LIPOSOME CONTAINING ETHYLENEDIAMINE TETRAACETIC ACID OR SALT THEREOF AND ERIBULIN OR PHARMACEUTICALLY ACCEPTABLE SALT THEREOF

Abstract

Provided are a liposome containing Eribulin or a pharmaceutically acceptable salt thereof and a preparation method therefor. An inner aqueous phase of the liposome contains Eribulin or a salt thereof, and one or more selected from ethylenediamine tetraacetic acid, phosphoric acid, pentetic acid, or salts thereof. Further provided are a pharmaceutical composition containing the aforementioned liposome and a preparation method therefor.

Claims

1. A liposome, containing eribulin or a pharmaceutically acceptable salt thereof as an active compound, and one or more selected from the group consisting of ethylenediaminetetraacetic acid, phosphoric acid, pentetic acid and salts thereof, in an inner aqueous phase.

2. The liposome according to claim 1, further containing an ammonium salt, wherein the ammonium salt is selected from the group consisting of ammonium sulfate, ethylammonium sulfate, ammonium ethylenediaminetetraacetate, ammonium chloride, ammonium hydroxide, ammonium acetate, ammonium phosphate, triethylammonium sucrose octasulfate, dextran ammonium sulfate and triethyldextran ammonium sulfate , in the inner aqueous phase.

3. The liposome according to claim 1, wherein the active compound is selected from eribulin mesylate.

4. The liposome according to claims 1, wherein the ethylenediaminetetraacetic acid, phosphoric acid, pentetic acid or salts thereof has a concentration selected from the group consisting of 1-300 mM.

5. The liposome according to claim 3, wherein the ammonium salt has a concentration selected from the group consisting of 10-400 mM.

6. The liposome according to claim 1, wherein the active compound has a concentration selected from the group consisting of 0.01-100 mg/mL .

7. The liposome according to claim 1, wherein the liposome is substantially free of citric acid in the inner aqueous phase.

8. The liposome according to claim 1, further containing a phospholipid selected from at least one of dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylcholine (DMPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), egg yolk phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), hydrogenated egg phosphatidylcholine (HEPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), palmitoyl oleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), dioleoyl phosphatidylglycerol (DOPG), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dipalmitoyl glycerophosphoglycerol (DPPG), dipalmitoyl phosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyristoyl phosphatidylserine (DMPS), distearoyl phosphatidylserine (DSPS), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA), 1,2-distearoyl-sn-glycero-3-phosphatidic acid (DSPA), dipalmitoyl phosphatidylinositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidylinositol (DOPI), dimyristoyl phosphatidylinositol (DMPI), and distearoyl phosphatidylinositol (DSPI).

9. (canceled)

10. The liposome according to claim 1, further containing a steroid.

11. (canceled)

12. The liposome according to claim 1, further containing a hydrophilic polymer-modified lipid, wherein the hydrophilic polymer-modified lipid is selected from the group consisting of polyethylene glycol-modified distearoyl phosphatidylethanolamine, polyethylene glycol-modified distearoyl phosphatidylglycerol and polyethylene glycol-modified cholesterol.

13–14. (canceled)

15. The liposome according to claim 10, wherein the phospholipid and the steroid are in a molar ratio selected from the group consisting of 1:0.01-1:1.

16. The liposome according to claim 13, wherein the phospholipid and the hydrophilic polymer-modified lipid are in a molar ratio selected from the group consisting of 20:1-1:1.

17-19. (canceled)

20. A pharmaceutical composition comprising the liposome according to claim 1 or prepared from the liposome according to claim 1 .

21. The pharmaceutical composition according to claim 20, further comprising a sugar or an electrolyte, which is selected from at least one of sodium chloride, trehalose and sucrose.

22. The pharmaceutical composition according to claim 21, wherein the sugar has a concentration selected from the group consisting of 2%-20%.

23. The pharmaceutical composition according to claim 20, wherein the buffer has a concentration selected from the group consisting of 1-300 mM .

24. The pharmaceutical composition according to claim 20, having a pH of 5.0-8.0.

25. A lyophilized composition obtained by freeze-drying the pharmaceutical composition according to claim 20, or reconstituted in a liquid medium to obtain the pharmaceutical composition according to claim 20, wherein the liquid medium used for the reconstitution is selected from the group consisting of normal saline, water for injection, a glucose injection, and a glucose and sodium chloride injection.

26-30. (canceled)

31. The pharmaceutical composition according to claim 20, wherein the pharmaceutical composition further comprises a buffer.

32. The pharmaceutical composition according to claim 31, wherein the buffer is at least one selected from the group consisting of an acetate buffer, a histidine buffer, a phosphate buffer, a succinate buffer and a 4-hydroxyethylpiperazine ethanesulfonate buffer.

Description

DETAILED DESCRIPTION

Example 1

[0119] TABLE-US-00001 Formula Amount HSPC 1215 mg CHOL 398.09 mg mPEG2000-DSPE 460.29 mg Eribulin mesylate 45 mg

[0120] The formula amount of lipid mixture was taken, added to absolute ethanol, and dissolved at 60° C. The solution of lipids in ethanol was injected into the inner aqueous phase solution of the liposome (300 mM ammonium sulfate/60 mM citric acid), and the resulting mixture was stirred for 15-30 min. Ethanol was removed under reduced pressure, and the residue was extruded and sized by an extruder to obtain a blank liposome.

[0121] The blank liposome was dialyzed (tangential flow filtration) against 180 mL of 0.9% sodium chloride/10 mM histidine buffer (pH = 7.5), and when most of the ammonium sulfate was removed from the outer aqueous phase, the pH of the outer aqueous phase of the liposomes was adjusted to 10.5 with sodium hydroxide.

[0122] The formula amount of eribulin mesylate was dissolved with an aqueous solution of 0.9% sodium chloride/10 mM histidine (pH 7.5), and the solution was stirred to obtain a 5 mg/mL eribulin mesylate solution.

[0123] The blank liposome (with outer aqueous phase at pH 10.5) was mixed with the eribulin mesylate solution, and the mixture was incubated at 60° C. to allow eribulin mesylate to be introduced into the liposome in the liposome. The outer aqueous phase of liposome was adjusted to pH 7.5, and the mixture was concentrated, and brought to a certain volume.

Determination of Encapsulation Efficiency

[0124] The liposome encapsulating the active ingredient was centrifuged at 13,000 rpm for 10-30 min, and the concentration of the active ingredient in the supernatant was determined by HPLC, and the corresponding value was calculated according to the encapsulation efficiency expression.

[0125] The encapsulation efficiency of eribulin mesylate was 95.70%.

Example 2

[0126] TABLE-US-00002 Formula Amount HSPC 1215 mg CHOL 398.09 mg mPEG2000-DSPE 460.29 mg Eribulin mesylate 45 mg

[0127] The formula amount of lipid mixture was taken, added to absolute ethanol, and dissolved at 60° C. The solution of lipids in ethanol was injected into the inner aqueous phase solution of the liposome (300 mM ammonium sulfate/60 mM ethylenediaminetetraacetic acid), and the resulting mixture was stirred for 15-30 min. Ethanol was removed under reduced pressure, and the residue was extruded and sized by an extruder to obtain a blank liposome.

[0128] The blank liposome was dialyzed (tangential flow filtration) against 180 mL of 0.9% sodium chloride/10 mM histidine buffer (pH = 7.5), and when most of the ammonium sulfate was removed from the outer aqueous phase, and the pH of the outer aqueous phase of the liposomes was adjusted to 10.5 with sodium hydroxide.

[0129] The formula amount of eribulin mesylate was dissolved with an aqueous solution of 0.9% sodium chloride/10 mM histidine (pH 7.5), and the solution was stirred to obtain a 5 mg/mL eribulin mesylate solution.

[0130] The blank liposome (with outer aqueous phase at pH 10.5) was mixed with the eribulin mesylate solution, and the mixture was incubated at 60° C. to introduce eribulin mesylate into the inner aqueous phase of the liposome. The outer aqueous phase of liposome was adjusted to pH 7.5, concentrated, and brought to a certain volume.

Determination of Encapsulation Efficiency

[0131] The encapsulation efficiency of eribulin mesylate was 96.85%.

Example 3

[0132] TABLE-US-00003 Formula Amount HSPC 1215 mg CHOL 398.09 mg mPEG2000-DSPE 460.29 mg Eribulin mesylate 45 mg

[0133] The formula amount of lipid mixture was taken, added to absolute ethanol, and dissolved at 60° C. The solution of lipids in ethanol was injected into the inner aqueous phase solution of the liposome (300 mM ammonium sulfate/60 mM phosphoric acid), and the resulting mixture was stirred for 15-30 min. Ethanol was removed under reduced pressure, and the residue was extruded and sized by an extruder to obtain a blank liposome.

[0134] The blank liposome was dialyzed (tangential flow filtration) against 180 mL of 0.9% sodium chloride/10 mM histidine buffer (pH = 7.5), and when most of the ammonium sulfate was removed from the outer aqueous phase, and the pH of the outer aqueous phase of the liposomes was adjusted to 10.5 with sodium hydroxide.

[0135] The formula amount of eribulin mesylate was dissolved with an aqueous solution of 0.9% sodium chloride/10 mM histidine (pH 7.5), and the solution was stirred to obtain a 5 mg/mL eribulin mesylate solution.

[0136] The blank liposome (with outer aqueous phase at pH 10.5) was mixed with the eribulin mesylate solution, and the mixture was incubated at 60° C. to introduce eribulin mesylate into the inner aqueous phase of the liposome. The outer aqueous phase of liposome was adjusted to pH 7.5, concentrated, and brought to a certain volume.

Determination of Encapsulation Efficiency

[0137] The encapsulation efficiency of eribulin mesylate was 99.46%.

Example 4

[0138] TABLE-US-00004 Formula Amount HSPC 1215 mg CHOL 398.09 mg mPEG2000-DSPE 460.29 mg Eribulin mesylate 45 mg

[0139] The formula amount of lipid mixture was taken, added to absolute ethanol, and dissolved at 60° C. The solution of lipids in ethanol was injected into the inner aqueous phase solution of the liposome (300 mM ammonium sulfate/60 mM tartaric acid), and the resulting mixture was stirred for 15-30 min. Ethanol was removed under reduced pressure, and the residue was extruded and sized by an extruder to obtain a blank liposome.

[0140] The blank liposome was dialyzed (tangential flow filtration) against 180 mL of 0.9% sodium chloride/10 mM histidine buffer (pH = 7.5), and when most of the ammonium sulfate was removed from the outer aqueous phase, and the pH of the outer aqueous phase of the liposomes was adjusted to 10.5 with sodium hydroxide.

[0141] The formula amount of eribulin mesylate was dissolved with an aqueous solution of 0.9% sodium chloride/10 mM histidine (pH 7.5), and the solution was stirred to obtain a 5 mg/mL eribulin mesylate solution.

[0142] The blank liposome (with outer aqueous phase at pH 10.5) was mixed with the eribulin mesylate solution, and the mixture was incubated at 60° C. to introduce eribulin mesylate into the inner aqueous phase of the liposome. The outer aqueous phase of liposome was adjusted to pH 7.5, concentrated, and brought to a certain volume.

Determination of Encapsulation Efficiency

[0143] The encapsulation efficiency of eribulin mesylate was 94.92%.

Example 5

[0144] TABLE-US-00005 Formula Amount HSPC 1215 mg CHOL 398.09 mg mPEG2000-DSPE 460.29 mg Eribulin mesylate 45 mg

[0145] The formula amount of lipid mixture was taken, added to absolute ethanol, and dissolved at 60° C. The solution of lipids in ethanol was injected into the inner aqueous phase solution of the liposome (300 mM ammonium sulfate/60 mM aconitic acid), and the resulting mixture was stirred for 15-30 min. Ethanol was removed under reduced pressure, and the residue was extruded and sized by an extruder to obtain a blank liposome.

[0146] The blank liposome was dialyzed (tangential flow filtration) against 180 mL of 0.9% sodium chloride/10 mM histidine buffer (pH = 7.5), and when most of the ammonium sulfate was removed from the outer aqueous phase, and the pH of the outer aqueous phase of the liposomes was adjusted to 10.5 with sodium hydroxide.

[0147] The formula amount of eribulin mesylate was dissolved with an aqueous solution of 0.9% sodium chloride/10 mM histidine (pH 7.5), and the solution was stirred to obtain a 5 mg/mL eribulin mesylate solution.

[0148] The blank liposome (with outer aqueous phase at pH 10.5) was mixed with the eribulin mesylate solution, and the mixture was incubated at 60° C. to introduce eribulin mesylate into the inner aqueous phase of the liposome. The outer aqueous phase of liposome was adjusted to pH 7.5, concentrated, and brought to a certain volume.

Determination of Encapsulation Efficiency

[0149] The encapsulation efficiency of eribulin mesylate was 98.02%.

Test Example 1

[0150] The samples obtained in Examples 1-5 described above were separately placed at 2-8° C. or 25° C. for investigating the stability of the encapsulation efficiency. The related data are shown in the following table:

TABLE-US-00006 Examples 1 2 3 4 5 Starting point (%) 95.70 96.85 99.46 94.92 98.02 2-8° C. 1 month (%) 95.31 96.84 N/A N/A 94.25 2 months (%) 98.04 97.29 98.80 95.35 89.93 3 months (%) 98.10 96.64 99.18 94.24 89.37 25 °C 15 days (%) 93.49 94.71 99.25 96.61 N/A 1 month (%) 81.43 93.31 93.64 92.01 73.40 2 months (%) 83.08 93.25 90.93 83.20 62.65 3 months (%) 44.06 91.64 91.45 74.67 56.29

Test Example 2

[0151] The samples obtained in Examples 1-5 described above were separately placed at 2-8° C. or 25° C. for investigating the content. The related data are shown in Table 2 below:

TABLE-US-00007 Examples 1 2 3 4 5 Starting point (%) 0.5368 0.5347 0.5715 0.5225 0.5136 2-8° C. 1 month (%) 0.5602 0.5454 N/A N/A 0.4804 2 months (%) 0.5401 0.5319 0.5799 0.5218 0.4922 3 months (%) 0.5586 0.5582 0.5606 0.5551 0.4927 25 °C 15 days (%) 0.5697 0.5593 0.5734 0.5256 N/A 1 month (%) 0.5437 0.5595 0.5512 0.5375 0.4961 2 months (%) 0.5577 0.5445 0.5544 0.5378 0.5005 3 months (%) 0.5859 0.5583 0.5333 0.5455 0.5117

[0152] Conclusion: as can be seen from Table 2, all of the liposomes with different compositions have good content stability. It is more important to determine whether the drug will be released from the liposome during the in vitro storage period, and it can be seen from Table 1 that the stability of the encapsulation efficiency of the liposome containing phosphoric acid or ethylenediammoniumtetraacetic acid in the inner aqueous phase is superior as compared to that of the liposome containing citric acid or tartaric acid or aconitic acid in the inner aqueous phase, especially under accelerated conditions (25° C.). Therefore, it is expected that the composition containing such liposomes is suitable for long-term storage under mild conditions.

Example 6

[0153] The pharmaceutical compositions (liposomes) A, B, C and D as shown in Table 3 below were prepared according to the method of Example 1. Female Balb/c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were subcutaneously inoculated with tumor cells (human pharyngeal squamous carcinoma cells FaDu) on the right dorsal side.

[0154] Reagent: pharmaceutical compositions (liposomes) A, B, C and D, eribulin mesylate injection (produced in-house)

[0155] Mode of administration: once a week, 0.15 mg/mL × 10 mL, intravenous injection.

[0156] After a certain period of time (0.5 h, 2 h, 6 h, 24 h and 48 h) after the administration, blood was collected or tumor tissues were removed. The blood samples were centrifuged within 1 h after the collection (centrifugation condition: about 11,000 rpm at 2-8° C. for 5 min), and could be initially placed on ice after the collection and placed in a refrigerator at -80° C. for long-term storage. After being removed, the tumor tissues were washed with PBS and wiped with absorbent paper, and the weight of the tissues was measured and recorded. The tissues were loaded into a test tube, cooled in ice water and stored at -80° C. for later analysis.

[0157] The amount of eribulin mesylate in plasma and tumor tissues was determined by LC/MS.

[0158] The results of plasma PK data and tumor tissue PK data for eribulin mesylate are shown in Tables 4 and 5, respectively.

TABLE-US-00008 Formula A B C HSPC 13.5 mg/mL 13.5 mg/mL 13.5 mg/mL CHOL 4.42 mg/mL 4.42 mg/mL 4.42 mg/mL mPEG2000-DSPE 5.11 mg/mL 5.11 mg/mL 5.11 mg/mL Drug/lipid ratio 1:27 1:27 1:27 Inner aqueous phase Ammonium sulfate/ethylenediaminetetraacetic acid Ammonium sulfate/phosphoric acid Ammonium sulfate/citric acid Outer aqueous phase Sodium chloride/histidine Sodium chloride/histidine Sodium chloride/histidine

TABLE-US-00009 Plasma PK data Group T.sub.max (h) C.sub.max (ug/mL) AUC.sub.0-t (h*ug/mL) T.sub.½ (h) Ratio Eribulin mesylate injection 0.5 0.154 0.654 3.38 -- A 0.5 20.3 304 8.17 465 B 0.5 21.1 285 8.71 436 C 0.5 21.1 307 8.94 469 Ratio: AUC.sub.liposome/AUC.sub.eribulin mesylate injection

TABLE-US-00010 Tumor tissue PK data Group T.sub.max (h) C.sub.max (ug/mL) AUC.sub.0-t (h*ug/mL) T.sub.½ (h) Ratio Eribulin mesylate injection 2 0.272 4.05 16.6 -- A 24 1.31 43 NA 10.6 B 6 1.2 40.6 39.6 10.0 C 24 0.936 36.7 NA 9.1 Ratio: AUC.sub.liposome/AUC.sub.eribulin mesylate injection

[0159] Conclusion: compared with eribulin mesylate, the pharmaceutical compositions A, B and C show increased AUC and significantly improved release half-cycle in plasma and tumor tissues. In tumor tissues, compared with the liposome with citric acid as the inner aqueous phase, the pharmaceutical compositions A and B have superior C.sub.max and higher AUC.sub.liposome/AUC.sub.eribulin .sub.mesylate .sub.injection.