CN114712309A

CN114712309A - Ginsenoside docetaxel liposome, preparation method and application thereof

Info

Publication number: CN114712309A
Application number: CN202011534465.7A
Authority: CN
Inventors: 王建新; 王丹; 陈颖江; 詹华杏
Original assignee: Shanghai Shensu Pharmaceutical Technology Co ltd
Current assignee: Shanghai Shensu Pharmaceutical Technology Co ltd
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2022-07-08
Anticipated expiration: 2040-12-22
Also published as: CN114712309B

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

Ginsenoside docetaxel liposome, preparation method and application thereof

Technical Field

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

Background

The liposome is a directional medicine-carrying system, belonging to a special dosage form of targeted medicine-feeding system, it can embed the medicine in the nano-grade particle whose diameter is similar to bilayer micro-vesicle in biological membrane structure, and can be fed into human body, and mainly phagocytized by reticuloendothelial system, and can change the in-vivo distribution of the encapsulated medicine, so that the medicine can be mainly accumulated in the targeted tissue, therefore the therapeutic index of medicine can be raised, therapeutic dose of medicine can be reduced and toxicity of medicine can be reduced.

The invention is a technical innovation based on the invention patent of Chinese countries such as CN201610693884.2, CN201811447245.3 and CN 201811447243.4. The three application patents all disclose the technical advantages that after the liposome taking ginsenoside as the membrane material is used for encapsulating paclitaxel and other therapeutic drugs, the related liposome has stable quality, obvious drug effect and the like.

CN201610693884.2 discloses a blank liposome taking ginsenoside Rg5 and derivatives thereof as membrane materials and application thereof, wherein the prescription not only comprises phospholipid, saponin, a medicament and a freeze-drying protective agent, but also can further comprise cholesterol, an antioxidant, soybean oil and/or sodium oleate and other auxiliary materials.

CN201811447245.3 discloses a blank liposome taking ginsenoside Rh5H and derivatives thereof as membrane materials and application thereof, and the patent further solves the hemolytic problem of ginsenoside on the basis of CN 201610693884.2. Similarly, the prescription can further comprise cholesterol, antioxidant, soybean oil and/or sodium oleate and other auxiliary materials besides phospholipid, saponin, medicament and freeze-drying protective agent.

CN201811447243.4 discloses a blank liposome taking ginsenoside Rg3 and derivatives thereof as membrane materials and application thereof. In the patent, the solubility problem of ginsenoside in chloroform is solved by ultrafine powder and other technical means of the saponins such as Rg3, Rh2 and the like, so that the problem that the saponins such as Rg3, Rh2 and the like need to form a film in chloroform is solved, and the Rg3 liposome with the quality meeting the standard is prepared.

The prior art still has some defects, such as large pressure required in the liposome production homogenization step, low speed of sterilization and filtration of a filter membrane, high retention rate and obviously poor product yield in part of schemes; 2-6 times of soybean oil is required to be added. However, the addition of soybean oil is not beneficial to freeze-drying of the preparation, and influences the long-term storage of the medicine.

The core of the compound preparation is the synergistic interaction of the medicines in vivo, and the clinical treatment effect of the medicines can be obviously improved. The reasonable proportion range of each functional component of the compound preparation is the core of the compound preparation, and particularly, the changes of medicine synergy, in-vivo medicine substitution, in-vivo tissue distribution, medicine effect and the like caused by the change of the functional components of the compound liposome are rarely related. Therefore, aiming at the compound ginsenoside docetaxel liposome (hereinafter referred to as Ginposome-DTX or Ginposome-CTX) for injection, the method determines how to select the most appropriate 'synergistic medicament and auxiliary material' ginsenoside and key auxiliary material 'phospholipid' and the related proportion thereof to prepare the composition with reasonable compatibility, small particle size, stable quality and optimal effect on medicament effect and toxicity, so that the composition with the specific proportion of the medicament and the key auxiliary material has innovation and uniqueness and has very important significance.

In the prescription screening, the change of any one factor in a plurality of factors such as medicines, phospholipid, saponin, a freeze-drying protective agent, a preparation process and the like can have fatal influence on the quality, the efficacy and the safety of the product. For example, the mass ratio of the saponin to the paclitaxel is increased, the quality stability and the synergistic anti-tumor effect of the compound liposome can be increased, the targeting distribution to tumor tissues is increased, but the accumulated toxicity of the saponin in a human body is also increased, and uncontrollable organ damage is caused; the appropriate mass ratio of the saponin to the paclitaxel has very important relevance to the stability, active targeting property, pharmacodynamics and safety of the liposome. Meanwhile, different freeze-drying protective agents are selected, so that the freeze-drying protective agent plays a vital role in preventing the lipid bilayer structure from being damaged in the liposome freeze-drying process and recovering the characteristics of the liposome after the freeze-drying medicament is redissolved. For example, in the selection of the lyoprotectant, different lyoprotectants have different effects on the lyophilization curve, and especially have important effects on the co-dissolution point of the compound liposome, whether the compound liposome is collapsed, whether the liposome is significantly changed after redissolving, the setting of the primary lyophilization temperature and time, the total lyophilization time, and the like.

The safety and the effectiveness of the medicine are two basic attributes of the medicine, one of the two basic attributes is not available, the approval and the use of the medicine are considered based on the risk-benefit ratio between the medicine and the medicine, and particularly, the core of the improved new medicine is to improve the effectiveness and the safety.

In toxicology research, pharmaceutics research plays an important role, and especially the influence of the selection of the prescription proportion and the preparation process on acute toxicity, long-term toxicity and accumulated toxicity of each functional organ directly determines whether the compound liposome meets the requirement of new drug declaration.

Therefore, the optimal proportion range of the phospholipid, the ginsenoside, the docetaxel and the carbohydrate component of the freeze-drying protective agent in the membrane components of the liposome prescription plays an important role in the good properties of the compound liposome, such as good pharmaceutical stability, in-vivo distribution, pharmacodynamics, toxicology and the like. However, this optimum ratio, the prior art does not provide any of the above components and ratios and any inferential relationship between process and pharmacological activity, pharmacokinetics and toxicology. Due to the large number of variables involved, screening must be done through a large number of experiments and creative efforts.

Therefore, how to select an optimal compound medicine compatibility and how to formulate an optimal preparation process are to produce the compound ginsenoside docetaxel liposome for injection with better drug effect, lower toxicity and quality and other indexes meeting the requirements of the medicine so as to meet the requirements of medicine declaration, and a great deal of research work and technical clearance are needed.

Disclosure of Invention

The invention aims to solve the technical problem of providing a (compound) ginsenoside docetaxel liposome, a preparation method and application thereof aiming at the defects of the existing docetaxel liposome; the composition has the advantages of stable property, small particle size, high drug encapsulation rate, good in vivo compatibility, good in vivo drug release, better drug effect, lower toxicity and reasonable compatibility; the preparation method has a good preparation process, the preparation conditions are easy to realize, and industrialization is facilitated; the optimization of the combination of the preparation process and the product performance is realized.

The invention solves the technical problems through the following technical scheme.

The invention provides a (compound) ginsenoside docetaxel liposome (named as Ginposome-DTX or Ginposome-CTX for short), 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 15-35 parts of freeze-drying protective agent; the ginsenoside docetaxel plastid does not contain one or more of cholesterol, soybean oil and sodium oleate.

In a certain aspect of the present invention, the ginsenoside docetaxel liposome is composed of the following components by mass: 8-18 parts of phospholipid, 1-1.5 parts of ginsenoside, 1 part of docetaxel and 15-35 parts of freeze-drying protective agent.

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

In one embodiment of the invention, the mass ratio of said docetaxel to said phospholipid may be 1:8 to 12, for example 1: 10; for example, the mass ratio of the docetaxel to the egg yolk lecithin is 1: 10.

In one embodiment of the present invention, the ginsenoside 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, more preferably 20(S) -ginsenoside Rg 3.

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

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

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

In one embodiment of the present invention, the mass ratio of the lyoprotectant to the docetaxel may be 20-35: 1; for example 25-35:1, further for example 25:1, 30:1 or 35: 1. For example, the mass ratio of the glucose to the docetaxel is 25: 1.

In a certain scheme of the invention, the particle size D90 of the ginsenoside docetaxel liposome is less than or equal to 150nm, and the entrapment rate is more than or equal to 98%.

In a certain scheme of the invention, the HPLC purity of the ginsenoside is more than or equal to 99%.

In a certain embodiment of the present invention, the ginsenoside docetaxel liposome comprises the following components by mass: 10 parts of phospholipid, 1 part or 1.5 parts of ginsenoside, 1 part of docetaxel and 25 parts of freeze-drying protective agent.

In a certain embodiment of the present invention, the ginsenoside docetaxel liposome may include the following components in percentage by mass:

10 parts of phospholipid, 1 part of 20(S) -ginsenoside Rg3, 1 part of docetaxel and 25 parts of freeze-drying protective agent;

or 10 parts of phospholipid, 1.5 parts of 20(S) -ginsenoside Rh2, 1 part of docetaxel and 25 parts of freeze-drying protective agent;

or 10 parts of phospholipid, 1 part of 20(S) -ginsenoside Rg3, 1 part of docetaxel and 30 parts of freeze-drying protective agent;

or 10 parts of phospholipid, 1.5 parts of 20(S) -ginsenoside Rh2, 1 part of docetaxel and 30 parts of freeze-drying protective agent.

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

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 invention also provides a blank liposome which comprises 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 liposomes do not contain one or more of cholesterol, soybean oil, sodium oleate.

In one embodiment of the present invention, the blank liposome may be loaded with a pharmaceutically active substance, preferably, the pharmaceutically active substance may be a taxane derivative; such as paclitaxel and/or docetaxel; and docetaxel, for example.

In a certain embodiment 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 one or more of egg yolk lecithin, soybean phospholipid, hydrogenated phospholipid and cephalin containing 0.01-10% distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000.

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

In one embodiment of the present invention, in the blank liposome, the ginsenoside 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, more preferably 20(S) -ginsenoside Rg 3.

In one embodiment of the present invention, the blank liposome may include 1 part, 1.5 parts or 2 parts of ginsenoside by mass.

In a certain embodiment 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 one embodiment of the present invention, the lyoprotectant may be 25-35 parts, such as 25 parts, 30 parts or 35 parts, of the blank liposome.

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

or 10 parts of soybean phospholipid, 2 parts of Rh2 and 20-35 parts of a freeze-drying protective agent;

or, 10 parts of egg yolk lecithin: 1.5 parts of pseudo Rg3 and 20-35 parts of a freeze-drying protective agent;

or, 10 parts of egg yolk lecithin: 1.5 parts of pseudo GQ and 20-35 parts of freeze-drying protective agent;

or, 10 parts of egg yolk lecithin: 1.5 parts of Rk1 and 20-35 parts of a freeze-drying protective agent;

or, 10 parts of egg yolk lecithin: 1.5 parts of Rp1 and 20-35 parts of a freeze-drying protective agent.

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

step 1, concentrating solution A1 of docetaxel, ginsenoside, phospholipid and 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 (high pressure homogenization) comprises the following steps:

and (3) homogenizing the liposome solution A2 obtained in the step (2) under high pressure, and controlling the particle size D90 to be less than 100nm to obtain a liposome solution A3 a.

Scheme 2 (high shear + extrusion process) comprises the following steps:

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;

wherein docetaxel, ginsenoside, phospholipid and lyoprotectant solution are as defined above for the (co-) 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, and preferably a mixed solvent of methanol and/or ethanol and chloroform and/or dichloromethane; for example, ethanol: 1-chloroform: 1 (volume ratio). The amount of the organic solvent is not particularly limited, and the organic solvent can be used for dissolving docetaxel, ginsenoside and phospholipid. For example, the mass-to-volume ratio of docetaxel to the organic solvent is 1g/60 to 120mL, e.g., 1g/80 mL.

In one embodiment of the present invention, in the step 1, the solution a1 is preferably prepared by dissolving docetaxel, ginsenoside, phospholipid, etc. in an organic solvent under heating; 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 in a water bath to 35-65 deg.C, for example 55 deg.C.

In one embodiment of the present invention, in the step 1, the concentration may be a concentration under reduced pressure; 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.

In one embodiment of the present invention, in the step 1, the concentration may be performed in a rotary evaporator at a rotation speed of 40-60 rp/min, for example, 50 rp/min.

In one embodiment of the present invention, in the 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 0.20-0.35mg/mL, such as 0.25 mg/mL.

In one embodiment of the present invention, in step 2, the hydration temperature may be 35-65 ℃, preferably 40-45 ℃.

In one embodiment of the invention, in the step 2, the hydration is performed in a rotary evaporation bottle at a rotation speed of 40-60 rp/min, for example 50 rp/min.

In one embodiment of the present invention, in the step 2, the hydration may be performed in a homogeneous solution, for example, for 2 to 4 hours.

In a certain embodiment of the present invention, in the step 2, the ratio of docetaxel: lyophilized protectant solution ═ 1 g: 100 mL.

In one embodiment of the present invention, in step 2, the volume of the lyoprotectant solution is the same as the volume of water.

In a certain embodiment of the present invention, in the embodiment 1 of the step 3, the high-pressure homogenization is a cooling and cutting cycle in a homogenizer using chilled water at a temperature of 0 to 10 ℃; preferably, the temperature of the liposome solution is ensured at 5-10 ℃.

In one embodiment of the present invention, in the embodiment 1 of step 3, the pressure for the high-pressure homogenization is between 800 and 1400bar, such as 1200 bar.

In one embodiment of the present invention, in the embodiment 1 of step 3, the number of times of the high pressure homogenization may be 3 to 4, for example, 4.

In one embodiment of the present invention, in the embodiment 2 of the step 3, the shearing may be performed at room temperature.

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

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

In one embodiment of the present invention, in the embodiment 2 of the step 3, the temperature of the extrusion is 35 to 45 ℃, for example, 40 ℃.

In one embodiment of the present invention, in the embodiment 2 of the step 3, the pore diameter of the extrusion plate is 150 nm.

In a certain embodiment of the present invention, in embodiment 2 of the step 3, the extrusion pressure is 600 to 800 psi; such as 800 psi.

In one embodiment of the present invention, in the embodiment 2 of the step 3, the number of times of the extrusion may be 3 to 4, for example, 4.

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

step 1-1, step 1-3 of the above-described method for preparing ginsenoside docetaxel liposomes, obtaining a liposome solution A3a or A3 b;

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

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

In the preparation method, the conditions and operations of sterile filtration and freeze drying can be the conditions and operations conventional in the processes in the field; in the present invention, the following are preferred:

in one embodiment of the present invention, in the step 2-1, a 0.22 μm filter membrane may be used for the sterile filtration.

In one embodiment of the present invention, in step 3-1, the freeze-drying may be performed in a vial, which may be a vial conventional in the art, such as a 30mL or 50mL vial.

In one embodiment of the present invention, in the step 3-1, the freeze-drying may sequentially comprise: 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 a shelf (about 1.5 hours) to below minus 25 +/-1 ℃, starting timing and preserving heat for 18 hours, quickly raising the temperature of the shelf to below minus 15 +/-1 ℃, preserving heat until ice crystals of the product completely disappear, and continuing preserving heat for 4 hours;

and c, after the heat preservation in the step b is finished, quickly 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 ℃.

In one embodiment of the present invention, step 3-1 may be followed by further post-treatment, and the conditions and operations of the post-treatment may be those conventional in the art; for example, 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.

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

In a certain scheme of the invention, the particle size D90 of the ginsenoside docetaxel liposome for injection is less than or equal to 150nm, and the entrapment rate is more than or equal to 98%. In one scheme of the invention, the purity of the ginsenoside is more than or equal to 99%.

The invention also provides an application of the ginsenoside docetaxel liposome, the blank liposome or the compound ginsenoside docetaxel liposome for injection in preparing a medicament for treating and/or preventing 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 corresponding to 90% of the cumulative percent particle size distribution for a sample. Its physical meaning is that the particles have a size less than 90% of its particle size.

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

the above preferred conditions may be combined arbitrarily to obtain preferred embodiments of the present invention without departing from the general knowledge in the art.

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

The positive progress effects of the invention are as follows: the compound ginsenoside docetaxel liposome provided by the invention has targeting effect on tumor cells, multi-drug resistance, synergy and attenuation and drug synergism. Taking the compound ginsenoside Rg3 docetaxel liposome for injection in the embodiment as an example, the drug effect is obviously superior to the technical scheme which is not in the protection range of the invention; the Rg3 is proved to have better effects of' medicines, auxiliary materials, membrane materials, targeting and the like in the compound ginsenoside Rg3 docetaxel liposome for injection, and have good medicine synergistic effect. Specifically, the method comprises the following steps:

(1) the drug effect is obviously improved. Particularly, the DTX-Rg3(1.0)/Lp group and the DTX-Rh2(1.5)/Lp group have optimal drug effects, wherein the tumor inhibition rate (68%) of the dose (10mg/kg) reaches or exceeds the tumor inhibition rate of the liposome group without the prescription of the invention with high dose (20mg/kg), so the tumor inhibition effect is improved by 2 times;

(2) glut1 significantly improved targeting. In a Glut1 targeting experiment of a tumor-bearing mouse, the Glut1 targeting of the ginsenoside liposome is improved by more than 4 times compared with the targeting of a common cholesterol liposome, while the Glut1 targeting of a common non-preferred ginsenoside liposome is improved by less than 2 times compared with the targeting of the common cholesterol liposome.

(3) The toxic and side effects are obviously reduced. According to the liposome prepared by the prescription, the acute toxicity (LD50 is more than or equal to 150mg/kg) of DTX-Rg3(1.0)/Lp and the acute toxicity (LD50 is more than or equal to 150mg/kg) of DTX-Rh2(1.5)/Lp (LD50 is more than or equal to 150mg/kg) are reduced by more than 6 times than that of a Taxotere group (LD50 is about 25mg/kg), the acute toxicity (LD) is reduced by 3-4 times than that of a common cholesterol docetaxel liposome (LD50 is between 25 and 50mg/kg), and the acute toxicity (LD50 is about 1.5 to 2 times than that of a non-prescription ginsenoside liposome (LD50 is about 50 to 100 mg/kg) of DTX-C-Rg3(1.8)/Lp, DTX-C-Rh2(2.0)/Lp and the like). In long-term toxicity experiments of SD rats, animal death occurs in the Taxotere group, which indicates that the toxicity effect of the Taxotere group is large. The toxicity of the ginsenoside Rg3 docetaxel liposome or the ginsenoside Rh2 docetaxel liposome is reduced by more than 4-6 times compared with that of Taxotere.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.

Experimental drugs and devices

Experimental drugs: the 20(S) -ginsenoside Rg3 (Rg 3 for short), ginsenoside Rg3 (Rg 3 for short), ginsenoside Rp1 (Rp 1 for short), ginsenoside GQ (GQ for short), ginsenoside Rk1 (Rk 1 for short), ginsenoside Rg5 (Rg 5 for short), 20(S) -ginsenoside Rh2 (Rh 2 for short), ginsenoside Rk2 (Rk 2 for short), 20(S) -ginsenoside Rg2 (Rg 2 for short), 20(S) -ginsenoside Rh1 (Rh 1 for short), 20(S) -protopanaxadiol (PPD for short), 20(S) -protopanaxatriol (PPT for short) and the like are commonly available in the art, for example, Shanghai Ben Zhi Sichuan Kangjin biosen, Shanghai bioscience, and the like.

Docetaxel injection: taxotere, manufacturer: aventis, UK, purchased from Shanghai Rad pharmacy.

The ginsenoside has the following molecular structural formula:

the test instrument: the instruments used in the following examples are owned instruments of Shanghai Benghai pharmaceutical science and technology Limited, the college of pharmacy of the university of Fudan, the model numbers and source information of which are as follows:

agilent liquid chromatography: agilent 1100, Autai 3300ELSD, Agilent technologies (China) Inc.;

evaporate the evaporimeter soon: ZX 98-15L, Shanghai Luyi Industrial and trade, Inc.;

ultrasonic cleaning machine (SB3200DT, Ningbo Xinzhi Biotech Co., Ltd.);

nitrogen blowing instrument (HGC-12A, constant Olympic technology development Co., Ltd., Tianjin);

probe ultrasound apparatus (JYD-650, Shanghai wisdom communication instruments, Inc., China);

high pressure homogenisers (B15, AVESTIN, canada);

micro-extruders (Mini-extruder, Avanti Polar Lipids Inc);

laser particle size analyzer (Nano ZS, marvin, uk);

malvern Nanosizer ZS90 (Malvern, uk);

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

enzyme-labeling instruments (Infinitie 200, Tecan tracing co., switzerland, Ltd);

flow cytometry (BD Biosciences, USA);

flow cytometry (CytoFlex S, Beckman Coulter, inc., USA);

inverted fluorescence microscopy (Leica, DMI 4000D, Germany);

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

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

confocal In Vivo Microscopy (IVM);

an upright two-photon microscope (DM 5500Q; Nikon);

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

biomacromolecule interaction instrument BiaCore T200 instrument (GE, USA);

clean bench (SW-CJ-1FD, air technologies, Inc., Antai, Suzhou);

20L rotary evaporator: R5002K, shanghai xiafeng industries ltd;

a freeze dryer: FD-1D-80, Shanghai Bilang instruments manufacturing Co., Ltd;

a freeze dryer: PDFD GLZ-1B, Pudong Freeze drying Equipment, Inc., Shanghai;

an electronic balance: CPA2250 (precision 0.00001g), sartorius trade ltd;

an electronic balance: JY3003 (precision 0.001g), Shanghai Shunhui constant-level scientific instruments, Inc.;

photoelectric microscopes (XDS-1B, Chongqing photoelectric Instrument Co., Ltd.);

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

Animals and cell lines

Animals: BALB/c nude mice, age of mice 3-4 weeks, produced by Shanghai pharmaceutical research institute of Chinese academy of sciences.

Tumor cell lines:

breast carcinoma orthotopic tumor 4T1 cell line, provided by the college of medicine of Zaudan university

Breast cancer MCF-7 cell line, available from the college of medicine of the university of regadenoson;

triple negative breast cancer MDA-MB-231 cell line provided by the college of medicine of Zadan university;

example 1 preparation of Compound ginsenoside Rg3 docetaxel liposome for injection

1. Prescription: 10g of yolk lecithin, 1g of docetaxel, 25g of glucose, 40ml of absolute ethanol, 40ml of chloroform and 200ml of water for injection, and ginsenoside Rg 31 g.

2. Film forming: preparing a mixed solvent of absolute ethyl alcohol and chloroform (1:1) according to the prescription amount for standby.

Adding docetaxel in a prescription amount into a mixed solvent for dissolving for later use, adding ginsenoside Rg3 and egg yolk lecithin in the prescription amount into the mixed solvent, heating and dissolving, transferring into a 1L rotary evaporation bottle, concentrating under reduced pressure, carrying out rotary evaporation at a water bath temperature of 55 ℃, a rotating speed of 50 r/min and a vacuum degree of-0.089-0.1 MPa until the solvent is completely volatilized.

3. Hydration: preparing a glucose solution: adding 25g anhydrous glucose into 100ml water for injection, stirring to dissolve, preparing into 0.25mg/ml glucose water solution, and heating in water bath at 40 deg.C for use.

Adding 100ml of water for injection into the rotary evaporation bottle after film formation, and hydrating and completely dissolving for about 2 hours at the water bath temperature of 40-45 ℃ and the rotating speed of 50 r/min.

Then 100ml of glucose aqueous solution is added, and the mixture is stirred evenly for standby.

4. High-pressure homogenization: transferring the hydrated solution to a homogenizer, using 0-10 ℃ chilled water for cold cutting circulation by the homogenizer, setting the homogenizing pressure at 1200bar, and carrying out circulation homogenization for 3-4 times until D90 is less than 100 nm.

5. And (3) degerming and filtering: the homogenized solution was sterile filtered through a 0.22 μm filter.

6. Subpackaging: and subpackaging the sterilized and filtered solution into 30 or 50ml penicillin bottles according to the set amount of 8-10 ml.

7. Pre-freezing: after the product is put into the box, the temperature of the shelf is reduced to about minus 10 ℃ at a constant speed, the temperature is preserved for 1 hour, the temperature of the shelf is increased to about minus 55 ℃, the temperature is preserved for 1 hour, the temperature preservation is finished, the temperature of the shelf is reduced to about minus 55 ℃, and the time is started to keep the temperature for about 3 hours after the temperature of the product reaches minus 45 ℃.

8. Primary drying: rapidly cooling the condenser to below-50 ℃, vacuumizing to below 10pa, raising the temperature of a shelf (about 1.5 hours) to-25 +/-1 ℃, starting timing and preserving heat for about 18 hours, rapidly raising the temperature of the shelf to-15 +/-1 ℃, preserving heat until ice crystals of the product completely disappear, and continuing to preserve heat for about 4 hours;

9. and (3) secondary drying: the temperature of the shelf is rapidly increased to about 15 ℃ within 1 hour, the shelf is kept warm for about 3 hours, then the temperature of the shelf is increased to about 25 ℃, and the shelf is kept warm for about 12 hours when the temperature of the product is increased to 25 ℃. And (5) after the heat preservation is finished, checking the vacuum degree condition, finishing the whole freeze-drying process, fully pressing the plug, and discharging from the box.

10. And (3) rolling and packaging: and (3) rolling and packaging the liposome to obtain the compound ginsenoside Rg3 docetaxel liposome for injection (formula 1).

Example 2 preparation of Compound ginsenoside Rg3 docetaxel liposome for injection

The prescription amount of the ginsenoside Rg3 in the example 1 is increased to 1.5g, and the compound ginsenoside Rg3 docetaxel liposome for injection is prepared in the same way as the example 1.

Example 3 preparation of Compound ginsenoside Rg3 docetaxel liposome for injection

1. Prescription: 10g of yolk lecithin, 31.5 g of ginsenoside Rg, 1g of docetaxel, 25g of glucose, 40ml of absolute ethyl alcohol, 40ml of chloroform and 200ml of water for injection.

2. Film forming: the same film formation method as in example 1.

3. Hydration: the same hydration method as in example 1.

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

Controlling the temperature of the liposome solution at 35-45 ℃, connecting an extrusion device, installing an extrusion plate with a 150nm aperture, and extruding under 800psi pressure.

5. The subsequent steps were the same as those of example 1.

EXAMPLE 4 preparation of Compound ginsenoside Rh2 docetaxel liposomes for injection

The ginsenoside Rg3 in example 1 was changed to ginsenoside Rh21.0 g, and the same preparation as in example 1 was carried out to obtain compound ginsenoside Rh2 docetaxel liposome for injection.

EXAMPLE 5 preparation of Compound ginsenoside Rh2 docetaxel liposomes for injection

Compound ginsenoside Rh2 docetaxel liposome for injection is prepared by changing ginsenoside Rg3 in example 1 to ginsenoside rh21.5g and the same as example 1.

Example 6 preparation of injectable Compound ginsenoside Rg3 docetaxel liposomes

The ginsenoside Rg3 in example 1 is changed into ginsenoside pseudo Rg 31.5 g, and the preparation method is otherwise the same as example 1 to prepare the compound ginsenoside pseudo Rg3 docetaxel liposome for injection.

Example 7 preparation of Compound ginsenoside Rg5 docetaxel liposomes for injection

The ginsenoside Rg3 in example 1 is changed into ginsenoside Rg 51.5 g, and the preparation method is otherwise the same as example 1 to prepare the compound ginsenoside Rg5 docetaxel liposome for injection.

EXAMPLE 8 preparation of Compound ginsenoside Rk1 docetaxel liposomes for injection

Compound ginsenoside Rk1 docetaxel liposome for injection is prepared by changing ginsenoside Rg3 in example 1 to ginsenoside rk11.5 g and the same as example 1.

EXAMPLE 9 preparation of Compound ginsenoside Rp1 docetaxel liposomes for injection

The ginsenoside Rg3 in example 1 is changed into ginsenoside Rp 11.5 g, and the preparation method is the same as that in example 1 to prepare the compound ginsenoside Rp1 docetaxel liposome for injection.

EXAMPLE 10 preparation of Compound ginsenoside pseudo GQ docetaxel liposomes for injection

The ginsenoside Rg3 in example 1 is changed to ginsenoside pseudo GQ 1.5g, and the preparation method is the same as that in example 1 to prepare the compound ginsenoside pseudo GQ docetaxel liposome for injection.

Effect example 1

(a) The following table shows the results of comparing the types of ginsenosides according to the following formula in the same manner as in example 1 (without the lyophilization step):

the series of experiments prove that the ginsenoside in the invention is as follows under the condition of not adding soybean oil or cholesterol and other auxiliary materials: when 7 Rg3, pseudo Rg3, Rh2, pseudo GQ, Rg5, Rk1, Rp1 and the like exist, the Ginposome-DTX or CTX has a better preparation process, the preparation conditions are easy to realize, and the industrialization is facilitated. When the ginsenoside is Rk2, Rg2, Rh1, PPD and PPT, other auxiliary materials are required to be added, and the preparation conditions are harsh.

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

remarking: egg yolk lecithin (EPC), soybean lecithin (SPC), cephalin (PE), Sphingomyelin (SM), hydrogenated phospholipid (HSPC), Phosphatidylserine (PS), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl lecithin (DOPC), distearoyl phosphatidylcholine (DSPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), dimyristoyl phosphatidylcholine (DMPC), polyethylene glycol 2000-distearoyl phosphatidylethanolamine (mPEG-DSPE), polyethylene glycol 2000-dioleoyl phosphatidylethanolamine (mPEG-DOPE).

Following the same procedure as in example 1 (without the need for the lyophilization step), the results of comparison of the phospholipid types and ratios are given in the following table:

the above experiment proves that docetaxel can be well wrapped without other auxiliary materials such as soybean oil or cholesterol, and the phospholipids which are easy to realize in the preparation process are egg yolk lecithin, soybean phospholipid, hydrogenated phospholipid, cephalin and mixed phospholipid (the 4 phospholipids contain 0.01-10% mPEG-DSPE). Other phospholipids and Rg3 can also wrap docetaxel well, but other auxiliary materials such as soybean oil and the like need to be added, or the homogenization pressure and the homogenization times are increased. The above experiments also illustrate that: in terms of film-forming properties, the phospholipid of the present invention: docetaxel 8-18: 1, the effect is better, and the ratio of the effective amount to the effective amount is preferably 8-12: 1.

(c) the optimum ratio of ginsenosides was compared in the same way as in example 1 (without the lyophilization step):

the experiments prove that under the condition of not adding other auxiliary materials such as soybean oil or cholesterol, the saponin of the invention: docetaxel 1-3: 1, the effect is better. However, as a result of the pharmacokinetic study of application example 4, the present invention only selects the following saponins: docetaxel 1-1.5: 1.

(d) The lyoprotectants were compared in the same manner as in example 1:

in the selection of the freeze-drying protective agent, different freeze-drying protective agents have obvious influence on the entrapment rate and the particle size distribution of the liposome after the product is redissolved, and have great influence on the economy of a freeze-drying curve. Through the experiment, under the condition that other auxiliary materials such as soybean oil and the like are not added, the freeze-drying protective agent is one or more of glucose, trehalose, sucrose, lactose and galactose, and the ratio of the docetaxel to the freeze-drying protective agent is as follows: when the freeze-drying protective agent/docetaxel is 25-35 times, for example, 15 times of glucose, the redissolution particle size of Ginposome-DTX is 200nm, which is between qualified and unqualified, and when the freeze-drying protective agent/docetaxel is 25 or 35 times, the freeze-drying protective agent/docetaxel has good matching property with docetaxel liposome. Due to comprehensive consideration of empirical data of the formulation, the ratio of the docetaxel to the lyoprotectant determined by the invention is as follows: the freeze-drying protective agent/docetaxel is 25-35 times.

Application example 1: cellular uptake assay for Glut1

1) Purpose of the experiment: the Glut1 targeting mechanism was demonstrated by the addition of glucose inhibitors and the like; the ginsenoside types and proportions and phospholipid types and proportions of the invention are verified by Glut1 targeting.

2) The experimental method comprises the following steps: to compare the uptake of 4T1 to each experimental group, the uptake mechanism of the combination preparation was investigated, and 4T1 cells were used at 2X 10⁵The cell density of (2) was inoculated in 12-well plates, and for the experimental group + glucose, the experimental group + phlorizin, and the experimental group + quercetin, the culture medium was replaced with 20mM glucose solution, phlorizin solution, and quercetin solution, respectively, after 12 hours. The three solutes should be dissolved in a glucose-free medium, incubated for 1 hour, added with each experimental drug (concentration of ultraviolet fluorescent developer is 100ng/ml), incubated for 4 hours, digested, washed with fresh PBS solution, and analyzed by flow cytometry.

3) The preparation method of the experimental group comprises the following steps: prepared according to the method of the invention example 1 (docetaxel is changed to coumarin without the need for a lyophilization step)

Using the results of the toxin generation experiments of example 4, the following experiments did not develop experiments in which the ginsenoside/docetaxel was over the prescription of 2.0.

Experimental groups:

prescription content	For short
		Egg yolk lecithin: cholesterol: the fluorescent probe is 9: 1.8: 1	C6-C/Lp
Egg yolk lecithin: rg 3: a fluorescent probe: cholesterol 9: 1.8: 1: 2.25	C6-C-Rg3/Lp
		Egg yolk lecithin: rg 3: the fluorescent probe is 9: 1.8: 1	C6-Rg3/Lp
Soybean lecithin: rh 2: a fluorescent probe: cholesterol 10: 2: 1: 2.25	C6-C-Rh2/Lp
		Soybean lecithin: rh 2: fluorescent probe 10: 2: 1	C6-Rh2/Lp
Soybean lecithin: rg 5: a fluorescent probe: soybean oil 4: 3: 1: 2	C6-Rg5/Lp
		Egg yolk lecithin: pseudo Rg 3: 10 for fluorescent probe: 1.5: 1	C6-pseudo Rg3(1.5)/Lp
Egg yolk lecithin: a pseudo GQ: fluorescent probe 10: 1.5: 1	C6-pseudo GQ (1.5)/Lp
		Egg yolk lecithin: rk 1:10 for fluorescent probe: 1.5: 1	C6-Rk1(1.5)/Lp
Egg yolk lecithin: rp 1: fluorescent probe 10: 1.5: 1	C6-Rp1(1.5)/Lp

The experimental results are as follows:

and (4) experimental conclusion:

1) under the same prescription, the Glut1 targeting property is greatly reduced after cholesterol is added.

2) The targeting property of the ginsenoside liposome with the non-optimal prescription is improved by more than 4 times relative to the cholesterol liposome, and the targeting property of the ginsenoside liposome with the non-optimal prescription is improved by about 2 times relative to the cholesterol liposome.

Application example 2: in vivo pharmacodynamic study of Breast cancer (MCF-7)

1) The test method comprises the following steps: injecting the tumor cell strain (MCF-7) into the subcutaneous part of the mouse to establish a subcutaneous tumor model. When the tumor volume reaches 100mm³(7 d post-inoculation), mice were randomized into groups (n-8 per group) and each group was injected tail vein with vehicle (5% glucose, Blank), DTX-C/Lp, DTX-Rg3(0.5)/Lp, DTX-Rg3(1.0)/Lp, DTX-Rg3(1.5)/Lp, DTX-Rg3(2.0)/Lp, DTX-Rh2(0.5)/Lp, DTX-Rh2(1.0)/Lp, DTX-Rh2(1.5)/Lp, DTX-Rh2(2.0)/Lp, doxycycline/Rg 3(1.8)/Lp (egg yolk lecithin: 3: docetaxel: 9: 1.8: 2.25: 1), DTX-C-Rh2 (2.0: Rh)/Lp (2.0: 2: soybean lecithin: 351: 1: 10: 1: 1.5: 1), the doses were given in the upper, middle and lower three groups (20mg, 10mg, 5mg) on docetaxel once every 7 days for up to day 28, with tumor length, width and recorded body weight being measured simultaneously with the administration. The formula for calculating the tumor volume (V) is that V ═ W²X L)/2. Length (L) is the longest diameter of a 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.

Remarking: docetaxel + Rg 3-20 mg/kg +30mg/kg, indicated drug concentration, as follows.

The toxicity results of application example 4 did not allow the study of the DTX-Rg3(2.0 or more)/Lp group.

2) The test results are as follows:

and (4) conclusion:

1) the DTX-Rg3(1.0)/Lp group and DTX-Rh2(1.5)/Lp group had the best drug effect, and the tumor completely disappeared on day 21. The tumor inhibition rate of the medium dose (docetaxel ═ 10mg/kg) in the two groups is basically consistent with that of the DTX-C-Rg3(1.8)/Lp group and the DTX-C-Rh2(2.0)/Lp group (docetaxel ═ 20 mg/kg). Namely: the tumor inhibiting effect is improved by more than about 2 times compared with the group without the invention.

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

3) The drug effect is not in linear relation with the ginsenoside ratio, and according to the prescription, when the ginsenoside Rg 3/docetaxel ratio is 1.0-1.5, the drug effect is optimal, preferably 1.0; the drug effect is best when the ratio of the ginsenoside Rh2 to the docetaxel is 1.0-2.0, and the optimal drug effect is 1.5.

Application example 3: acute toxicity (LD50) study (SD rats)

1) The experimental method comprises the following steps: 160-260 g of rats aged 6-9 weeks, 6 rats in each group, and the administration mode is as follows: slow rest push (about 1mL/min), dosing frequency: 3 times per day.

The docetaxel dose of the test sample is set to be 25, 50, 100 and 150 mg/kg/day, and the test sample contains Rg3 of 25, 50, 100 and 150 mg/kg/day respectively; rh2 was 37.5, 75, 150 and 225 mg/kg/day, respectively. And a solvent control group (5% glucose injection), a commercial positive control group (Taxotere group), an Rg3 liposome group and a DTX-C-Rg3/Lp group are arranged at the same time, and the slow static pushing (about 1mL/min) is carried out for 3 times/day, wherein the interval of each administration is at least 4 h.

2) Grouping experiments: the total 13 groups, 5% glucose group, DTX-C/Lp, DTX-C-Rg3(1.8)/Lp, and DTX-C-Rh2(2.0)/Lp were identical to the pharmacodynamic experimental groups, and the other group names are shown in the following table.

3) The preparation method of the experimental group comprises the following steps: prepared according to the method of example 1 according to the prescription requirements.

3) The results of the experiment are as follows:

remarking: the toxicity results of application example 4 did not allow the study of the DTX-Rg3(2.0 or more)/Lp group.

The experiments show that the technical scheme of the invention has excellent pharmaceutics and Glut1 targeting property, so that the toxicity reduction effect of the related preparation is optimal, and the toxicity is generally reduced by about 4-6 times compared with cholesterol liposome group (DTX-C/Lp) and docetaxel injection (Taxotere); 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 to 2 times.

Application example 4: pharmacokinetic (TK) study

1. Purpose of the experiment: the cumulative toxicity of each experimental group was studied.

2. The experimental method comprises the following steps: the TK group in the experiment is provided with 8 groups, namely a solvent control group (5% glucose injection), a commercial positive control group Taxotere group (10mg/kg), a test article 1(DTX-Rg3(1.0)/Lp group: 10mg/kg and 20mg/kg), a test article 2(DTX-Rh2(1.5)/Lp group: 10mg/kg and 20mg/kg), and a test article 3(DTX-Rh2(2.0)/Lp group, 10mg/kg and 20 mg/kg). Each group had 10 SD rats, half male and half female, for a total of 80. Intravenous injection, 1 time per week, continuous administration for four weeks, collecting whole blood before and 1hr after D1 and D29 administration for the solvent control group, collecting whole blood before and 3min, 15min, 30min, 1hr, 3hr, 6hr and 24hr after D1 and D29 administration for the test article 1, test article 2, test article 3 and the commercial positive control group, respectively, collecting whole blood into a test tube containing EDTA-K2 anticoagulant, placing on crushed ice, and centrifugally collecting plasma for analysis and detection.

The LC-MS/MS method is adopted to detect the concentrations of docetaxel, 20(S) -Rg3 and 20(S) -Rh2 in blood plasma, and the lower limit of the quantification of the docetaxel analysis method is as follows: 25.000ng/mL, and the lower limit of the quantitative determination of the analytical methods of Rg3 and Rh2 is 50.000 ng/mL. A non-compartmental model of WinNonlin software is adopted to analyze plasma concentration data, and parameters are calculated to research the pharmacokinetic characteristics of a test sample, so that reference is provided for clinical tests.

3. Experimental results, under the test conditions:

1) after D1 and D29 administration, the exposure (in AUC (0-t)) of ginsenoside Rg3 and Rh2 in blood plasma of male and female SD rats after intravenous injection of test article 1, test article 2 and test article 3 increases with the increase of the administration dosage, and the increase is higher than the increase of the dosage.

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

3) Following D1 and D29 administration, the exposure of docetaxel to plasma (in AUC (0-t) and Cmax) in male and female SD rats after i.v. administration of test article 1 increased with increasing dose, with a higher increase in dose.

4) After administration of D1 and D29, exposure (in Cmax) of ginsenoside Rg3 and Rh2 to plasma of male and female SD rats after intravenous injection of test article 2 is proportional to the dose administered.

5) Under the test condition, after continuous administration, the ginsenoside Rg3 and Rh2 in the blood plasma have no accumulation tendency after the male and female SD rats are administered with the test article 1 and the test article 2 by intravenous injection; after the male and female SD rats are intravenously administered with the test sample 1, the test sample 2 and the test sample 3, docetaxel in blood plasma basically has no accumulation tendency; after the male and female SD rats are subjected to intravenous injection to the test article 3, the exposure amount of docetaxel and ginsenoside Rh2 in blood plasma slightly accumulates; there was no accumulation of exposed amount of docetaxel in plasma of a commercial control group administered by intravenous injection to male and female SD rats.

And (4) conclusion: the DTX-Rg3(1.0)/Lp group had no risk of cumulative toxicity.

The DTX-Rh2(1.5)/Lp group had no risk of cumulative toxicity.

The DTX-Rh2(2.0)/Lp group was at a slight risk of cumulative toxicity.

Application example 5: long-term toxicity study in SD rats

1) The experimental method comprises the following steps: the dosage of the drug is as follows, wherein the dosage of the drug is 160-260 g of rats, 6-9 weeks old, 6 rats in each group: slow rest push (about 1mL/min), dosing frequency: 1/week (D1, D8, D15, D22, D29).

2) Grouping experiments: a5% glucose group (vehicle control group), a Taxotere group (positive control group), a Taxotere + Rg3/Lp group, a DTX-Rg3/Lp group and a DTX-Rh2/Lp group are arranged, and the total number of the groups is 5.

The dose of docetaxel in the test sample is set to be 5, 10 and 20mg/kg, and the mass ratio of docetaxel to Rg3 in the test sample is 1: 1.0, the mass ratio of docetaxel to Rh2 is 1:1.5, so the Rg3 content is respectively 5, 10 and 20mg/kg, and the Rh2 content is respectively 7.5, 15 and 30 mg/kg. (obtained by the preparation method of example 1)

The dose settings for each group were as follows:

3) the experimental results are as follows:

results 1: results of Long-term toxicity test in Taxotere group (examination results on day 30)

Dosage to be administered	5mg/kg	10mg/kg	20mg/kg
				Death rate of animals	Without death	Without death	33.3％
Mean body weight change in animals	+15％	+2％	-18％
				Condition of liver tissue damage	Without damage	Of moderate degree	Severe degree
Condition of renal tissue injury	Without damage	Of moderate degree	Severe degree
				Condition of spleen tissue damage	Without damage	Of moderate degree	Severe degree
Cardiac tissue damage condition	Without damage	Of moderate degree	Severe degree

Results 2: results of Long-term toxicity test in Taxotere group + Rg3/Lp group (examination on day 30)

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

Results 4: DTX-Rh2/Lp group Long-term toxicity test results (examination results on day 30)

The long-term toxicity test results of the rats show that:

1) rats in the Taxotere group died, lost weight severely, and each organ severely damaged, indicating high toxicity.

2) No rat death occurred in both the DTX-Rg3/Lp group and the DTX-Rh2/Lp group, the body weight was hardly reduced, and the organs were mainly slightly injured, indicating that the long-term toxicity was greatly reduced.

Application example 6: in vivo research of compound ginsenoside docetaxel liposome for injection on triple negative breast cancer (MDA-MB-231)

Animals: BALB/c nude mice, age 3-4 weeks, produced by Shanghai institute of pharmaceuticals.

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

Provided by the Shanghai pharmaceutical research institute of the Chinese academy of sciences.

Transplantation tumor model: inoculating the above cell strains under the right axillary skin of nude mice with the cell inoculation amount of 5 × 10⁶After the formation of the graft tumor, the tumor was used after 1 passage in nude mice.

The experimental method comprises the following steps: injecting the tumor cell strain into the subcutaneous part of a mouse to establish a subcutaneous tumor model. When the tumor volume reaches 100mm³At 7d post-inoculation, mice were randomized into 4 groups (n-8 groups) of treatment with tail vein injections of vehicle (5% glucose), Taxotere, DTX-Rg3/Lp (20mg/kg docetaxel, 20mg/kg ginsenoside Rg3), DTX-Rh2/Lp (20mg/kg docetaxel, 30mg/kg ginsenoside Rh2), administered once every 7 days for up to day 28 with simultaneous tumor length, width and weight recordings. The formula for calculating tumor volume (V) is: v ═ W2 × L)/2. Length (L) is the longest diameter of a 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 pharmacodynamic experimental method, the data of the in vivo pharmacodynamics research aiming at the triple negative breast cancer MDA-MB-231 is as follows.

The results show that: aiming at triple negative breast cancer MDA-MB-231 tumor-bearing mice, under the same dose, the Taxotere group has a general tumor inhibition effect (the tumor inhibition rate is 68 percent), the DTX-Rg3/Lp group and the DTX-Rh2/Lp group have the best effect, and the tumors completely disappear on the 28 th day. The experimental results show that: the compound preparation has obvious drug effect and obvious drug synergistic effect.

Claims

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

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 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;

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.