Liposome of irinotecan or irinotecan hydrochloride and preparation method thereof

Abstract

A liposome of irinotecan or irinotecan hydrochloride and its preparation method are disclosed. The liposome contains irinotecan or irinotecan hydrochloride, neutral phospholipid and cholesterol, wherein the weight ratio of the cholesterol to the neutral phospholipid is 1:3 to 1:5. The liposome is prepared by an ion gradient method.

Claims

1. A liposome, comprising: irinotecan hydrochloride, hydrogenated soybean phosphatidylcholine, polyethylene glycol 2000-distearoyl phosphatidyl ethanolamine, cholesterol, and ethylene diamine tetraacetic acid disodium, wherein the weight ratio of the cholesterol to the hydrogenated soybean phosphatidylcholine is about 1:4, and there is no significant change in the particle size and encapsulation efficiency of the liposome after the liposome is stored at 25 C. for 60 days.

2. The liposome according to claim 1, wherein the liposome is prepared by an ion gradient method.

3. The liposome according to claim 2, wherein the liposome has an ion gradient formed between the internal water phase and the external water phase of the liposome.

4. The liposome according to claim 1, wherein the weight ratio of the polyethylene glycol 2000-distearoyl phosphatidyl ethanolamine to the irinotecan hydrochloride is 0.2 to 0.4.

5. A liposome injection comprising the liposome of irinotecan hydrochloride according to claim 1.

6. The liposome according to claim 1, wherein the weight ratio of the hydrogenated soybean phosphatidylcholine to the irinotecan hydrochloride is 2.5:1 to 4:1.

7. The liposome according to claim 3, wherein the internal water phase of the liposome has a higher ion concentration than the external water phase of the liposome.

8. A method of preparing a liposome of irinotecan hydrochloride, comprising: 1) preparing a blank liposome comprising a neutral phospholipid, polyethylene glycol 2000-distearoyl phosphatidyl ethanolamine, and cholesterol, wherein the weight ratio of the cholesterol to the neutral phospholipid is 1:4, and the neutral phospholipid consists of hydrogenated soybean phosphatidylcholine; 2) replacing the external water phase of the blank liposome with a solution containing ethylene diamine tetraacetic acid disodium and a salt to obtain a blank liposome having an ionic gradient between the internal water phase and the external water phase of the blank liposome; 3) preparing an aqueous solution of the irinotecan hydrochloride; 4) mixing the aqueous solution with the blank liposome having the ionic gradient in a dispersion; and 5) incubating the dispersion with heating and stirring to obtain a preparation comprising the liposome of irinotecan hydrochloride, wherein the weight ratio of the neutral phospholipid to the irinotecan hydrochloride in the liposome is 2:1 to 4:1, and there is no significant change in the particle size and encapsulation efficiency of the liposome after the liposome is stored at 25 C. for 60 days.

9. The method according to claim 8, further comprising: 6) removing non-encapsulated irinotecan hydrochloride from the preparation comprising the liposome or irinotecan hydrochloride to obtain a preparation with the non-encapsulated irinotecan hydrochloride being removed; and 7) concentrating the preparation with non-encapsulated irinotecan hydrochloride being removed.

10. The method according to claim 8, wherein the blank liposome has a desired particle size and is prepared using a method selected from the group consisting of methods A, B, C and D: A. dissolving the neutral phospholipid and the cholesterol in anhydrous ethanol or a mixed solvent of anhydrous ethanol and tert-butyl alcohol to obtain a solution; mixing the solution with a buffer to obtain a mixture; obtaining a crude blank liposome after removing the ethanol from the mixture through a reduced pressure distillation; and obtaining the blank liposome from the crude blank liposome using at least one of a high-pressure homogenizer and an extrusion equipment; B. dissolving the neutral phospholipid and the cholesterol in chloroform or a mixed solvent of chloroform and methanol to obtain a solution; forming a lipid film from the solution using rotary evaporation; obtaining a crude blank liposome after hydrating the lipid film with a buffer; and preparing the blank liposome from the crude blank liposome using at least one of a high-pressure homogenizer and an extrusion equipment; C. mixing the neutral phospholipid, the cholesterol and a buffer to obtain a mixture; preparing the blank liposome from the mixture using at least one of a high-pressure homogenizer and an extrusion equipment; and D. dissolving the neutral phospholipid and the cholesterol in anhydrous ethanol or a mixed solvent of anhydrous ethanol and tert-butyl alcohol to obtain a solution; mixing the solution with a buffer to obtain a mixture; and preparing the blank liposome from the mixture using at least one of a high-pressure homogenizer and an extrusion equipment; wherein the buffer is selected from the group consisting of a buffer comprising Na.sup.+, K.sup.+, Fe.sup.2+,Ca.sup.2+, Ba.sup.2+, Mn.sup.2+, Mg.sup.2+, Li.sup.+, NH.sup.4+, H.sup.+ion salts and a mixture thereof.

11. The liposome injection according to claim 5, wherein the amount of the ethylene diamine tetraacetic acid disodium is more than 0% (w/v), but less than or equal to 0.5% (w/v), of the liposome injection.

12. The liposome injection according to claim 5, wherein the injection is a liquid injection or a lyophilized powder for injection.

13. The liposome injection according to claim 5, wherein the injection comprises an osmotic pressure regulator selected from the group consisting of glucose, sucrose, sorbitol, mannitol, sodium chloride, glycerine, histidine, histidine hydrochloride, glycine, glycine hydrochloride, lysine, serine, glutamic acid, arginine, valine and a mixture thereof; and the amount of the osmotic pressure regulator is more than 0% (w/v), but less than or equal to 5% (w/v), of the liposome injection.

14. The liposome injection according to claim 5, wherein the injection further comprises an antioxidant selected from the group consisting of a water-soluble antioxidant and an oil-soluble antioxidant; wherein the oil-soluble antioxidant is selected from the group consisting of -tocopherol, -tocopherol succinate, -tocopherol acetate and a mixture thereof; the water-soluble antioxidant is selected from the group consisting of ascorbic acid, sodium bisulfite, sodium sulfite, sodium pyrosulfite, L-cysteine and a mixture thereof; and the amount of the antioxidant is more than 0% (w/v), but less than or equal to 0.5% (w/v), of the liposome injection.

15. The liposome injection according to claim 12, wherein the injection is the lyophilized powder for injection comprising a lyoprotectant, and the lyophilized powder is prepared by freeze-drying.

16. The liposome injection according to claim 5, comprising: TABLE-US-00022 irinotecan hydrochloride 1 part by weight; hydrogenated soybean 3.4-3.8 parts by weight; phosphatidylcholine polyethylene glycol 2000-distearoyl 0.34-0.38 part by weight; phosphatidyl ethanolamine Cholesterol 0.8-0.95 part by weight; and ethylene diamine tetraacetic acid 0.05-0.09 part by weight disodium wherein the weight ratio of the cholesterol to the hydrogenated soybean phosphatidylcholine is 1:4.

17. A method of preparing a liposome injection comprising a liposome of irinotecan hydrochloride, the method comprising preparing the liposome using the method of claim 8.

18. The method according to claim 17, further comprising: adjusting the concentration of the irinotecan hydrochloride in the injection, metering the volume of the injection, sterilizing the injection by filtration, filling the sterilized injection to vials and sealing the vials to obtain a liquid injection; or adding a lyoprotectant to the injection, adjusting the concentration of the irinotecan hydrochloride in the injection, metering the volume of the injection, sterilizing the injection by filtration, filling the sterilized injection to vials, sealing the vials, freeze-drying the sealed vials to obtain a lyophilized power for injection.

19. A liposome, consisting essentially of: irinotecan hydrochloride, hydrogenated soybean phosphatidylcholine, polyethylene glycol 2000-distearoyl phosphatidyl ethanolamine, cholesterol, ethylene diamine tetraacetic acid disodium, ammonium sulfate injectable water, and one or more salts wherein the weight ratio of the cholesterol to the hydrogenated soybean phosphatidylcholine is about 1:4, and there is no significant change in the particle size and encapsulation efficiency of the liposome after the liposome is stored at 25 C. for 60 days.

20. The liposome of claim 19, wherein the weight ratio of the hydrogenated soybean phosphatidylcholine to the irinotecan hydrochloride is 2.5:1 to 4:1.

21. The liposome of claim 20, wherein the weight ratio of the polyethylene glycol 2000-distearoyl phosphatidyl ethanolamine to the irinotecan hydrochloride is 0.2 to 0.4.

22. A method of treating a tumor in a subject in need thereof, comprising administering to the subject an effective amount of the liposome of claim 1.

23. A method of treating a tumor in a subject in need thereof, comprising administering to the subject an effective amount of the liposome of claim 19.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the particle size distribution of liposome injection of irinotecan or irinotecan hydrochloride according to the present invention.

(2) FIG. 2 shows the morphology of liposome injection of irinotecan or irinotecan hydrochloride according to the present invention.

(3) FIG. 3 shows the results of in vivo anticancer effect test of liposome injection of irinotecan or irinotecan hydrochloride according to the present invention.

PREFERRED EMBODIMENTS

(4) The following examples are intended to further illustrate the invention, but are in no way intended to limit the scope thereof.

EXAMPLE 1

(5) Formulation:

(6) TABLE-US-00004 irinotecan hydrochloride 0.28 g 0.28 g 0.28 g 0.28 g 0.28 g hydrogenated soybean 1 g 1 g 1 g 1 g 1 g phosphatidylcholine cholesterol 0.4 g 0.33 g 0.25 g 0.2 g 0.167 g DSPE-PEG2000 0.1 g 0.1 g 0.1 g 0.1 g 0.1 g ammonium sulfate 5 g 5 g 5 g 5 g 5 g sodium chloride 0.45 g 0.45 g 0.45 g 0.45 g 0.45 g cholesterol:phospholipid 1:2.5 1:3 1:4 1:5 1:6 injectable water up to the required volume

(7) Preparation Method: ydrogenated soybean phosphatidylcholine (HSPC) and cholesterol (CHOL) of the formulation amount were dissolved in an adequate amount of anhydrous ethanol, the resulting lipid solution was mixed with ammonium sulfate solution (100 ml), ethanol was removed by reduced pressure distillation, and then the crude blank liposome was obtained. After 5 cycles homogenization in high-pressure homogenizer (1000 bar), the particle size of liposome was controlled by extruding the liposome on extrusion equipment (two 0.1 m extrusion membranes on extrusion equipment, five times extrusion), and then DSPE-PEG.sub.2000 aqueous solution was added. Under stirring, the mixture was incubated for 20 minutes. The blank liposome was dialyzed by using tangential flow ultrafiltration device with continuous supplementary of injectable water in the course, then the blank liposome was obtained finally.

(8) Irinotecan hydrochloride aqueous solution was prepared with injectable water and was added to the dispersion of blank liposome with ion gradient above according to the weight ratio of irinotecan hydrochloride to HSPC 1:3.5. Under stirring, the mixture was heated to 60 C. and incubated for 20 minutes, and then the drug-loaded liposome was obtained. The non-encapsulated drug was removed by using tangential flow ultrafiltration device. 0.45 g sodium chloride was added to adjust the osmotic pressure after the sample was concentrated to about 50 ml. After the drug concentration was adjusted by diluting to the metered volume, the liposome was sterilized by filtration with 0.22 m filter, filled under the protection of nitrogen, and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was obtained finally.

(9) The change in particle size of each formulation was shown in the table below. The results indicated that the particle size of the sample was the smallest when the weight ratio of phospholipid to cholesterol was 4:1.

(10) TABLE-US-00005 HSPC:CHOL Preparation procedure Average particle size 6:1 After homogenization 138.7 0.1 m 92.26 five times extrusion 5:1 After homogenization 136.2 0.1m 89.5 five times extrusion 4:1 After homogenization 123.4 0.1 m 87.26 five times extrusion 3:1 After homogenization 145.1 0.1 m 93.4 five times extrusion 2.5:1 After homogenization 142 0.1 m 98.56 five times extrusion

(11) The stability of the sample prepared was investigated at 25 C. at various weight ratios of phospholipid to cholesterol. The results were shown in the table below. After stored at 25 C. for 60 days, the particle size and encapsulation efficiency of the sample had no significant changes when the weight ratio of phospholipid to cholesterol was 4:1. However, for the samples having other weight ratios of phospholipid to cholesterol, the size of the sample increased and the encapsulation efficiency declined. Therefore, the stability of the sample was better when the weight ratio of phospholipid to cholesterol was 4:1.

(12) TABLE-US-00006 Storage Particle time Encapsulation size (z-v) Potential Content Total Lysophospholipid HSPC:CHOL (25 C., day) Appearance efficiency % nm (mv) (mg/ml) impurities % (mg/ml) 6:1 0 Off-white 98.86 92.3 30.5 5.05 0.58 0.39 suspension 30 Off-white 98.56 94.3 26.8 5.04 0.75 0.56 suspension 60 Off-white 98.20 95.9 24.9 5.06 0.85 0.66 suspension 4:1 0 Off-white 99.37 87.3 32.1 5.10 0.55 0.40 suspension 30 Off-white 99.25 87.5 30.9 5.11 0.64 0.50 suspension 60 Off-white 99.18 87.8 28.6 5.09 0.70 0.62 suspension 2.5:1.sup. 0 Off-white 99.27 98.5 35.8 5.12 0.60 0.38 suspension 30 Off-white 98.75 100.2 28.6 5.09 0.73 0.51 suspension 60 Off-white 98.19 101.7 25.3 5.07 0.84 0.67 suspension

(13) Conclusions: Taking into account all the indexes, it can obtain better results when the ratio of cholesterol to phospholipid was 1:35, most preferably 1:4.

EXAMPLE 2

(14) Formulation:

(15) TABLE-US-00007 irinotecan hydrochloride 0.28 g hydrogenated soybean 1 g phosphatidylcholine (HSPC) polyethylene glycol 2000-distearoyl 0.1 g phosphatidylethanolamine (DSPE-PEG.sub.2000) cholesterol 0.25 g ammonium sulfate 5 g ethylene diamine tetraacetic acid disodium 0.02 g sodium chloride 0.45 g injectable water up to the required volume

(16) Preparation Method:

(17) Hydrogenated soybean phosphatidylcholine and cholesterol of the formulation amount were dissolved in an adequate amount of anhydrous ethanol, the resulting lipid solution was mixed with ammonium sulfate solution (100 ml), anhydrous ethanol was removed by reduced pressure distillation, and then the crude blank liposome was obtained. After 5 cycles homogenization in high-pressure homogenizer (1000 bar), the particle size of liposome was controlled by extruding the liposome on extrusion equipment (two 0.1 m extrusion membrane on extrusion equipment, five times extrusion), and then DSPE-PEG.sub.2000 aqueous solution was added. Under stirring, the mixture was incubated for 20 minutes. The blank liposome was dialyzed by using tangential flow ultrafiltration device with continuous supplementary of injectable water in the course, then the blank liposome was obtained finally.

(18) Irinotecan hydrochloride aqueous solution was prepared with injectable water and was added to the dispersion of blank liposome with ion gradient above according to the weight ratio of irinotecan hydrochloride to HSPC 1:3.5. Under stirring, the mixture was heated to 60 C. and incubated for 20 minutes, and then the drug-loaded liposome was obtained. The non-encapsulated drug was removed by using tangential flow ultrafiltration device. 0.45 g sodium chloride was added to adjust the osmotic pressure after the sample was concentrated to about 50 ml. After the drug concentration was adjusted by diluting to the constant volume, the liposome was sterilized by filtration with 0.22 m filter, filled under the protection of nitrogen, and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was obtained finally.

EXAMPLE 3

(19) The formulation and preparation method of blank liposome were as same as Example 2, except that the weight ratio of irinotecan hydrochloride to HSPC was 1:1.5, 1:2, 1:3.5, 1:4 and 1:5 in the liposome preparing process. The encapsulation efficiency and particle size of liposome sample of irinotecan hydrochloride were shown in the table below:

(20) TABLE-US-00008 Encapsulation Drug-loaded Particle efficiency content size CPT11:HSPC (%) (mg/ml) (nm) 1:1.5 83.2 5.11 87.1 1:2 90.8 5.15 86.5 1:3.5 99.4 5.08 85.9 1:4 99.1 4.81 85.4 1:5 99.4 4.25 86.7

(21) It was shown that the encapsulation efficiency significantly reduced when the weight ratio of irinotecan hydrochloride to HSPC was 1:1.5, and the drug-loaded content decreased remarkably when the ratio was 1:5. It's not suitable for preparing formulations used in clinical application in both conditions. The encapsulation efficiency and drug-loaded content were higher when the ratio was 1:21:4.

EXAMPLE 4

(22) The formulation and preparation method of blank liposome and drug-loaded liposome were as same as Example 2, except that HSPC in the formulation was replaced by high-purity egg phosphatidylcholine (EPC), high purity soybean phosphatidylcholine (SPC) respectively. The stability of resulting liposome sample was investigated at 25 C. and the results were shown in the table below. The test results showed that the stability of liposome sample prepared by HSPC was the best and the main indexes had no remarkable change when stored at 25 C. for 2 months.

(23) TABLE-US-00009 PC Encapsulation Drug-loaded Particle Compo- efficiency content size Time sition (%) (mg/ml) (nm) 0 M HSPC 99.4 5.08 85.9 EPC 99.5 5.10 87.5 SPC 99.2 5.01 86.9 1 M HSPC 99.5 5.10 85.5 EPC 92.4 5.07 88.2 SPC 93.9 5.05 87.3 2 M HSPC 98.7 5.07 86.5 EPC 85.8 5.06 93.2 SPC 89.6 5.02 91.5

EXAMPLE 5

(24) Formulation:

(25) TABLE-US-00010 irinotecan hydrochloride 0.28 g hydrogenated soybean 1 g phosphatidylcholine (HSPC) polyethylene glycol 2000-distearoyl 0.1 g phosphatidylethanolamine (DSPE-PEG.sub.2000) cholesterol 0.25 g saline solution 50 ml injectable water up to the required volume

(26) Preparation Method <1>:

(27) Ethanol injection method: hydrogenated soybean phosphatidylcholine, DSPE-PEG.sub.2000 and cholesterol of the formulation amount were dissolved in an adequate amount of anhydrous ethanol, the resulting lipid solution was injected into saline solution of irinotecan hydrochloride. Ethanol was removed by reduced pressure distillation, and then the crude blank liposome was obtained. The particle size of the liposome was controlled by extruding the liposome on extrusion equipment (two 0.1 m extrusion membrane on extrusion equipment, five times extrusion) after 5 cycles homogenization in high-pressure homogenizer (1000 bar). The drug concentration was adjusted by diluting to metered volume, the liposome was sterilized by filtration with 0.22 m filter, filled under the protection of nitrogen, and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was obtained finally.

(28) Preparation Method <2>:

(29) Film dispersion method: hydrogenated soybean phosphatidylcholine, DSPE-PEG.sub.2000 and cholesterol of the formulation amount were dissolved in an adequate amount of chloroform and the resulting lipid solution was prepared to film by rotary evaporator then the chloroform was removed. Saline solution of irinotecan hydrochloride was added and the mixture was incubated for 1 h. The particle size of the liposome was controlled by extruding the liposome on extrusion equipment (two 0.1 m extrusion membrane on extrusion equipment, five times extrusion) after 5 cycles homogenization in high-pressure homogenizer (1000 bar). The drug concentration was adjusted by diluting to metered volume, the liposome was sterilized by filtration with 0.22 m filter, filled under the protection of nitrogen, and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was obtained finally.

(30) The encapsulation efficiency and particle size of the irinotecan hydrochloride liposome prepared by Preparation method <1>, <2> and Example 2 were determined.

(31) TABLE-US-00011 Encapsulation Particle size Sample efficiency (%) (nm) Example 2 99.4 85.9 Preparation method <1> 15.3 87.9 Preparation method <2> 17.8 90.2

(32) It was shown that the target product could be prepared by passive drug-loaded methods such as ethanol injection method and film dispersion method when preparing liposome of irinotecan hydrochloride. But the liposome prepared by these methods had low encapsulation efficiency and only a small amount of the drug can be loaded into the liposome. In contrast, the sample prepared by active drug-loaded method (Example 2) had high encapsulation efficiency and drug-loaded content. In addition, the sample prepared by active drug-loaded method had small and uniform particle size. So in the present invention, the active drug-loaded method was used to prepare the liposome. It had extremely good results to prepare the liposome of irinotecan hydrochloride by ion gradient method.

EXAMPLE 6

(33) TABLE-US-00012 Formulation Formulation 1 Formulation 2 Formulation 3 Formulation 4 HSPC 1 g 1 g 1 g 1 g Cholesterol 250 mg 250 mg 250 mg 250 mg PEG.sub.2000-DSPE 0.1 g 0.1 g 0.1 g 0.1 g Vitamin E / 0.02 g / 0.02 g EDTA-2Na / / 0.02 g 0.02 g Ammonium sulfate 100 ml 100 ml 100 ml 100 ml solution (300 mM) Irinotecan hydrochloride 0.3 g 0.3 g 0.3 g 0.3 g

(34) Preparation Method:

(35) Blank liposome: the lipid ethanol solution was injected, and the solution was homogenized under 1000 bar, 6 times; extruded 3 times in 200 nm, 5 times in 100 nm; PEG.sub.2000-DSPE was added and the mixture was incubated for 30 min at 60 C. Then the mixture was dialyzed 3 times with tangential flow device, 50 ml every time, wherein Vitamin E(VE) was added to phospholipid organic solvent and EDTA was added to ammonium sulfate solution.

(36) Drug-loaded liposome: about 10 mg/ml of irinotecan hydrochloride aqueous solution was prepared and added to the blank liposome, then the mixture was incubated at 60 C. for 15 min. The sample was concentrated to approximately 50 ml by using tangential flow device and 5 mg/ml of sample was obtained.

(37) The results of stability were shown in the table below. All indexes of the sample had no remarkable change when EDTA was added alone. It improved the stability of the liposome significantly. But other stabilizers did not significantly improve the stability of the liposome.

(38) TABLE-US-00013 Storage Particle time Encapsulation size (z-v) Content Total Lysophospholipid Sample (25 C., day) Appearance efficiency % nm mg/ml impurities % (mg/ml) HSPC 0 Off-white 99.70 85.6 5.42 0.65 0.40 suspension 30 Off-white 91.51 87.7 5.40 0.74 0.65 suspension HSPC + 0 Off-white 97.10 89.0 5.01 0.48 0.35 VE suspension 30 Off-white 93.49 93.4 5.03 0.56 0.43 suspension HSPC + 0 Off-white 95.67 87.2 4.94 0.56 0.38 EDTA suspension 30 Off-white 95.67 86.5 4.98 0.60 0.40 suspension HSPC + 0 Off-white 98.92 89.2 5.55 0.61 0.39 VE + suspension EDTA 30 particle 87.31 99.7 5.51 0.61 0.47 precipitation

EXAMPLE 7

(39) Formulation (1):

(40) TABLE-US-00014 irinotecan hydrochloride 0.5 g hydrogenated soybean 1.5 g phosphatidylcholine cholesterol 0.4 g manganese sulfate 10 g mannitol 2.5 g injectable water up to the required volume

(41) Preparation Method:

(42) Hydrogenated soybean phosphatidylcholine and cholesterol of the formulation amount were dissolved in an adequate amount of anhydrous ethanol and the resulting lipid solution was mixed with manganese sulfate solution (100 ml). After anhydrous ethanol was removed by reduced pressure distillation, the crude blank liposome was obtained. The particle size of the liposome was controlled by extruding the liposome on extrusion equipment (two 0.1 m extrusion membrane on extrusion equipment, five times extrusion). The blank liposome was dialyzed by using tangential flow ultrafiltration device with continuous supplementary of injectable water in the course, then the blank liposome was obtained. Irinotecan hydrochloride aqueous solution was prepared with injectable water and was added to the dispersion of blank liposome with ion gradient. Under stirring, the mixture was heated to 50 C. and incubated for 20 minutes, and then the drug-loaded liposome was obtained. The non-encapsulated drug was removed by using tangential flow ultrafiltration device and then 2.5 g mannitol was added to adjust the osmotic pressure. After the drug concentration was adjusted by diluting to the constant volume, the liposome was sterilized by filtration with 0.22 m filter, and then filled under the protection of nitrogen, and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was obtained finally. The particle size of the liposome was measured by the nano particle size analyzer (89.3 nm), and the encapsulation efficiency was 97.5%.

(43) Formulation (2):

(44) TABLE-US-00015 irinotecan hydrochloride 1 g hydrogenated egg 3.45 g lecithin (HEPC) cholesterol 0.8 g magnesium sulfate 10 g histidine 2.5 g injectable water up to the required volume

(45) Preparation Method:

(46) Hydrogenated egg lecithin and cholesterol of the formulation amount were dissolved in an adequate amount of anhydrous ethanol and the resulting lipid solution was mixed with manganese sulfate solution (100 ml). The particle size of the liposome was controlled by extruding the liposome on extrusion equipment (two 0.1 m extrusion membrane on extrusion equipment, five times extrusion). The blank liposome was dialyzed by using tangential flow ultrafiltration device with continuous supplementary of injectable water in the course, then the blank liposome was obtained. Irinotecan hydrochloride aqueous solution was prepared with injectable water and was added to the dispersion of blank liposome with ion gradient. Under stirring, the mixture was heated to 50 C. and incubated for 20 minutes, and then the drug-loaded liposome was obtained. The non-encapsulated drug was removed by using tangential flow ultrafiltration device and the sample was concentrated to about 50 ml. Then 2.5 g histidine was added to adjust the osmotic pressure. After the drug concentration was adjusted by diluting to the metered volume, the liposome was sterilized by filtration with 0.22 m filter, and then filled under the protection of nitrogen, and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was obtained finally. The particle size of the liposome was measured by the nano particle size analyzer (87.6 nm), and the encapsulation efficiency was 98.1%.

(47) Formulation (3):

(48) TABLE-US-00016 irinotecan hydrochloride 0.3 g hydrogenated soybean 1 g phosphatidylcholine (HSPC) polyethylene glycol 2000-distearoyl 0.05 g phosphatidylethanolamine (DSPE-PEG.sub.2000) cholesterol 0.25 g ammonium sulfate 5 g sodium chloride 0.45 g injectable water up to the required volume

(49) Preparation Method:

(50) Hydrogenated soybean phosphatidylcholine and cholesterol of the formulation amount were dissolved in an adequate amount of anhydrous ethanol and the resulting lipid solution was mixed with ammonium sulfate solution (100 ml). After anhydrous ethanol was removed by reduced pressure distillation, the crude blank liposome was obtained. After 5 cycles homogenization in high-pressure homogenizer (1000 bar), DSPE-PEG.sub.2000 aqueous solution was added. Under stirring, the mixture was incubated for 20 minutes. The blank liposome was dialyzed by using tangential flow ultrafiltration device with continuous supplementary of injectable water in the course, then the blank liposome was obtained. Irinotecan hydrochloride aqueous solution was prepared with injectable water and was added to the dispersion of blank liposome with ion gradient. Under stirring, the mixture was heated to 60 C. and incubated for 20 minutes, and then the drug-loaded liposome was obtained. The non-encapsulated drug was removed by using tangential flow ultrafiltration device and the sample was concentrated to about 50 ml. Then 0.45 g sodium chloride was added to adjust the osmotic pressure. After the drug concentration was adjusted by diluting to the metered volume, the liposome was sterilized by filtration with 0.22 m filter, and then filled under the protection of nitrogen, and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was obtained finally. The particle size of the liposome was measured by the nano particle size analyzer (87.3 nm), and the encapsulation efficiency was 99.2%.

EXAMPLE 8

(51) Formulation:

(52) TABLE-US-00017 irinotecan hydrochloride 0.5 g hydrogenated soybean 1 g phosphatidylcholine (HSPC) myocardial phospholipids (CL) 0.5 g polyethylene glycol 5000-distearoyl 0.5 g phosphatidylethanolamine (DSPE-PEG.sub.5000) -Tocopherol 0.05 g cholesterol 0.35 g citric acid 5.76 g sodium chloride about 3.6 g injectable water up to the required volume

(53) Preparation Method:

(54) Hydrogenated soybean phosphatidylcholine, myocardial phospholipid, DSPE-PEG.sub.5000, cholesterol and -tocopherol of the formulation amount were dissolved in an adequate amount of anhydrous ethanol and the resulting lipid solution was mixed with citric acid solution (100 ml). After anhydrous ethanol was removed by reduced pressure distillation, the crude blank liposome was obtained. After 5 cycles homogenization in high-pressure homogenizer (1000 bar), the blank liposome was dialyzed by using tangential flow ultrafiltration device with continuous supplementary of sodium chloride solution (0.9%, 400 ml) in the course, then the blank liposome was obtained. Irinotecan hydrochloride aqueous solution was prepared with injectable water and was added to the dispersion of blank liposome with ion gradient. Under stirring, the mixture was heated to 60 C. and incubated for 20 minutes, and then the drug-loaded liposome was obtained. The non-encapsulated drug was removed by using tangential flow ultrafiltration device and the sample was concentrated to about 50 ml. After the drug concentration was adjusted by diluting to the constant volume, the liposome was sterilized by filtration with 0.22 m filter, and then filled under the protection of nitrogen, and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was obtained finally. The particle size of the liposome was measured by the nano particle size analyzer (85.8 nm), and the encapsulation efficiency was 98.6%.

EXAMPLE 9

(55) Formulation:

(56) TABLE-US-00018 irinotecan hydrochloride 0.8 g dipalmitoyl phosphatidyl choline (DPPC) 2 g dipalmitoyl phosphatidylglycerol (DPPG) 0.2 g cholesterol 0.5 g ascorbic acid 0.05 g ethylene diamine tetraacetic acid disodium 0.05 g ammonium sulfate 5 g sodium chloride about 3.6 g injectable water up to the required volume

(57) Preparation Method:

(58) DPPC, DPPG and cholesterol of the formulation amount were dissolved in an adequate amount of anhydrous ethanol and the resulting lipid solution was mixed with ammonium sulfate solution (100 ml, containing ethylene diamine tetraacetic acid disodium). After ethanol was removed by reduced pressure distillation, the crude blank liposome was obtained. After 5 cycles homogenization in high-pressure homogenizer (1000 bar), the blank liposome was dialyzed by using tangential flow ultrafiltration device with continuous supplementary of sodium chloride solution (0.9%, 400 ml) in the course, then the blank liposome was obtained. Irinotecan hydrochloride aqueous solution was prepared with injectable water and was added to the dispersion of blank liposome with ion gradient. Under stirring, the mixture was heated to 60 C. and incubated for 20 minutes, and then the drug-loaded liposome was obtained. The non-encapsulated drug was removed by using tangential flow ultrafiltration device and the sample was concentrated to about 50 ml. After the drug concentration was adjusted by diluting to the constant volume, the liposome was sterilized by filtration with 0.22 m filter, and then filled under the protection of nitrogen, and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was obtained finally. The particle size of the liposome was measured by the nano particle size analyzer (89.4nm), and the encapsulation efficiency was 97.2%.

EXAMPLE 10

(59) Formulation:

(60) TABLE-US-00019 irinotecan hydrochloride 0.5 g hydrogenated soybean 1 g phosphatidylcholine (HSPC) polyethylene glycol 5000-distearoyl 0.1 g phosphatidylethanolamine (DSPE-PEG.sub.5000) -tocopherol 0.05 g cholesterol 0.3 g ammonium sulfate 5 g sodium chloride about 3.6 g sucrose 2 g mannitol 1 g injectable water up to the required volume

(61) Preparation Method:

(62) Hydrogenated soybean phosphatidylcholine, cholesterol and -tocopherol of the formulation amount were dissolved in an adequate amount of anhydrous ethanol and the resulting lipid solution was mixed with ammonium sulfate solution (100 ml). After ethanol was removed by reduced pressure distillation, the crude blank liposome was obtained. After 5 cycles homogenization in high-pressure homogenizer (1000 bar), the liposome was extruded on extrusion equipment (five 100 nm extrusion membrane on extrusion equipment, five times extrusion). Then DSPE-PEG.sub.5000 aqueous solution was added, and the mixture was incubated under stirring for 20 minutes. The blank liposome was dialyzed by using tangential flow ultrafiltration device with continuous supplementary of sodium chloride solution (0.9%, 400 ml) in the course, then the blank liposome was obtained. Irinotecan hydrochloride aqueous solution was prepared with injectable water and was added to the dispersion of blank liposome with ion gradient. Under stirring, the mixture was heated to 60 C. and incubated for 20 minutes, and then the drug-loaded liposome was obtained. The non-encapsulated drug was removed by using tangential flow ultrafiltration device and the sample was concentrated to about 50 ml. Then sucrose and mannitol were added to the mixture and mixed homogeneously. After the drug concentration was adjusted by diluting to the constant volume, the liposome was sterilized by filtration with 0.22 m filter, and then filled into penicillin bottle and freeze-dried. The liposome lyophilized powder for injection of irinotecan hydrochloride was obtained finally. The particle size of the liposome was measured (90.8 nm) after hydration of the lyophilized powder for injection, and the encapsulation efficiency was 97.5%.

Experiment 1

(63) Taking the product of Example 2 as an example to study the physicochemical characteristics of the product obtained according to the present invention:

(64) [Particle size distribution]: Appropriate amount of the sample was diluted with water then measured by Dynamic Light Scattering (DLS) method. Detective wavelength: =633 nm; detective angle: 173; detective temperature: 25 C. The particle size was represented by intensity. The particle size distribution was shown in FIG. 1. The average particle size was 85.9 nm.

(65) [Morphology]: Appropriate amount of the diluted sample was drawn, a copper mesh was placed on a clean filter paper, the sample was dropped onto the copper mesh, dyed with phosphotungstic acid, and observed with transmission electron microscope (TEM, JEM2010, Japan Electronics Co., Ltd.) after dry. The morphology was shown in FIG. 2. The appearance of irinotecan hydrochloride liposome was typical bilayer structure and the majority of the particle size was below 200 nm. It's consistent with the result measured by dynamic light scattering.

(66) [Encapsulation efficiency]: Method for determination of drug content: Column: Agilent ZORBAX Eclipse XDB-C18 (4.6150 mm, 5 m) mobile phase: acetonitrile 0.05M KH.sub.2PO.sub.4 buffer solution (pH value was adjusted to 4, containing 1% triethylamine)=20:80; column temperature: 40 C.; injection volume: 20 L; flow rate: 1.0 mL/min.

(67) Method for detecting encapsulation efficiency:

(68) 1 mL sample solution was pipeted into a 10 mL volumetric flask and was diluted with water to the mark. Then it was shaken homogeneously and ultrafiltered with 8010 ultrafilter (MILLIPORE company). The initial filtrate was discarded and the subsequent filtrate was reserved as the sample solution. 20 L solution of the sample and the control were pipeted into liquid chromatography and the chromatogram was recorded. The free drug content of the formulation was calculated by external standardization method, recorded as W. The total amount of drug in this product was calculated by a content determination method, recorded as W.sub.0. The encapsulation efficiency was calculated by the follow equation:

(69) $\mathrm{Encapsulation}\mathrm{Efficiency}=\frac{W_0-W}{W_0}100\%$

(70) Results of the determination: The encapsulation efficiency of the product was 99.4%.

(71) [Impact Factors Test]: The impact factors were investigated by placing the product under different conditions. The results were shown in the table below:

(72) TABLE-US-00020 Storage time Particle Content Encapsulation Total impurities Lysophospholipid Conditions (day) Appearance pH size (nm) (%) efficiency (%) (%) (mg/ml) Illumination 0 Off-white suspension 6.39 85.9 98.14 99.40 0.43 0.19 4500 lx 5 Earth yellow suspension 6.30 86.3 78.99 99.11 14.4 0.23 500 lx 10 Earth yellow suspension 6.40 86.5 76.39 99.20 19.5 0.30 40 C. 0 Off-white suspension 6.39 85.9 98.14 99.40 0.43 0.19 5 Off-white suspension 6.35 87.1 98.77 99.29 0.45 0.29 10 Off-white suspension 6.47 88.7 98.86 96.82 0.55 0.44 Low 3 cycles Off-white suspension 6.41 89.1 100.07 99.16 0.44 0.38 temperature Freeze-thaw 3 cycles White suspension 6.38 110.5 95.22 99.28 0.46 0.23

(73) The result was shown that the sample was light-sensitive. Under a bright light, the appearance of the sample turned yellow, the content decreased and related substances were significantly increased. The encapsulation efficiency and particle size of the sample had no remarkable change at 40 C., while related substances were increased a little. Big size particles were generated in the sample under low temperature or freeze-thaw conditions. Considering the instability of the phospholipid under high temperature and the test results of the impact factors test, the product should be stored under low-temperature and dark conditions.

(74) [Antitumor Therapeutic Efficacy Test In Vivo]

(75) Drug name: The liposome of Irinotecan hydrochloride (CPT-11 liposome) (prepared according to Example 2) was provided by Shanghai Hengrui Pharmaceutical Co., LTD. The injection of Irinotecan hydrochloride (CPT-11) was provided by Jiangsu Hengrui Medicine Co., LTD.

(76) Preparation methods: The drug was diluted with saline solution to required concentration.

(77) Experimental animals: BALB/cA-nude mice, 6-7 weeks, , purchased from Shanghai Slac Laboratory Animal Co., LTD. Certificate No.: SCXK (Shanghai) 2007-0005. Environment: SPF level.

(78) Experimental Protocol:

(79) Nude mice were subcutaneously inoculated Ls-174t human colon cancer cell. After tumors grew to the 150-300 mm.sup.3, mice were randomly divided into teams (d0). Dosage and dosage regimens were shown in the table below. The volume of tumors and the weight of the mice were measured and recorded for 2-3 times per week. The tumor volume (V) was calculated by the follow equation:
V=ab.sup.2 wherein a, b represent length and width respectively.

(80) TABLE-US-00021 Average Average tumor tumor Tumor volume volume inhibition (mm.sup.3) (mm.sup.3) RTV % T/C rate (%) P value Partial Number Drug Administration Route D0 SD D12 SD D12 SD D12 D12 D12 regression of animals Vehicle D0, 3 IV 219.8 37.2 2013.7 303.1 9.4 2.3 100 0 0 8 CPT-11 D0, 3 IV 212.2 42.1 732.2 162.6 3.5 0.7 38 62 0.000 0 13 liposomes 1.0 mg/kg CPT-11 D0, 3 IV 205.0 49.0 265.1 122.9 1.3 0.4 13 87 0.000 4 13 liposomes 3.0 mg/kg CPT-11 D0, 3 IV 204.6 44.7 844.4 197.5 4.2 0.9 45 55 0.000 0 14 10 mg/kg D0: the first administration time; RTV: relative tumor volume; P value means relative to the control. Control group n = 12, treatment group n = 6.

(81) Results:

(82) CPT-11 liposome and CPT-11 both inhibited the growth of Ls-174t human colon cancer in nude mice significantly. CPT-11 liposome was dose-dependent in inhibiting the growth of Ls-174t. 4/14 tumor regressed partially when CPT-11 liposome was administrated in high-dose (3 mg/kg). The therapeutic efficacy of CPT-11 liposome was equivalent to CPT-11 (10 mg/kg) when CPT-11 liposome was administrated in low-dose (1 mg/kg). It was indicated that the therapeutic efficacy of CPT-11 liposome may have prompted at least 10 times than the CPT-11 injection. The detailed results were shown in FIG. 3.