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1. WO2010078045 - METHOD OF MAKING SMALL LIPOSOMES

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WHAT IS CLAIMED IS:

1. A method for producing a preparation of liposomes of constrained particle size, said method comprising the steps of a) providing a substantially continuously flowing stream of water, b) providing a substantially continuously flowing stream of an organic solvent, said organic solvent containing, dissolved therein, at least one lipid, said lipid or lipids being capable of forming liposomes, c) substantially continuously mixing said stream of water and said stream of organic solvent, so as to obtain a mixture, and d) cooling said mixture, and e) allowing liposomes to form within said mixture, the ratio of the flow rate of the stream of water to the flow rate of the stream of organic solvent, and the rate of cooling of said mixture, being controlled so as to obtain a preparation of liposomes such that at least about 90% of the liposomes are of a particle size less than about 200 nm.

2. The method of claim 1 wherein the ratio of the flow rate of the stream of water to the flow rate of the stream of organic solvent is at least about 2:1.

3. The method of claim 1 wherein the ratio of the flow rate of the stream of water to the flow rate of the stream of organic solvent is not more than about 19:1. 4. The method of claim 1 wherein the ratio of the flow rate of the stream of water to the flow rate of the stream of organic solvent is at least about 3:1 and not more than about 19:1.

5. The method of claim 1 wherein the ratio of the flow rate of the stream of water to the flow rate of the stream of organic solvent is at least about 3:1 and not more than about 9:1.

6. The method of claim 1 wherein the ratio of the flow rate of the stream of water to the flow rate of the stream of organic solvent is about 4:1.

7. The method of any one of claims 1-6 wherein the rate of cooling is on average at least about 4°C per hour. 8. The method of any one of claims 1-7 wherein the mixture is cooled byat least about 200C in not more than about 2 hours. 9. The method of any one of claims 1-8 wherein said organic solvent stream is, prior to said mixing, at a temperature at least 100C above the transition temperature of said lipids.

10. The method of any one of claims 1-9 wherein at least one lipid is a phospholipid.

11. The method of claim 10 wherein at least one phospholipid is DPPC.

5 12. The method of any one of claims 1-11 wherein at least one lipidis a sterol.

13. The method of claim 12 wherein said sterol is cholesterol.

14. The method of any one of claims 1-13 wherein the organic solvent is tert-butanol.

15. The method of any one of claims 1-14 wherein the mixture is cooled from at least about 55°C to not more than about 35°C in not more than about 2 hours.

10 16. The method of any one of claims 1-15 wherein the organic solvent further contains, dissolved therein, a bioactive agent.

17. The method of claim 16 wherein the bioactive agent is a MUC-I peptide, or a glycosylated and/or lipidated derivative of such a peptide.

18. The method of claim 17 wherein the bioactive agent has the amino acid sequence 15 of SEQ ID NO:l.

19. The method of claim 18 wherein the agent is lipidated at the lysine.

20. The method of any one of claims 17-19 wherein the agent is palmitoylated.

21. The method of claim 17 wherein the bioactive agent has the amino acid sequence of SEQ ID NO:2.

20 22. The method of claim 21 wherein the bioactive agent is lipidated at the two final serines.

23. The method of claim 22 in which the bioactive agent is glycosylated.

24. The method of claim 23 in which the glycosylation pattern is the same as that defined for BGLP40.

25 25. The method of any one of claims 1-24, further comprising providing means for inducing turbulence in said stream of water, said stream of organic solvent, or in a stream of mixture resulting from said mixing.