Docetaxel liposome, preparation and application thereof

Abstract

The invention discloses a docetaxel-based liposome drug delivery system as well as a preparation method and application thereof, and belongs to the technical field of anti-tumor nano-drugs. The delivery system is composed of an active pharmaceutical ingredient docetaxel and a specific excipient, and comprises egg yolk lecithin, phosphatidylserine, taraxasterol, protamine oligopeptide, a necessary stabilizer and a necessary pH regulator. Through an optimized preparation process, the nano drug delivery system with good stability is successfully constructed. Experiments show that the liposome is high in encapsulation efficiency and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drug preparations, and in particular relates to docetaxel liposomes, a preparation and applications thereof. Background Art

[0002] Docetaxel is a taxane anti-tumor drug with the molecular formula: C43H53NO14, chemical name: (2R, 3S)-N-carboxy-3-phenylisoserine-N-tert-butyl ester-13-ester-5β, 20-epoxy-1, 2α, 4, 7β, 10β, 13α-hexahydroxytaxane-11-ene-9-one-4-acetate-2-benzoate, and the structural formula is as follows: Developed by the French company Sanofi, the original manufacturer received approval in the 1990s, though its core patent has since expired. It was approved by the US FDA in 1995 and launched that same year in Mexico, South Africa, and Canada for breast cancer. This approval was subsequently expanded to include lung and prostate cancers, as well as for the treatment of metastatic breast cancer and metastatic non-small cell lung cancer after chemotherapy failure and platinum-based treatment failure. Subsequently, it was launched in 33 countries, including the UK, France, Italy, Germany, and Japan, becoming the most effective single-agent, first-choice chemotherapy for metastatic breast cancer and non-small cell lung cancer. In March 2006, the US FDA approved it for the treatment of advanced gastric cancer.

[0003] Chinese patent CN101322689B discloses a method for preparing long-circulating docetaxel liposomes and their lyophilized powder injection. The liposomes increase the solubility of the drug, solving the stability issues of docetaxel lyophilized powder and the toxicity issues caused by composite solvents. They increase the drug's circulation time in the body, have reduced toxicity compared to pegylated long-circulating liposomes, and enhance the inhibitory effect on tumor cells.

[0004] Chinese patent CN101810574B discloses a method for preparing a stable docetaxel microemulsion injection and a lyophilized emulsion. By selecting a suitable oil phase and emulsifier and optimizing the preparation process, the prepared docetaxel microemulsion and lyophilized emulsion have the characteristics of high drug loading, good stability, easy industrial production, and strong clinical applicability.

[0005] Chinese patent CN115887692B discloses a PEG-modified lyophilized docetaxel liposome powder and its preparation method. This invention, through the encapsulation of docetaxel in long-circulating liposomes, can overcome the shortcomings of conventional liposomes and lyophilized powders, enhancing the solubility and safety of docetaxel and changing its distribution in the body, thus achieving good targeting. The PEG-modified long-circulating liposomes enhance the stability of the docetaxel liposomes in plasma and prolong their retention time in the blood, facilitating the EPR effect and achieving high efficacy and low toxicity.

[0006] Chinese patent CN114712309B discloses a ginsenoside docetaxel liposome, its preparation method, and application. The ginsenoside docetaxel liposomes described in this invention have enhanced active targeting of Glut1; their efficacy is more than 2-fold higher than conventional ginsenoside docetaxel liposomes; their toxicity is more than 1.5-fold lower than conventional ginsenoside docetaxel liposomes and more than 4-6-fold lower than conventional cholesterol docetaxel liposomes; and the risk of cumulative toxicity in the body is low.

[0007] Currently, the development of docetaxel liposome formulations still faces certain technical bottlenecks, primarily manifested in insufficient formulation stability and high process complexity. These issues may affect the consistency of the product's clinical efficacy. The physicochemical stability of existing liposome membrane materials needs to be improved, which may lead to fluctuations in drug loading; the complex preparation process also poses challenges to industrial production. Therefore, the development of novel docetaxel liposome formulations with enhanced stability and simplified processing is of great significance. Summary of the Invention

[0008] The present invention aims to address the problems of poor stability and complex preparation processes in existing docetaxel liposomes. A lyophilized docetaxel liposome powder is provided. The liposomes contain, by weight, 0.5-2.0 parts docetaxel, 3-5.5 parts egg yolk lecithin, 2-3 parts phosphatidylserine, 1.5-3.5 parts taraxasterol (CAS No. 114977-28-5), 2-5 parts protamine short peptide, 8-13 parts protective agent, and an appropriate amount of a pH adjuster. More preferably, the liposomes contain, by weight, 1 part docetaxel, 4.4 parts egg yolk lecithin, 2.5 parts phosphatidylserine, 2.5 parts taraxasterol, 3.5 parts protamine short peptide, 10 parts protective agent, and an appropriate amount of a pH adjuster. The protamine short peptide is a peptide fragment produced by enzymatic digestion of natural protamine, with a molecular weight between 1 and 3 kDa.

[0009] Furthermore, the present invention also provides a method for preparing a lyophilized powder of liposomes containing docetaxel: 1) Dissolve egg yolk lecithin, phosphatidylserine, and taraxasterol in an organic solvent, place in an eggplant-shaped bottle, and set aside; 2) Dissolve docetaxel in N,N-dimethylformamide and add the pH adjuster to an eggplant-shaped flask. Rotary evaporate under reduced pressure in a water bath for 10-20 minutes until a thin film forms. Rotary evaporate until the solvent is completely evaporated and set aside. 3) Add an appropriate amount of protamine short peptide protective agent aqueous solution to hydrate and heat, add it to the eggplant-shaped flask in step 2), and ultrasonically vibrate for 1-5 minutes; then add an appropriate amount of protective agent aqueous solution, stir evenly, and set aside; 4) The hydrated solution is transferred to a homogenizer, cooled to 0-5°C, subjected to high-pressure homogenization, filtered, packaged, and subjected to gradient freeze-drying to obtain a lyophilized powder containing docetaxel liposomes.

[0010] Furthermore, in the method for preparing docetaxel liposome lyophilized powder, the organic solvent is preferably a mixture of ethanol and tert-butanol, with a dosage ratio of 2:1, and the liposome formation is better. At the same time, single-factor comparative experiments of the same type of organic solvents were conducted, using ethanol and butanol, ethanol, ethanol and tert-butanol, methanol, methanol and tert-butanol, and tert-butanol as dissolving organic solvents. The results showed that ethanol and tert-butanol as solvents had better encapsulation efficiency and / or drug loading. Gradient calculations were performed in the dosage and found that when the dosage of the two was 2:1, the technical effects of liposome formation, as well as the final encapsulation efficiency and / or drug loading were better than other ratios.

[0011] Furthermore, in the method for preparing the docetaxel liposome lyophilized powder, the pH of the pH adjuster is 4.0-6.0, more preferably 5; the pH adjuster is selected from citric acid. Citric acid is used to stably maintain the optimal pH range of 4.0-6.0 in the docetaxel liposome lyophilized powder, preventing docetaxel hydrolysis and inhibiting phospholipid oxidation, thereby improving stability. Citric acid also reduces free radical damage and enhances lipid membrane integrity. Its good water solubility and lyophilization compatibility also help maintain the reconstitution properties of the lyophilized powder, ultimately achieving long-term storage stability and clinical safety.

[0012] Furthermore, in the preparation method of docetaxel liposome lyophilized powder, the protective agent aqueous solution is prepared by mixing glycine with one of trehalose, glucose, maltose, raffinose, and sucrose and adding the mixture to water for injection and dissolving. Furthermore, glycine and trehalose are added to water for injection and dissolved; glycine and glucose are added to water for injection and dissolved; glycine and maltose are added to water for injection and dissolved; glycine and raffinose are added to water for injection and dissolved; and glycine and sucrose are added to water for injection and dissolved.

[0013] More preferably, the protective agent in the preparation method of docetaxel liposome lyophilized powder is selected from glycine and trehalose. The protective agent is selected from glycine and trehalose in a ratio of 1:7-12. More preferably, the protective agent is selected from glycine and trehalose in a ratio of 1:9. The specific preparation method is as follows: 1g of glycine and 7g of trehalose are added to 200ml of water for injection and dissolved, or 1g of glycine and 12g of trehalose are added to 200ml of water for injection and dissolved, or 1g of glycine and 9g of trehalose are added to 200ml of water for injection and dissolved, and then set aside.

[0014] Further, the gradient freeze-drying described in the preparation method of docetaxel liposome lyophilized powder is as follows: after the filled product is put into the box, it is cooled to -50±5°C for pre-freezing and kept for 2-5 hours; heated to -15±5°C, kept warm for 1-3 hours, and then cooled to -30±5°C and kept warm for 1-3 hours; the cold trap is opened and the vacuum is opened; the partition temperature is raised to -15--10°C and then kept for 5-10 hours; the partition temperature is raised to -5-0°C and then kept for 3-5 hours; the partition temperature is then raised to 30-35°C and then kept for 3-5 hours; after the freeze-drying is completed, nitrogen is filled under vacuum and the product is removed from the pot.

[0015] Furthermore, in a preferred embodiment of the present invention, the liposome lyophilized powder containing docetaxel comprises, in parts by weight, 1 g of docetaxel, 4.4 g of egg yolk lecithin, 2.5 g of phosphatidylserine, 2.5 g of taraxasterol, 3.5 g of protamine short peptide, 10 g of a protective agent, and an appropriate amount of a pH adjuster; the preparation method of the lyophilized powder is: 1) Preparation of protective agent aqueous solution: Dissolve 1g glycine and 9g trehalose in 200ml water for injection and set aside. 2) Dissolve egg yolk lecithin, phosphatidylserine, and taraxasterol in a 2:1 weight ratio of ethanol to tert-butanol (60 ml total), place in an eggplant-shaped bottle, and set aside. 3) Dissolve docetaxel in N,N-dimethylformamide, adjust the pH to 5.0 with citric acid, and add the solution to the eggplant-shaped flask from step 2). Rotary evaporate under reduced pressure in a 40-50°C water bath for 10-20 minutes until a thin film forms. Rotary evaporate until the solvent is completely evaporated and set aside. 4) Add the protamine short peptide (3.5% by mass) to 100 ml of protective agent aqueous solution, heat to 30-40°C, then add it to the eggplant-shaped flask in step 3) and ultrasonically shake for 1-5 minutes; then add 100 ml of protective agent aqueous solution, stir evenly, and set aside; 5) The hydrated solution was transferred to a homogenizer and cooled to 0-5°C. High-pressure homogenization was performed (parameters 1200-1500 bar, 3 times, 5 minutes each time), filtered, and packaged. The resulting product was placed in a box and pre-frozen at -50±5°C for 2-5 hours. The temperature was raised to -15±5°C and kept warm for 1-3 hours, and then the temperature was lowered to -30±5°C and kept warm for 1-3 hours. The cold trap was opened and the vacuum was turned on. The temperature of the partition was raised to -15-10°C and kept warm for 5-10 hours. The temperature of the partition was raised to -5-0°C and kept warm for 3-5 hours. The temperature of the partition was raised to 30-35°C and kept warm for 3-5 hours. After lyophilization, nitrogen was filled under vacuum to obtain docetaxel liposome lyophilized powder.

[0016] Furthermore, the docetaxel liposome lyophilized powder of the present invention is an anti-tumor drug, and the docetaxel liposome lyophilized powder of the present invention can be used for tumor treatment.

[0017] Furthermore, the tumor described in the present invention is any one of breast cancer, non-small cell lung cancer, prostate cancer, gastric cancer, head and neck squamous cell carcinoma, ovarian cancer, and pancreatic cancer.

[0018] Compared with the prior art, the present invention has the following significant technical advantages: (1) During the liposome construction process, each component optimizes the carrier performance through intermolecular synergy. Egg yolk phosphatidylcholine, as a matrix material, forms a bilayer structure through self-assembly, constructing the basic skeleton for drug encapsulation. Phosphatidylserine effectively improves the physical stability and phase transition properties of the lipid membrane by regulating the molecular arrangement density. Excipients such as taraxasterol, protamine short peptides, and protective agents interact specifically with the main phospholipids, enhancing the integrity and thermodynamic stability of the membrane structure at the molecular level, thereby improving the structural uniformity and storage stability of the entire drug delivery system.

[0019] (2) The physical properties of the docetaxel liposomes and their lyophilized powder of the present invention are excellent. The technical effects of Examples 1-5 are significantly better than those of other examples. By optimizing the formula ratio (2.5-3 mg of egg yolk lecithin and 2.5-3.5 mg of phosphatidylserine), a high encapsulation rate of more than 98% (up to 98.5 ± 2.3%) and particle size control (D90 < 130 nm) were achieved. The synergistic effect of protamine short peptide and taraxasterol significantly enhanced the stability of the lipid membrane. In the absence of protamine phosphatidylserine or taraxasterol, the encapsulation rate of the liposomes was significantly reduced, confirming that the excipients of the present invention are an integral part of the entire invention process.

[0020] (3) The process of the present invention is advanced, using an ethanol-tert-butanol mixed solvent and a gradient freeze-drying process, optimizing the synergistic effect of the protective agent system and the protamine short peptide, and through staged temperature-controlled freeze-drying and nitrogen protection, the process has good reproducibility and is easy to achieve industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : The content (%) of docetaxel liposome lyophilized powder in the relevant examples of the present invention.

[0022] Figure 2 : Dissolution (%) of the liposome freeze-dried powder of the relevant embodiments of the present invention and the commercially available docetaxel liposome freeze-dried powder.

[0023] Figure 3 : Related substances (%) of docetaxel liposome lyophilized powder according to the relevant embodiments of the present invention.

[0024] Figure 4: The permeability (%) of the docetaxel liposome lyophilized powder according to the relevant examples of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose and technical solution of the present invention more clear, the present invention is further described below in conjunction with the embodiments, but the scope of protection of the present invention is not limited to these embodiments, and the embodiments are only used to illustrate the present invention. It should be understood by those skilled in the art that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the scope of protection of the present invention.

[0026] Example 1: Lyophilized powder containing docetaxel liposomes, the formula and preparation process are as follows: Preparation method: 1) Preparation of protective agent aqueous solution: Dissolve 1g glycine and 9g trehalose in 200ml water for injection and set aside. 2) Dissolve egg yolk lecithin, phosphatidylserine, and taraxasterol in a 2:1 weight ratio of ethanol to tert-butanol (60 ml total), place in an eggplant-shaped bottle, and set aside. 3) Dissolve docetaxel in N,N-dimethylformamide, adjust the pH to 5.0 with citric acid, and add the solution to the eggplant-shaped flask prepared in step 2). Rotary evaporate under reduced pressure in a water bath at 40-50°C for 15 minutes until a thin film forms. Rotary evaporate until the solvent is completely evaporated and set aside. 4) Add the protamine short peptide to 100 ml of protective agent aqueous solution (mass fraction of protamine short peptide is 3.5%), hydrate it and heat it to 30°C, then add it to the eggplant-shaped flask in step 3), and ultrasonically vibrate for 1 minute; then add 100 ml of protective agent aqueous solution, stir evenly, and set aside; 5) The hydrated solution was transferred to a homogenizer and cooled to 0°C. High-pressure homogenization was performed (parameters: 1200 bar, 3 times, 5 min each time), filtered, and packaged. The resulting product was placed in a box, cooled to -50±5°C for pre-freezing, and held for 2 hours; heated to -15±5°C, held for 2 hours, and then cooled to -30±5°C and held for 2 hours; the cold trap was opened and the vacuum was turned on; the temperature of the partition was raised to -15-10°C and held for 10 hours; the temperature of the partition was raised to -5-0°C and held for 3 hours; the temperature of the partition was then raised to 30°C and held for 3 hours; after lyophilization, nitrogen was filled under vacuum to obtain docetaxel liposome lyophilized powder.

[0027] Example 2: Lyophilized powder containing docetaxel liposomes, the formula and preparation process are as follows: Preparation method: 1) Preparation of protective agent aqueous solution: Dissolve 1g glycine and 7g trehalose in 200ml water for injection and set aside. 2) Dissolve egg yolk lecithin, phosphatidylserine, and taraxasterol in a 2:1 weight ratio of ethanol to tert-butanol (60 ml total), place in an eggplant-shaped bottle, and set aside. 3) Dissolve docetaxel in N,N-dimethylformamide, adjust the pH to 4.0 with citric acid, and add the solution to the eggplant-shaped flask from step 2). Rotary evaporate under reduced pressure in a water bath at 40-50°C for 10 minutes until a thin film forms. Rotary evaporate until the solvent is completely evaporated and set aside. 4) Add the protamine short peptide to 100ml of protective agent aqueous solution (mass fraction of protamine short peptide is 2%), hydrate it, heat it to 30-40℃, then add it to the eggplant-shaped flask in step 3), and ultrasonically vibrate for 1 minute; then add 100ml of protective agent aqueous solution, stir evenly, and set aside; 5) The hydrated solution was transferred to a homogenizer and cooled to 0-5°C, subjected to high-pressure homogenization (parameter 1200 bar, treatment 3 times, each time 5 minutes), filtered, and packaged. The resulting product was placed in a box, cooled to -50±5°C for pre-freezing, and maintained for 3.5 hours; heated to -15±5°C, maintained for 2 hours, and then cooled to -30±5°C and maintained for 2 hours; opened the cold trap and started the vacuum; heated the partition to -15--10°C and maintained for 7.5 hours; heated the partition to -5-0°C and maintained for 4 hours; then heated the partition to 30-35°C and maintained for 4 hours; after lyophilization, the product was filled with nitrogen under vacuum to obtain docetaxel liposome lyophilized powder.

[0028] Example 3: Lyophilized powder containing docetaxel liposomes, the formula and preparation process are as follows: Preparation method: 1) Preparation of protective agent aqueous solution: Dissolve 1g glycine and 12g trehalose in 200ml water for injection and set aside. 2) Dissolve egg yolk lecithin, phosphatidylserine, and taraxasterol in a 2:1 weight ratio of ethanol to tert-butanol (60 ml total), place in an eggplant-shaped bottle, and set aside. 3) Dissolve docetaxel in N,N-dimethylformamide, adjust the pH to 6.0 with citric acid, and add the solution to the eggplant-shaped flask from step 2). Rotary evaporate under reduced pressure in a 40-50°C water bath for 20 minutes until a thin film forms. Rotary evaporate until the solvent is completely evaporated and set aside. 4) Add the protamine short peptide to 100ml of protective agent aqueous solution (mass fraction of protamine short peptide is 5%), hydrate it, heat it to 30-40℃, then add it to the eggplant-shaped flask in step 3), and ultrasonically shake it for 1-5 minutes; then add 100ml of protective agent aqueous solution, stir evenly, and set aside; 5) The hydrated solution was transferred to a homogenizer and cooled to 0-5°C. High-pressure homogenization was performed (parameters 1200-1500 bar, 3 times, 5 minutes each time), filtered, and packaged. The resulting product was placed in a box, cooled to -50±5°C for pre-freezing, and held for 5 hours; heated to -15±5°C, held for 3 hours, and then cooled to -30±5°C and held for 3 hours; the cold trap was opened and the vacuum was turned on; the temperature of the partition was raised to -15--10°C and held for 10 hours; the temperature of the partition was raised to -5-0°C and held for 5 hours; the temperature of the partition was raised to 30-35°C and held for 5 hours; after lyophilization, nitrogen was filled under vacuum to obtain docetaxel liposome lyophilized powder.

[0029] Example 4: Lyophilized powder containing docetaxel liposomes, the formula and preparation process are as follows: The preparation method is the same as described in Example 1.

[0030] Example 5: Lyophilized powder containing docetaxel liposomes, the formula and preparation process are as follows: The preparation method is the same as described in Example 1.

[0031] Example 6: Lyophilized powder containing docetaxel liposomes, the formula and preparation process are as follows: The preparation method is the same as described in Example 1.

[0032] Example 7: Lyophilized powder containing docetaxel liposomes, the formula and preparation process are as follows: The preparation method is the same as described in Example 1.

[0033] Example 8: Lyophilized powder containing docetaxel liposomes, the formula and preparation process are as follows: The preparation method is the same as described in Example 1.

[0034] Example 9: Lyophilized powder containing docetaxel liposomes, the formula and preparation process are as follows: The preparation method is the same as described in Example 1.

[0035] Example 10: Lyophilized powder containing docetaxel liposomes, the formula and preparation process are as follows: The preparation method is the same as described in Example 1.

[0036] Example 11: Lyophilized powder containing docetaxel liposomes, the formula and preparation process are as follows: The preparation method is the same as described in Example 1.

[0037] Example 12: Lyophilized powder containing docetaxel liposomes, the formula and preparation process are as follows: Preparation method: 1) Preparation of protective agent aqueous solution: Dissolve 1g glycine and 9g trehalose in 200ml water for injection and set aside. 2) Dissolve egg yolk lecithin, phosphatidylserine, and taraxasterol in 60 ml of ethanol, place in an eggplant-shaped bottle, and set aside. 3) Dissolve docetaxel in chloroform, adjust the pH to 5.0 with hydrochloric acid, and add to the eggplant-shaped flask from step 2). Rotary evaporate under reduced pressure in a 40-50°C water bath for 15 minutes until a thin film forms. Rotary evaporate until the solvent is completely evaporated and set aside. 4) Add the protamine short peptide to 100 ml of protective agent aqueous solution (mass fraction of protamine short peptide is 3.5%), hydrate it and heat it to 30°C, then add it to the eggplant-shaped flask in step 3), and ultrasonically vibrate for 1 minute; then add 100 ml of protective agent aqueous solution, stir evenly, and set aside; 5) The hydrated solution was transferred to a homogenizer, cooled to 0°C, and subjected to high-pressure homogenization (parameter 1200 bar, 3 times, 5 min each time), filtered, aliquoted, and dried at a gradient temperature to obtain docetaxel liposome lyophilized powder.

[0038] Confirmatory trial 1: encapsulation efficiency of docetaxel liposomes The appearance, encapsulation efficiency and particle size of the lyophilized powder prepared in the above examples were tested. Samples were taken on day 0, and the encapsulation efficiency of each sample was determined three times. The encapsulation efficiency of docetaxel liposomes was determined by ultrafiltration centrifugation and HPLC, as shown in Table 1.

[0039] Table 1 Encapsulation efficiency of docetaxel liposomes As shown in Table 1, the lyophilized powders containing docetaxel liposomes in Examples 1-5 are superior to those in other Examples in terms of appearance, encapsulation efficiency, and particle size range. This indicates that phosphatidylserine, taraxasterol, protamine short peptide, and the protective agent can synergistically stabilize the lipid membrane, further facilitating the encapsulation of docetaxel.

[0040] Confirmatory test 2: related substances, permeability and content The docetaxel liposome lyophilized powder of Examples 1-12 and commercially available docetaxel lyophilized powder and commercially available docetaxel liposome lyophilized powder were placed at a temperature of 40±2° C. and a relative humidity of 75%±5%. Samples were taken at the 0th month, the 1st month, the 2nd month, the 3rd month, and the 6th month, and the docetaxel content was determined according to the high performance liquid chromatography method (General Chapter 0512) of the Chinese Pharmacopoeia.

[0041] Test solution: Take an appropriate amount of the product, accurately weigh it, dissolve it in the mobile phase, and quantitatively dilute it to a solution containing approximately 0.1 mg per 1 ml. Reference solution: Take a docetaxel reference substance, accurately weigh it, dissolve it in the mobile phase, and quantitatively dilute it to a solution containing approximately 0.1 mg per 1 ml.

[0042] System suitability solution: Dissolve an appropriate amount of docetaxel reference substance in the mobile phase and dilute to a mixed solution containing 50 μg of each substance per 1 mL. Chromatographic conditions: Octadecylsilane bonded silica gel as the filler; 0.043 mol / L ammonium acetate-acetonitrile (55:45 volume ratio) as the mobile phase; detection wavelength at 232 nm; injection volume 10 μL.

[0043] Determination method: Accurately measure the test solution and reference solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the peak area according to the external standard method.

[0044] Table 2 Content of docetaxel liposome lyophilized powder from Figure 1 As can be seen from Table 2, Examples 1-3 performed best, with the content remaining above 97% after 6 months, with a decrease of less than 2.5%, while Examples 7, 9-10: the content decreased to 91.82%-95.85% after 6 months, with a decrease of 3.5%-7.5%. After 6 months of accelerated experiments, Examples 11-12 showed significant degradation, with the content only within 85%, which did not meet the requirements for pharmaceutical use.

[0045] Table 3 Dissolution of docetaxel liposome lyophilized powder (%) from Figure 2As can be seen from Table 3, the sustained release of the Example 1 group was superior, reflecting a longer retention time in the body and sustained efficacy. This was significantly superior to the normal group (95.95%) and the Example 6 group (95.09%), demonstrating a more ideal controlled release capability. The Example 7 group exhibited significantly slower dissolution, reaching only 66.67% after 80 hours, potentially impacting drug bioavailability. The dissolution profiles of the normal group and the Example 6 group were similar, but the Example 6 group exhibited slightly slower dissolution in the early stages (1-24 hours), demonstrating a certain improvement.

[0046] Table 4 Related substances of docetaxel liposome lyophilized powder (%) from Figure 3 The stability analysis shown in Table 4 shows that Examples 1-3 performed best, with the lowest initial impurities and significantly better stability than other example groups; Examples 4-5 were second, with impurities of 0.52%-0.66% after 6 months, which still met general requirements; while Examples 7 and 9-12 had poor stability, among which Example 7 had a faster impurity growth rate, and Example 12 was the worst, with impurities reaching 1.15% after 6 months.

[0047] Verification Example 3: Permeability Experiment Leakage rate refers to the proportion of drug that leaks from the liposomes during storage or reconstitution, reflecting the physical stability of the formulation. Ultrafiltration tubes with a molecular weight cutoff (e.g., 10 kDa) are used for centrifugation to separate the free drug (filtrate) from the liposomes (retained fraction), and drug concentration is measured by HPLC / UV. This directly reflects the integrity of the liposome membrane and predicts the risk of drug leakage during storage. The permeability is calculated as follows: Table 5 Permeability of docetaxel liposome lyophilized powder (%) from Figure 4 As can be seen from Table 5, Examples 1-3 showed the slowest permeability growth, reaching only 4.6%-5.4% at 12 months, indicating that their formulations significantly enhanced lipid membrane stability. Examples 4-5 showed slightly higher permeabilities (6.1%-6.3% at 12 months), possibly due to differences in the protective agent or process, but still superior to Examples 6-12. Examples 6-12 showed poor stability, with permeabilities soaring to 11.5%-16.5% at 12 months, with Examples 6 (16.5%) and 7 (14.2%) showing particularly high permeabilities.

Claims

1. A lyophilized powder containing docetaxel liposomes, characterized in that: The docetaxel liposomes comprise, by weight, 0.5-2.0 parts of docetaxel, 3-5.5 parts of egg yolk lecithin, 2-3 parts of phosphatidylserine, 1.5-3.5 parts of taraxasterol, 2-5 parts of protamine short peptide, 8-13 parts of a protective agent, and an appropriate amount of a pH adjuster; further preferably, the docetaxel liposomes comprise, by weight, 1 part of docetaxel, 4.4 parts of egg yolk lecithin, 2.5 parts of phosphatidylserine, 2.5 parts of taraxasterol, 3.5 parts of protamine short peptide, 10 parts of a protective agent, and an appropriate amount of a pH adjuster.

2. The freeze-dried powder according to claim 1, characterized in that The preparation method of the freeze-dried powder is: 1) Dissolve egg yolk lecithin, phosphatidylserine, and taraxasterol in an organic solvent, place in an eggplant-shaped bottle, and set aside; 2) Dissolve docetaxel in N,N-dimethylformamide, add pH adjuster to an eggplant-shaped flask, and rotary evaporate under reduced pressure in a water bath for 10-20 minutes until a thin film forms. Rotary evaporate until the solvent is completely evaporated and set aside. 3) Add an appropriate amount of protamine short peptide protective agent aqueous solution to hydrate and heat, add it to the eggplant-shaped flask in step 2), and ultrasonically vibrate for 1-5 minutes; then add an appropriate amount of protective agent aqueous solution, stir evenly, and set aside; 4) The hydrated solution is transferred to a homogenizer, cooled to 0-5°C, subjected to high-pressure homogenization, filtered, packaged, and subjected to gradient freeze-drying to obtain a lyophilized powder containing docetaxel liposomes.

3. The freeze-dried powder according to claim 2, characterized in that The organic solvent is a mixture of ethanol and tert-butanol, with a usage ratio of 2:

1.

4. The freeze-dried powder according to claim 1, characterized in that The pH of the pH regulator is 4.0-6.0, and the pH regulator is selected from citric acid.

5. The freeze-dried powder according to claim 1, characterized in that The protective agent aqueous solution is prepared by mixing glycine with one of trehalose, glucose, maltose, raffinose and sucrose and adding the mixture to water for injection for dissolution. Preferably, the protective agent is selected from glycine and trehalose.

6. The freeze-dried powder according to claim 1, characterized in that The ratio of the protective agent selected from glycine and trehalose is 1:7-12. More preferably, the ratio of the protective agent selected from glycine and trehalose is 1:

9.

7. The freeze-dried powder according to claim 2, characterized in that The gradient freeze-drying process comprises the following steps: after the filled product is put into the box, the temperature is lowered to -50±5°C for pre-freezing and maintained for 2-5 hours; the temperature is raised to -15±5°C, kept warm for 1-3 hours, and then the temperature is lowered to -30±5°C and kept warm for 1-3 hours; the cold trap is opened and the vacuum is opened; the partition temperature is raised to -15-10°C and then maintained for 5-10 hours; the partition temperature is raised to -5-0°C and then maintained for 3-5 hours; the partition temperature is then raised to 30-35°C and then maintained for 3-5 hours; after the freeze-drying is completed, nitrogen is filled under vacuum and the product is taken out of the pot.

8. The freeze-dried powder according to claim 1, characterized in that Docetaxel liposome lyophilized powder contains, in parts by weight, 1 g of docetaxel, 4.4 g of egg yolk lecithin, 2.5 g of phosphatidylserine, 2.5 g of taraxasterol, 3.5 g of protamine short peptide, 10 g of a protective agent, and an appropriate amount of a pH adjuster. The preparation method of the lyophilized powder is as follows: 1) Preparation of protective agent aqueous solution: Dissolve 1g glycine and 9g trehalose in 200ml water for injection and set aside. 2) Dissolve egg yolk lecithin, phosphatidylserine, and taraxasterol in a 2:1 weight ratio of ethanol to tert-butanol (60 ml total), place in an eggplant-shaped bottle, and set aside. 3) Dissolve docetaxel in N,N-dimethylformamide, adjust the pH to 5.0 with citric acid, and add the solution to the eggplant-shaped flask from step 2). Rotary evaporate under reduced pressure in a 40-50°C water bath for 10-20 minutes until a thin film forms. Rotary evaporate until the solvent is completely evaporated and set aside. 4) Add the protamine short peptide (3.5% by mass) to 100 ml of protective agent aqueous solution, heat to 30-40°C, then add it to the eggplant-shaped flask in step 3) and ultrasonically shake for 1-5 minutes; then add 100 ml of protective agent aqueous solution, stir evenly, and set aside; 5) The hydrated solution is transferred to a homogenizer and cooled to 0-5°C. High-pressure homogenization is performed (parameters 1200-1500 bar, 3 times, 5 minutes each time), filtered, and packaged. The resulting product is placed in a box and cooled to -50±5°C for pre-freezing, and kept for 2-5 hours. Raise the temperature to -15±5°C, keep warm for 1-3 hours, then cool to -30±5°C and keep warm for 1-3 hours; open the cold trap and start the vacuum; raise the temperature of the partition to -15--10°C, and keep warm for 5-10 hours; raise the temperature of the partition to -5-0°C, and keep warm for 3-5 hours; then raise the temperature of the partition to 30-35°C, and keep warm for 3-5 hours; after the freeze-drying is completed, fill with nitrogen under vacuum to obtain docetaxel liposome freeze-dried powder.

9. Use of the freeze-dried powder according to any one of claims 1 to 8 in the preparation of anti-tumor drugs.

10. The use according to claim 9, wherein the tumor is any one of breast cancer, non-small cell lung cancer, prostate cancer, gastric cancer, head and neck squamous cell carcinoma, ovarian cancer, and pancreatic cancer.

Images