Ultrasound contrast agents and process for the preparation thereof
Method for preparing a lyophilized matrix and, upon reconstitution of the same, a respective injectable contrast agent comprising a liquid aqueous suspension of gas-filled microbubbles stabilized predominantly by a phospholipid and comprising a ligand agent. The method comprises preparing an emulsion from an aqueous medium, comprising a phospholipid and a water immiscible organic solvent. A suspension of a compound comprising the ligand agent or a precursor thereof is then added to emulsion. The emulsion is then freeze-dried and subsequently reconstituted in an aqueous suspension of gas-filled microbubbles.
1 . A method for preparing an aqueous suspension of gas-filled microbubbles comprising a ligand agent, which comprises the steps of
a) preparing an aqueous-organic emulsion comprising i) an aqueous medium, ii) an organic solvent substantially immiscible with water, iii) an amphiphilic material comprising a phospholipid and iv) a lyoprotecting agent;
b) adding a precursor of said ligand agent with said aqueous-organic emulsion;
c) converting said precursor into said ligand agent;
d) lyophilizing said mixture, to obtain a lyophilized matrix;
e) contacting said lyophilized matrix with a biocompatible gas; and
f) reconstituting said lyophilized matrix by dissolving it in a physiologically acceptable aqueous carrier liquid, to obtain said suspension of gas-filled microbubbles.
2 . A method according to claim 1 , wherein said precursor comprises a phospholipid.
3 . A method according to claim 1 , wherein said precursor comprises a hydrophilic polymer covalently bound to said phospholipid.
4 . A method according to any one of claims 2 or 3 , wherein said phospholipid is a phosphoethanolamine.
5 . A method according to any one of claims 2 or 3 , wherein said hydrophylic polymer is a polyethyleneglycol.
6 . A method according to claim 1 , wherein said precursor is added in a molar amount of from about 0.1% to about 10% of a molar amount of said amphiphilic material.
7 . A method according to claim 1 , wherein step c comprises adding a compound comprising the ligand agent to the emulsion comprising said precursor.
8 . A method according to claim 7 , wherein the compound comprising the ligand agent is added in a molar ratio of from about 1:2 to about 1:10000 with respect to the precursor in the emulsion.
9 . A method according to claim 8 , wherein said molar ratio is of from about 1:5 to 1:1000.
10 . A method according to claim 1 , wherein said ligand agent comprises a targeting ligand.
11 . A method according to claim 10 , wherein said targeting ligand is an antibody or a fragment thereof.
12 . A method according to claim 1 wherein said ligand agent comprises a moiety of an affinity binding pair.
13 . A method according to claim 12 wherein said moiety is avidin or streptavidin.
14 . A method according to any one of claims 12 or 13 , which comprises the further step of adding a targeting ligand, comprising a complementary moiety of said affinity binding pair, to the suspension of gas-filled microbubbles obtained in step f).
15 . A method according to claim 14 , wherein the targeting ligand is an antibody or a fragment thereof.
16 . A method according to claim 14 , wherein said targeting ligand is added in a molar ratio of from about 1:2 to about 20:1 with respect to the ligand agent.
17 . A method according to claim 1 wherein said biocompatible gas comprises SF 6 or a perfluorocarbon, optionally in admixture with air, nitrogen, oxygen or carbon dioxide.
18 . A method according to claim 1 wherein said perfluorocarbon is perfluoropropane or perfluorobutane.
19 . A method for preparing a lyophilized precursor of gas-filled microbubbles comprising a ligand agent, comprising the steps of
a) preparing an aqueous-organic emulsion comprising i) an aqueous medium, ii) an organic solvent substantially immiscible with water, iii) an amphiphilic material comprising a phospholipid and iv) a lyoprotecting agent;
b) adding a precursor of the ligand agent to said aqueous-organic emulsion;
c) converting said precursor of the ligand agent into said ligand agent;
d) lyophilizing said mixture, to obtain a lyophilized matrix containing said precursor of gas-filled microbubbles.
20 . A method according to claim 19 , wherein said precursor of the ligand agent comprises a phospholipid.
21 . A method according to claim 19 , wherein said precursor of the ligand agent comprises a hydrophilic polymer covalently bound to said phospholipid.
22 . A method according to any one of claims 20 or 21 , wherein said phospholipid is a phosphoethanolamine.
23 . A method according to any one of claims 20 or 21 , wherein said hydrophylic polymer is a polyethyleneglycol.
24 . A method according to claim 19 , wherein said precursor of the ligand agent is added in a molar amount of from about 0.1% to about 10% of a molar amount of said amphiphilic material.
25 . A method according to claim 19 , wherein step c comprises adding a compound comprising the ligand agent to the emulsion comprising said precursor of the ligand agent.
26 . A method according to claim 21 , wherein the compound comprising the ligand agent is added in a molar ratio of from about 1:2 to about 1:10000 with respect to the precursor in the emulsion.
27 . A method according to claim 19 , wherein said ligand agent comprises a targeting ligand.
28 . A method according to claim 27 , wherein said targeting ligand is an antibody or a fragment thereof.
29 . A method according to claim 19 , wherein said ligand agent comprises a moiety of an affinity binding pair.
30 . A method according to claim 29 wherein said moiety is avidin or streptavidin.
31 . A method according to any one of claims 1 or 19 , wherein said precursor of the ligand agent is converted into the ligand agent before adding said precursor to the aqueous-organic emulsion.
32 . A method according to any one of claims 1 or 19 , wherein said precursor of the ligand agent is added to said aqueous-organic emulsion in the form of an aqueous micellar suspension of said ligand agent.
33 . A method according to claim 32 wherein said precursor of the ligand agent is converted into the ligand agent in said micellar suspension, before the addition thereof to the aqueous-organic emulsion.
34 . A method for preparing a suspension of gas-filled microbubbles comprising a targeting ligand bound to an amphiphilic compound by means of an affinity binding pair, which comprises the steps of:
a) contacting a lyophilized precursor of gas-filled microbubbles, obtained according to method comprising the steps of:
preparing an aqueous-organic emulsion comprising i) an aqueous medium, ii) an organic solvent substantially immiscible with water, iii) an amphiphilic material comprising a phospholipid and iv) a lyoprotecting agent;
adding a precursor of a moiety of an affinity binding pair to said aqueous-organic emulsion;
converting said precursor of the moiety of the affinity binding pair into said moiety; and
lyophilizing said mixture, to obtain said lyophilized precursor;
with a biocompatible gas;
b) reconstituting said lyophilized precursor by dissolving it in a physiologically acceptable aqueous carrier liquid, to obtain said suspension of gas-filled microbubbles;
c) adding a targeting ligand, comprising a complementary moiety of said affinity binding pair, to the suspension of gas-filled microbubbles.
35 . A method according to claim 15 , wherein said targeting ligand is added in a molar ratio of from about 1:2 to about 20:1 with respect to the ligand agent.