Docetaxel palmitate liposome and preparation method thereof

Abstract

The present invention relates to the technical field of medicine, which provides a liposomal docetaxel palmitate formulation and a method for preparing the said formulation. The liposomal docetaxel palmitate formulation contains docetaxel palmitate as the main drug, a chelating agent, lecithin and DSPE-PEG2000. It is characterized in that docetaxel is prepared into a docetaxel palmitate lipophilic prodrug, thus overcoming the defect that docetaxel cannot be processed into liposomes due to poor hydrophilicity and hydrophobicity. The liposome prescription of this invention contains a chelating agent with a substantive effect that can prolong the action time of the drug in the body, improve the anti-tumor effect and be prepared smoothly. Therefore, the chelating agent in the formulation is the core technical feature of the present invention. The purpose of the present invention is to develop a more efficient docetaxel palmitate liposome with no solubilizer so that it can be prepared more easily

Claims

1. A docetaxel palmitate liposome, which contains docetaxel palmitate as the main drug, a chelating agent, lecithin and DSPE-PEG2000, with a respective content of 0.1-2%, 0.001-1%, 1-10% and 0.05-1%.

2. The docetaxel palmitate liposome according to claim 1, wherein the liposome is a freeze-dried powder injection.

3. The docetaxel palmitate liposome according to claim 1, wherein the liposome is a liposome solution for injection.

4. The docetaxel palmitate liposome according to claim 1, which is specifically formulated by the following: TABLE-US-00004 Docetaxel palmitate 0.1-1% g/mL; Lecithin 1-10% g/mL; DSPE-PEG2000 0.05-1.0% g/mL; Cholesterol 0~1% g/mL; Chelating agent 0.001-1% g/mL; Lyophilized protective agent 0~40% g/mL; pH is adjusted to 3.5~9.0 using a pH adjuster; and the remaining is the water for injection.

5. The docetaxel palmitate liposome according to claim 1, which is specifically formulated by the following: TABLE-US-00005 Docetaxel palmitate 0.1-0.8% g/mL; Lecithin 2-7% g/mL; DSPE-PEG2000 0.1-0.8% g/mL; Cholesterol 0~0.6% g/mL; Chelating agent 0.005-0.8% g/mL; Lyophilized protective agent 5~35% g/mL; pH is adjusted to 3.5~8.0 using a pH adjuster; and the remaining is the water for injection.

6. The docetaxel palmitate liposome according to claim 1, which is specifically formulated by the following: TABLE-US-00006 Docetaxel palmitate 0.2-0.7% g/mL; Lecithin 3-6% g/mL; DSPE-PEG2000 0.2-0.7% g/mL; Cholesterol 0~0.5% g/mL; Chelating agent 0.01-0.5% g/mL; Lyophilized protective agent 10~30% g/mL; pH is adjusted to 3.5~7.0 using a pH adjuster; and the remaining is the water for injection.

7. The docetaxel palmitate liposome according to claim 1, wherein the lecithin is selected from high purity egg yolk lecithin (EPCS), hydrogenated soybean lecithin (HSPC), dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, egg yolk lecithin, soy lecithin, phosphatidylserine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, phospholipids, one or more of acylethanolamine and sphingomyelin.

8. The docetaxel palmitate liposome according to claim 1, is characterized in that the chelating agent is selected from one or more of the following: citric acid, disodium citrate, trisodium citrate, lactic acid, sodium lactate, malic acid, sodium malate, ethylenediamine tetraacetic acid, disodium ethylenediamine tetraacetate and trisodium ethylenediamine tetraacetate.

9. The docetaxel palmitate liposome according to claim 4, wherein the freeze-dried protective agent is selected from one or more of the following: trehalose, sucrose, maltose, lactose, mannitol, glucose, sorbitol, xylitol, erythritol, and threonine.

10. The docetaxel palmitate liposome according to claim 4, wherein the pH adjusting agent is one or more of sodium hydroxide and hydrochloric acid.

11. The docetaxel palmitate liposome according to claim 1, wherein the liposome has a particle size of 50-150 nm.

12. The preparation method of docetaxel palmitate liposomes according to claim 1, wherein the preparation method is as follows: Weigh the prescription amount of docetaxel palmitate, cholesterol, phospholipid, DSPE-PEG2000, chelating agent, put them in an organic solvent for injection and dissolve them by heating at 25-70 C. to obtain the organic phase; heat a proper amount of water to 25-70 C. to obtain the water phase; pour the organic phase into the water phase under stirring and mix them well to obtain crude liposomes; emulsify the crude liposomes and place them under high pressure; perform homogenization and emulsification in a homogenizer, or place them in an extruder to extrude through extruded membranes with different pore diameters, or extrude after high-pressure homogenization to obtain a liposome solution; lyophilized protective agent, place it in the above liposome solution and dissolve it by stirring and dilute it to the full volume with water for injection; adjust the pH value with a pH adjuster; finally, sterilize, pack and seal it through a 0.22 m filter membrane to obtain so-called liposomes of liposome docetaxel palmitate. A powder form of docetaxel palmitate liposomes can also be prepared by lyophilization.

13. The method for preparing docetaxel palmitate liposomes according to claim 12, wherein the organic solvent for injection is selected from one, two or more of the following: propylene glycol, absolute ethanol, and tert-butanol at a dose of 1-10% g/mL.

14. The method for preparing docetaxel palmitate liposomes according to claim 12, wherein the organic solvent for injection can be retained in liposomes or in crude liposomes. After emulsification, it can be removed by ultrafiltration, or freeze-drying.

15. The method for preparing docetaxel palmitate liposomes according to claim 12, characterized in that the crude liposomes are emulsified, and the pore size of the extruded membrane is selected from among 0.8 m, 0.6 m, 0.4 m, 0.2 m, 0.1 m and 0.05 m by one, two or more in turn through extrusion of large pores to small pores.

16. The method for preparing docetaxel palmitate liposomes according to claim 12, wherein the chelating agent is dissolved in an oil phase, an aqueous phase or a liposome solution.

17. The method for preparing docetaxel palmitate liposomes according to claim 12, wherein the lyophilized protective agent is dissolved in a liposome solution, or an aqueous phase.

18. The docetaxel palmitate liposome according to claim 2, which is specifically formulated by the following: TABLE-US-00007 Docetaxel palmitate 0.1-1% g/mL; Lecithin 1-10% g/mL; DSPE-PEG2000 0.05-1.0% g/mL; Cholesterol 0~1% g/mL; Chelating agent 0.001-1% g/mL; Lyophilized protective agent 0~40% g/mL; pH is adjusted to 3.5~9.0 using a pH adjuster; and the remaining is the water for injection.

19. The docetaxel palmitate liposome according to claim 3, which is specifically formulated by the following: TABLE-US-00008 Docetaxel palmitate 0.1-1% g/mL; Lecithin 1-10% g/mL; DSPE-PEG2000 0.05-1.0% g/mL; Cholesterol 0~1% g/mL; Chelating agent 0.001-1% g/mL; Lyophilized protective agent 0~40% g/mL; pH is adjusted to 3.5~9.0 using a pH adjuster; and the remaining is the water for injection.

20. The docetaxel palmitate liposome according to claim 2, which is specifically formulated by the following: TABLE-US-00009 Docetaxel palmitate 0.1-0.8% g/mL; Lecithin 2-7% g/mL; DSPE-PEG2000 0.1-0.8% g/mL; Cholesterol 0~0.6% g/mL; Chelating agent 0.005-0.8% g/mL; Lyophilized protective agent 5~35% g/mL; pH is adjusted to 3.5~9.0 using a pH adjuster; and the remaining is the water for injection.

Description

SPECIFIC IMPLEMENTATION MODES

[0061] The following is a detailed description about the present invention in conjunction with specific embodiments. It should be understood that the following examples are only used to illustrate the present invention and not to limit the scope of the present invention.

Example 1

Preparation of Docetaxel Palmitate Liposomes

[0062] [01] The organic phase was prepared with the prescription amount of 0.5 g docetaxel palmitate, 3 g high-purity egg yolk lecithin (EPCS), 0.3 g DSPE-PEG2000 and 4 g absolute ethanol. The mixture was dissolved by heating at 50 C. 0.05 g disodium ethylenediaminetetraacetic acid was put in 90 g water for injection and heated at 50 C. The resulting mixture was stirred to obtain an aqueous phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were then placed in an extruder and separated through extruded membranes with a pore diameter of 0.4 m, 0.1 m and 0.05 m to obtain liposome solution. The solution was diluted to 100 ml with water for injection, and the phase. The pH value was adjusted to 4.50 with hydrochloric acid. The liposomes were filtrated and sterilized through a 0.22 m nylon syringe filter. The obtained filtrate was then separately packaged and cap-sealed to obtain docetaxel palmitate liposome solution with a mean particle size of 92.4 nm.

Example 2

Preparation of Docetaxel Palmitate Liposomes

[0063] [02] The organic phase was prepared with the prescription amount of 0.3 g docetaxel palmitate, 3 g high-purity egg yolk lecithin (EPCS), 0.2 g DSPE-PEG2000, 0.1 g citric acid and 4 g propylene glycol. The mixture was dissolved by heating at 60 C. 70 g water for injection was heated at 60 C. to obtain a water phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were then placed in an extruder and sequentially passed through a nylon syringe filter of 0.4 m, 0.2 m, 0.1 m and 0.05 m to obtain liposome solution. 15 g saccharose and 5 g mannitol were dissolved in the liposome solution by stirring and diluted to 100 mL with water for injection. The pH value was adjusted to 5.50 with natrium hydroxydatum. The liposomes were filtrated and sterilized through a 0.22 m nylon syringe filter, and the obtained filtrate was separately packaged, freeze-dried and cap-sealed to obtain a liposomal docetaxel palmitate freeze-dried powder with a mean particle size of 86.6 nm.

Example 3

Preparation of Docetaxel Palmitate Liposomes

[0064] [03] The organic phase was prepared with the prescription amount of 0.7 g docetaxel palmitate, 6 g high-purity egg yolk lecithin (EPCS), 0.5 g DSPE-PEG2000, 0.3 g citric acid and 6 g absolute ethanol. The mixture was dissolved by heating at 45 C. 65 g water for injection was heated to 45 C. to obtain a water phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were then placed in an extruder and sequentially passed through a nylon syringe filter with a pore diameter of 0.6 m, 0.4 m and 0.1 m to obtain liposome solution. 20 g trehalose was dissolved in the liposome solution by stirring and diluted to 100 mL with water for injection. The pH value was adjusted to 6.20 with natrium hydroxydatum. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter. Then, the filtrate was separately packaged, freeze-dried and cap-sealed to obtain a liposomal docetaxel palmitate freeze-dried powder with a mean particle size of 120.7 nm.

Example 4

Preparation of Docetaxel Palmitate Liposomes

[0065] [04] The organic phase was prepared with the prescription amount of 0.3 g docetaxel palmitate, 5 g high-purity egg yolk lecithin (EPCS), 0.3 g DSPE-PEG2000, 0.1 g malic acid,0.2 g citric acid and 5 g absolute ethanol. The mixture was dissolved by heating at 65 C. 70 g water for injection was heated to 65 C. to obtain a water phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were homogenized and emulsified by using a high pressure homogenizer, and then sequentially extruded with the extrusion film with a pore diameter of 0.1 m and 0.05 m to obtain liposome solution. 10 g saccharose and 5 gtrehalose were dissolved in the liposome solution by stirring and diluted to 100 ml with water for injection. The pH value was adjusted to 6.0 with natrium hydroxydatum. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter, and the filtrate was then separately packaged, freeze-dried and cap-sealed to obtain a liposomal docetaxel palmitate freeze-dried powder with a mean particle size of 50.36 nm.

Example 5

Preparation of Docetaxel Palmitate Liposomes

[0066] [05] The organic phase was prepared with the prescription amount of 0.2 g docetaxel palmitate, 3 g high-purity egg yolk lecithin (EPCS), 0.2 g DSPE-PEG2000, 0.01 g citric acid and 3 g absolute ethanol. The mixture was dissolved by heating at 65 C. 75 g water for injection was heated to 50 C. to obtain a water phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were homogenized and emulsified solution by using a high pressure homogenizer to obtain liposome. 10 g saccharose and 5 mannitol were dissolved in the liposome solution by stirring and diluted to 100 ml with water for injection. The pH value was adjusted to 7.0 with natrium hydroxydatum. The liposome was filtrated and sterilized through a 0.22 lam nylon syringe filter. Then, the filtrate was separately packaged, freeze-dried and cap-sealed to obtain docetaxel palmitate liposome solution with a mean particle size of 60.7 nm.

Example 6

Preparation of Docetaxel Palmitate Liposomes

[0067] [06] The organic phase was prepared with the prescription amount of 0.7 g docetaxel palmitate, 6 g high-purity egg yolk lecithin (EPCS), 0.7 g DSPE-PEG2000, 0.5 g cholesterol, 0.5 g citric acid and 6 g absolute ethanol. The mixture was dissolved by heating at 55 C. 80 g water for injection was heated to 55 C. to obtain a water phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were homogenized and emulsified by using a high-pressure homogenizer to obtain liposome solution. The obtained liposome solution was diluted to 100 ml with water for injection. The pH value was adjusted to 4.80 with natrium hydroxydatum. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter. Then, the filtrate was separately packaged, and cap-sealed to obtain docetaxel palmitate liposome solution with a mean particle size of 80.4 nm.

Example 7

Preparation of Docetaxel Palmitate Liposomes

[0068] [07] The organic phase was prepared with the prescription amount of 0.7 g docetaxel palmitate 2 g egg yolk lecithin, 1 g hydrogenated soy lecithin (HSPC), 0.5 g DSPE-PEG2000, 0.1 g cholesterol and 4 g absolute ethanol. The mixture was dissolved by heating at 55 C. 0.2 g trisodium citrate, 10 g trehalose, 12 g mannitol, 8 g glucose and 90 g water for injection were mixed and heated to 55 C. to obtain an aqueous phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were separated via extruded membranes with a pore diameter of 0.8 m, 0.4 m and 0.2 m to obtain liposome solution. The obtained liposome solution was diluted to 100 ml with water for injection. The pH value was adjusted to 4.50 with hydrochloric acid regulator. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter. Then, the filtrate was separately packaged, freeze-dried and cap-sealed to obtain a liposomal docetaxel palmitate freeze-dried powder with a mean particle size of 150.0 nm.

Example 8

Preparation of Docetaxel Palmitate Liposomes

[0069] [08] The organic phase was prepared with the prescription amount of 0.8 g docetaxel palmitate, 3 g dipalmitoylphosphatidylcholine (DPPC), 3 g phosphatidylcholine, 1 g egg yolk lecithin, 0.8 g DSPE-PEG2000 and 8 g propylene glycol. The mixture was dissolved by heating at 70 C. 80 g water for injection was heated to 55 C. to obtain a water phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were separated through the extruded membranes with a pore diameter of 0.8 m, 0.4 m, 0.2 m and 0.1 m to obtain liposome solution and propylene glycol was removed by ultrafiltration. 0.8 g trisodium citrate was placed in the liposome solution after ultrafiltration, stirred thoroughly and then diluted to 100 ml with water for injection. The pH value was adjusted to 9.0 with hydrochloric acid. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter, and the filtrate was separately packaged and cap-sealed to obtain docetaxel palmitate liposome solution with a mean particle size of 145.2 nm.

Example 9

Preparation of Docetaxel Palmitate Liposomes

[0070] [09] The organic phase was prepared with the prescription amount of 0.1 gdocetaxel palmitate, 2 g soy lecithin, 0.1 g DSPE-PEG2000 and 6 g absolute ethanol. The mixture was stirred to dissolve by heating at 25 C. 0.5 g trisodium diaminetetraacetic acid, 0.5 g disodium citrate and 80 g water for injection were heated at 25 C. and stirred thoroughly to obtain the water phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes. The crude liposome was separated through extruded membranes with a pore diameter of 0.6 m, 0.2 m, 0.1 m and 0.05 m to obtain liposome solution, which was then diluted to 100 ml with water for injection. The pH value was adjusted to 3.50 with hydrochloric acid. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter, and the filtrate was separately packaged and cap-sealed to obtain docetaxel palmitate liposome solution with a mean particle size of 118.8 nm.

Example 10

Preparation of Docetaxel Palmitate Liposomes

[0071] [10] The organic phase was prepared with the prescription amount of 1.0 g docetaxel palmitate, 10 g high-purity egg yolk lecithin (EPCS), 1.0 g DSPE-PEG2000, 1 g cholesterol and 10 g absolute ethanol. The mixture was heated at 60 C. while stirring. 74 g water for injection was heated at 60 C. to obtain the water phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were homogenized and emulsified by using a high pressure homogenizer to obtain liposome solution, and absolute ethyl alcohol was removed by ultrafiltration. 0.5 g natrium lacticum and 0.5 g natrium malicum were placed in the liposome solution after ultrafiltration, stirred thoroughly and then diluted to 100 ml with water for injection. The pH value was adjusted to 5.0 with hydrochloric acid. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter, and the filtrate was separately packaged and cap-sealed to obtain docetaxel palmitate liposome solution with a mean particle size of 130.2 nm.

Example 11

Preparation of Docetaxel Palmitate Liposomes

[0072] [11] The organic phase was prepared with the prescription amount of 0.1 g docetaxel palmitate, 1 g distearoylphosphatidylcholine, 0.05 g DSPE-PEG2000 and 1 g absolute ethanol. The mixture was heated at 55 C. while stirring. 95 g water for injection was heated at 55 C. to obtain the aqueous phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes. The crude liposome was homogenized and emulsified by using a high pressure homogenizer to obtain liposome solution. 0.001 g natrium lacticum was placed in the liposome solution, stirred thoroughly and then diluted to 100 ml with water for injection. The pH value was adjusted to 8.0 with natrium hydroxydatum. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter, and the filtrate was separately packaged, freeze-dried and cap-sealed to obtain a liposomal docetaxel palmitate freeze-dried powder with a mean particle size of 90.7 nm.

Example 12

Preparation of Docetaxel Palmitate Liposomes

[0073] [12] The organic phase was prepared with the prescription amount of 0.1 g docetaxel palmitate, 1 g phosphatidylethanolamine, 1 g dimyristoylphosphatidylcholine, 0.5 g DSPE-PEG2000, 0.005 g ethylenediaminetetraacetic acid and 4 g absolute ethanol. The mixture was dissolved by heating at 55 C. 5 g trehalose was put in 70 g water for injection and heated at 55 C. The mixture was dissolved by stirring to obtain an aqueous phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were then placed in an extruder and separated through extruded membranes with a pore diameter of 0.8 m, 0.6 m, 0.4 m and 0.1 m to obtain liposome solution. The obtained solution was diluted to 100 ml with water for injection. The pH value was adjusted to 7.5 with sodium hydroxide. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter, and the filtrate was separately packaged, freeze-dried and cap-sealed to obtain a liposomal docetaxel palmitate freeze-dried powder with a mean particle size of 104.3 nm.

Example 13

Preparation of Docetaxel Palmitate Liposomes

[0074] [13] The organic phase was prepared with the prescription amount of 0.2 g docetaxel palmitate, 1 g phosphatidylserine, 1 g sphingomyelin, 0.2 g DSPE-PEG2000 and 0.01 g citric acid in a mixed solvent of 2 g absolute ethanol and 4 g propylene glycol. 50 g water was heated to 70 C. to obtain a water phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were then homogenized and emulsified with a high-pressure homogenizer to obtain a liposome solution. 15 g sucrose, 15 g mannitol, 5 g erythritol and 5 g threonine were dissolved in the liposome solution by stirring and then diluted to 100 ml with water for injection. The pH value was adjusted to 6.0 with sodium hydroxide. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter, and the filtrate was separately packaged, freeze-dried and cap-sealed to obtain a liposomal docetaxel palmitate freeze-dried powder with a mean particle size of 78.4 nm.

Example 14

Preparation of Docetaxel Palmitate Liposomes

[0075] [14] The organic phase was prepared with the prescription amount of 0.4 g docetaxel palmitate, 5 g high-purity egg yolk lecithin (EPCS), 0.5 g DSPE-PEG2000 and 0.01 g citric acid. The mixture was dissolved by heating at 50 C. 50 g water was heated to 50 C. to obtain a water phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were then homogenized and emulsified with a high-pressure homogenizer, placed in an extruder, and separated through extruded membranes with a pore diameter of 0.2 m, 0.1 m, and 0.05 m to obtain liposome solution. 10 g xylitol, 15 g sorbitol and 10 g mannitol were dissolved in the liposome solution by stirring and diluted to 100 ml with water for injection. The pH value was adjusted to 6.0 with sodium hydroxide. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter and the filtrate was separately packaged, freeze-dried and cap-sealed to obtain a liposomal docetaxel palmitate freeze-dried powder with a mean particle size of 140.7 nm.

Example 15

Preparation of Docetaxel Palmitate Liposomes

[0076] [15] The organic phase was prepared with the prescription amount of 0.3 g docetaxel palmitate, 3 g high-purity egg yolk lecithin (EPCS), 0.1 g DSPE-PEG2000 and 5 g tert-butanol. The mixture was dissolved by heating at 45 C. 80 g water was heated to 45 C. to obtain a water phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were then homogenized and emulsified with a high-pressure homogenizer to obtain liposome solution. 0.1 g trisodium citrate, 13 g sucrose and 5 g mannitol were dissolved in the liposome solution by stirring and then diluted to 100 ml with water for injection. The pH value was adjusted to 5.5 with hydrochloric acid. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter, and the filtrate was separately packaged, freeze-dried and cap-sealed to obtain a liposomal docetaxel palmitate freeze-dried powder injection with a mean particle size of 105.3 nm.

Example 16

Preparation of Docetaxel Palmitate Liposomes

[0077] [16] The organic phase was prepared with the prescription amount of 0.3 g docetaxel palmitate, high purity egg yolk lecithin (EPCS) 1.5 g, hydrogenated soybean lecithin (HSPC) 0.5 g, 0.1 g DSPE-PEG2000 and 6 g absolute ethanol. The mixture was dissolved by heating at 55 C. 60 g water was heated to 55 C. to obtain a water phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were then homogenized and emulsified with a high-pressure homogenizer to obtain liposome solution, with absolute ethanol removed by ultrafiltration. 0.3 g disodium edetate, 19 g trehalose and 5 g lactose were then dissolved in the liposome solution by stirring and diluted to 100 ml with water for injection. The pH value was adjusted to 7.0 with sodium hydroxide. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter, and the filtrate was separately packaged, freeze-dried and cap-sealed to obtain a liposomal docetaxel palmitate freeze-dried powder injection with a mean particle size of 89.4 nm.

Example 17

Preparation of Docetaxel Palmitate Liposomes

[0078] [17] The organic phase was prepared with the prescription amount of 0.3 g docetaxel palmitate lipid, 3 g high-purity egg yolk lecithin (EPCS), 0.7 g DSPE-PEG2000, 0.1 g citric acid and 4 g propylene glycol. The mixture was dissolved by heating at 70 C. 10 g sucrose and 5 g trehalose were put in 70 g water for injection and heated at 70 C. The obtained mixture was dissolved by stirring to obtain an aqueous phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes, which were then placed in an extruder and separated through extruded membranes with a pore diameter of 0.8 m, 0.6 m, 0.4 m, and 0.1 m to obtain liposome solution. It was diluted to 100 ml with water for injection. The pH value was adjusted to 6.0 with sodium hydroxide. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter. Then, the filtrate was separately packaged, freeze-dried and cap-sealed to obtain a liposomal docetaxel palmitate freeze-dried powder with a mean particle size of 113.6 nm.

Example 18

Preparation of Docetaxel Palmitate Liposomes

[0079] [18] Organic phase was prepared with the prescription amount of docetaxel palmitate 0.3 g, 3 g high-purity egg yolk lecithin (EPCS), 0.5 g DSPE-PEG2000, 0.01 g citric acid and 4 g propylene glycol. The mixture was dissolved by heating at 60 C. 17 g sucrose, 5 g mannitol was put in 65 g water for injection then heated at 70 C., in which the mixture was dissolved by stilling to obtain an aqueous phase. The organic phase was injected into the water phase under stirring conditions to obtain crude liposomes. The crude liposome was placed in an extruder and separated through extruded membranes with a pore diameter of 0.6 m, 0.4 m, 0.1 m, and 0.05 m to obtain liposome solution. The obtained solution was then diluted to 100 ml with water for injection. The pH value was adjusted to 6.7 with sodium hydroxide. The liposome was filtrated and sterilized through a 0.22 m nylon syringe filter, and the obtained filtrate was separately packaged, freeze-dried and cap-sealed to obtain a liposomal docetaxel palmitate freeze-dried powder with a mean particle size of 106.3 nm.

Example 19

Effect of Chelating Agent on Anti-Tumor Effect of Docetaxel Palmitate Liposome

[0080] [19] The chelating agent contained in the prescription was the key to the substantial effect of the docetaxel palmitate liposome of the present invention. In order to further verify the superiority of the chelating agent in the present invention, multiple parallel comparisons were used to preparate liposomes containing the chelating agent and chelating agent-free liposomes under the same processing conditions, using mouse S180 sarcoma as a tumor model. The anti-tumor effects were compared between docetaxel palmitate liposomes containing and those without the chelating agent. The experimental design and results were shown below.

1. Sample Source

[0081] The commercially available docetaxel injection was used as a positive control drug, and the chelating agent-containing docetaxel palmitate liposome prepared in Example 1 was used as the test preparation. By strictly following the prescription of Example 1 in the process, liposomes without the chelating agent were prepared in parallel as control.

2. Establishment of the Mouse S180 Tumor Model and Design of the Dosing Regimen

[0082] Mouse ascites tumor S180 cells were cultured in DMEM medium at 37 C. and 5% CO.sub.2, and passaged at a mean interval of 2 days. When cells grew to the logarithmic growth phase, they were injected into the abdominal cavity of the mice under aseptic conditions at an adjusted concentration of 510.sup.7 cells/mL. When obvious ascites was observed in about a week, ascites was drawn from the tumor-bearing mice aseptically and diluted with NS at an appropriate ratio of 1:5. The diluted ascites (0.2 mL) was inoculated into the mouse abdominal cavity. When the second-generation ascites was visible in about a week, it was drawn from the tumor-bearing mice aseptically, diluted with NS at a 1:5 ratio and prepared into a S180 cell suspension, which was then injected into the left armpit of the mice subcutaneously, 0.2 mL per mouse.

[0083] 24 h after inoculation, the ICR mice were weighed and randomly divided into four groups (n=8/group): a blank control group, a commercial docetaxel injection group, a chelating agent-free docetaxel group, and a chelating agent-containing docetaxel group. The mice in the three docetaxel groups received 10 mg/kg docetaxel-based injection via the tail vein each time, and the mice in the blank control group received 0.2 ml NS daily, for a total of four administrations. On the third day of drug withdrawal, the mice were sacrificed and weighed, and the tumors were removed and weighed to calculate the tumor inhibition rate using the following equation:

Tumor inhibition rate=(tumor weight in NS group-tumor weight in the drug administration group)/tumor weight in NS group100%

3. The Anti-Tumor Effect

[0084] Using mouse S180 sarcoma as a model, the anti-tumor effects of docetaxel palmitate liposomes with and without the chelating agent and the commercial docetaxel injection were investigated. The results are shown in Table 1.

TABLE-US-00001 TABLE 1 Docetaxel palmitate liposomes with and without the chelating agent and comparative results of the anti- tumor effect of the commercial docetaxel injection Mean tumor Tumor Group weight (g) inhibition rate Blank control 1.17 0.49 / Commercial docetaxel injection 0.44 0.21 62.39% Docetaxel palmitate liposomes without 0.30 0.11 74.36% the chelating agent Docetaxel palmitate liposomes with 0.21 0.06 82.05% the chelating agent

Result Analysis:

[0085] 1. The anti-tumor effect of docetaxel palmitate liposomes with and without the chelating agent was significantly better than that of the commercial docetaxel injection, indicating that docetaxel was successfully developed into a prodrug docetaxel. The anti-tumor effect was significantly improved after modification with cypalmitate liposome, which is an important aspect of the substantial effect of the present invention.

[0086] 2. The anti-tumor effects of docetaxel palmitate liposomes with and without the chelating agent were compared in parallel. The results showed that the anti-tumor effect of the liposomes with the chelating agent was better than that of the liposomes without the chelating agent.

[0087] In conclusion, the anti-tumor effect of docetaxel palmitate liposomes containing the chelating agent is improved as compared with those without, which is the embodiment of the substantial effect and the core technical feature of the present invention.

Example 20

Effect of the Chelating Agent on Pharmacokinetics of Docetaxel Palmitate Liposomes In Vivo

[20] 1. Sample Source

[0088] A commercially available docetaxel injection was used as the reference preparation, the docetaxel palmitic acid liposome containing the chelating agent prepared completely according to the preparation process described in Example 1 was used as the chelating agent-containing docetaxel palmitic acid liposome sample, and the docetaxel palmitic acid liposome without containing the chelating agent prepared completely according to the preparation process described in Example 1 was used as the chelating agent-free docetaxel palmitic acid liposome sample.

2. Pharmacokinetic Test Design

[0089] Eighteen SD male rats were equally randomized into three groups: a commercial docetaxel injection group, a group of docetaxel palmitic acid liposome without the chelating agent, and a group of docetaxel palmitic acid liposome containing the chelating agent. Before the experiment, the animals were fasted overnight with free access to drinking water, and then received 10 mg/kg docetaxel-based injection via the tail vein. At 0.033, 0.083, 0.167, 0.25, 0.5, 0.75, 1 1.5, 2, 3, 4, 6, 8, 10, 12 and 24 h after drug administration, 0.5 mL blood was drawn from the orbital venous plexus, placed in a centrifuge tube containing heparin sodium, shaken well, and centrifuged at 4500 rpm for 10 min. 150 l plasma was taken, stored at 20 C., and processed according to the conventional method. The plasma concentration of docetaxel was determined by high performance liquid chromatography (HPLC).

3. Results and Analysis

[0090] DAS 2.0 software was used for model fitting, and the pharmacokinetic parameters were calculated. The in vivo pharmacokinetic results of the commercial docetaxel injection, chelating agent-containing docetaxel palmitate liposomes and chelating agent-free docetaxel palmitate liposomes are shown in Table 2.

TABLE-US-00002 TABLE 2 Comparison of the main pharmacokinetic parameters between the commercial docetaxel injection group, chelating agent- containing docetaxel palmitate liposome group and chelating agent-free docetaxel palmitate liposome group Docetaxel Docetaxel palmitate palmitate Commercial liposome liposome docetaxel without the with the Index Unit injection chelating agent chelating agent AUC.sub. g/mL*h 4.729 18.210 22.112 C.sub.max g/mL 6.921 3.061 2.824 T.sub.1/2 h 5.902 9.315 10.425 CL L/h/kg 2.115 0.549 0.494 MRT h 1.232 3.744 4.891

Result Analysis:

[0091] 1. AUCs and T of docetaxel palmitate liposomes were significantly larger than those of the commercially available docetaxel palmitate injections. The results of this experiment demonstrated that the docetaxel into prodrug liposome preparation was able to delay the metabolism of the drug in the body and prolong the action time remarkably.

[0092] 2. Compared with docetaxel palmitate liposomes without chelating agents, AUC(x) was increased and T was prolonged in docetaxel palmitate liposomes containing the chelating agent. From the perspective of the pharmacological effect, liposomes containing the chelating agent had a better anti-tumor effect, perhaps because they have a relatively longer action time in the body.

[0093] In conclusion, after addition of the chelating agent to the formulation of docetaxel palmitate liposomes, the in vivo action time of the drug was prolonged and the anti-tumor effect was improved, indicating that the chelating agent in the prescription plays a particularly important role in the docetaxel palmitate liposome of the present invention and is the key technical feature of the prevent invention.

Example 21

Effect of the Chelating Agent on the Characteristics of the Docetaxel Palmitate Liposome Formulation

[0094] [21] As the research object of the present invention is a kind of liposome which cannot be sterilized at high temperature during the production process, sterilization is usually affected by filtration via a 0.22 m filter membrane. In the actual production process, the large liposome particle size or uneven PDI often results in poor sterilization and filtration, which seriously affects the production efficiency. For this reason, we paid special attention to the smoothness of filtration and sterilization of the docetaxel palmitate liposomes during the research process and found that addition of the chelating agent could make the filtration process more smooth, because both the particle size and PDI of the liposomes with the chelating agent are slightly smaller than those without the chelating agent. The experimental design and results are shown below.

[0095] In Example 1 for instance, 1000 ml docetaxel palmitate liquid liposomes with and without the chelating agent were prepared completely according to the recipe described in Example 1 by using an 11 mm plate filter and a 0.22 m polyethersulfone membrane. The filtration volume was recorded. The particle size and PDS of the docetaxel palmitate liquid liposomes with and without the chelating agent were measured and the results are shown in Table 3

TABLE-US-00003 TABLE 3 Comparison of the characteristics of the docetaxel palmitate liquid liposomes with and without the chelating agent Docetaxel palmitate Docetaxel palmitate liposomes without liposomes with Index the chelating agent the chelating agent Mean particle size 102.1 nm 92.4 nm PDI 0.237 0.124 Filter volume 625 ml 867 ml

Result Analysis:

[0096] The docetaxel palmitate liposome of the present invention has smaller particle size, narrower distribution, and smoother sterilization filtration. It can be seen that after addition of the chelating agent, the basic properties of the formulation are significantly improved and the production is implemented more smoothly, which further reflects the superiority of the chelating agent contained in the prescription.

[0097] We have described the preferred embodiment of the present invention in detail but the present invention is not limited to the embodiment described. Technicians and researchers who are familiar with the trade can make various equivalents as long as they do not violate the spirit of the present invention and these equivalent variations or replacements are all included in the scope defined by the claims of this application.