Method for the preparation of stable suspensions of hollow gas-filled microspheres suitable for ultrasonic echography

• US 5,310,540 A
• Filed: 05/06/1992
• Issued: 05/10/1994
• Est. Priority Date: 10/05/1990
• Status: Expired due to Term

• Current Assignee: Bracco Suisse SA
• Original Assignee: Sintetica SA
• Inventors: Giddey, Claude, Dove, Georges
• Primary Examiner(s): Raymond, Richard L.
• Application Number:
  US07/855,032
• Time in Patent Office: 734 Days
• Field of Search: 424/9, 424/450, 128/662.02, 128/660.01
• US Class Current: 424/9.52
• CPC Class Codes: A61K 49/223 Microbubbles, hollow microspheres, free gas bubbles, gas microspheres

Abstract

A viscous solution of a filmogenic protein is whipped into a foam and the foam is subjected to shear to reduce the size of the foam bubbles to a range (about 0.5-10 μm) suitable for echography. The proteins include animal and vegetal proteins or partial hydrolyzates thereof with foaming properties. The suspensions of microspheres can be stabilized by heat or by cross-linkers.

Claims

1. A method for preparing stable aqueous suspensions of air or gas filled microbubbles suitable for ultrasonic applications in living organisms, said method comprising the steps of:

a) converting a viscous aqueous solution of a filmogenic protein into a foam of air or gas filled bubbles by whipping with air or a pharmaceutically acceptable gas to produce microbubbles bound by a membrane of said protein, and

(b) subjecting, simultaneously or subsequently, the foaming solution to high shear by forcing it through narrow openings or channels for a time sufficient to reduce the size of the bubbles in the foam to values in a range of about 0.5-10 μ

m suitable for injection.

2. The method of claim 1, wherein said openings or channels contain baffling means.

3. The method of claim 1, in which the foam is subjected to high shear in a homogenizer and emulsifier apparatus comprising a tubular head with parallel axially oriented slits at the periphery and an axially rotating cutter in the center the action of which causes air from the surroundings to be admixed with the liquid and the air-liquid mixture to be forced radially through the slits, thus subjecting the foam to a high shear effect.

4. The method of claim 3, in which the temperature is kept below 50°

C. during foaming and bubble size reduction to avoid protein denaturation by the heat developed by the homogenizer-emulsifier apparatus.

5. The method of claim 1, in which the resulting suspension has a concentration of microbubbles in the range of 10⁸ -10⁹ /ml.

6. The method of claim 1, in which the resulting suspension is further diluted with water or a physiologically acceptable liquid to decrease the viscosity to a few cP so as to make it directly injectable into the blood-stream of living bodies.

7. The method of claim 6, in which the diluted solution is freeze-dried into a solid which can be kept unchanged for an indefinite period in dryness and which will regenerate the suspension of microbubbles upon admixing with a corresponding quantity of water or a physiologically acceptable liquid suitable for injections.

8. The method of claim 6, in which the diluting liquid comprises a stabilizer to increase the storage stability of the diluted suspension.

9. The method of claim 8, wherein the stabilizer is an organic aldehyde.

10. The method of claim 6, which comprises heating the diluted solution to temperatures between 60°

and 120°

C. for a few seconds to a few minutes to effect hardening of the membrane protein by denaturation.

11. The method of claim 1, in which the foam is subjected to high shear by forcing it through a mineral or organic material with a plurality of baffled channels providing attrition effects on the bubbles.

12. The method of claim 5, in which the resulting suspension is further heated at 60°

-120°

C. for a few seconds to a few minutes to modify the properties of the bubble bounding protein by progressive denaturation.

13. An aqueous composition to be converted by the method of claim 1 to a suspension of microbubbles suitable for ultrasonic echography, comprising, in solution, 0.1-5% by weight of a filmogenic protein and 40-80% by weight of a water-soluble polyol viscosity enhancer to raise the ambient temperature viscosity to about 100-600 cP.

14. The composition of claim 13, comprising surfactants as foam enhancers and cross-linking agents for increasing stability of the foam.

15. The composition of claim 13, in which the polyol viscosity enhancer has at least four OH groups per molecule.

16. The composition of claim 15, in which the viscosity enhancer is selected from the group consisting of polyalcohols, mono-, poly- and oligosaccharides and mixtures thereof.

17. The composition of claim 16, in which the viscosity enhancer is selected from the group consisting of glucose, fructose, mannose, dextrose, dextran, polydextrose, sugar syrups, corn syrup, polyglycerol with at least four glycerol units, and mixtures thereof.

18. The composition of claim 13, in which a stabilizing agent is present.

19. The composition of claim 18, in which the stabilizing agent is formaldehyde or glutaraldehyde.

20. The suspension of microbubbles which results from carrying out the method of claim 1, said suspension being stable for at least four weeks.

21. The suspension of claim 20, in which stability is further increased by heating to 60°

-120°

C. for a few seconds to a few minutes to effect hardening of the filmogenic protein.

22. A method of conducting an echographic investigation of a living body, said method comprising the steps of:

a) diluting a suspension of air or gas filled microbubbles produced according to the method of claim 19 with a physiologically acceptable liquid to obtain a suspension with a viscosity of about 2-30 cP;

b) injecting the diluted suspension obtained into the blood-stream of said body, and

c) obtaining an echographic response generated by said body.