Liposomes as contrast agents for ultrasonic imaging and apparatus and methods for preparing the same

• US 5,571,497 A
• Filed: 06/06/1995
• Issued: 11/05/1996
• Est. Priority Date: 12/22/1989
• Status: Expired due to Term

• Current Assignee: Lantheus Medical Imaging, Inc. (Lantheus Holdings, Inc.), Imarx Therapeutics, Inc. (Sycamore Entertainment Group, Inc.)
• Original Assignee: Imarx Pharmaceutical Corp. (Sycamore Entertainment Group, Inc.)
• Inventors: Unger, Evan C.
• Primary Examiner(s): Dees, Jose G.
• Assistant Examiner(s): Cebulak, Mary
• Application Number:
  US08/468,056
• Time in Patent Office: 518 Days
• Field of Search: 424/9.51
• US Class Current: 423/87
• CPC Class Codes: A61K 41/0028 Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
• CPC Class Codes: A61K 41/0052 Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
• CPC Class Codes: A61K 47/6911 the form being a liposome
• CPC Class Codes: A61K 47/6941 the form being a granulate or an agglomerate
• CPC Class Codes: A61K 49/1815 compo-inhalant, e.g. breath tests
• CPC Class Codes: A61K 49/223 Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
• CPC Class Codes: A61K 49/227 Liposomes, lipoprotein vesicles, e.g. LDL or HDL lipoproteins, micelles, e.g. phospholipidic or polymeric
• CPC Class Codes: A61K 9/127 Liposomes
• CPC Class Codes: A61K 9/1277 Processes for preparing; Proliposomes
• CPC Class Codes: A61K 9/1278 Post-loading, e.g. by ion or pH gradient

Abstract

Liposomes suitable as ultrasound contrast agents which contain media of various types including gases, gaseous precursors activated by pH, temperature or pressure, as well as other solid or liquid contrast enhancing agents, are described. Methods of using the same as ultrasound contrast agents are also disclosed. The present invention also comprises novel methods for synthesizing liposomes having encapsulated therein gases.

Claims

1. A method of synthesizing a liposome having encapsulated therein a gas comprising the steps of:

(a) placing a liquid media containing liposomes in a vessel,

(b) pressurizing said vessel with a cooled gas, thereby dissolving a first portion of said gas into said liquid media, so that a portion of said dissolved gas enters said liposomes, and

(c) rapidly depressurizing said vessel by releasing a second portion of said gas from said vessel, thereby causing said dissolved gas having entered said liposomes to form bubbles within said liposomes,

and further comprising the step of sizing said liposomes.

2. The method according to claim 1 wherein said gas is selected from the group consisting of nitrogen, oxygen, carbon dioxide, xenon, argon, neon and helium.

3. The method according to claim 1 wherein said liquid media is a phosphate buffered saline solution.

4. The method of according to claim 1 wherein said liposomes comprise one or more lipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.

5. The method according to claim 1 wherein the step of pressurizing said vessel comprises the step of pressurizing said vessel to a pressure between about 50 and about 120 psi.

6. The method according to claim 1 wherein the temperature of said cooled gas prior to said pressurization of said vessel is between about 1°

to about 4°

C.

7. The method according to claim 1 wherein the step of depressurizing said vessel is performed in less than about 5 seconds.

17. The method according to claim 1, wherein the step of sizing said liposomes is performed prior to placing said liquid media in said vessel, whereby said liposomes are pre-sized.

18. The method according to claim 17, wherein the step of pre-sizing said liposomes comprises flowing said liquid media through a filter.

19. The method according to claim 18, wherein said filter has a pore size in the range of about 0.015 to 10 microns.

20. The method according to claim 18, wherein the step of flowing said liquid media through a filter comprises discharging said liquid media from a syringe.

21. The method according to claim 1, wherein the step of sizing said liposomes is performed after said bubbles have been formed therein.

22. The method according to claim 21, wherein the step of sizing said liposomes comprises flowing said bubble containing liposomes through a filter.

23. The method according to claim 22, wherein said filter has a pore size in the range of about 0.015 to 10 microns.

24. The method according to claim 22, wherein the step of flowing said bubble containing liposomes through a filter comprises discharging said bubble containing liposomes from said vessel into a syringe.

53. A method according to claim 4 wherein said liposomes further comprise a polymer on the surface of said liposomes.

54. A method according to claim 53 wherein said polymer comprises polyethylene glycol.

8. A method of synthesizing a liposome having encapsulated therein a gas comprising the steps of:

(a) placing a liquid media containing liposomes in a vessel,

(b pressurizing said vessel with a gas,

(c bubbling said gas through said liquid media, thereby dissolving a portion of said gas in said liquid media, so that a portion of said dissolved gas enters said liposomes, and

(d) depressurizing said vessel,

and further comprising the step of sizing said liposomes.

9. The method according to claim 8 wherein the step of depressurizing said vessel further comprises the step of rapidly releasing a portion of said liquid media from said vessel.

10. The method according to claim 9 wherein:

(a) said vessel is approximately cylindrically shaped, said vessel having an inlet port, an outlet port, and a bottom, a tube disposed in said vessel, said tube having first and second ends, said first end of said tube disposed in said bottom of said vessel, said second end of said tube in flow communication with said inlet port, and

(b) the step of pressurizing said vessel and bubbling said gas comprises the steps of introducing said gas into said inlet port under pressure and allowing said gas to flow through said tube, said gas discharging from said tube at said first end of said tube.

11. The method according to claim 10 wherein:

(a) said second end of said tube is in flow communication with said outlet port, and

(b) the step of depressurizing said vessel comprises the step of allowing said pressurizing gas to force said liquid to flow through said tube and out said cutlet port.

12. The method according to claim 10 wherein said bottom of said vessel curves convexly inward, said first end of said tube being closest to the most inward point on said convex bottom.

13. The method according to claim 8 wherein said gas is selected from a group consisting of nitrogen, oxygen, carbon dioxide, xenon, argon, neon and helium.

14. The method according to claim 8 wherein the step of pressurizing said vessel comprises the step of pressurizing said vessel to a pressure in approximately the 50-120 psi range.

15. The method according to claim 8 wherein said liquid media is a phosphate buffered saline solution.

16. The method of according to claim 8 wherein said liposomes comprise one or more lipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.

35. The method according to claim 8, wherein the step of sizing said liposomes is performed prior to placing said liquid media in said vessel, whereby said liposomes are pre-sized.

36. The method according to claim 35, wherein the step of pre-sizing said liposomes comprises flowing said liquid media through a filter.

37. The method according to claim 36, wherein said filter has a pore size in the range of about 0.015 to 10 microns.

38. The method according to claim 36, wherein the step of flowing said liquid media through a filter comprises discharging said liquid media from a syringe.

39. The method according to claim 8, wherein the step of sizing said liposomes is performed after said dissolved gas has entered therein.

40. The method according to claim 39, wherein the step of sizing said liposomes comprises flowing said bubble containing liposomes through a filter.

41. The method according to claim 40, wherein said filter has a pore size in the range of about 0.015 to 10 microns.

42. The method according to claim 40, wherein the step of flowing said bubble containing liposomes through a filter comprises discharging said bubble containing liposomes from said vessel into a syringe.

55. A method according to claim 16 wherein said liposomes further comprise a polymer on the surface of said liposomes.

56. A method according to claim 55 wherein said polymer comprises polyethylene glycol.

25. A method of synthesizing a liposome having encapsulated therein a gas comprising the steps of:

(a) flowing a liquid media containing liposomes through a filter,

(b) placing said liquid media containing liposomes in a vessel,

(c) pressurizing said vessel with a cooled gas, thereby dissolving a first portion of said gas into said liquid media, so that a portion of said dissolved gas enters said liposomes, and

(d) rapidly depressurizing said vessel by releasing a second portion of said gas from said vessel, thereby causing said dissolved gas having entered said liposomes to form bubbles within said liposomes.

57. A method according to claim 25 wherein said liposomes comprise one or more lipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.

58. A method according to claim 57 wherein said liposomes further comprise a polymer on the surface of said liposomes.

59. A method according to claim 58 wherein said polymer comprises polyethylene glycol.

26. A method of synthesizing a liposome having encapsulated therein a gas comprising the steps of:

(a) placing a liquid media containing liposomes in a vessel,

(b) pressurizing said vessel with a cooled gas, thereby dissolving a first portion of said gas into said liquid media, so that a portion of said dissolved gas enters said liposomes, and

(c) rapidly depressurizing said vessel by releasing a second portion of said gas from said vessel, thereby causing said dissolved gas having entered said liposomes to form bubbles within said liposomes,

wherein said liquid media contains non-liposomal solids, and further comprising the step of removing said non-liposomal solids from said liquid media.

27. The method according to claim 26, wherein the step of removing said non-liposomal solids from said liquid media comprises the step of flowing said liquid media through a filter prior to placing said liquid media in said vessel.

28. The method according to claim 26 wherein the step of removing said non-liposomal solids from said liquid media comprises the step of flowing said liposomes through a filter after said bubbles have been formed therein.

60. A method according to claim 26 wherein said liposomes comprise one or more lipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.

61. A method according to claim 60 wherein said liposomes further comprise a polymer on the surface of said liposomes.

62. A method according to claim 61 wherein said polymer comprises polyethylene glycol.

29. A method of synthesizing a liposome having encapsulated therein a gas comprising the steps of:

(a) placing a liquid media containing liposomes in a vessel by discharging said liquid media from a syringe,

(b) pressurizing said vessel with a cooled gas, thereby dissolving a first portion of said gas into said liquid media, so that a portion of said dissolved gas enters said liposomes, and

(c) rapidly depressurizing said vessel by releasing a second portion of said gas from said vessel, thereby causing said dissolved gas having entered said liposomes to form bubbles within said liposomes.

63. A method according to claim 29 wherein said liposomes comprise one or more lipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.

64. A method according to claim 63 wherein said liposomes further comprise a polymer on the surface of said liposomes.

65. A method according to claim 64 wherein said polymer comprises polyethylene glycol.

30. A method of synthesizing a liposome having encapsulated therein a gas comprising the steps of:

(a) placing a liquid media containing liposomes in a vessel,

(b) pressurizing said vessel with a cooled gas, thereby dissolving a first portion of said gas into said liquid media, so that a portion of said dissolved gas enters said liposomes,

(c) rapidly depressurizing said vessel by releasing a second portion of said gas from said vessel, thereby causing said dissolved gas having entered said liposomes to form bubbles within said liposomes, and

(d) directing said bubble containing liposomes from said vessel into a syringe.

66. A method according to claim 30 wherein said liposomes comprise one or more lipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.

67. A method according to claim 66 wherein said liposomes further comprise a polymer on the surface of said liposomes.

68. A method according to claim 67 wherein said polymer comprises polyethylene glycol.

31. A method of synthesizing a liposome having encapsulated therein a gas comprising the steps of:

(a) placing a liquid media containing liposomes in a vessel,

(b) pressurizing said vessel with a cooled gas, thereby dissolving a first portion of said gas into said liquid media, so that a portion of said dissolved gas enters said liposomes,

(c) rapidly depressurizing said vessel by releasing a second portion of said gas from said vessel, thereby causing said dissolved gas having entered said liposomes to form bubbles within said liposomes and causing said dissolved gas in said liquid media external to said liposomes to form bubbles in said external media, and

(d) decavitating said liquid media containing liposomes.

32. The method according to claim 31, wherein the step of decavitating said external bubbles comprises flowing said liquid media through a filter.

33. The method according to claim 32, wherein said filter has a pore size in the range of about 0.015 to 10 microns.

34. The method according to claim 32, wherein the step of flowing said liquid media through a filter comprises discharging said liquid media from said vessel into a syringe.

69. A method according to claim 31 wherein said liposomes comprise one or more lipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.

70. A method according to claim 69 wherein said liposomes further comprise a polymer on the surface of said liposomes.

71. A method according to claim 70 wherein said polymer comprises polyethylene glycol.

43. A method of synthesizing a liposome having encapsulated therein a gas comprising the steps of:

(a) flowing a liquid media containing liposomes through a filter,

(b) placing said liquid media containing liposomes in a vessel,

(c) pressurizing said vessel with a gas,

(d) bubbling said gas through said liquid media, thereby dissolving a portion of said gas in said liquid media, so that a portion of said dissolved gas enters said liposomes, and

(e) depressurizing said vessel.

72. A method according to claim 43 wherein said liposomes comprise one or more lipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.

73. A method according to claim 72 wherein said liposomes further comprise a polymer on the surface of said liposomes.

74. A method according to claim 73 wherein said polymer comprises polyethylene glycol.

44. A method of synthesizing a liposome having encapsulated therein a gas comprising the steps of:

(a) placing a liquid media containing liposomes in a vessel,

(b) pressurizing said vessel with a gas,

(c) bubbling said gas through said liquid media, thereby dissolving a portion of said gas in said liquid media, so that a portion of said dissolved gas enters said liposomes, and

(d) depressurizing said vessel, wherein said liquid media contains non-liposomal solids, and further comprising the step of removing said non-liposomal solids from said liquid media.

45. The method according to claim 44, wherein the step of removing said non-liposomal solids from said liquid media comprises the step of flowing said liquid media through a filter prior to placing said liquid media in said vessel.

46. The method according to claim 44, wherein the step of removing said non-liposomal solids from said liquid media comprises the step of flowing said liposomes through a filter after said dissolved gas has entered therein.

75. A method according to claim 44 wherein said liposomes comprise one or more lipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.

76. A method according to claim 75 wherein said liposomes further comprise a polymer on the surface of said liposomes.

77. A method according to claim 76 wherein said polymer comprises polyethylene glycol.

47. A method of synthesizing a liposome having encapsulated therein a gas comprising the steps of:

(a) placing a liquid media containing liposomes in a vessel by discharging said liquid media from a syringe,

(b) pressurizing said vessel with a gas,

(c) bubbling said gas through said liquid media, thereby dissolving a portion of said gas in said liquid media, so that a portion of said dissolved gas enters said liposomes, and

(d) depressurizing said vessel.

78. A method according to claim 47 wherein said liposomes comprise one or more lipids selected from the group consisting of phosphatidylcholine phosphatidylethanolamine and phosphatidic acid

79. A method according to claim 78 wherein said liposomes further comprise a polymer on the surface of said liposomes.

80. A method according to claim 79 wherein said polymer comprises polyethylene glycol.

48. A method of synthesizing a liposome having encapsulated therein a gas comprising the steps of:

(a) placing a liquid media containing liposomes in a vessel,

(b) pressurizing said vessel with a gas,

(c) bubbling said gas through said liquid media, thereby dissolving a portion of said gas in said liquid media, so that a portion of said dissolved gas enters said liposomes,

(d) depressurizing said vessel, and

(e) directing said liposomes in which said dissolved gas has entered from said vessel into a syringe.

81. A method according to claim 48 wherein said liposomes comprise one or more lipids selected from the consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.

82. A method according to claim 81 wherein said liposomes further comprise a polymer on the surface of said liposomes.

83. A method according to claim 82 wherein said polymer comprises polyethylene glycol.

49. A method of synthesizing a liposome having encapsulated therein a gas comprising the steps of:

(a) placing a liquid media containing liposomes in a vessel,

(b) pressurizing said vessel with a gas,

(c) bubbling said gas through said liquid media, thereby dissolving a portion of said gas in said liquid media, so that a portion of said dissolved gas enters said liposomes,

(d) depressurizing said vessel, and

(e) decavitating said liquid media containing liposomes.

50. The method according to claim 49, wherein the step of decavitating said external bubbles comprises flowing said liquid media through a filter.

51. The method according to claim 50, wherein said filter has a pore size in the range of about 0.015 to 10 microns.

52. The method according to claim 50, wherein the step of flowing said liquid media through a filter comprises discharging said liquid media from said vessel into a syringe.

84. A method according to claim 49 wherein said liposomes comprise one or more lipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.

85. A method according to claim 84 wherein said liposomes further comprise a polymer on the surface of said liposomes.

86. A method according to claim 85 wherein said polymer comprises polyethylene glycol.