 1, the ginsenoside and the phospholipid are added to docetaxel The solution with the organic solvent is heated and dissolved to obtain solution A1. 10. The preparation method of ginsenoside docetaxel liposomes as claimed in claim 8, characterized in that, in the step 1, the organic solvent is methanol and/or ethanol and chloroform and/or dihydrogen. Mixed solvents of methyl chloride; And/or, in the step 1, the concentration under reduced pressure is vacuum=-0.08mpa~-0.1mpa; And/or, in the step 1, the concentration is carried out in a rotary evaporator, and the rotation speed is 50 rpm; And/or, in the step 2, the concentration of the lyophilized protective agent solution is 0.25 mg/mL; And/or, in the step 2, the hydration temperature is 40-45°C; And/or, in the step 2, the hydration is carried out in a rotary evaporator bottle, and the rotation speed is 50 rpm; And/or, in the step 2, the hydration can be as long as the solution is uniform, 2-4 hours; And/or, in the solution 1 of step 3, the high-pressure homogenization is to use a 0-10°C refrigerated water cooling cycle in a homogenizer; and ensure that the temperature of the liposome solution is 5-10 degrees; And/or, in the solution 1 of step 3, the pressure of the high-pressure homogenization is 1200 bar; And/or, in the solution 2 of step 3, the shearing speed is 2000 rpm; And/or, in the solution 2 of step 3, the extrusion temperature is 40°C. 11. The preparation method of ginsenoside docetaxel liposomes as claimed in claim 10, characterized in that, in the step 1, the organic solvent is ethanol: chloroform with a volume ratio of 1:1. Mixed solvents; And/or, adding the ginsenoside and the phospholipid to the solution of docetaxel and the organic solvent, and heating and dissolving it; the heating is to heat the water bath to 35-65°C; And/or, in the step 1, the concentration is concentration under reduced pressure; the concentration under reduced pressure is vacuum=-0.089~-0.1MPa. 12. The preparation method of ginsenoside docetaxel liposomes as claimed in claim 11, characterized in that in the step 1, the concentration is until all solvents are completely volatilized; the total concentration time is as low as in 4 hours. 13. A method for preparing ginsenoside docetaxel liposome for injection, which includes the following steps; Step 1-1. The liposome solution A3a or A3b obtained in the preparation method of ginsenoside docetaxel liposomes according to any one of claims 7-12; Step 2-1. Sterilize and filter the liposome solution A3a or A3b to obtain liposome solution A4; Step 3-1: freeze-dry the liposome solution A4 to obtain ginsenoside docetaxel liposome for injection. 14. The preparation method of ginsenoside docetaxel liposome for injection according to claim 13, characterized in that, In the step 2-1, the sterilization filtration uses a 0.22 μm filter membrane; And/or, in the step 3-1, the freeze-drying includes in sequence: pre-freezing, primary drying, and secondary drying; specifically, the following steps are included: step a, quantitatively dispensing the lipids packed in the vials Plastid solution A4 is placed in the freeze-drying box, where the temperature of the freeze-dried shelf is lowered to -10±1°C at a constant speed and kept for 1 hour, and then the shelf temperature is raised to -13±1°C, kept for 1 hour, and then The shelf temperature continues to drop to -55±1℃. After the product temperature reaches -45±1℃, start the timer and continue to keep warm for 3 hours; Step b. After the insulation in step a is completed, quickly lower the condenser temperature to below -50±1℃, evacuate to below 10pa, raise the temperature of the shelf to -25±1℃ for 1.5 hours, and then start timing the insulation for 18 After an hour, quickly raise the shelf temperature to -15±1℃, keep it warm until the ice crystals of the product completely disappear, and then continue to keep it warm for 4 hours; Step c. After the heat preservation in step b is completed, quickly raise the shelf temperature to 15±1°C within 1 hour, keep it warm for 3 hours, then raise the shelf temperature to 30±1°C, and raise the temperature of the finished product to 25±1°C. At ℃, the heat preservation period is over after 12 hours; And/or, step 3-1 further includes post-processing, and the post-processing includes the following steps: after the heat preservation in step c is completed, fully press the plug, take it out of the box, and roll the cap. 15. A compound ginsenoside docetaxel liposome for injection, which is prepared by the preparation method of ginsenoside docetaxel liposome for injection according to claim 13 or 14. 16. A ginsenoside docetaxel liposome as described in any one of claims 1-4, or a blank liposome as claimed in claim 5, or an injection as claimed in claim 15 Application of compound ginsenoside docetaxel liposomes in the preparation of drugs for the treatment and/or prevention of cancer. 17. The application according to claim 16, wherein the cancer is one or more of breast cancer, ovarian cancer, lung cancer, gastric cancer, head and neck cancer and esophageal cancer.