CN114712309B - Ginsenoside docetaxel liposome, and preparation method and application thereof - Google Patents

Ginsenoside docetaxel liposome, and preparation method and application thereof Download PDF

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CN114712309B
CN114712309B CN202011534465.7A CN202011534465A CN114712309B CN 114712309 B CN114712309 B CN 114712309B CN 202011534465 A CN202011534465 A CN 202011534465A CN 114712309 B CN114712309 B CN 114712309B
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ginsenoside
docetaxel
liposome
parts
phospholipid
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CN114712309A (en
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王建新
王丹
陈颖江
詹华杏
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Shanghai Shensu Pharmaceutical Technology Co ltd
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Shanghai Shensu Pharmaceutical Technology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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 parts 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 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 in vivo accumulated toxicity is low.

Description

Ginsenoside docetaxel liposome, and 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 high-efficiency low-toxicity compound ginsenoside docetaxel liposome for injection, and a preparation method and application thereof.
Background
The liposome is a directional drug-carrying system, belongs to a special dosage form of a targeted drug-carrying system, and can embed drugs in nano-sized particles, wherein the particles are similar to double-molecular layer microvesicles in a biological membrane structure, enter a human body to be mainly phagocytized by reticuloendothelial systems, and change the in-vivo distribution of the encapsulated drugs, so that the drugs are mainly accumulated in 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 invention is a technical innovation based on the Chinese patent applications of CN201610693884.2, CN201811447245.3, CN201811447243.4 and the like. All three application patents disclose the technical advantages that the liposome taking the ginsenoside as the membrane material has stable quality, obvious drug effect and the like of the related liposome after the chemotherapy drugs such as paclitaxel and the like are entrapped in the liposome.
CN201610693884.2 discloses a blank liposome and application using ginsenoside Rg5 and its derivatives as membrane material, and the prescription can further comprise other adjuvants such as cholesterol, antioxidant, soybean oil and/or sodium oleate, besides phospholipid, saponin, medicine and freeze-drying protective agent.
CN201811447245.3 discloses a blank liposome taking ginsenoside Rh5H and derivatives thereof as membrane materials and application, and the patent further solves the problem of hemolysis of ginsenoside on the basis of CN 201610693884.2. In addition to phospholipids, saponins, medicines and freeze-drying protective agents, the prescription can also further comprise cholesterol, antioxidants, soybean oil, sodium oleate and other auxiliary materials.
CN201811447243.4 discloses a blank liposome taking ginsenoside Rg3 and derivatives thereof as membrane materials and application thereof. According to the patent, the problem of solubility of ginsenoside in chloroform is solved by technical means such as superfine powder and the like of the ginsenoside such as Rg3, rh2 and the like, so that the problem that the ginsenoside such as Rg3, rh2 and the like must be formed into a film in chloroform is solved, and Rg3 liposome with quality meeting the standard is prepared.
The prior art still has some defects, such as larger pressure required by liposome production homogenizing step in partial scheme, slow speed of filter membrane sterilization and filtration, high retention rate and obviously worse product yield; it is necessary to add 2-6 times the amount of soybean oil. However, the addition of soybean oil is not beneficial to freeze-drying the preparation, and affects the long-term storage of the medicine.
The core of the compound preparation is the synergistic interaction of the medicines in the body, 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 especially the change 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 all rarely involved. Therefore, aiming at compound ginsenoside docetaxel liposome (hereinafter referred to as "ginmosome-DTX" or "ginmosome-CTX") for injection, the main drug "docetaxel" is determined how to select the most suitable "synergistic drug and auxiliary material" ginsenoside and key auxiliary material "phospholipid" and related proportion thereof, and the preparation method has reasonable compatibility, small particle size, stable quality, optimal effect of drug effect and toxicity, so that the composition with the specific proportion of the drug and the key auxiliary material has innovation and uniqueness and very important significance.
In prescription screening, any one of a plurality of factors such as medicines, phospholipids, saponins, freeze-drying protective agents, 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 taxol is increased, so that the mass stability and the synergistic anti-tumor effect of the compound liposome can be increased, the targeting distribution of tumor tissues can be increased, but the accumulated toxicity of the saponin in a human body can be increased, and uncontrollable organ injury can be caused; the proper mass ratio of the saponin to the taxol has very important relevance to the stability, the active targeting, the pharmacodynamics and the safety of the liposome. Meanwhile, different freeze-drying protective agents are selected, so that the method plays a vital role in avoiding damage to lipid bilayer structures in the liposome freeze-drying process and recovering the characteristics of the liposome after re-dissolving the freeze-dried medicine. For example, in the selection of lyoprotectants, different lyoprotectants have different effects on the lyophilization curve, especially in many aspects such as the co-melting point of the compound liposome, whether collapse, whether the liposome changes significantly after reconstitution, the primary lyophilization temperature and time setting, the total lyophilization time, and the like.
The safety and effectiveness of the medicine are two basic properties of the medicine, the approval and the use of the medicine are considered based on the risk-benefit ratio between the two, and especially the improvement of the new medicine is the core of improving the effectiveness and the safety.
In toxicology research, the pharmaceutics research plays a vital role, and especially, the selection of prescription proportion and preparation process has various effects on acute toxicity, long-term toxicity and accumulated toxicity of various functional organs, so that whether the compound liposome meets the requirements of new drug declaration can be directly determined.
Therefore, the optimal proportion range of phospholipid, ginsenoside, docetaxel and saccharide components of the freeze-drying protective agent in the prescription membrane components of the liposome plays an important role in the properties of good pharmaceutical stability, in-vivo distribution, pharmacodynamics, toxicology and the like of the formed compound liposome. However, this optimum ratio does not give any deduction of the above components and ratios and process to pharmacological activity, drug generation and toxicology. Because of the large number of variables involved, screening must be done through extensive experimentation and creative effort.
Therefore, how to select an optimal compound medicine compatibility and how to formulate an optimal preparation process so as to produce the compound ginsenoside docetaxel liposome for injection, wherein the compound ginsenoside docetaxel liposome has better medicine effect, lower toxicity and quality and other indexes which can meet the medicine requirements, so as to meet the medicine declaration requirements, and a great deal of research work and technical attack are needed.
Disclosure of Invention
Aiming at the defects of the existing docetaxel liposome, the invention provides a (compound) ginsenoside docetaxel liposome, a preparation method and application thereof; the medicine has stable property, small particle size, high medicine encapsulation efficiency, good in vivo compatibility, good in vivo medicine release, better medicine effect, lower toxicity and reasonable compatibility; the preparation method has a good preparation process, and the preparation conditions are easy to realize, so that the 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 (called 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 one scheme of the invention, 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 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 distearoyl phosphatidylethanolamine-methoxypolyethylene glycol 2000 (mPEG 2000-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 docetaxel to phospholipid may be 1:8-12, such as 1:10; for example, the mass ratio of docetaxel to 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-Rg 3, 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 Rg3.
In one embodiment of the present invention, the mass ratio of the docetaxel to the ginsenoside may be 1:1 or 1:1.5.
In one scheme of the invention, the mass ratio of the docetaxel to the 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, and for example 25:1, 30:1 or 35:1. For example, the mass ratio of the glucose to the docetaxel is 25:1.
In one scheme of the invention, the particle size D90 of the ginsenoside docetaxel liposome is less than or equal to 150nm, and the encapsulation rate is more than or equal to 98%.
In one embodiment of the invention, the HPLC purity of the ginsenoside is greater than or equal to 99%.
In one scheme of the invention, the ginsenoside docetaxel liposome comprises the following components in percentage by mass: 10 parts of phospholipid, 1 part or 1.5 parts of ginsenoside, 1 part of docetaxel and 25 parts of lyoprotectant.
In one embodiment of the present invention, the ginsenoside docetaxel liposome may comprise the following components in mass fraction:
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 lyoprotectant;
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 lyoprotectant.
In one embodiment of the present invention, the ginsenoside docetaxel liposome may comprise the following components in mass fraction:
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 liposome does not contain one or more of cholesterol, soybean oil and sodium oleate.
In one embodiment of the present invention, the empty 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 also for example docetaxel.
In one 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 distearoyl phosphatidyl ethanolamine-methoxy polyethylene glycol 2000 of 0.01-10%.
In one embodiment of the present invention, the number of phospholipids in the empty liposome is 10.
In one embodiment of the present invention, the ginsenoside may be one or more of 20 (S) -ginsenoside Rg3, ginsenoside pseudo-Rg 3, 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 Rg3.
In one embodiment of the present invention, the mass ratio of the ginsenoside in the empty liposome may be 1 part, 1.5 parts or 2 parts.
In one embodiment of the present invention, the lyoprotectant in the blank liposome 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, e.g., 25 parts, 30 parts, or 35 parts, in the blank liposome.
In one embodiment of the present invention, the blank liposome may comprise the following components in mass fraction: 9 parts of egg yolk lecithin, 1.8 parts of Rg3 and 20-35 parts of freeze-drying protective agent;
or 10 parts of soybean phospholipid, 2 parts of Rh2 and 20-35 parts of lyoprotectant;
or, 10 parts of egg yolk lecithin: 1.5 parts of pseudo Rg3 and 20-35 parts of 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 lyoprotectant;
or, 10 parts of egg yolk lecithin: 1.5 parts of Rp1 and 20-35 parts of lyoprotectant.
The invention also provides a preparation method of the ginsenoside docetaxel liposome, which comprises the following steps of;
step 1, concentrating a solution A1 of docetaxel, ginsenoside, phospholipid and an organic solvent to form a film;
step 2, after the membrane obtained in the step 1 is subjected to heat preservation and hydration in water, uniformly mixing the membrane 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) includes the steps of:
And (3) homogenizing the liposome solution A2 obtained in the step (2) under high pressure, and controlling the particle size D90 to be smaller than 100nm to obtain a liposome solution A3a.
Scheme 2 (high shear + extrusion process) includes the steps of:
shearing the liposome solution A2 obtained in the step 2, extruding through a 150nm aperture extrusion plate, and controlling the particle diameter D90 to be smaller than 100nm to obtain a liposome solution A3b;
wherein, the definition of docetaxel, ginsenoside, phospholipid and lyoprotectant solution is the same as that of the ginsenoside docetaxel liposome (compound) described above.
In a certain embodiment of the present invention, in the 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 and chloroform and/or dichloromethane; for example ethanol: chloroform = 1:1 (volume ratio). The amount of the organic solvent is not particularly limited, and may be used in order to dissolve docetaxel, ginsenoside, and phospholipid. For example, the mass to volume ratio of docetaxel to the organic solvent is 1g/60-120mL, for example 1g/80mL.
In one embodiment of the present invention, in the step 1, the solution A1 is preferably obtained by dissolving docetaxel, ginsenoside, phospholipid, etc. in an organic solvent under heating; for example, the ginsenoside and the phospholipid are added into the solution of docetaxel and the organic solvent, and the mixture is heated and dissolved to obtain the ginsenoside and the phospholipid; the heating may be in a water bath to a temperature of 35-65deg.C, for example 55deg.C.
In one embodiment of the present invention, in the step 1, the concentrating may be reduced pressure concentrating; the reduced pressure concentration can be vacuum= -0.08MPa to-0.1 MPa, for example, -0.089 to-0.1 MPa; 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, and the rotation speed may be 40-60 rp/min, for example, 50rp/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 the step 2, the concentration of the lyoprotectant solution may be 0.20-0.35mg/mL, for example 0.25mg/mL.
In one embodiment of the present invention, the hydration temperature in step 2 may be 35-65deg.C, preferably 40-45deg.C.
In one embodiment of the present invention, in the step 2, the hydration is performed in a rotary evaporator, and the rotation speed is 40-60 rp/min, for example, 50rp/min.
In one embodiment of the present invention, in the step 2, the hydration is performed uniformly in a solution, for example, for 2 to 4 hours.
In one embodiment of the present invention, in the step 2, the docetaxel is: lyoprotectant solution = 1g:100mL.
In one embodiment of the present invention, in the step 2, the volume of the lyoprotectant solution is the same as the volume of the water.
In one embodiment of the present invention, in the embodiment 1 of the step 3, the high-pressure homogenization is performed by using a freezing water cutting cycle at 0 to 10 ℃ in a homogenizer; preferably, the temperature of the liposome solution is ensured at 5-10 ℃.
In one embodiment of the present invention, in embodiment 1 of step 3, the high-pressure homogenizing pressure is between 800 and 1400bar, for example 1200bar.
In one embodiment of the present invention, in embodiment 1 of the step 3, the number of times of high-pressure homogenization may be 3 to 4, for example, 4.
In one embodiment of the present invention, in embodiment 2 of step 3, the shearing may be performed at room temperature.
In a certain aspect of the present invention, in the aspect 2 of the step 3, the rotation speed of the shearing is 1500-2200 rp/min; for example 2000rp/min.
In a certain scheme of the invention, in the scheme 2 of the step 3, the shearing time is 5-10 min; for example 5min.
In one embodiment of the present invention, in embodiment 2 of step 3, the extrusion temperature is 35-45 ℃, for example 40 ℃.
In one embodiment of the present invention, in embodiment 2 of the step 3, the aperture of the extruded plate is 150nm.
In one aspect of the invention, in the step 3, in the step 2, the extrusion pressure is 600 to 800psi; such as 800psi.
In a certain embodiment of the present invention, in the embodiment 2 of the step 3, the number of extrusion times may be 3 to 4, for example, 4 times.
The invention also provides a preparation method of the ginsenoside docetaxel liposome for injection, which comprises the following steps of;
step 1-1, step 1-3 in the preparation method of the ginsenoside docetaxel liposome described above, to obtain liposome solution A3a or A3b;
step 2-1, sterilizing and filtering the liposome solution A3a or A3b to obtain a liposome solution A4;
and 3-1, freeze-drying the liposome solution A4 to obtain the ginsenoside docetaxel liposome for injection.
In the preparation method, the conditions and operations of sterilization filtration and freeze drying can be the conventional conditions and operations in the art; the following are preferred in the present invention:
in one embodiment of the present invention, in the step 2-1, the sterilization filtration may use a 0.22 μm filter.
In one embodiment of the present invention, in step 3-1, the freeze-drying may be performed in a penicillin bottle, which may be a penicillin bottle conventional in the art, such as a 30mL or 50mL penicillin bottle.
In one embodiment of the present invention, in the step 3-1, the freeze-drying may sequentially include: prefreezing, primary drying and secondary drying; specifically, the method can comprise the following steps:
step a, quantitatively split charging liposome solution A4 in a penicillin bottle into a freeze drying box, wherein the temperature of a freeze-dried shelf is reduced to minus 10+/-1 ℃ uniformly, the temperature is kept for 1 hour, the temperature of the shelf is increased to minus 13+/-1 ℃, the temperature is kept for 1 hour, the temperature of the shelf is reduced to minus 55+/-1 ℃, and after the temperature of a product reaches minus 45+/-1 ℃, the time counting is started, and the temperature is kept for 3 hours;
b, after the heat preservation in the step a is finished, quickly reducing the temperature of a condenser to below-50+/-1 ℃, vacuumizing to below 10pa, raising the temperature of a shelf (about 1.5 hours) to-25+/-1 ℃, starting timing and preserving heat for 18 hours, quickly raising the temperature of the shelf to-15+/-1 ℃, preserving heat until ice crystals of a product completely disappear, and continuously preserving heat for 4 hours;
and c, after the heat preservation in the step b is finished, rapidly (within 1 hour) raising the temperature of the shelf to 15+/-1 ℃, 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, the step 3-1 may further include a post-treatment, where the conditions and operations of the post-treatment may be those conventional in the art; for example, the post-processing includes the steps of: c, after the heat preservation in the step c is finished, fully pressing the plug, and discharging the plug from the box; and (5) capping.
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 one scheme of the invention, the particle size D90 of the ginsenoside docetaxel liposome for injection is less than or equal to 150nm, and the encapsulation 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 application of the ginsenoside docetaxel liposome, or the blank liposome, or the compound ginsenoside docetaxel liposome for injection in preparation of medicines for treating and/or preventing cancers.
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 a sample having a cumulative particle size distribution percentage of 90%. Its physical meaning is that its particle size is less than 90% of its particle size.
The prescription abbreviations of the present invention are explained as follows:
the above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: the compound ginsenoside docetaxel liposome provided by the invention has the targeting effect on tumor cells, the anti-multi-drug resistance effect, the synergy and attenuation effects and the drug synergy effect. Taking the compound ginsenoside Rg3 docetaxel liposome for injection in the example as an example, the drug effect is obviously better than that of the technical scheme which is not in the scope of the invention; the Rg3 plays a better role in various roles such as medicine, auxiliary materials, membrane materials, target heads and the like in the compound ginsenoside Rg3 docetaxel liposome for injection, and plays a good role in medicine synergism. Specifically:
(1) The drug effect is obviously improved. Especially, the drug effect of the DTX-Rg3 (1.0)/Lp group and the DTX-Rh2 (1.5)/Lp group is optimal, wherein the tumor inhibition rate (68%) of the dosage (10 mg/kg) reaches or exceeds the tumor inhibition rate of the high dosage (20 mg/kg) of the liposome group which is not the prescription of the invention, so that the tumor inhibition effect is improved by 2 times;
(2) Glut1 targeting is significantly improved. In Glut1 targeting experiments of tumor-bearing mice, glut1 targeting of the ginsenoside liposome is improved by more than 4 times compared with that of common cholesterol liposome, and Glut1 targeting of common non-preferred ginsenoside liposome is improved by less than 2 times compared with that of common cholesterol liposome.
(3) The toxic and side effects are obviously reduced. The acute toxicity (LD 50 is more than or equal to 150 mg/kg) of DTX-Rg3 (1.0)/Lp and DTX-Rh2 (1.5)/Lp (LD 50 is more than or equal to 150 mg/kg) of the liposome prepared according to the prescription of the invention are reduced by more than 6 times compared with that of a Taxote group (LD 50 is about 25 mg/kg), 3-4 times compared with that of a common cholesterol docetaxel liposome (LD 50 is between 25 and 50 mg/kg), and about 1.5-2 times compared with that of a non-prescription ginsenoside liposome such as DTX-C-Rg3 (1.8)/Lp and DTX-C-Rh2 (2.0)/Lp and the like (LD 50=50-100 mg/kg). In the SD rat long-term toxicity experiment, animal death occurs in the Taxote group, which indicates that the Taxote group has large toxic effect. 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 Taxote.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Experimental drugs and devices
Experimental drugs: 20 (S) -ginsenoside Rg3 (abbreviated as Rg 3), ginsenoside pseudo Rg3 (abbreviated as pseudo Rg 3), ginsenoside Rp1 (abbreviated as Rp 1), ginsenoside pseudo GQ (abbreviated as pseudo GQ), ginsenoside Rk1 (abbreviated as Rk 1), ginsenoside Rg5 (abbreviated as Rg 5), 20 (S) -ginsenoside Rh2 (abbreviated as Rh 2), ginsenoside Rk2 (abbreviated as Rk 2), 20 (S) -ginsenoside Rg2 (abbreviated as Rg 2), 20 (S) -ginsenoside Rh1 (abbreviated as Rh 1), 20 (S) -protopanaxadiol (abbreviated as PPD), 20 (S) -protopanaxatriol (abbreviated as PPT) and the like are commercially available in the field, such as Shanghai-Benzisu medical science and technology Co, suzhou star-Japan Jikinson biological Co Ltd, shanghai-Litsea leaf biological science and technology Co.
Docetaxel injection: taxote, manufacturer: aventis, england, purchased from Shanghai Lei Dagavage, inc.
The molecular structural formula of the ginsenoside is as follows:
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test instrument: the instruments used in the following examples were the instruments and equipment owned by Shanghai Bensu medical science and technology Co., ltd, university of double denier medical college, and the equipment model and source information were as follows:
agilent liquid chromatography: agilent 1100 set, autai 3300ELSD, agilent technologies (China) Inc.;
spin-on evaporator: ZX98-1 5L, shanghai Lu Yi Goodyear Co., ltd;
ultrasonic cleaning machine (SB 3200DT, ningbo Xinzhi biotechnology Co., ltd.);
nitrogen blowing instrument (HGC-12A, heng ao technology development Co., tianjin);
probe ultrasonic instrument (JYD-650, shanghai Zhi Xin instruments Co., ltd., china);
a high pressure homogenizer (B15, AVESTIN, canada);
mini-extruders (Avanti Polar Lipids Inc);
laser particle size analyzer (Nano ZS, markov in the united kingdom);
malvern particle sizer Malvern Nanosizer ZS (malvern, uk);
microplate reader (Thermo Scientific, waltham, MA, USA);
microplate reader (infinie 200, tecan tracking co., 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 living microscope (Confocal intravital microscopy, IVM);
a front two-photon microscope (DM 5500Q; nikon);
a small animal living body optical imaging system (in vivo imaging system, IVIS) (PerkinElmer, USA);
biomacromolecule interactor BiaCore T200 instrument (GE, USA);
clean bench (SW-CJ-1 FD, air technologies Co., ltd.);
20L rotary evaporator: R5002K, shanghai xiafeng real company limited;
freeze dryer: FD-1D-80, shanghai Bilang instruments Co., ltd;
freeze dryer: PDFD GLZ-1B, shanghai Pudong freeze drying Equipment Co., ltd;
an electronic balance: CPA2250 (precision 0.00001 g), sidoris (Shanghai) trade Co., ltd;
an electronic balance: JY3003 (precision 0.001 g), shanghai Shunyu Hengping scientific instruments Co., ltd;
photo-electric microscope (XDS-1B, chongqing photo-electric instruments Co., ltd.);
Cell incubator (CCL-170B-8, singapore ESCO).
Animals and cell lines
Animals: BALB/c nude mice are 3-4 weeks old and produced by Shanghai pharmaceutical research institute, national academy of sciences.
Tumor cell lines:
breast cancer in-situ tumor 4T1 cell line provided by university of double denier pharmacy
Breast cancer MCF-7 cell line, supplied by university of double denier pharmacy;
a triple negative breast cancer MDA-MB-231 cell line provided by the university of double denier pharmaceutical college;
example 1 preparation of Compound ginsenoside Rg3 docetaxel liposome for injection
1. Prescription: egg yolk lecithin 10g, ginsenoside Rg 31 g, docetaxel 1g, glucose 25g, absolute ethyl alcohol 40ml, chloroform 40ml and water for injection 200ml.
2. Film forming: preparing a mixed solvent of absolute ethyl alcohol and chloroform (1:1) with a prescription amount for standby.
Adding docetaxel with a prescription amount into a mixed solvent for dissolving for standby, adding ginsenoside Rg3 with a prescription amount and egg yolk lecithin into the mixed solvent, heating for dissolving, transferring into a 1L rotary steaming bottle, concentrating under reduced pressure, carrying out water bath at a temperature of 55 ℃ and a rotating speed of 50 revolutions per minute, carrying out rotary steaming until the solvent is completely volatilized, wherein the vacuum degree is-0.089 to-0.1 MPa.
3. Hydration: preparing a glucose solution: 25g of anhydrous glucose is added into 100ml of water for injection, and is prepared into 0.25mg/ml glucose aqueous solution after stirring and dissolution, and the mixture is heated in a water bath at 40 ℃ for standby.
100ml of water for injection is added into a rotary steaming bottle after film formation, the water bath temperature is 40-45 ℃, the rotating speed is 50 revolutions per minute, and the water is hydrated and completely dissolved for about 2 hours.
Then 100ml of glucose aqueous solution is added and stirred uniformly for standby.
4. Homogenizing under high pressure: transferring the hydrated solution to a homogenizer, performing water cooling cutting circulation by using a freezing water at 0-10 ℃, setting the homogenizing pressure to 1200bar, and circularly homogenizing for 3-4 times until D90 is less than 100nm.
5. And (3) sterilizing and filtering: the homogenized solution was sterilized by filtration through a 0.22 μm filter.
6. And (5) subpackaging: the sterilized and filtered solution is packaged into 30 or 50ml penicillin bottles according to the installation quantity of 8-10 ml.
7. Pre-freezing: after the product enters the box, the temperature of the shelf is reduced to about minus 10 ℃ uniformly, the temperature is kept for 1 hour, the temperature of the shelf is increased to about minus 55 ℃ for 1 hour, the heat preservation is finished, the temperature of the shelf is reduced to about minus 55 ℃ continuously, and after the temperature of the product reaches minus 45 ℃, the timing is started to keep the temperature for about 3 hours continuously.
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 the ice crystals of the product completely disappear, and continuously preserving heat for about 4 hours;
9. And (3) secondary drying: the temperature of the shelf is quickly (within 1 hour) raised to about 15 ℃, the temperature is kept for about 3 hours, then the temperature of the shelf is raised to about 25 ℃, and when the temperature of the product is raised to 25 ℃, the temperature is kept for about 12 hours. 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. Capping and packaging: rolling the liposome, and packaging to obtain compound ginsenoside Rg3 docetaxel liposome for injection (prescription 1).
Example 2 preparation of Compound ginsenoside Rg3 docetaxel liposome for injection
The prescription amount of ginsenoside Rg3 in example 1 is increased to 1.5g, and the compound ginsenoside Rg3 docetaxel liposome for injection is prepared in the same way as in example 1.
Example 3 preparation of Compound ginsenoside Rg3 docetaxel liposome for injection
1. Prescription: egg yolk lecithin 10g, ginsenoside Rg 3.5 g, docetaxel 1g, glucose 25g, absolute ethyl alcohol 40ml, chloroform 40ml and water for injection 200ml.
2. Film forming: the film formation method was the same as in example 1.
3. Hydration: the hydration method of example 1 was repeated.
4. High speed shear and extrusion: the liposome solution was rapidly sheared at 2000rp/min for 5min at room temperature.
The liposome solution temperature was controlled at 35-45 ℃, the extrusion device was connected, a 150nm pore size extrusion plate was mounted, and extrusion was performed at 800psi pressure.
5. The subsequent steps are the same as those of example 1.
Example 4 preparation of Compound ginsenoside Rh2 docetaxel liposome for injection
A compound ginsenoside Rh2 docetaxel liposome for injection was prepared in the same manner as in example 1 except that ginsenoside Rg3 in example 1 was changed to ginsenoside Rh 2.0 g.
EXAMPLE 5 preparation of Compound ginsenoside Rh2 docetaxel liposome for injection
A compound ginsenoside Rh2 docetaxel liposome for injection was prepared in the same manner as in example 1 except that ginsenoside Rg3 in example 1 was changed to ginsenoside Rh 2.5 g.
EXAMPLE 6 preparation of Compound ginsenoside pseudo Rg3 docetaxel liposome for injection
The compound ginsenoside Rg3 docetaxel liposome for injection was prepared by changing ginsenoside Rg3 in example 1 to ginsenoside pseudo Rg 3.5 g and the same procedure as in example 1.
EXAMPLE 7 preparation of Compound ginsenoside Rg5 docetaxel liposome for injection
The compound ginsenoside Rg5 docetaxel liposome for injection was prepared in the same manner as in example 1 except that ginsenoside Rg3 in example 1 was changed to ginsenoside Rg 5.5 g.
EXAMPLE 8 preparation of Compound ginsenoside Rk1 docetaxel liposome for injection
The compound ginsenoside Rk1 docetaxel liposome for injection was prepared in the same manner as in example 1 except that ginsenoside Rg3 in example 1 was changed to ginsenoside Rk 1.5 g.
Example 9 preparation of Compound ginsenoside Rp1 docetaxel liposome for injection
The compound ginsenoside Rp1 docetaxel liposome for injection was prepared in the same manner as in example 1 except that the ginsenoside Rg3 in example 1 was changed to ginsenoside Rp 1.5 g.
EXAMPLE 10 preparation of Compound ginsenoside pseudo GQ docetaxel liposome for injection
A compound ginsenoside pseudo GQ docetaxel liposome for injection was prepared in the same manner as in example 1 except that ginsenoside Rg3 in example 1 was changed to ginsenoside pseudo GQ 1.5 g.
Effect example 1
(a) The comparison of the types of ginsenosides according to the following table was carried out in the same manner as in example 1 (without the lyophilization step), as follows:
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the series of experiments prove that under the condition that other auxiliary materials such as soybean oil or cholesterol are not added, the ginsenoside in the invention is as follows: when Rg3, pseudo Rg3, rh2, pseudo GQ, rg5, rk1, rp1 and the like are 7, the Ginposome-DTX or CTX has a better preparation process, and the preparation conditions are easy to realize and are beneficial to industrialization. When ginsenoside is Rk2, rg2, rh1, PPD and PPT, other auxiliary materials are required to be added, and the preparation conditions are relatively harsh.
(b) The types and proportions of phospholipids were compared:
remarks: egg yolk lecithin (EPC), soybean lecithin (SPC), cephalin (PE), sphingomyelin (SM), hydrogenated lecithin (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).
The comparison of the phospholipid species and proportions is carried out in the same manner as in example 1 (without the need for a lyophilization step), as shown in the following table:
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the experiment proves that docetaxel can be well coated without adding 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% of mPEG-DSPE). Other phospholipids and Rg3 can also well encapsulate docetaxel, but other auxiliary materials such as soybean oil and the like are added, or the homogenization pressure and the homogenization times are improved. The above experiments are also illustrated: in terms of film forming properties, the phospholipids of the present invention: docetaxel = 8-18:1, the effect is better, preferably 8-12:1.
(c) The optimum proportions of ginsenoside were compared in the same manner as in example 1 (no lyophilization step was required):
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the experiment proves that under the condition of not adding other auxiliary materials such as soybean oil or cholesterol, the saponin in the invention: docetaxel = 1-3:1, the effect is better. However, as a result of the toxicity and pharmacokinetics study in application example 4, only saponins were selected: docetaxel = 1-1.5: 1.
(d) The lyoprotectants were compared in the same manner as in example 1:
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in the selection of the freeze-drying protective agent, different freeze-drying protective agents have obvious influence on the encapsulation rate of liposome after product re-dissolution and the particle size distribution, 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 docetaxel to the freeze-drying protective agent is as follows: the lyoprotectant/docetaxel=25-35 times, for example 15 times of glucose, the re-dissolved particle size of ginnocome-DTX is 200nm, between pass and fail, and at 25 or 35 times, it has good matching with docetaxel liposome. Because of comprehensive consideration of empirical data of pharmaceutics, the ratio of docetaxel to lyoprotectant determined by the invention is as follows: lyoprotectant/docetaxel=25-35 fold amount.
Application example 1: cell uptake assay for Glut1
1) The purpose of the experiment is as follows: the Glut1 targeting mechanism is proved by adding glucose inhibitors and the like; the ginsenosides and the proportion, the phospholipid and the proportion of the ginsenosides are verified by Glut1 targeting.
2) The experimental method comprises the following steps: to compare the uptake of 4T1 into each experimental group, the uptake mechanism of the compound preparation was examined, and 4T1 cells were grown at 2X 10 5 Is inoculated in 12-well plates, and 20mM glucose solution, phlorizin solution and quercetin solution are used to replace the culture medium after 12 hours for experimental group + glucose, experimental group + phlorizin and experimental group + quercetin group, respectively. The three solutes should be dissolved in glucose-free medium, after incubation for 1 hour, each experimental group of drugs (ultraviolet fluorescent developer concentration 100 ng/ml) was added, after incubation 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: preparation according to the method of example 1 of the present invention (docetaxel is changed to coumarin without the need for lyophilization step)
Using the results of the poison experiment of example 4, the following experiment was not conducted for a prescription of more than 2.0 ginsenoside/docetaxel.
Experimental group:
prescription content Short for short
Egg yolk lecithin: cholesterol: fluorescent probe = 9:1.8:1 C6-C/Lp
Egg yolk lecithin: rg3: fluorescent probe: cholesterol=9: 1.8:1:2.25 C6-C-Rg3/Lp
Egg yolk lecithin: rg3: fluorescent probe = 9:1.8:1 C6-Rg3/Lp
Soybean phospholipid: rh2: fluorescenceOptical probe: cholesterol=10: 2:1:2.25 C6-C-Rh2/Lp
Soybean phospholipid: rh2: fluorescent probe = 10:2:1 C6-Rh2/Lp
Soybean phospholipid: rg5: fluorescent probe: soybean oil = 4:3:1:2 C6-Rg5/Lp
Egg yolk lecithin: pseudo Rg3: fluorescent probe = 10:1.5:1 C6-pseudo Rg3 (1.5)/Lp
Egg yolk lecithin: pseudo GQ: fluorescent probe = 10:1.5:1 C6-pseudo GQ (1.5)/Lp
Egg yolk lecithin: rk1: fluorescent probe = 10:1.5:1 C6-Rk1(1.5)/Lp
Egg yolk lecithin: rp1: fluorescent probe = 10:1.5:1 C6-Rp1(1.5)/Lp
The experimental results are as follows:
conclusion of experiment:
1) Under the same prescription, after cholesterol is added, the Glut1 targeting is greatly reduced.
2) The targeting of the prescription of the invention is improved by more than 4 times compared with cholesterol liposome, and the targeting of the non-preferential prescription of ginsenoside liposome is improved by about 2 times compared with cholesterol liposome.
Application example 2: in vivo pharmacodynamic study of breast cancer (MCF-7)
1) The test method comprises the following steps: the tumor cell line (MCF-7) was injected subcutaneously into mice to establish a subcutaneous tumor model. When the tumor volume reaches 100mm 3 At (7 d post inoculation), mice were treated with random groups (n=8 per group), each with tail intravenous injection of 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, DTX-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 phospholipids: rh2: cholesterol docetaxel=10:1:2.25:1), doses were given once every 7 days for up to day 28, with tumor length, width and record body weight measured at the same time as the administration. The formula for calculating the tumor volume (V) is V= (W) 2 X 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 at day 28, all animals were sacrificed and tumors were removed for imaging and histological examination.
Remarks: docetaxel + rg3 = 20mg/kg +30mg/kg, representing drug concentration, the same applies below.
As a result of the toxicity generation in example 4, no study was conducted on the DTX-Rg3 (2.0 or more)/Lp group in this example.
2) The test results are as follows:
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conclusion:
1) The DTX-Rg3 (1.0)/Lp group and DTX-Rh2 (1.5)/Lp group had the best efficacy, and the tumor completely disappeared on day 21. The tumor inhibition rate of the medium dose of the two groups (docetaxel=10 mg/kg) is basically consistent with that of the high dose group of DTX-C-Rg3 (1.8)/Lp group and 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 which is not provided by the invention.
2) Animal death occurred in the DTX-C/Lp group, indicating a large toxic effect.
3) The ratio of the medicine effect to the ginsenoside does not have a linear relation, and the medicine effect is optimal, preferably 1.0 when the ratio of the ginsenoside Rg 3/docetaxel is 1.0-1.5 according to the prescription; the ratio of ginsenoside Rh 2/docetaxel is preferably 1.5 when the ratio is 1.0-2.0.
Application example 3: acute toxicity (LD 50) study (SD rat)
1) The experimental method comprises the following steps: rats 160-260 g, 6-9 weeks old, 6 per group, mode of administration: slow static push (about 1 mL/min), dosing frequency: 3 times per day.
The docetaxel dosage of the test sample is set to 25, 50, 100 and 150 mg/kg/day, and the Rg3 content of the test sample is respectively 25, 50, 100 and 150 mg/kg/day; rh2 content was 37.5, 75, 150 and 225 mg/kg/day, respectively. A vehicle control group (5% glucose injection), a commercially available positive control group (Taxote group), an Rg3 liposome group and a DTX-C-Rg3/Lp group are simultaneously arranged, and slow static pushing (about 1 mL/min) is carried out for 3 times/day, and the dosing interval is at least 4 hours.
2) Experimental grouping: the total of 13 groups, 5% glucose group, DTX-C/Lp, DTX-C-Rg3 (1.8)/Lp group, DTX-C-Rh2 (2.0)/Lp group and pharmacodynamic experimental groups were identical, 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 recipe, following the procedure of example 1.
3) The experimental results are shown in the following table:
remarks: as a result of the toxicity generation using example 4, no study was conducted on the DTX-Rg3 (2.0 or more)/Lp group in this example.
The experiment shows that the technical scheme of the invention has excellent pharmaceutics and Glut1 targeting, so that the toxicity reducing effect of related preparations is optimal, and compared with cholesterol liposome (DTX-C/Lp) and docetaxel injection (Taxote), the toxicity is generally reduced by about 4-6 times; the toxicity is generally reduced by 1.5-2 times relative to DTX-C-Rg3 (1.8)/Lp group and DTX-C-Rh2 (2.0)/Lp group.
Application example 4: toxicity Kinetics (TK) study
1. The purpose of the experiment is as follows: the cumulative toxicity of each experimental group was studied.
2. The experimental method comprises the following steps: the TK group of the experiment was 8 groups, namely, a vehicle control group (5% glucose injection), a commercial positive control group Taxote group (10 mg/kg), a test 1 (DTX-Rg 3 (1.0)/Lp group: 10mg/kg and 20 mg/kg), a test 2 (DTX-Rh 2 (1.5)/Lp group: 10mg/kg and 20 mg/kg), a test 3 (DTX-Rh 2 (2.0)/Lp group, 10mg/kg and 20 mg/kg), respectively. 10 SD rats in each group, each half of the male and female rats, and total 80 rats. Intravenous administration, 1 time per week, continuous administration for four weeks, collecting whole blood before and 1hr after administration of D1 and D29, collecting whole blood before and 3min, 15min, 30min, 1hr, 3hr, 6hr, and 24hr before and after administration of D1 and D29, respectively, collecting whole blood in test tube containing EDTA-K2 anticoagulant, placing on crushed ice, and centrifuging to collect blood plasma for analysis and detection.
The concentration of docetaxel and 20 (S) -Rg3 and 20 (S) -Rh2 in blood plasma is detected by adopting an LC-MS/MS method, and the quantitative lower limit of the docetaxel analysis method is as follows: the lower limit of quantification for Rg3 and Rh2 assays was 50.000ng/mL at 25.000 ng/mL. And analyzing the plasma concentration data by adopting a non-atrioventricular model of WinNonlin software, performing parameter calculation, researching the toxicity and pharmacokinetics characteristics of a test sample, and providing reference for clinical experiments.
3. Experimental results, under the current experimental conditions:
1) After administration of D1 and D29, the exposure of ginsenoside Rg3 and Rh2 (in terms of AUC (0-t)) in plasma of the female SD rats after intravenous administration of the test 1, the test 2 and the test 3 increased with the increase of the administration dose, and the increase range was higher than the increase of the dose.
2) After D1 administration, the exposure amounts (in Cmax) of ginsenoside Rg3 and Rh2 in the plasma after intravenous administration of sample 1, sample 2 and sample 3 to male and female SD rats were proportional to the administered dose.
3) Following D1 and D29 administration, the exposure to docetaxel in the plasma (in AUC (0-t) and Cmax) following intravenous administration to test 1 in female and male SD rats increased with increasing doses, with a higher magnitude than the dose.
4) After D1 and D29 administration, the exposure amounts (in Cmax) of ginsenoside Rg3 and Rh2 in the plasma after intravenous administration of sample 2 to male and female SD rats were directly proportional to the administered dose.
5) Under the test condition, after continuous administration, the ginsenoside Rg3 and Rh2 in the plasma of the male and female SD rats after intravenous injection administration of the test article 1 and the test article 2 have no accumulation tendency basically; docetaxel in the plasma after intravenous injection administration of the test article 1, the test article 2 and the test article 3 in the female and male SD rats has no substantial tendency to accumulate; the exposure of docetaxel and ginsenoside Rh2 in the plasma after intravenous injection of the test 3 is slightly accumulated in the male and female SD rats; there was no accumulation of docetaxel exposure in plasma of the commercial control group given by intravenous injection to male and female SD rats.
Conclusion: the DTX-Rg3 (1.0)/Lp group had no cumulative toxicity risk.
The DTX-Rh2 (1.5)/Lp group had no cumulative toxicity risk.
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) The experimental method comprises the following steps: rats 160-260 g, 6-9 weeks old, 6 per group, mode of administration: slow static push (about 1 mL/min), dosing frequency: 1 time per week (D1, D8, D15, D22, D29).
2) Experimental grouping: a total of 5% glucose (vehicle control), taxote (positive control), taxote+Rg3/Lp, DTX-Rg3/Lp, DTX-Rh2/Lp were set.
The docetaxel dosage of 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, thus containing Rg3 of 5, 10 and 20mg/kg, respectively, and Rh2 of 7.5, 15 and 30mg/kg, respectively. (prepared by the method of example 1)
The dose administered for each group was set as follows:
3) The experimental results are as follows:
results 1: long-term toxicity test results of the Taxote group (test results on day 30)
Dosage for administration 5mg/kg 10mg/kg 20mg/kg
Proportion of death of animals No death No death 33.3％
Average weight change of animals +15％ +2％ -18％
Liver tissue injury condition No damage Moderate degree Heavy weight
Condition of injury to renal tissue No damage Moderate degree Heavy weight
Injury of spleen tissue No damage Moderate degree Heavy weight
Cardiac tissue injury condition No damage Moderate degree Heavy weight
Results 2: long-term toxicity test results (day 30 test results) of Taxote group + Rg3/Lp group
Results 3: long-term toxicity test results (test results on day 30) of DTX-Rg3/Lp group
Results 4: long-term toxicity test results of DTX-Rh2/Lp group (test results on day 30)
The long-term toxicity test results of the rats show that:
1) The Taxote group showed death of rats, severe weight loss, severe injury of each organ, indicating high toxicity.
2) No rat death occurred in both DTX-Rg3/Lp and DTX-Rh2/Lp groups, body weight was hardly reduced, and each organ was mainly slightly damaged, indicating a significant reduction in long-term toxicity.
Application example 6: in vivo study of compound ginsenoside docetaxel liposome for injection on triple negative breast cancer (MDA-MB-231)
Animals: BALB/c nude mice, mice aged 3-4 weeks, shanghai pharmaceutical research institute.
Tumor cell lines: triple negative breast cancer MDA-MB-231 cell strain
Is provided by Shanghai pharmaceutical research institute of Chinese sciences.
Transplantation tumor model: the cell strains are inoculated under the armpit skin on the right side of the nude mice, and the cell inoculation amount is 5 multiplied by 10 6 And/or, the mice are used after 1 generation of transplanted tumor.
The experimental method comprises the following steps: the tumor cell line was injected subcutaneously into mice to establish a subcutaneous tumor model. When the tumor volume reaches 100mm 3 (7 d after inoculation) mice were treatedTreatment was randomized into 4 groups (n=8 per group), each with tail vein injection of blank solvent (5% glucose), taxotere group, DTX-Rg3/Lp group (20 mg/kg docetaxel meter, 20mg/kg ginsenoside Rg3 meter), DTX-Rh2/Lp group (20 mg/kg docetaxel meter, 30mg/kg ginsenoside Rh2 meter), administered once every 7 days for up to day 28, with tumor length, width and recorded body weight measured simultaneously with administration. 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 at day 28, all animals were sacrificed and tumors were removed for imaging and histological examination.
Triple negative breast cancer MDA-MB-231: according to the in vivo pharmacodynamics experimental method, the study data of in vivo pharmacodynamics for triple negative breast cancer MDA-MB-231 are as follows.
The results show that: for the MDA-MB-231 tumor-bearing mice with triple negative breast cancer, the tumor inhibition effect of the Taxote group is general (tumor inhibition rate is 68%), the DTX-Rg3/Lp group and the DTX-Rh2/Lp group have the best effect, and tumors completely disappear on the 28 th day. The experimental result shows that: the compound preparation has obvious drug effect and obvious drug synergistic effect.

Claims (17)

1. A ginsenoside docetaxel liposome, 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; the ginsenoside docetaxel plastid 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, or one or more of egg yolk lecithin, soybean lecithin, hydrogenated phospholipid and cephalin containing distearoyl phosphatidylethanolamine-methoxy polyethylene glycol 2000 with the content of 0.01-10%; 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; the freeze-drying 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 mass ratio of the freeze-drying protective agent to the docetaxel is (25-35): 1.
2. The ginsenoside docetaxel liposome of claim 1, wherein,
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 less than or equal to 150nm.
3. The ginsenoside docetaxel liposome of claim 2, wherein,
the mass ratio of docetaxel to the phospholipid is 1:10;
and/or the mass ratio of the lyoprotectant to the docetaxel is 30:1.
4. The ginsenoside docetaxel liposome of claim 1, wherein,
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 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 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 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 lyoprotectant.
5. A blank liposome, which is characterized by comprising the following components in percentage by mass: 8-18 parts of phospholipid, 1-1.5 parts of ginsenoside and 20-35 parts of freeze-drying protective agent; the blank liposome 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 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; the freeze-drying protective agent is one or more of trehalose, glucose, sucrose, lactose and galactose.
6. The blank liposome of claim 5,
the phospholipid is 10 parts.
7. A method for preparing the 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; the organic solvent is one or more of methanol, ethanol, chloroform and dichloromethane;
step 2, after the membrane obtained in the step 1 is subjected to heat preservation and hydration in water, uniformly mixing the membrane with a freeze-drying protective agent solution to obtain a liposome solution A2;
step 3, which is scheme 1 or scheme 2;
scheme 1 includes the steps of:
homogenizing the liposome solution A2 obtained in the step 2 under high pressure, and controlling the particle size D90 to be smaller than 100nm to obtain liposome solution A3a;
scheme 2 includes the steps of:
and (3) shearing the liposome solution A2 obtained in the step (2), extruding through a 150nm aperture extrusion plate, and controlling the particle size D90 to be smaller than 100nm to obtain a liposome solution A3b.
8. The method for preparing ginsenoside docetaxel liposome of claim 7, wherein,
the mass volume ratio of docetaxel to the organic solvent is 1mg/80mL;
And/or, in the step 1, the solution A1 is obtained by heating and dissolving docetaxel, ginsenoside and phospholipid in an organic solvent;
and/or, in the step 1, the concentration is reduced pressure concentration;
and/or, in the step 1, the concentration is performed in a rotary steaming bottle, and the rotating speed is=40-60 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 ℃;
and/or, in the step 2, the hydration is carried out in a rotary steaming bottle, and the rotating speed is 40-60 rpm;
and/or, in the step 2, the hydration is carried out by uniformly dissolving;
and/or, in the step 2, the docetaxel is: lyoprotectant 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 scheme 1 of the step 3, the high-pressure homogenization is carried out by using a freezing water-cooling cutting cycle at 0-10 ℃ in a homogenizer;
And/or, in scheme 1 of step 3, the high pressure homogenizing pressure is between 800 and 1400 bar;
and/or, in the scheme 1 of the step 3, the number of times of high-pressure homogenization is 3-4 times;
and/or, in said step 3, in scheme 2, said shearing is performed at room temperature;
and/or, in the step 3 of the scheme 2, the shearing speed is 1500-2200 rpm;
and/or, in the step 3 of the scheme 2, the shearing time is 5-10 min;
and/or, in the step 3, in the scheme 2, the extrusion temperature is 35-45 ℃;
and/or, in the step 3 of the solution 2, the aperture of the extrusion plate is 150nm;
and/or, in said step 3, in scheme 2, said extruding is at a pressure of 600 to 800psi;
and/or, in the scheme 2 of the step 3, the number of times of extrusion is 3-4.
9. The method of claim 7, wherein in step 1, the ginsenoside and the phospholipid are added into a solution of docetaxel and the organic solvent, and the solution is heated and dissolved to obtain a solution A1.
10. The method for preparing ginsenoside docetaxel liposome according to claim 8, wherein in the step 1, the organic solvent is a mixed solvent of methanol and/or ethanol and chloroform and/or dichloromethane;
And/or, in the step 1, the reduced pressure concentration is vacuum= -0.08mpa to-0.1 mpa;
and/or, in the step 1, the concentration is carried out in a rotary steaming bottle, and the rotating speed is 50rpm;
and/or, in the step 2, the concentration of the lyoprotectant solution is 0.25mg/mL;
and/or, in the step 2, the hydration temperature is 40-45 ℃;
and/or, in the step 2, the hydration is carried out in a rotary steaming bottle, and the rotating speed is 50rpm;
and/or, in the step 2, the hydration is carried out by uniformly dissolving for 2-4 hours;
and/or, in the scheme 1 of the step 3, the high-pressure homogenization is carried out by using a freezing water-cooling cutting cycle at 0-10 ℃ in a homogenizer; and ensuring that the temperature of the liposome solution is 5-10 ℃;
and/or, in the step 3 of the scheme 1, the high pressure homogenizing pressure is 1200bar;
and/or, in the step 3 of the scheme 2, the shearing speed is 2000rpm;
and/or, in the step 3, in the scheme 2, the extrusion temperature is 40 ℃.
11. The method for preparing ginsenoside docetaxel liposome of claim 10, wherein in the step 1, the organic solvent is ethanol: volume ratio of chloroform = 1: 1;
And/or adding the ginsenoside and the phospholipid into the solution of docetaxel and the organic solvent, and heating and dissolving to obtain the ginsenoside and the phospholipid; the heating is to heat to 35-65 ℃ in a water bath;
and/or, in the step 1, the concentration is reduced pressure concentration; the reduced pressure concentration is vacuum= -0.089 to-0.1 MPa.
12. The method for preparing ginsenoside docetaxel liposome according to claim 11, wherein in the step 1, the concentration is performed until the solvent is completely volatilized; the total concentration time was less than 4 hours.
13. A preparation method of ginsenoside docetaxel liposome for injection, which comprises the following steps of;
step 1-1, liposome solution A3a or A3b obtained in the method for preparing ginsenoside docetaxel liposome according to any one of claims 7-12;
step 2-1, sterilizing and filtering the liposome solution A3a or A3b to obtain a liposome solution A4;
and 3-1, freeze-drying the liposome solution A4 to obtain the ginsenoside docetaxel liposome for injection.
14. The method for preparing ginsenoside docetaxel liposome for injection according to claim 13, wherein,
In the step 2-1, a filter membrane with the diameter of 0.22 mu m is adopted for the sterilization filtration;
and/or, in the step 3-1, the freeze drying sequentially comprises: prefreezing, primary drying and secondary drying; specifically, the method comprises the following steps: step a, quantitatively split charging liposome solution A4 in a penicillin bottle into a freeze drying box, wherein the temperature of a freeze-dried shelf is reduced to minus 10+/-1 ℃ uniformly, the temperature is kept for 1 hour, the temperature of the shelf is increased to minus 13+/-1 ℃, the temperature is kept for 1 hour, the temperature of the shelf is reduced to minus 55+/-1 ℃, and after the temperature of a product reaches minus 45+/-1 ℃, the time counting is started, and the temperature is kept for 3 hours;
b, after the heat preservation in the step a is finished, quickly reducing the temperature of a condenser to below-50+/-1 ℃, vacuumizing to below 10pa, raising the temperature of a shelf to-25+/-1 ℃ for 1.5 hours, starting timing and preserving heat for 18 hours, quickly raising the temperature of the shelf to-15+/-1 ℃, preserving heat until ice crystals of a product completely disappear, and continuously preserving heat for 4 hours;
c, after the heat preservation in the step b is finished, rapidly increasing the temperature of the shelf to 15+/-1 ℃ within 1 hour, preserving the heat for 3 hours, then increasing the temperature of the shelf to 30+/-1 ℃, and finishing the heat preservation for 12 hours when the temperature of the product is increased to 25+/-1 ℃;
And/or, the step 3-1 is further followed by 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 plug from the box; and (5) capping.
15. A compound ginsenoside docetaxel liposome for injection, which is prepared by the preparation method of the ginsenoside docetaxel liposome for injection according to claim 13 or 14.
16. Use of a ginsenoside docetaxel liposome according to any one of claims 1-4, or a blank liposome according to claim 5, or a compound ginsenoside docetaxel liposome for injection according to claim 15, in the preparation of a medicament for treating and/or preventing cancer.
17. The use of 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.
CN202011534465.7A 2020-12-22 2020-12-22 Ginsenoside docetaxel liposome, and preparation method and application thereof Active CN114712309B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109833298A (en) * 2017-11-29 2019-06-04 厦门本素药业有限公司 Using Hydrolizates as novel blank liposome, preparation method and the application of membrane material
CN111939130A (en) * 2015-08-19 2020-11-17 上海本素医药科技有限公司 Docetaxel liposome and preparation method thereof
CN111973557A (en) * 2018-11-29 2020-11-24 上海参素药物技术有限公司 Docetaxel liposome, preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111939130A (en) * 2015-08-19 2020-11-17 上海本素医药科技有限公司 Docetaxel liposome and preparation method thereof
CN109833298A (en) * 2017-11-29 2019-06-04 厦门本素药业有限公司 Using Hydrolizates as novel blank liposome, preparation method and the application of membrane material
CN111973557A (en) * 2018-11-29 2020-11-24 上海参素药物技术有限公司 Docetaxel liposome, preparation method and application thereof

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