Methods for making ultrasound contrast agents
Provided herein are improved methods for preparing phospholipid formulations including phospholipid UCA formulations.
1. A method of ultrasound contrast imaging a subject comprising
(a) forming a phospholipid solution that comprises
(i) a phospholipid blend comprising one or more phospholipids and a non-aqueous solvent, wherein the phospholipid blend is formed in the presence of one or two but not all three of methanol, toluene and methyl t-butyl ether (MTBE), and
(ii) calcium and/or magnesium divalent metal cation present at a total divalent metal cation concentration of less than 0.7 parts per million (ppm),
(b) combining the phospholipid solution with an aqueous solution to form a phospholipid suspension,
(c) activating the phospholipid suspension with a perfluorocarbon gas to form lipid-encapsulated gas microspheres,
(d) administering the lipid-encapsulated gas microspheres to a subject, and
(e) obtaining an ultrasound image of the subject.
2. The method of claim 1 , wherein the one or more phospholipids comprise (a) DPPC and MPEG-5000-DPPE or (b) DPPA, DPPC and MPEG-5000-DPPE.
3. The method of claim 1 , wherein the one or more phospholipids comprise DPPA, DPPC and MPEG-5000-DPPE.
4. The method of claim 2 , wherein DPPC has a combined calcium and magnesium concentration of less than 90 ppm, and MPEG5000-DPPE has a combined calcium and magnesium concentration of less than 115 ppm.
5. The method of claim 1 , wherein the non-aqueous solvent comprises propylene glycol.
6. The method of claim 1 , wherein the non-aqueous solvent has a combined calcium and magnesium concentration of less than 0.7 ppm.
7. The method of claim 1 , wherein the non-aqueous solvent comprises propylene glycol and glycerol.
8. The method of claim 1 , wherein the one or more phospholipids comprise DPPA, DPPC and MPEG-5000-DPPE in molar percentages of 5-15%, 77-90% and 5-15%, respectively.
9. The method of claim 1 , wherein the one or more phospholipids comprise DPPA, DPPC and MPEG-5000-DPPE in a mole % ratio of 10 to 82 to 8 (10:82:8).
10. The method of claim 1 , wherein the perfluorocarbon gas is perfluoropropane gas.
11. The method of claim 1 , wherein the one or more phospholipids comprise DPPC and MPEG-5000-DPPE, the phospholipid solution comprises DPPC, MPEG5000-DPPE and propylene glycol, the aqueous solution comprises water, propylene glycol and glycerol, and the phospholipid suspension comprises DPPC, MPEG5000-DPPE, propylene glycol, water and glycerol.
12. The method of claim 1 , wherein the aqueous solution further comprises one or more salts.
13. The method of claim 1 , wherein the aqueous solution further comprises one or more buffers.
14. The method of claim 3 , wherein the MPEG5000-DPPE has a combined calcium and magnesium concentration of less than 115 ppm.
15. The method of claim 1 , wherein the phospholipid blend is formed in the presence of methanol.
16. The method of claim 2 , wherein the phospholipid blend is formed in the presence of methanol.
17. The method of claim 3 , wherein the phospholipid blend is formed in the presence of methanol.
18. The method of claim 1 , wherein the phospholipid blend is formed in the presence of toluene.
19. The method of claim 2 , wherein the phospholipid blend is formed in the presence of toluene.
20. The method of claim 3 , wherein the phospholipid blend is formed in the presence of toluene.
21. The method of claim 1 , wherein the phospholipid blend is formed in the presence of methanol and toluene.
22. The method of claim 2 , wherein the phospholipid blend is formed in the presence of methanol and toluene.
23. The method of claim 3 , wherein the phospholipid blend is formed in the presence of methanol and toluene.
24. The method of claim 1 , wherein the phospholipid blend is formed in the presence of MTBE.
25. The method of claim 2 , wherein the phospholipid blend is formed in the presence of MTBE.
26. The method of claim 3 , wherein the phospholipid blend is formed in the presence of MTBE.
27. A method of ultrasound contrast imaging a subject comprising
(a) forming a phospholipid blend comprising one or more phospholipids, in the presence of one or two but not all three of methanol, toluene and methyl t-butyl ether (MTBE),
(b) combining the phospholipid blend with a non-aqueous solvent to form a phospholipid solution having calcium and/or magnesium divalent metal cation present at a total divalent metal cation concentration of less than 0.7 parts per million (ppm),
(c) combining the phospholipid solution with an aqueous solution to form a phospholipid suspension,
(d) activating the phospholipid suspension with a perfluorocarbon gas to form lipid-encapsulated gas microspheres,
administering the lipid-encapsulated gas microspheres to a subject, and obtaining an ultrasound image of the subject.