Means of preparation and applications of liposomes containing high concentrations of entrapped ionic species

• US 4,310,506 A
• Filed: 02/20/1980
• Issued: 01/12/1982
• Est. Priority Date: 02/22/1979
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Gamble, Ronald C., Baldeschwieler, John D., Mauk, Marcia R.
• Primary Examiner(s): Nucker, Christine M.
• Application Number:
  US06/123,162
• Time in Patent Office: 692 Days
• Field of Search: 424/1, 424/1.5, 424/9, 424/35, 252/316, 252/319
• US Class Current: 424/1.21
• CPC Class Codes: A61K 2123/00 Preparations for testing in vivo
• CPC Class Codes: A61K 51/1234 Liposomes
• CPC Class Codes: A61K 9/1278 Post-loading, e.g. by ion or pH gradient
• CPC Class Codes: Y10T 428/2984 Microcapsule with fluid core [includes liposome]

Abstract

Unilamellar vesicles comprising a lipid bilayer, an ionophore being incorporated in said lipid bilayer, a chelating agent entrapped within the vesicles, and an effective amount of physiologically compatible cation bound to said chelating agent within the vesicles. Unilamellar vesicles are loaded by incubating the vesicles with the cation, terminating the incubation and recovering the loaded vesicles by chromatography. In some applications of the invention, the cation may be a radioactive tracer, in which case the vesicles can be administered to the human host and observed by scintillation techniques to produce a radioimage which is useful for diagnostic purposes.

Claims

1. Vesicles comprising a lipid bilayer, an ionophore being incorporated in said lipid bilayer, a chelating agent entrapped within the vesicles, and an effective amount of physiologically compatible cation bound to said chelating agent within the vesicles.

2. The vesicles of claim 1 wherein the ionophore is [6S-[6α

(2S*,3S*)-,8β

(R*),9β

,11α

]]-5-(methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7-dioxaspiro[5,5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid.

3. The vesicles of claim 1 wherein the cation is selected from the group consisting of all bivalent and trivalent cations.

4. The vesicles of claim 1 wherein the cation is a radioactive tracer.

5. The vesicles of claim 1 wherein the cation is a radioactive tracer selected from the group consisting of:

¹¹¹ In, ⁴⁵ Ca, ⁵¹ Cr, ⁹⁹ Tc, ⁶⁷ Ga, ⁵⁷ Co, and ⁶⁵ Zn.

6. The vesicles of claim 1 wherein the bilayer includes cholesterol.

7. The vesicles of claim 1 wherein the bilayer includes L-α

-distearoyl phosphatidylcholine (DSPC).

8. The vesicles of claim 1 wherein the bilayer includes L-α

-dipalmitoyl phosphatidylcholine (DPPC).

9. The vesicles of claim 1 wherein the chelating agent is selected from the group consisting of nitrilotriacetic acid, diethylenetriaminepentaacetic acid, diaminocyclohexanetetraacetic acid, iminodiacetic acid and ethylenediaminetetraacetic acid.

11. The method of claim 8 wherein the ionophore is [6S-[6α

(2S*,3S*),8β

(R*),9β

,11α

]]-5-(methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7-dioxaspiro[5,5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid.

10. The method comprising administering to the mammalian host vesicles comprising a lipid bilayer, an ionophore being incorporated in said lipid bilayer, a chelating agent entrapped within the vesicles, and an effective amount of physiologically compatible radioactive tracer bound to said chelating agent within the vesicles, and observing at least some body portion by scintillation counting technique to observe the radioimage produced.

12. The method of claim 10 wherein the tracer is selected from the group consisting of:

¹¹¹ In, ⁴⁵ Ca, ⁵¹ Cr, ⁹⁹ Tc, ⁶⁷ Ga, ⁵⁷ Co, and ⁶⁵ Zn.

13. The method of claim 10 wherein the bilayer includes cholesterol.

14. The method of claim 10 wherein the bilayer includes L-α

-distearoyl phosphatidylcholine (DSPC).

15. The method of claim 10 wherein the bilayer includes L-α

-dipalmitoyl phosphatidylcholine (DPPC).

16. The method of claim 10 wherein the chelating agent is selected from the group consisting of nitrilotriacetic acid, diethylenetriaminepentaacetic acid, diaminocyclohexanetetraacetic acid, iminodiacetic acid, and ethylenediaminetetraacetic acid.

17. The method of determining the distribution and condition of vesicles within a mammal, said vesicles comprising a lipid bilayer, an ionophore being incorporated in said lipid bilayer, a chelating agent entrapped within the vesicles, and an effective amount of physiologically compatible radioactive tracer bound to said chelating agent within the vesicles, said method comprising:

(a) determining the initial rotational correlation time of the radioactive tracer through measurement of the time-integrated perturbation factor of said vesicles,

(b) injecting said vesicles into said mammal,

(c) observing by scintillation techniques the distribution of said vesicles, and

(d) determining any change in said time-integrated perturbation factor of the whole body or of parts by appropriate shielding.

18. The method of claim 17 wherein the ionophore is [6S-[6α

(2S*,3S*),8β

(R*),9β

,11α

]]-5-(methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl-1,7-dioxaspiro[5,5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid.

19. The method of claim 17 wherein the tracer is selected from the group consisting of:

¹¹¹ In, ⁴⁵ Ca, ⁵¹ Cr, ⁹⁹ Tc, ⁶⁷ Ga, ⁵⁷ CO, and ⁶⁵ Zn.

20. The method of claim 17 wherein the bilayer includes cholesterol.

21. The method of claim 17 wherein the bilayer includes L-α

-distearoyl phosphatidylcholine (DSPC).

22. The method of claim 17 wherein the bilayer includes L-α

-dipalmitoyl phosphatidylcholine (DPPC).

23. The method of claim 17 wherein the chelating agent is selected from the group consisting of nitrilotriacetic acid, diethylenetriaminepentaacetic acid, diaminocyclohexanetetraacetic acid, iminodiacetic acid, and ethylenediaminetetraacetic acid.

24. The method of loading lipid vesicles comprising:

(1) incubating vesicles comprising a lipid bilayer, an ionophore in said lipid bilayer and a chelating agent entrapped within said vesicles, with,

(2) a physiologically compatible cation,

(3) terminating said incubation, and

(4) recovering the loaded vesicles by chromatography.

25. The method of claim 24 wherein the ionophore is [6S-[6α

(2S*,3S*),8β

(R*),9β

,11α

]]-5-(methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7-dioxaspiro[5,5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid.

26. The method of claim 24, wherein the cation is selected from the group consisting of all bivalent and trivalent cations.

27. The method of claim 24, wherein the cation is a radioactive tracer.

28. The method of claim 24 wherein the cation is a radioactive tracer selected from the group consisting of:

¹¹¹ In, ⁴⁵ Ca, ⁵¹ Cr, ⁹⁹ Tc, ⁶⁷ Ga, ⁵⁷ Co, and ⁶⁵ Zn.

29. The method of claim 24 wherein the bilayer includes cholesterol.

30. The method of claim 24 wherein the bilayer includes L-α

-distearoyl phosphatidylcholine (DSPC).

31. The method of claim 24 wherein the bilayer includes L-α

-dipalmitoyl phosphatidylcholine (DPPC).

32. The method of claim 24 wherein the chelating agent is selected from the group consisting of nitrilotriacetic acid. diethylenetriaminepentaacetic acid, diaminocyclohexanetetraacetic acid, iminodiacetic acid, and ethylenediaminetetraacetic acid.