Encapsulation of bioactive agents

• US 6,309,569 B1
• Filed: 05/13/1999
• Issued: 10/30/2001
• Est. Priority Date: 05/13/1998
• Status: Expired due to Term

• Current Assignee: Health Protection Agency (Government of United Kingdom)
• Original Assignee: Microbiological Research Authority
• Inventors: Jones, David Hugh, Farrar, Graham Henry, Tinsley-Bown, Anne Margaret
• Primary Examiner(s): Nutter, Nathan M.
• Application Number:
  US09/310,936
• Time in Patent Office: 901 Days
• Field of Search: 264/4.1, 264/4.3, 264/4.33, 264/4.6, 427/213.3, 427/213.31, 427/213.36
• US Class Current: 264/4.1
• CPC Class Codes: A61K 9/1647 Polyesters, e.g. poly(lactide-co-glycolide)

Abstract

Bioactive agent is encapsulated in a polymer microparticle in a (water-in-oil)-in-water emulsion-based method, and using a solvent that comprises ethyl acetate. Also described are microparticles comprising low inherent viscosity (i.v.) PLG, some with i.v. less than 0.5dl/g, and methods for their preparation. DNA release is modified through use of low i.v. PLG. A particle production method for scale-up uses a blender that avoids excessive shear damage to DNA being encapsulated.

Claims

1. A method for encapsulating DNA in a polymer microparticle, comprising:

dissolving polymer in a solvent to form a polymer solution;

preparing an aqueous solution of DNA;

combining the polymer and aqueous solutions with agitation to form a water-in-oil emulsion;

adding the water-in-oil emulsion to a further aqueous phase with agitation to form a (water-in-oil)-in-water emulsion;

adding the (water-in-oil)-in-water emulsion to excess of an aqueous phase to extract the solvent, thereby forming polymer microparticles of a size up to 10 microns in diameter, said microparticles containing DNA in aqueous solution;

wherein the solvent comprises ethyl acetate.

2. A method according to claim 1, wherein the ratio of solvent:

aqueous phase in formation of the water-in-oil emulsion is in the range of 4;

1 to 20;

1 (volume;

volume).

4. A method according to claim 1 wherein the polymer is PLG and the solvent comprises a mixture of ethyl acetate and a co-solvent which increases the amount of PLG that can be dissolved in the solvent provided that the major proportion of the mixture is ethyl acetate.

5. A method according to claim 1 comprising maintaining the agitation of the polymer and aqueous solutions to form the water-in-oil emulsion over a prolonged period of time so as to obtain microparticles the majority of which are of a size up to 10 microns in diameter.

6. A method according to claim 1 comprising maintaining the agitation of the polymer and aqueous solutions to form the water-in-oil emulsion over a prolonged period of time so as to obtain microparticles the majority of which are of a size up to 10 microns in diameter and more than 1 micron in diameter.

11. The method of claim 4, wherein the co-solvent comprises propylene carbonate.

12. The method of claim 4, wherein the co-solvent comprises acetone.

3. A method for encapsulating a bioactive agent in a polymer microparticle, comprising:

dissolving polymer in a solvent to form a polymer solution;

preparing an aqueous solution of a bioactive agent;

combining the polymer and aqueous solutions with agitation to form a water-in-oil emulsion;

adding the water-in-oil emulsion to a further aqueous phase with agitation to form a (water-in-oil)-in-water emulsion;

adding the (water-in-oil)-in-water emulsion to excess of an aqueous phase to extract the solvent, thereby forming polymer microparticles of a size up to 10 microns in diameter said microparticles containing bioactive agent in aqueous solution;

wherein the solvent comprises ethyl acetate, wherein the ratio of solvent;

aqueous phase in formation of the water-in-oil emulsion is in the range of 5;

1 to 12;

1 (volume;

volume).

7. A method for encapsulating DNA in a polymer microparticle, comprising:

dissolving polymer in a solvent to form a polymer solution;

preparing an aqueous solution of DNA;

combining the polymer and DNA solutions with agitation to form a water-in-oil emulsion;

adding the water-in-oil emulsion to a further aqueous phase with agitation to form a (water-in-oil)-in-water emulsion;

adding the (water-in-oil)-in-water emulsion to excess of an aqueous phase to extract the solvent, thereby forming polymer microparticles of a size up to 10 microns in diameter, said microparticles containing DNA in aqueous solution;

wherein the DNA comprises a coding sequence that is expressed in a recipient following oral administration, and wherein the solvent comprises ethyl acetate.

8. A method of making a microparticle for use as or in a vaccinating composition, comprising following the method according to claim 7 wherein the microparticle contains DNA coding for an antigen.

13. A method according to claim 7 wherein the polymer is PLG and the solvent comprises a mixture of ethyl acetate and a co-solvent which increases the amount of PLG that can be dissolved in the solvent, provided that the major proportion of the mixture is ethyl acetate.

14. A method according to claim 7 comprising maintaining the agitation of the polymer and aqueous solutions to form the water-in-oil emulsion over a prolonged period of time so as to obtain microparticles the majority of which are of a size up to 10 microns in diameter.

15. A method according to claim 7 comprising maintaining the agitation of the polymer and,aqueous solutions to form the water-in-oil emulsion over a prolonged period of time so as to obtain microparticles the majority of which are of a size up to 10 microns in diameter and more than 1 micron in diameter.

9. A method for encapsulating a bioactive agent in a polymer microparticle, comprising:

dissolving polymer in a solvent to form a polymer solution;

preparing an aqueous solution of a bioactive agent;

combining the polymer and aqueous solutions with agitation to form a water-in-oil emulsion;

adding the water-in-oil emulsion to a further aqueous phase with agitation to form a (water-in-oil)-in-water emulsion;

adding the (water-in-oil)-in-water emulsion to excess of an aqueous phase to extract the solvent, thereby forming microparticles of a size up to 10 microns in diameter, said microparticles containing bioactive agent in aqueous solution;

wherein the agitation steps comprise agitating the solutions or emulsions in the blending compartment of a blender, and using a blending blade which both blends and mixes the contents of the blending compartment.

10. A method according to claim 9 wherein the blending is continued for a prolonged period of time so as to obtain microparticles the majority of which are of a size up to 10 microns in diameter.