A PEG-modified docetaxel liposome lyophilized powder and preparation method thereof

Abstract

The present invention provides a PEG-modified docetaxel liposome lyophilized powder and a preparation method thereof. The composition of the liposome lyophilized powder according to mass percentage is: 0.01%-10% docetaxel; 10%-45% lipid membrane material; the balance is other excipients; the other excipients are PEG materials, antioxidants and lyophilization protectants, and the amount of PEG material added accounts for 0.01%~10% of the total mass of the lyophilized powder. The present invention can improve the deficiencies of ordinary liposomes and injections and other dosage forms by encapsulating docetaxel through long-circulating liposomes, enhance the solubility and safety of docetaxel, change the distribution of docetaxel in the body, and help to achieve good targeting; polyethylene glycol-modified long-circulating liposomes enhance the stability of docetaxel liposomes in plasma, prolong the retention time in the blood, help to exert the EPR effect, and achieve the purpose of high efficiency and low toxicity.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a PEG-modified docetaxel liposome freeze-dried powder and a preparation method thereof. Background Art

[0002] Docetaxel is a new generation of taxane anticancer drugs obtained by semi-synthesis of natural compounds extracted from the needles of Taxus chinensis. Docetaxel can both enhance tubulin polymerization and inhibit microtubule depolymerization, leading to the formation of stable non-functional microtubule bundles in tumor cells, thereby destroying the mitosis of tumor cells. Docetaxel is currently the first-line drug for the treatment of tumors such as colon cancer, breast cancer, non-small cell lung cancer, ovarian cancer, and melanoma. Docetaxel has poor water solubility and is a fat-soluble compound. Currently, the only dosage forms available in China are injections, which are Dopafil and Taxotere. However, the injection contains a large amount of ethanol and Tween as cosolvents. It is easy to cause adverse reactions such as hemolysis and allergies, and anti-allergic drugs need to be given in advance to slow down allergic reactions. In addition, docetaxel has poor distribution and organization. While killing tumor cells, the drug will also kill normal cells, causing toxic side effects in tissues and organs such as neutropenia, peripheral neurotoxicity, and cardiovascular reactions, making the patient's compliance poor.

[0003] In 1965, British scientist Bangham A first proposed the concept of liposomes. Due to the hydration of liposomes, they form a lipid membrane with a closed concentric spherical structure, encapsulating the liquid solvent. The basic composition of a liposome is phospholipids - the molecules contain a hydrophilic "head" composed of phosphate groups, connected to a non-polar hydrophobic "tail" composed of two hydrocarbon chains. The amphiphilic structure of phospholipids allows them to be arranged in an ordered structure - so that the hydrophobic tail cannot come into contact with water.

[0004] Hydrophilic drugs are encapsulated in the aqueous phase, and lipophilic drugs are encapsulated in the phospholipid bilayer. Liposomes have a series of characteristics such as sustained release, improved drug stability, reduced drug toxicity, cell affinity and tissue compatibility, and targeting. However, it was later discovered that when this traditional liposome preparation enters the body, the phospholipid components fuse and aggregate, and can exchange with apolipoprotein A-1 (the main component of high-density lipoprotein) in the blood, and activate the complement system, causing the encapsulated drug to leak and the liposome to lyse; and it is susceptible to adsorption by opsonins in plasma (such as immunoglobulins, fibronectin, complement molecules C3, CS, C-reactive protein, etc.), so that some liposomes are quickly cleared by the reticuloendothelial system as foreign bodies, and accumulate in large quantities in organs such as the liver and spleen, making it difficult for drugs to reach effective therapeutic concentrations at the target site, which to some extent limits its clinical application. Long-circulating liposomes, also known as stealth liposomes, are surface-modified liposomes. Since the hydrophilic groups on its surface are highly hydrated, they can effectively prevent many different components in the blood, especially opsonins, from binding to it, thereby reducing the body's clearance of liposomes. Therefore, there is a great demand for improving the stability, targeting, and efficacy of docetaxel liposomes and reducing toxicity.

[0005] Patent CN102085189A discloses a docetaxel liposome sterile lyophilized preparation and a preparation method thereof. During freeze drying in this preparation method, the amount of organic solution exceeds 10%, which makes freeze drying difficult, severely damages the freeze drying system, and makes post-treatment of the dried organic solvent gas difficult. Once the production is scaled up, it will be a big problem both from the perspective of environmental protection and cost. In addition, the PEGylated distearoylphosphatidylethanolamine (PEG-DSPE) used is extremely expensive, which will also increase the difficulty of industrialization. Moreover, it has been verified by the present invention that the effect of modifying liposomes with PEG-DSPE is not as good as that of PEG4000.

[0006] Patent CN105188675A discloses a modified docetaxel liposome preparation, and provides a PEG-modified docetaxel liposome, which optimizes the liposome structure: first forming a first liposome having a lipid bilayer containing phosphatidylcholine lipids and sterols, loading docetaxel into the first liposome, and finally embedding the PEG-lipid into the bilayer structure to form a mixture containing the loaded liposome and the PEG-lipid. This method does not further prepare its liposome into a lyophilized powder, so the liposome still inevitably has problems such as enlarged particle size or drug leakage during storage, and the membrane materials used are mostly synthetic phospholipids, which are all imported products, expensive and most of them are not injection-grade pharmaceutical excipients, the cost is still high, and the safety is worth considering.

[0007] Therefore, further development of more stable docetaxel preparations with better performance and therapeutic effects remains an urgent need in this field. Summary of the invention

[0008] In view of this, the purpose of the present invention is to provide a PEG-modified docetaxel liposome lyophilized powder and a preparation method thereof. By adopting a thin film dispersion method to prepare PEG-modified docetaxel liposomes and freeze-drying, the prescription is reasonable and effective, and the preparation process is stable and feasible. Encapsulation of docetaxel by long-circulating liposomes can improve the shortcomings of ordinary liposomes and injection solutions, enhance the solubility and safety of docetaxel, change the distribution of docetaxel in the body, and help achieve good targeting; polyethylene glycol-modified long-circulating liposomes enhance the stability of docetaxel liposomes in plasma, prolong the retention time in the blood, and help exert the EPR effect to achieve the purpose of high efficiency and low toxicity. The technical scheme of the present invention is:

[0009] In a first aspect, the present invention provides a PEG-modified docetaxel liposome lyophilized powder, wherein the composition of the liposome lyophilized powder is as follows in terms of mass percentage: 0.01%-10% docetaxel or its derivatives; 10%-45% lipid membrane material; the remainder is other auxiliary materials; the other auxiliary materials are PEG material, antioxidant, and lyophilization protectant, and the amount of the PEG material added accounts for 0.01%~10% of the total mass of the lyophilized powder.

[0010] Furthermore, the mass percentage of each component in the other auxiliary materials to the lyophilized powder is: 50%-85% lyophilization protectant, 0.01%-10% PEG material, and the balance is antioxidant.

[0011] Furthermore, the lipid membrane material is one or a mixture of natural soybean lecithin, egg yolk lecithin, sphingomyelin, cholesterol, hydrogenated soybean lecithin, dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, 1-palmitoyl-2-oleoyl lecithin, distearoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, and phosphatidylethanolamine.

[0012] Furthermore, the lyophilization protective agent is one or a mixture of glucose, mannose, mannitol, trehalose, lactose, sucrose, and dextran; preferably sucrose.

[0013] Furthermore, the antioxidant is one or a combination of α-tocopherol, vitamin E, threonine, lysine, glycine, and vitamin C; preferably, vitamin E and glycine or threonine are combined in a mass ratio of 1:5-10.

[0014] Furthermore, the PEG material at least includes PEG4000.

[0015] Optionally, the PEG material also includes at least one of PEG400, PEG1500, PEG2000, and PEG6000.

[0016] In a second aspect, the present invention provides a method for preparing a PEG-modified docetaxel liposome lyophilized powder, comprising loading a PEG material on a film containing docetaxel or a derivative thereof, and then granulating and lyophilizing.

[0017] Furthermore, the preparation method comprises:

[0018] (1) Film formation: according to the composition of docetaxel liposome lyophilized powder, docetaxel or its derivative, lipid film material and antioxidant are mixed with an organic solvent, and then a lyophilization protective agent is added after mixing. After mixing, the organic solvent is removed to obtain a dry film;

[0019] (2) Hydration: Add the dry film into a hydration medium containing PEG material and stir at a certain temperature to obtain a liposome suspension;

[0020] (3) Granulation: The liposome suspension is granulated, sterilized and freeze-dried in sequence.

[0021] Furthermore, the organic solvent is one or a mixture of the following anhydrous solvents: ethanol, dichloromethane, chloroform, preferably ethanol.

[0022] Furthermore, the removal of the organic solvent is carried out by distilling it off under reduced pressure at 20°C to 80°C.

[0023] Furthermore, the hydration medium is one of the following groups: purified water, physiological saline, pH 3-8 buffered saline solution, threonine solution, glycine solution.

[0024] Furthermore, the hydration process is carried out under constant temperature stirring at 20°C to 80°C and 2400 to 3000 rpm for 20 to 40 minutes.

[0025] Furthermore, the liposome suspension is sorted by microfluidization, high-pressure homogenization, ultrasound or extrusion in sequence.

[0026] Preferably, the antioxidant is a combination of vitamin E and glycine or threonine in a mass ratio of 1:5-10, wherein vitamin E is added during the film-forming process, and glycine or threonine forms a hydration medium with water.

[0027] More preferably, the antioxidant is a combination of vitamin E and glycine in a mass ratio of 1:5-10, wherein vitamin E is added during the film-forming process, and glycine forms a hydration medium with water.

[0028] Advantages of the present invention:

[0029] The docetaxel liposomes of the present invention are surface modified by introducing PEG. On the one hand, the hydrophilicity of PEG will lead to the formation of a hydration film on the surface of the liposomes, thereby blocking the van der Waals force of mutual attraction between the liposomes, providing a large steric hindrance for the liposomes, covering the binding sites of the hydrophobicity of the surface, thereby improving the physical, chemical and biological stability of the liposomes and the encapsulated objects; on the other hand, the PEG-modified liposomes can change the distribution of docetaxel in the body, reduce the recognition and uptake by the mononuclear macrophage system such as the liver and spleen in the body, and can greatly improve its targeting, so that most of the drugs accumulate in the target organs, and can use the "enhanced penetration and retention" effect for the special structure of blood vessels in tumor tissues to passively retain liposomes with smaller particle sizes in tumor tissues, thereby significantly prolonging the in vivo circulation time of the liposomes, and minimizing the side effects of the drugs on non-lesion sites, increasing the efficacy of the drugs, and achieving the purpose of high efficiency and low toxicity. In addition, the freeze-dried powder of the present invention has stable and uniform quality, simple preparation process, is conducive to large-scale industrial production, reduces production costs, improves stability, and improves the safety of clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a photo of the solid tumor of nude mice bearing human breast cancer MDA-MB-231 excised in Example 9 of the present invention. DETAILED DESCRIPTION

[0031] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0032] The present invention provides a PEG-modified docetaxel liposome lyophilized powder and a preparation method thereof. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0033] Example 1

[0034] This embodiment provides a method for preparing a PEG-modified docetaxel liposome lyophilized powder, comprising the following steps:

[0035] First, 2 mg of docetaxel, 40 mg of cholesterol, 369.2 mg of soy lecithin, 30.8 mg of phosphatidylethanolamine, and 1 mg of vitamin E in a prescribed amount were placed in a round-bottom flask, and an appropriate amount of ethanol was added to the mixture to completely dissolve it into a clear solution, 0.6 g of sucrose was added thereto and evenly dispersed, and the organic solvent was removed by heating at 60° C. under reduced pressure to obtain a dry film;

[0036] 5 mg of glycine and 15 mg of PEG4000 were added to a certain amount of purified water, and then added to the dry powder obtained above, and magnetically stirred at a high speed of 2500 r / min in a 40°C water bath for 30 minutes to obtain a liposome suspension with uneven particle size; then the particle size was reduced to 70-120 nm using an ultrasonic cell crusher), and the intermediate was sterilized by filtration using a 0.22 μm microporous filter membrane at room temperature and packaged in 40 mL vials; the drug-containing liposome solution was vacuum freeze-dried to remove moisture to obtain a white lyophilized powder.

[0037] Example 2

[0038] This embodiment provides a method for preparing a PEG-modified docetaxel liposome lyophilized powder, comprising the following steps:

[0039] First, 30 mg of docetaxel, 40 mg of cholesterol, 240 mg of soy lecithin, 160 mg of egg yolk lecithin (PL-100M), and 0.1 mg of vitamin E were placed in a round-bottom flask, and an appropriate amount of ethanol was added to the mixture to completely dissolve it into a clear solution, 1.6 g of sucrose was added thereto and evenly dispersed, and the organic solvent was removed by heating at 60° C. under reduced pressure to obtain a dry film;

[0040] 10 mg of glycine and 40 mg of PEG2000 were added to a certain amount of purified water, and then added to the dry powder obtained above, and magnetically stirred at a high speed of 3000 r / min in a 40°C water bath for 30 min to obtain a liposome suspension with uneven particle size; the particle size was reduced to 70-120 nm using an ultrasonic cell crusher, and the intermediate was sterilized by filtering with a 0.22 μm microporous filter membrane at room temperature and dispensed into 40 mL vials; the drug-containing liposome solution was freeze-dried in vacuum to remove moisture to obtain a white freeze-dried powder.

[0041] Example 3

[0042] This embodiment provides a method for preparing a PEG-modified docetaxel liposome lyophilized powder, comprising the following steps:

[0043] First, 100 mg of docetaxel, 40 mg of cholesterol, 240 mg of soy lecithin, 160 mg of egg yolk lecithin (PL-100M), and 10 mg of vitamin E were placed in a round-bottom flask, and an appropriate amount of ethanol was added to the mixture to completely dissolve it into a clear solution, 0.5 g of trehalose and 0.5 g of sucrose were added thereto and evenly dispersed, and the organic solvent was removed by heating at 60° C. under reduced pressure to obtain a dry film;

[0044] 30 mg PEG6000 was added to a certain amount of purified water, and then added to the dried powder after rotary evaporation. The mixture was magnetically stirred at a high speed of 2500 r / min in a 40°C water bath for 30 min to obtain a liposome suspension with uneven particle size. The particle size was reduced to 70-120 nm using an ultrasonic cell crusher, and the intermediate was sterilized by filtering with a 0.22 μm microporous filter membrane at room temperature and dispensed into 40 mL vials. The drug-containing liposome solution was freeze-dried in vacuum to remove moisture to obtain a white freeze-dried powder.

[0045] Example 4

[0046] This embodiment provides a method for preparing a PEG-modified docetaxel liposome lyophilized powder, comprising the following steps:

[0047] First, 20 mg of docetaxel, 40 mg of cholesterol, 400 mg of soy lecithin, and 1 mg of vitamin E in a prescribed amount were placed in a round-bottom flask, and an appropriate amount of ethanol was added to the mixture to completely dissolve it into a clear solution, 1 g of sucrose was added thereto and evenly dispersed, and the organic solvent was removed by heating at 40° C. under reduced pressure to obtain a dry film;

[0048] 10 mg of glycine and 20 mg of PEG4000 were added to a certain amount of purified water, and then added to the dried powder after rotary evaporation. The mixture was magnetically stirred at a speed of 2500 r / min in a 40°C water bath for 30 min to obtain a liposome suspension with uneven particle size. The particle size was reduced to 70-120 nm using an ultrasonic cell crusher, and the intermediate was sterilized by filtering with a 0.22 μm microporous filter membrane at room temperature and dispensed into 40 mL vials. The drug-containing liposome solution was freeze-dried in vacuum to remove moisture to obtain a white freeze-dried powder.

[0049] Example 5

[0050] This embodiment provides a method for preparing a PEG-modified docetaxel liposome lyophilized powder, comprising the following steps:

[0051] First, 60 mg of docetaxel, 40 mg of cholesterol, 400 mg of soy lecithin, and 1 mg of vitamin E in a prescribed amount are placed in a round-bottom flask, and an appropriate amount of ethanol is added to the mixture to completely dissolve it into a clear solution, 0.8 g of sucrose and 0.8 g of mannose are added thereto and evenly dispersed, and the organic solvent is removed by heating at 60° C. and reducing pressure to obtain a dry film;

[0052] 10 mg PEG400 was added to a certain amount of purified water, and then added to the dried powder after rotary evaporation. The mixture was magnetically stirred at a high speed of 2400 r / min in a 40°C water bath for 30 min to obtain a liposome suspension with uneven particle size. The particle size was reduced to 70-120 nm using an ultrasonic cell crusher, and the intermediate was sterilized by filtering with a 0.22 μm microporous filter membrane at room temperature and dispensed into 40 mL vials. The drug-containing liposome solution was freeze-dried in vacuum to remove moisture to obtain a white freeze-dried powder.

[0053] Example 6

[0054] This embodiment provides a method for preparing a PEG-modified docetaxel liposome lyophilized powder, comprising the following steps:

[0055] First, 4 g of docetaxel, 800 mg of cholesterol, 8 g of soy lecithin, and 20 mg of vitamin E (20 times the amount of the prescription) were placed in a round-bottom flask, and an appropriate amount of ethanol was added to the mixture to completely dissolve it into a clear solution, and 32 g of mannose was added thereto and evenly dispersed, and the organic solvent was removed by heating at 40° C. under reduced pressure to obtain a dry film;

[0056] Add 2g PEG4000 to a certain amount of purified water, and add it to the dry powder, stir magnetically at a speed of 3000r / min in a 40°C water bath for 30min until hydration is complete, and obtain a liposome suspension with uneven particle size; use a microjet high-pressure homogenizer to reduce the particle size to 70-120nm, filter the intermediate with a 0.22μm microporous filter membrane at room temperature for sterilization, and distribute it into 40mL vials; vacuum freeze-dry the drug-containing liposome solution to remove moisture to obtain a white lyophilized powder.

[0057] Example 7

[0058] The PEG-modified docetaxel liposome lyophilized powder obtained in Examples 1 to 6 was subjected to the following tests:

[0059] (1) Encapsulation efficiency, determination method: microcolumn centrifugation method. This method is a conventional method for testing encapsulation efficiency in this field and will not be explained in detail here.

[0060] (2) Particle size and dispersion coefficient, determination method: The freeze-dried powder obtained in the above examples was first reconstituted with 10 mL of water for injection, then diluted 10 times with water for injection, and then measured using a ZetaSizer nano Malvern particle size analyzer.

[0061] (3) Evaluation of formulation stability: The lyophilized product prepared in Example 4 was stored at 2°C-8°C for 12 months. The appearance, particle size, and encapsulation efficiency were tested at 0, 1, 3, 6, and 12 months to evaluate the formulation.

[0062] (4) In vitro cell experiment: B16 melanoma cells were cultured at 7.0×10 4 The cells were inoculated at a concentration of 5 μg / mL in a 96-well culture plate, with 100 μL of cell suspension in each well. 90 μL of 5 μg / mL liposome lyophilized powder of Examples 1 to 6 diluted with blank culture medium was added to the drug-treated group 24 hours after plating. 90 μL of blank culture medium was added to the control group 24 hours after plating. After incubation for 48 hours, 10 μL of CCK8 solution was added to each well, and the cells were incubated in a light-proof incubator for 2 hours. The absorbance was measured at a wavelength of 450 nm using a microplate reader, and the cell proliferation inhibition rate was calculated.

[0063]

[0064] (5) In vivo efficacy study: Tumor cells were subcutaneously inoculated into C57BL / 6 mice. When the tumor volume grew to 50-100 nm, the mice were divided into groups and given 11.7 mg / kg of medication once every three days for a total of 7 times. After the experiment, the tumors were removed and weighed.

[0065] The above test analysis results are shown in Table 1 and Table 2:

[0066] Table 1 Comparison of preparation indicators

[0067] index Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Encapsulation rate (%) 97±1.9 93±1.8 91±1.2 98±1.5 93±2.5 97±0.8 Particle size (nm) 110±2.6 120±3.3 120±1.9 111±4.2 125±3.5 116±3.6 Dispersion coefficient 0.114±0.015 0.170±0.013 0.206±0.020 0.101±0.011 0.169±0.024 0.098±0.012 Difficulty of passing the membrane easy easy generally easy easy easy

[0068] Table 2 Stability results of PEGylated docetaxel liposome lyophilized powder formulation

[0069] Measurement items 0 month January March June December Appearance Comply with regulations Comply with regulations Comply with regulations Comply with regulations Comply with regulations Encapsulation rate% 98.28 98.05 98.63 98.97 98.62 Average particle size (nm) 111 116 115 113 117

[0070] Table 3 Comparison of in vivo and in vitro efficacy

[0071] index Control group Docetaxel Injection Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Cell proliferation inhibition rate (%) 0 53±1.3 67±1.6 58±1.5 50±0.9 70±1.8 62±2.3 67±2.0 Average tumor weight (g) 4.2432±0.52 2.0345±1.03 1.5876±0.99 1.8594±0.86 2.0074±1.33 1.4925±0.78 1.7063±1.69 1.5991±1.29

[0072] From the above data, it can be seen that after adding PEG4000 to the lyophilized powder, the particle size of the preparation obtained is less than 120nm (Example 1, Example 4, Example 6), which helps the liposomes to target and enter the tumor site and enrich; and the liposome polydispersity coefficient is low (PDI<0.2), indicating that the prepared liposomes have uniform particle size distribution, good stability, and are more conducive to being taken up by tumor cells, thereby achieving better anti-tumor effects, and the encapsulation rate is greater than 95%, with a light blue opalescence, which meets the requirements of the pharmacopoeia and can effectively exert the drug effect. The appearance and color of the lyophilized sample are uniform, the pores are dense, loose and full, and the stability is good. And from the in vitro and in vivo efficacy experiments, it can be seen that after adding PEG4000 to the lyophilized powder, the cell proliferation inhibition rate is increased, the average tumor weight is reduced, and the tumor inhibition effect is exerted. While ensuring the original efficacy of the drug, the solubility and safety of docetaxel are enhanced, and the distribution of docetaxel in the body is changed, which is conducive to achieving good targeting.

[0073] Example 8

[0074] According to Example 4, lyophilized powder of docetaxel liposomes containing DSPE-PEG2000, PEG4000, PEG2000, PEG400 and PEG-free was prepared, and then reconstituted with 10 mL of water for injection to obtain a solution concentration of 2 mg / mL. 20 SD rats were selected and randomly divided into 5 groups, 3 rats in each group, and 1 rat was reserved. All groups were injected with liposomes through the tail vein, and the dosage was 11.7 mg / kg. 0.4 mL of blood was collected from the retroorbital venous plexus 5min, 15min, 30min, 1.0h, 2.0h, 3.0h, 4.0h, and 8.0h after the first administration. After the blood sample was processed, HPLC was performed for detection; the second administration was performed 3 days after the first administration, and 0.4 mL of blood was collected from the retroorbital venous plexus 5min, 15min, 30min, 1.0h, 2.0h, 3.0h, 4.0h, and 8.0h after the second administration. After the blood sample was processed, HPLC was performed for detection. The test results were input into DAS software for data processing to obtain relevant pharmacokinetic parameters (Table 4). From the results, it can be seen that the half-life of docetaxel liposomes containing PEG4000 doubled after two administrations of the same concentration relative to the first administration, and the maximum blood drug concentration and the area under the drug-dose curve remained basically unchanged, indicating that docetaxel liposomes containing PEG4000 increased the residence time in plasma and reduced the clearance rate of the drug in the body.

[0075] Table 4 In vivo pharmacokinetic parameters of docetaxel liposomes containing different PEG

[0076]

[0077] Example 9

[0078] According to Example 4, PEG-modified docetaxel liposomes were prepared to investigate their inhibitory effect on the growth of nude mice bearing human breast cancer MDA-MB-231 tumors. The specific operation was as follows:

[0079] 1. Inoculation of MDA-MB-231 tumor strain

[0080] The cells were cultured normally, and the nude mice were free to eat and drink, with 12h cycle light, and adapted to the environment for about two weeks. The human breast cancer MDA-MB-231 cells in good logarithmic growth phase were taken out, digested with 0.05% trypsin, centrifuged at 1000r / min for 10min, collected, and mixed with complete culture medium. 10μl of cell suspension was taken and counted under a microscope using a hemocytometer, and the concentration of the cell suspension was adjusted to 2.5×10 7 The skin of the right forelimb axillary area of ​​nude mice was disinfected and 0.2 ml (5 × 10 6 The above operations were performed under sterile conditions.

[0081] After the tumor was implanted in nude mice, the tumor was grown to 100 mm 3 After about 1 week, the mice were randomly divided according to the balance of tumor volume and body weight, and animal experiments were carried out according to the dosing schedule.

[0082] 2. Animal grouping

[0083] Group A: saline group, blank control

[0084] Group B: conventional docetaxel liposome group

[0085] Group C: PEG4000-containing docetaxel liposome group

[0086] Group D: PEG400 docetaxel liposome group

[0087] Group E: DSPE-PEG2000 docetaxel liposome group

[0088] All five groups received intravenous injections. Since the clinical equivalent dose of docetaxel is around 17 mg / kg, the injection dose for all five groups was 20 mg / kg.

[0089] 3. Dosage regimen

[0090] After the tumor was implanted in nude mice, the tumor was grown to 100 mm 3After about 15 days, the animals were randomly divided into 5 groups according to the balance of tumor volume and body weight, namely, normal saline group, ordinary liposome group, DSPE-PEG2000 docetaxel liposome group, PEG4000 docetaxel liposome group, and PEG400 docetaxel liposome group, with 10 animals in each group. Each drug-treated group was administered once through the tail vein on the 1st day, 11th day, and 22nd day after grouping. The animals were free to eat and drink during the experiment.

[0091] 4. Efficacy evaluation index

[0092] Observe and record the weight of each group of nude mice every day, observe their survival status (nude mouse activity, urine color and defecation status, etc.), record abnormal survival status and activity before and after administration, and record tumor volume twice a week. The animals were killed on the 26th day after the first administration, the tumor tissue was removed, the tumor weight was weighed, and the tumor growth inhibition rate was calculated. The calculation formula of tumor growth inhibition rate (TIR) ​​and tumor volume is as follows:

[0093]

[0094] The longest diameter of the tumor is the length, and the shortest diameter is the width. Tumor volume = length * width 2 *0.5

[0095] Table 5 Tumor inhibition rate of PEGylated docetaxel liposomes on nude mice bearing breast cancer MDA-MB-231 tumor

[0096] Group Tumor growth inhibition rate (%) Blank control — Liposomal docetaxel 56.74 PEG4000 docetaxel liposome 80.94 PEG400 docetaxel liposome 71.86 DSPE-PEG2000 docetaxel liposome 58.93

[0097] Table 6 Effects of PEGylated docetaxel liposomes on body weight of nude mice bearing breast cancer MDA-MB-231 tumor

[0098] Group Initial weight (g) / Final weight (g) Blank control 21.11 / 16.44 Liposomal docetaxel 21.09 / 16.13 PEG4000 docetaxel liposome 20.88 / 17.37 PEG400 docetaxel liposome 21.21 / 18.17 DSPE-PEG2000 docetaxel liposome 21.26 / 18.44

[0099] The results show that at the same dose (20 mg / kg), the tumor inhibition effect of PEG-modified docetaxel liposomes was better than that of ordinary docetaxel liposomes, and the body weight was significantly higher than that of the ordinary docetaxel liposomes group (such as Figure 1 Among them, the tumor inhibition rate of PEG4000-modified docetaxel liposomes was the best, indicating that PEG4000-modified docetaxel has low toxicity and is a relatively safe preparation.

[0100] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A PEG-modified docetaxel liposome lyophilized powder, characterized in that: The liposome freeze-dried powder is composed of: 0.01%-10% docetaxel in terms of mass percentage; 10%-45% lipid membrane material; the balance is other excipients; the other excipients are PEG materials, antioxidants and lyophilization protective agents, and the amount of PEG material added accounts for 0.01%~10% of the total mass of the lyophilized powder; the lipid membrane material includes soybean lecithin and cholesterol; the lyophilization protective agent is one or a mixture of glucose, mannose, mannitol, trehalose, lactose, sucrose, and dextran; the antioxidant is one or a combination of vitamin E, threonine, lysine, glycine, and vitamin C; the PEG material is PEG4000.

2. The PEG-modified docetaxel liposome lyophilized powder according to claim 1, characterized in that: The mass percentage of each component in the other auxiliary materials to the lyophilized powder is as follows: 50%-85% lyophilization protectant, 0.01%-10% PEG material, and the balance is antioxidant.

3. The PEG-modified docetaxel liposome lyophilized powder according to claim 1 or 2, characterized in that: The freeze-drying protective agent is sucrose.

4. The PEG-modified docetaxel liposome lyophilized powder according to claim 1 or 2, characterized in that: The antioxidant is a combination of vitamin E and glycine in a mass ratio of 1:5-10; or, the antioxidant is a combination of vitamin E and threonine in a mass ratio of 1:5-10.

5. The method for preparing a PEG-modified docetaxel liposome lyophilized powder according to any one of claims 1 to 4, characterized in that: The preparation method comprises: (1) Film formation: according to the composition of docetaxel liposome lyophilized powder, docetaxel, lipid film material and antioxidant are mixed with an organic solvent, and then a lyophilization protective agent is added after mixing. After mixing, the organic solvent is removed to obtain a dry film; (2) Hydration: Add the dry film into a hydration medium containing PEG material and stir at a certain temperature to obtain a liposome suspension; (3) Granulation: The liposome suspension is granulated, sterilized and freeze-dried in sequence.

6. The method for preparing a PEG-modified docetaxel liposome lyophilized powder according to claim 5, characterized in that: The hydration medium is one of the following groups: purified water, physiological saline, pH 3-8 buffered salt solution, threonine solution, glycine solution.

7. The method for preparing a PEG-modified docetaxel liposome lyophilized powder according to claim 5, characterized in that: The hydration process is carried out under constant temperature stirring at 20°C to 80°C and 2400 to 3000 rpm for 20 to 40 minutes.

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