METHOD FOR PREPARING LIPOSOME

Abstract

A method for preparing a liposome, comprising the step of: (1) dissolving a substance to be encapsulated and phospholipid in an organic solvent to obtain an organic phase, and then mixing the organic phase with water to obtain a liposome feed liquid; (2) extruding the liposome feed liquid obtained in step (1) by means of a polycarbonate membrane; and (3) lyophilizing same.

Claims

1. A method for preparing a liposome, comprising (1) dissolving moexitecan and a phospholipid in an organic solvent to obtain an organic phase, and then mixing the organic phase with an aqueous phase to obtain a liposome liquid; (2) extruding the liposome liquid through a polycarbonate membrane; and (3) lyophilizing, wherein the phospholipid is a combination of yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine and a weight ratio of yolk phosphatidylcholine to hydrogenated soybean phosphatidylcholine of 3:1; and the organic solvent is selected from the group consisting of anhydrous ethanol and 95% ethanol.

2. The method of claim 1, wherein a lyoprotectant is added to the aqueous phase in step (1) or before performing the lyophilization in step (3).

3. The method of claim 1, wherein a weight ratio of moexitecan to the phospholipid in step (1) is 1:1-100.

4. The method of claim 1, wherein a weight ratio of moexitecan to the organic solvent in step (1) is 1:9-50.

5. The method of claim 1, wherein the aqueous phase comprises water as a major component or substantially consists of water.

6. The method of claim 1, wherein the aqueous phase further comprises a metal ion chelating agent, which is selected from the group consisting of disodium edetate, sodium calcium edetate, 1,2-diaminocyclohexane tetraacetic acid, diethylenetriamine pentaacetic acid, trisodium N-(2-hydroxyethyl)-ethylenediamine triacetate, and N-di(2-hydroxyethyl)glycine.

7. The method of claim 1, wherein the organic phase is mixed with the aqueous phase in step (1) at a temperature of 55-65 C.

8. The method of claim 1, wherein a temperature of the liposome liquid in step (2) is controlled at 55-65 C.

9. The method of claim 2, wherein the lyoprotectant is one or more selected from the group consisting of mannitol, glucose, galactose, sucrose, lactose, maltose, and mycose.

10. The method of claim 1, wherein the organic phase in step (1) further comprises an antioxidant, which is one or more selected from the group consisting of sodium sulfite, sodium bisulfite, sodium pyrosulfite, sodium thiosulfate, vitamin C, ascorbyl palmitate, tert-butyl-4-hydroxyanisole, di-tert-butyl-4-hydroxytoluene, vitamin E acetate, cysteine, and methionine.

11. The method of claim 1, wherein a pH regulator may be further added before performing the lyophilization in step (3), and the pH regulator is selected from the group consisting of hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, citric acid, tartaric acid, maleic acid, sodium hydroxide, sodium bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium citrate.

12. The method of claim 1, wherein the polycarbonate membrane has a pore size of 0.1 m or 0.2 m.

13. The method of claim 1, wherein a weight ratio of moexitecan to the phospholipid in step (1) is 1:15-50; a weight ratio of moexitecan to the organic solvent in step (1) is 1:9-50; the aqueous phase is water for injection; the organic phase is mixed with the aqueous phase in step (1) at a temperature of 55-65 C.; a pore size of the polycarbonate membrane is 0.1 m or 0.2 m; a temperature of the liposome liquid in step (2) is controlled at 55-65 C.; the lyoprotectant is selected from sucrose or a combination of sucrose and mannitol, wherein a weight ratio of sucrose to mannitol is 2:1.

14. A liposome prepared by the method of claim 1, wherein the liposome has an entrapment efficiency >99%, and is reconstituted after the addition of water or an aqueous solvent, and the reconstituted liposome has a particle size of 50-400 nm.

15. The liposome of claim 14, wherein the liposome has a particle size distribution index of below 0.18.

16. The liposome of claim 14, wherein the reconstituted liposome has a particle size of 100-250 nm.

17. A method for preparing a liposome, comprising (1) dissolving moexitecan and a phospholipid in an organic solvent to obtain an organic phase, and then mixing the organic phase with an aqueous phase to obtain a liposome liquid; (2) extruding the liposome liquid obtained in step (1) through a polycarbonate membrane; and (3) adding water for injection, sterilizing by filtration, subpackaging and lyophilizing, wherein the phospholipid is a combination of yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine, and wherein a lyoprotectant is added to the aqueous phase in step (1) or before performing the sterilization by filtration in step (3), wherein a weight ratio of yolk phosphatidylcholine to hydrogenated soybean phosphatidylcholine is 3:1.

18. The method of claim 17, wherein the organic solvent is selected from the group consisting of anhydrous ethanol and 95% ethanol.

19. The method of claim 17, wherein the polycarbonate membrane has a pore size of 0.1 m or 0.2 m.

20-21. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1 is a graph showing the tissue distribution of moexitecan in vivo after intravenous injection of 30 mg/kg moexitecan liposome into a rat.

[0052] FIG. 2 is a graph showing the tissue distribution of an active metabolite SN38 in vivo after intravenous injection of 30 mg/kg moexitecan liposome into a rat.

[0053] FIG. 3 is a graph showing the distribution of a fluorescent label IR623 and an IR623-labelled moexitecan liposome in a mouse; and

[0054] FIG. 4 is a graph showing the distribution of a fluorescent label IR623 and an IR623-labelled moexitecan liposome in a tumor site and each visceral organ in a mouse.

EXAMPLES

[0055] The specific preparation methods according to the present application are illustrated by the following examples, but the protection scope of the present application is not limited thereto. All equivalent replacements or modifications made by those skilled in the art within the technical scope disclosed in the present application according to the technical solutions and inventive concepts of the present application shall fall within the protection scope of the present application.

Example 1

[0056] Formula:

TABLE-US-00001 1 kg of formulation amount Formulated amount Moexitecan 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean phosphatidylcholine l0 g Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g

[0057] Preparation process: formulated amounts of moexitecan, yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were dissolved in a formulated amount of anhydrous ethanol under heating at 60 C., and used as an organic phase; 70% of the formulation amount of water for injection was heated to 60 C., and used as an aqueous phase; the organic phase was added to the aqueous phase upon shearing the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was extruded through a 0.1 m polycarbonate membrane 3 times; a formulated amount of sucrose was added; and then the resulting mixture was diluted to 1000 g by adding water for injection again, subpackaged into vials for injection (penicillin bottle) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes. The entrapment efficiency measured by the ultrafiltration method was more than 99%.

Example 2

[0058] Formula:

TABLE-US-00002 15 kg of formulation amount Formulated amount Moexitecan 30 g Yolk phosphatidylcholine 450 g Hydrogenated soybean phosphatidylcholine 150 g Anhydrous ethanol 900 g Sucrose 900 g Water for injection Adding to 15 kg

[0059] Preparation process: formulated amounts of moexitecan, yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were dissolved in a formulated amount of anhydrous ethanol under heating at 60 C., and used as an organic phase; 70% of the formulation amount of water for injection was heated to 60 C., and used as an aqueous phase; the organic phase was added to the aqueous phase upon shearing the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was extruded through a 0.2 m polycarbonate membrane 3 times; a formulated amount of sucrose was added; and then the resulting mixture was diluted to 15 kg by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 3

[0060] Formula:

TABLE-US-00003 1 kg of formulation amount Formulated amount Moexitecan 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean phosphatidylcholine 10 g Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g

[0061] Preparation process: formulated amounts of moexitecan, yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were dissolved in a formulated amount of anhydrous ethanol under heating at 60 C., and used as an organic phase; a formulated amount of sucrose was added to 70% of the formulation amount of water for injection and dissolved under heating at 60 C. to obtain a clear solution as an aqueous phase; the organic phase was added to the aqueous phase upon shearing or stirring the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was extruded through a 0.4 m polycarbonate membrane 3 times; and the extruded feed liquid was diluted to 1000 g by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 4

[0062] Formula:

TABLE-US-00004 1 kg of formulation amount Formulated amount Moexitecan 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean phosphatidylcholine 10 g Anhydrous ethanol 60 g Sucrose 40 g Mannitol 20 g Water for injection Adding to 1000 g

[0063] Preparation process: formulated amounts of moexitecan, yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were dissolved in a formulated amount of anhydrous ethanol under heating at 60 C., and used as an organic phase; 70% of the formulation amount of water for injection was heated to 60 C., and used as an aqueous phase; the organic phase was added to the aqueous phase upon shearing or stirring the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was extruded through a 0.2 m polycarbonate membrane 3 times; formulated amounts of sucrose and mannitol were added; and then the resulting mixture was diluted to 1000 g by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 5

[0064] Formula:

TABLE-US-00005 1 kg of formulation amount Formulated amount Moexitecan 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean phosphatidylcholine 10 g BHT 0.1 g Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g

[0065] Preparation process: formulated amounts of BHT and moexitecan were added to a formulated amount of anhydrous ethanol, and dissolved under heating at 60 C. to obtain a clear solution, then formulated amounts of yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were added, and dissolved under heating at 60 C. to obtain a clear solution as an organic phase; 70% of the formulation amount of water for injection was heated to 60 C., and used as an aqueous phase; the organic phase was added to the aqueous phase upon shearing or stirring the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was extruded through a 0.2 m polycarbonate membrane to obtain a liposome feed liquid having a certain particle size and a certain particle size distribution; a formulated amount of sucrose was added; and then the resulting mixture was diluted to 1000 g by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 6

[0066] Formula:

TABLE-US-00006 1 kg of formulation amount Formulated amount Moexitecan 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean phosphatidylcholine 10 g Disodium edetate 0.1 g Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g

[0067] Preparation process: a formulated amount of moexitecan was added to a formulated amount of anhydrous ethanol, and dissolved under heating at 60 C. to obtain a clear solution; then formulated amounts of yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were added, and dissolved under heating at 60 C. to obtain a clear solution as an organic phase; a formulated amount of disodium edetate was dissolved in 70% of the formulation amount of water for injection under heating at 60 C., and used as an aqueous phase; the organic phase was added to the aqueous phase upon shearing or stirring the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was extruded through a 0.1 m polycarbonate membrane to obtain a liposome feed liquid having a certain particle size and a certain particle size distribution; a formulated amount of sucrose was added; and then the resulting mixture was diluted to 1000 g by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 7

[0068] Formula:

TABLE-US-00007 1 kg of formulation amount Formulated amount Moexitecan 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean phosphatidylcholine 10 g Hydrochloric acid or sodium hydroxide Appropriate amount Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g

[0069] Preparation process: a formulated amount of moexitecan was fully dissolved in a formulated amount of anhydrous ethanol under heating at 60 C. to obtain a clear solution; formulated amounts of yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were added and fully dissolved under heating at 60 C. to obtain a clear solution as an organic phase; 70% of the formulation amount of water for injection was kept warm at 60 C., and used as an aqueous phase; the organic phase was added to the aqueous phase upon shearing or stirring the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was extruded through a 0.2 m polycarbonate membrane to obtain a liposome feed liquid having a certain particle size and a certain particle size distribution; a formulated amount of sucrose was added; hydrochloric acid or sodium hydroxide was added to adjust the pH to 5; and then the resulting mixture was diluted to 1000 g by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 8

[0070] Formula:

TABLE-US-00008 1 kg of formulation amount Formulated amount Moexitecan 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean phosphatidylcholine 10 g Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g

[0071] Preparation process: a formulated amount of moexitecan was fully dissolved in a formulated amount of anhydrous ethanol under heating at 60 C. to obtain a clear solution; formulated amounts of yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were added and fully dissolved under heating at 60 C. to obtain a clear solution as an organic phase; a formulated amount of sucrose was dissolved in 70% of the formulation amount of water for injection under heating at 60 C. to obtain a solution as an aqueous phase; the organic phase was added to the aqueous phase upon shearing or stirring the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was extruded through a 0.1 m polycarbonate membrane to obtain a liposome feed liquid having a certain particle size and a certain particle size distribution; the extruded feed liquid was diluted to 1000 g by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 9

[0072] Formula:

TABLE-US-00009 1 kg of formulation amount Formulated amount Moexitecan 2 g Yolk phosphatidylcholine 30 g Hydrogenated yolk phosphatidylcholine 10 g Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g

[0073] Preparation process: a formulated amount of moexitecan was fully dissolved in a formulated amount of anhydrous ethanol under heating at 60 C. to obtain a clear solution; formulated amounts of yolk phosphatidylcholine and hydrogenated yolk phosphatidylcholine were added and fully dissolved under heating at 60 C. to obtain a clear solution as an organic phase; 70% of the formulation amount of water for injection was kept warm at 60 C., and used as an aqueous phase; the organic phase was added to the aqueous phase upon shearing or stirring the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was extruded through a 0.1 m polycarbonate membrane to obtain a liposome feed liquid having a certain particle size and a certain particle size distribution; a formulated amount of sucrose was added; and then the resulting mixture was diluted to 1000 g by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 10

[0074] Formula:

TABLE-US-00010 1 kg of formulation amount Formulated amount Moexitecan 2 g Yolk phosphatidylcholine 22.5 g Hydrogenated soybean phosphatidylcholine 7.5 g Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g

[0075] Preparation process: specific operation steps are identical to those in Example 2. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 11

[0076] Formula:

TABLE-US-00011 1 kg of formulation amount Formulated amount Moexitecan 2 g Yolk phosphatidylcholine 150 g Hydrogenated soybean phosphatidylcholine 50 g Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g

[0077] Preparation process: specific operation steps are identical to those in Example 2. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 12

[0078] Formula:

TABLE-US-00012 1 kg of formulation amount Formulated amount Moexitecan 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean phosphatidylcholine 10 g Anhydrous ethanol 18 g Sucrose 60 g Water for injection Adding to 1000 g

[0079] Preparation process: specific operation steps are identical to those in Example 2. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 13

[0080] Formula:

TABLE-US-00013 1 kg of formulation amount Formulated amount Moexitecan 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean phosphatidylcholine 10 g Anhydrous ethanol 100 g Sucrose 60 g Water for injection Adding to 1000 g

[0081] Preparation process: specific operation steps are identical to those in Example 2. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 14

[0082] Formula:

TABLE-US-00014 1 kg of formulation amount Formulated amount Paclitaxel 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean phosphatidylcholine 10 g Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g

[0083] Note: except that moexitecan in the formula was replaced with paclitaxel, each formula and each process in Examples 2-13 were also applicable to this example.

[0084] Preparation process: formulated amounts of paclitaxel, yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were dissolved in a formulated amount of anhydrous ethanol under heating at 60 C., and used as an organic phase; 70% of the formulation amount of water for injection was heated to 60 C., and used as an aqueous phase; the organic phase was added to the aqueous phase upon shearing the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was extruded through a 0.1 m polycarbonate membrane 3 times; a formulated amount of sucrose was added, and then the resulting mixture was diluted to 1000 g by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 15

[0085] Formula:

TABLE-US-00015 1 kg of formulation amount Formulated amount Docetaxel 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean phosphatidylcholine 10 g Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g

[0086] Note: except that moexitecan in the formula was replaced with docetaxel, each formula and each process in Examples 2-13 were also applicable to this example.

[0087] Preparation process: formulated amounts of docetaxel, yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were dissolved in a formulated amount of anhydrous ethanol under heating at 60 C., and used as an organic phase; 70% of the formulation amount of water for injection was heated to 60 C., and used as an aqueous phase; the organic phase was added to the aqueous phase upon shearing the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was extruded through a 0.1 m polycarbonate membrane 3 times; a formulated amount of sucrose was added; and then the resulting mixture was diluted to 1000 g by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 16

[0088] Formula:

TABLE-US-00016 1 kg of formulation amount Formulated amount Tacrolimus 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean phosphatidylcholine 10 g Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g Note: except that moexitecan in the formula was replaced with tacrolimus, each formula and each process in Examples 2-13 were also applicable to this example.

[0089] Preparation process: formulated amounts of tacrolimus, yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were dissolved in a formulated amount of anhydrous ethanol under heating at 60 C., and used as an organic phase; 70% of the formulation amount of water for injection was heated to 60 C., and used as an aqueous phase; the organic phase was added to the aqueous phase upon shearing the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was extruded through a 0.1 m polycarbonate membrane 3 times; a formulated amount of sucrose was added; and then the resulting mixture was diluted to 1000 g by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Example 17

[0090] Formula:

TABLE-US-00017 1 kg of formulation amount Formulated amount Alprazolam 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean phosphatidylcholine 10 g Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g Note: except that moexitecan in the formula was replaced with alprazolam, each formula and each process in Examples 2-13 were also applicable to this example.

[0091] Preparation process: formulated amounts of alprazolam, yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were dissolved in a formulated amount of anhydrous ethanol under heating at 60 C., and used as an organic phase; 70% of the formulation amount of water for injection was heated to 60 C., and used as an aqueous phase; the organic phase was added to the aqueous phase upon shearing the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was extruded through a 0.1 m polycarbonate membrane 3 times; a formulated amount of sucrose was added; then the resulting mixture was diluted to 1000 g by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes. The entrapment efficiency measured by ultrafiltration method was more than 99%.

Comparison Example 1: Film Dispersion Method

[0092] Formula:

TABLE-US-00018 100 g of Formulated formulation amount amount Moexitecan 0.2 g Yolk phosphatidylcholine 3 g Hydrogenated soybean 1 g phosphatidylcholine Anhydrous ethanol 6 g Sucrose 6 g Water for injection Adding to 100 g

[0093] Preparation process: formulated amounts of moexitecan, yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were dissolved in a formulated amount of anhydrous ethanol under heating at 60 C., and used as an organic phase; the organic phase was placed in a pear-shaped flask, which was then placed in a rotary evaporator, and subsequently rotary-evaporated at 60 C. under reduced pressure to removed ethanol, so that the organic phase formed a thin film; 70% of the formulation amount of water for injection was heated to 60 C., and used as an aqueous phase; the aqueous phase was added to the flask having the thin film formed by rotary evaporation to form a liposome feed liquid after hydration; the resulting liposome feed liquid was extruded through a 0.2 m polycarbonate membrane; a formulated amount of sucrose was added; and then the resulting mixture was diluted to 100 g by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes.

[0094] Results: hydration cannot be carried out smoothly, and it is difficult to form a homogenous liposome feed liquid. Furthermore, the resulting liposome feed liquid cannot be extruded through the polycarbonate membrane, and settled and layered after being left to stand. Therefore, the film dispersion method was not suitable for preparing liposomes of moexitecan.

Comparison Example 2: Micro-Jet Homogenization Method

[0095] Formula:

TABLE-US-00019 1 kg of Formulated formulation amount amount Moexitecan 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean 10 g phosphatidylcholine Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g

[0096] Preparation process: formulated amounts of moexitecan, yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were dissolved in a formulated amount of anhydrous ethanol under heating at 60 C., and used as an organic phase; 70% of the formulation amount of water for injection was heated to 60 C., and used as an aqueous phase; the organic phase was added to the aqueous phase upon shearing the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was homogenized by micro-jet; a formulated amount of sucrose was added; and then the resulting mixture was diluted to 1000 g by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube, and then lyophilized to obtain liposomes.

[0097] Results: the resulting liposome sample was difficultly reconstituted, and the reconstituted liposomes had very large particle size and very wide particle size distribution.

Comparison Example 3: Micelle Preparation 1

[0098]

TABLE-US-00020 1 kg of Formulated formulation amount amount Moexitecan 2 g Cremophor 30 g Glycerol 10 g Anhydrous ethanol 58 g

[0099] Preparation process: formulated amounts of moexitecan, cremophor and glycerol were fully dissolved in a formulated amount of anhydrous ethanol under heating at 45 C. in a water bath to obtain a clear solution; and the resulting clear solution was sterilized by filtration, and then subpackaged to obtain the micelle preparation.

Comparison Example 4: Emulsion

[0100]

TABLE-US-00021 1 kg of Formulated formulation amount amount Moexitecan 1 g Vitamin E 50 g F68 (poloxamer 188) 20 g Water 1000 Anhydrous ethanol 10 g

[0101] Preparation process: {circle around (1)} formulated amounts of moexitecan and vitamin E were fully dissolved in a formulated amount of anhydrous ethanol under heating to obtain a clear solution; {circle around (2)} a formulated amount of F68 was fully dissolved in a formulated amount of water to obtain a clear solution; {circle around (3)} the solution obtained from {circle around (1)} was added to a half of the solution obtained from {circle around (2)} upon shearing the half of the solution obtained from {circle around (2)}, and after fully shearing, the other half of the solution obtained from {circle around (2)} was added thereto, and fully mixed under shearing; and {circle around (4)} the solution obtained from {circle around (3)} was homogenized under high pressure 10 times, and then subpackaged to obtain emulsion.

Comparison Example 5: Micelle Preparation 2

[0102]

TABLE-US-00022 1 kg of Formulated formulation amount amount Moexitecan 5 g Adding tert-butanol to 1000 g

[0103] Preparation process: a formulated amount of moexitecan was fully dissolved in a formulated amount of tert-butanol under heating to obtain a clear solution, and then the clear solution was sterilized by filtration, subpackaged, and then lyophilized to obtain moexitecan powders.

TABLE-US-00023 1 kg of Formulated formulation amount amount ELP (polyoxylethylene 315 g castor oil ether (35)) Glycerol 105 g Anhydrous ethanol 610 g (pharmaceutical grade)

[0104] Preparation process: formulated amounts of ELP, glycerol and anhydrous ethanol were uniformly mixed, sterilized by filtration, and then subpackaged to obtain a special solvent.

[0105] Usage: the moexitecan powders were dissolved in a 100-fold amount of the special solvent to obtain an injection, which was diluted and then administered to a subject.

Comparison Example 6: High-Pressure Homogenization Method

[0106] Formula:

TABLE-US-00024 1 kg of Formulated formulation amount amount Moexitecan 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean 10 g phosphatidylcholine Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g

[0107] Preparation process: formulated amounts of moexitecan, yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were dissolved in a formulated amount of anhydrous ethanol under heating at 60 C., and used as an organic phase; 70% of the formulation amount of water for injection was heated to 60 C., and used as an aqueous phase; the organic phase was added to the aqueous phase upon shearing the aqueous phase to obtain a liposome feed liquid; and the resulting liposome feed liquid was homogenized under high pressure. The homogenized samples still had a large particle size of more than 500 nm, and the high-pressure homogenizer was difficult to normally operate.

Comparison Example 7

[0108] Formula:

TABLE-US-00025 1 kg of Formulated formulation amount amount Moexitecan 2 g Yolk phosphatidylcholine 30 g Hydrogenated soybean 10 g phosphatidylcholine Anhydrous ethanol 60 g Sucrose 60 g Water for injection Adding to 1000 g

[0109] Preparation process: formulated amounts of moexitecan, yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine were dissolved in a formulated amount of anhydrous ethanol under heating at 60 C., and used as an organic phase; 70% of the formulation amount of water for injection was heated to 60 C., and used as an aqueous phase; the organic phase was added to the aqueous phase upon shearing the aqueous phase to obtain a liposome feed liquid; the resulting liposome feed liquid was extruded through a 0.1 m polycarbonate membrane 3 times; a formulated amount of sucrose was added; and then the resulting mixture was diluted to 1000 g by adding water for injection again, subpackaged into vials for injection (penicillin bottles) made from neutral borosilicate glass tube.

[0110] Results: The appearance of the sample was changed significantly after it was placed at 40 C. for 15 days. That is, a white emulsion became a yellow emulsion.

Example 18: Stability Test

[0111] Ten batches of pharmaceutical compositions comprising moexitecan were prepared in accordance with the formulas and preparation processes in Examples 1-7 and Comparison Examples 3, 4, 5 and 7. Each batch of samples was stored at 40 C. for 15 days. The appearance of and related substances in the samples were detected, and compared with those on the 0th day. The results were shown as follows.

TABLE-US-00026 0th day 40 C. 15th day Total Total Example Appearance impurity (%) Appearance impurity (%) Example 1 White solid 1.15 White solid 1.65 Example 2 White solid 1.14 White solid 1.63 Example 3 White solid 1.17 White solid 1.68 Example 4 White solid 1.12 White solid 1.59 Example 5 White solid 1.15 White solid 1.65 Example 6 White solid 1.13 White solid 1.67 Example 7 White solid 1.14 White solid 1.63 Comparison Colorless 1.11 Yellow 45.54 Example 3 clear liquid liquid Comparison White 1.77 Yellow 43.13 Example 4 emulsion liquid Comparison White 1.13 White 1.63 Example 5 powder powder Comparison White 1.19 Yellow 45.07 Example 7 emulsion liquid

Example 19: Long-Term Stability Test

[0112] Seven batches of pharmaceutical compositions comprising moexitecan were prepared in accordance with the formulas and preparation processes in Examples 1-7. Each batch of pharmaceutical compositions was stored at 6 C. and 25 C., respectively. The samples stored at 6 C. were taken at the 3rd, 6th, 9th and 12th months, respectively, to detect related substances, and the samples stored at 25 C. were taken at the 1st, 2nd, 3rd and 6th months, respectively, to detect related substances, both of which were compared with those on the 0th day. The results were shown as follows.

TABLE-US-00027 Related substances- total impurity (%) 25 C. 25 C. 25 C. 25 C. 0th 1st 2nd 3rd 6th Example month month month month month Example 1 1.15 1.17 1.20 1.23 1.44 Example 2 1.14 1.15 1.19 1.22 1.46 Example 3 1.17 1.16 1.18 1.21 1.47 Example 4 1.12 1.15 1.18 1.21 1.45 Example 5 1.15 1.16 1.19 1.24 1.48 Example 6 1.13 1.16 1.19 1.23 1.49 Example 7 1.12 1.14 1.18 1.23 1.51

TABLE-US-00028 Related substances- total impurity (%) 6 C. 6 C. 6 C. 6 C. 0th 3rd 6th 9th 12th Example month month month month month Example 1 1.15 1.15 1.14 1.16 1.24 Example 2 1.14 1.15 1.15 1.16 1.21 Example 3 1.17 1.16 1.17 1.18 1.20 Example 4 1.12 1.13 1.11 1.16 1.21 Example 5 1.15 1.15 1.14 1.19 1.22 Example 6 1.13 1.13 1.14 1.20 1.24 Example 7 1.13 1.12 1.14 1.19 1.22

Example 20: Measurement of Particle Size

[0113] Ten batches of pharmaceutical compositions comprising moexitecan were prepared in accordance with the formulas and preparation processes in Examples 1-9 and Comparison Example 2. One vial of pharmaceutical composition for each batch was reconstituted in water, and then sampled to measure the particle size with a nanometer particle size analyzer. The results were shown as follows.

TABLE-US-00029 Particle size Particle size after before lyophilization lyophilization and reconstitution Average Average particle Dispersion particle Dispersion Example size coefficient size coefficient Example 1 95.8 0.127 109.0 0.212 Example 2 178.5 0.183 204.5 0.199 Example 3 351.3 0.233 399.1 0.353 Example 4 128.0 0.132 149.6 0.224 Example 5 178.5 0.174 184.5 0.193 Example 6 149.6 0.089 176.5 0.104 Example 7 233.9 0.164 245.7 0.188 Example 8 96.5 0.106 119.8 0.140 Example 9 95.2 0.085 122.9 0.118 Comparison 91.0 0.511 7998.1 2.022 Example 2

Example 21: Toxicity Text

[0114] One batch of pharmaceutical compositions comprising moexitecan was prepared in accordance with the formula and preparation process in Example 2, and one batch of micelle preparations comprising moexitecan was prepared in accordance with the formula and preparation process in Comparison Example 5. The resulting two batches of pharmaceutical preparations were subjected to acute toxicity test in mice, acute toxicity test in rats, and toxicity test in rats after administration for 4 weeks at the same dosage. The results of toxicity tests for the two pharmaceutical preparations were compared, and the results were shown as follows:

TABLE-US-00030 Toxicity in Acute Acute rats after toxicity toxicity administration Example in mice in rats for 4 weeks Example 2 No significant No significant No significant abnormality after abnormality after abnormality after administration administration administration Mice mortality: Five days after Rat mortality: 0% 40% administration, No significant about 1% reduction abnormality in in body weight the lungs after Rat mortality: 0% gross dissection Comparison Significant Significant Significant Example 5 abnormality after abnormality after abnormality after administration administration administration Mice mortality: Five days after Rat mortality: 75% 65% administration, Significant about 10% reduction abnormality in the in body weight lungs after gross Rat mortality: 30% dissection

Example 22: Pharmacodynamic Test

[0115] One batch of pharmaceutical compositions comprising moexitecan was prepared in accordance with the formula and preparation process in Example 2, and one batch of micelle preparations comprising moexitecan was prepared in accordance with the formula and preparation process in Comparison Example 5. The resulting two batches of pharmaceutical preparations were subjected to a pharmacodynamic test in nude mice with NCI-H292 lung cancer, i.e., inhibitory effect on xenograft tumor growth. Results of the pharmacodynamic test for the two pharmaceutical preparations were shown as follows.

TABLE-US-00031 Inhibitory effect on xenograft tumor growth in nude mice Example with NCI-H292 lung cancer Example 2 The pharmaceutical preparation obtained in Example 2 is superior to that obtained in Comparison Comparison Example 5 at Example 5 the same dosage of 10 mg/kg.

Example 23: Long-Term Toxicity Test

[0116] One batch of pharmaceutical compositions comprising moexitecan was prepared in accordance with the formula and preparation process in Example 2, and one batch of micelle preparations comprising moexitecan was prepared in accordance with the formula and preparation process in Comparison Example 5. The resulting two batches of pharmaceutical preparations were subjected to a long-term toxicity test in rats at a dosage of 60, 30 or 10 mg/kg. The test results of the two pharmaceutical preparations were compared, and shown as follows.

TABLE-US-00032 Example Rats Example 2 Death: 3/6 deaths in the 60 mg/kg group, and no deaths in other groups Other symptoms: myelosuppression was found in each group, and showed dose-dependency The toxicity was reduced by about 5 times, compared with the toxicity of the pharmaceutical preparation obtained in Comparison Example 5. Comparison Death: 6/6 deaths in the 60 mg/kg group, Example 5 5/6 deaths in the 30 mg/kg group, and no deaths in the 10 mg/kg group. Other symptoms: myelosuppression was found in each group, and showed dose-dependency

Example 24: Tissue Distribution

[0117] One batch of pharmaceutical compositions comprising moexitecan was prepared in accordance with the formula and preparation process in Example 2. 18 SD rats were divided into three groups with 6 rats (3 female ones and 3 male ones) in each group. Rats in each group were intravenously injected via tail vein with a pharmaceutical composition comprising moexitecan at a dosage of 30 mg/kg. The rats were anesthetized at 15 min, 2 h and 6 h after administration, and then taken blood samples and tissues. The blood samples and tissues were respectively treated to obtain blood plasma and tissue homogenate samples, and moexitecan and its active metabolite SN-38 (chemical name: 20(s)-7-ethyl-10-hydroxycamptothecine) in the blood plasma and tissue homogenate samples were determined using an LC-MS/MS method. The results were shown in FIG. 1 and FIG. 2. FIG. 1 showed that moexitecan was mainly distributed in the organs, such as the rectum, liver, lung, blood plasma, colon, kidney, ovary, and heart. FIG. 2 showed that SN-38 was mainly distributed in the organs, such as the colon, rectum, liver, lung, blood plasma, ovary, jejunum, ileum, duodenum, and kidney. The concentrations of moexitecan and SN-38 in the rectum are very high. The concentration of moexitecan in the colon is lower than that in the blood plasma, but the concentration of the active metabolite SN-38 of moexitecan in the colon is highest, indicating the concentrated distribution of the pharmaceutical composition according to the present application in a special organ or tissue. Moexitecan and SN-38 both had the lowest concentrations in the cerebrum and testis.

Example 25: In Vivo Targeting Research

[0118] A batch of pharmaceutical compositions (particle size: about 100 nm) comprising moexitecan and fluorescence probe (IR623) was prepared according to the formula (additionally adding about 0.8% (w/w, by weight of the total amount of phospholipids in the formula as 100%) DSPE conjugated with a fluorescence probe IR623 (added and dissolved in an organic phase)) and the preparation process in Example 1. A batch of pharmaceutical compositions (particle size: about 400 nm) comprising moexitecan and fluorescence probe (IR623) was prepared according to the formula (additionally adding about 0.8% (w/w, by weight of the total amount of phospholipids in the formula as 100%) DSPE conjugated with a fluorescence probe IR623 (added and dissolved in an organic phase)) and the preparation process in Example 3. The two batches of pharmaceutical compositions were used for an in vivo targeting research in nude mice bearing intestinal cancer HT29 using near-infrared in vivo imaging technique, and were compared with the in vivo targeting of the fluorescence probe IR623. The results were shown in FIG. 3 and FIG. 4.

[0119] Results: there were obvious fluorescence signals in the abdomen at 0.5 h after a free fluorescence probe was injected into mice via tail vein. The fluorescence signals gradually weakened over time, and were metabolized to the outside (results as shown in FIG. 3). For mice injected with moexitecan liposomes containing the fluorescence probe and having a particle size of about 100 nm, fluorescence signals spread throughout the body at 0.5 h, began to concentrate at a tumor site at 4 h, were strongest at the tumor site at 8 h, began to weaken at the tumor site after 8 h, and still were present at the tumor site at 48 h (results as shown in FIG. 3). For mice injected with moexitecan liposomes containing the fluorescence probe and having a particle size of about 400 nm, fluorescence signals were obvious in the abdomen at 0.5 h, enhanced in the abdomen at 4 h, and still concentrated in the abdomen thereafter (results as shown in FIG. 3).

[0120] The tumor-bearing mice were dissected at 48 h after drug injection. Each visceral organ (tumor, liver, spleen, kidney and intestine) in the body was excised, and the fluorescence distribution in each visceral organ was observed using an in-vivo imager. It can be seen from FIG. 4 that the fluorescence in the organs of mice injected with the free fluorescent probe was very weak, and almost completely metabolized. Among the organs of mice injected with moexitecan liposomes containing the fluorescence probe and having a particle size of about 100 nm, the fluorescence in the tumor was stronger than that in other organs. Among the organs of mice injected with moexitecan liposomes containing the fluorescence probe and having a particle size of about 400 nm, the fluorescence in the liver was strongest.

[0121] It therefore can be concluded that the moexitecan liposomes containing the fluorescence probe and having a particle size of about 100 nm had passive tumor targeting, and the moexitecan liposomes containing the fluorescence probe and having a particle size of about 400 were mainly accumulated in the liver. Fluorescence probes were excreted mainly through intestinal and renal metabolism.