Freeze-dried product and gas-filled microvesicles suspension

1. A suspension of gas-filled microvesicles comprising a freeze-dried product dissolved in a pharmaceutically acceptable liquid carrier, said freeze-dried product comprising 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dipalmitoylphosphatidylglycerol sodium salt (DPPG-Na), palmitic acid and polyethylene glycol 4000 (PEG4000), wherein said suspension has a value of critical pressure (Pc50) of at least 20 kPa, and wherein the microvesicles are filled with a SF 6 gas.

2. The suspension according to claim 1 , wherein said suspension has a value of Pc50 of at least 22 kPa.

3. The suspension according to claim 1 , wherein said suspension has a value of Pc50 of at least 25 kPa.

4. The suspension according to claim 1 , wherein said suspension has a value of Pc50 of from 20 kPa to 32 kPa.

5. The suspension according to claim 1 , wherein the weight ratio DSPC/DPPG-Na/palmitic acid is of 4.75/4.75/1.

6. The suspension of claim 5 , wherein the weight ratio between DSPC/DPPG-Na/palmitic acid and PEG4000 is of 0.017:1.

7. A freeze-dried product obtained according to a method comprising:

a. preparing a liquid mixture comprising (i) an amphiphilic material comprising 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dipalmitoylphosphatidylglycerol (DPPG) and palmitic acid; and (ii) a polyethylene glycol as freeze-drying protecting component in a solvent;

b. sampling said liquid mixture into a glass vial;

c. freeze-drying the liquid mixture to remove said solvent and obtain a freeze-dried product; and

d. after completion of the freeze-drying of step c:

saturating a headspace of said vial with a SF 6 gas; and

heating said freeze-dried product at ambient pressure at a temperature higher than 35° C. and lower than the melting point of the polyethylene glycol freeze-drying protecting component, for a period of time of from eight to twenty hours, wherein the freeze-dried product of step c has not been reconstituted prior to step d, and wherein the freeze-dried product is suitable for the preparation of a suspension of stabilized gas-filled microbubbles.

8. A method of diagnosing which comprises

(i) administering to a patient the suspension of gas-filled microvesicles according to claim 1 ;

(ii) applying an ultrasound beam to the patient; and detecting an ultrasound signal from a region of interest in said patient.

9. The method according to claim 8 , wherein the region of interest in said patient is the heart.

10. The method according to claim 8 , wherein the region of interest in said patient is the liver.

11. The method according to claim 8 , wherein the region of interest in said patient is the urinary tract.

12. The method according to claim 8 , wherein said suspension has a value of Pc50 of from 20 kPa to 32 kPa.

13. The method according to claim 8 , wherein the weight ratio DSPC/DPPG-Na/palmitic acid is of 4.75/4.75/1.

14. The method according to claim 13 , wherein the weight ratio between DSPC/DPPG-Na/palmitic acid and PEG4000 is of 0.017:1.

15. The suspension of gas-filled microvesicles according to claim 1 obtained according to a method comprising:

a. preparing a liquid mixture comprising (i) an amphiphilic material comprising 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),

dipalmitoylphosphatidylglycerol sodium salt (DPPG-Na) and palm itic acid; and

(ii) a polyethylene glycol as freeze-drying protecting component in a solvent;

b. sampling said liquid mixture into a glass vial;

c. freeze-drying the liquid mixture to remove said solvent and obtain a freeze-dried product;

d. after completion of the freeze-drying of step c:

saturating a headspace of said vial with a SF 6 gas; and

heating said freeze-dried product at ambient pressure at a temperature higher than 35° C. and lower than the melting point of the polyethylene glycol freeze-drying protecting component, for a period of time of from eight to twenty hours, wherein the freeze-dried product of step c has not been reconstituted prior to step d; and

e. reconstituting the freeze-dried product with a pharmaceutically acceptable liquid carrier in the presence of said SF 6 gas under gentle agitation to obtain the suspension of gas-filled microvesicles.

16. The freeze-dried product according to claim 7 wherein the glass vial is stoppered and sealed after saturating the headspace of said vial with said SF 6 gas.

17. A method of diagnosing which comprises

(i) reconstituting the freeze-dried product according to claim 7 with a pharmaceutically acceptable liquid carrier in the presence of said SF 6 gas under gentle agitation to obtain a suspension of gas-filled microvesicles;

(ii) administering to a patient the suspension of gas-filled microvesicles;

(iii) applying an ultrasound beam to the patient and detecting an ultrasound signal from a region of interest in said patient.

18. The method according to claim 17 , wherein the region of interest in said patient is the heart.

19. The method according to claim 17 , wherein the region of interest in said patient is the liver.

20. The method according to claim 17 , wherein the region of interest in said patient is the urinary tract.