Liposomes for in-vivo delivery

Patent metadata

Field	Value
Publication number	US-9655848-B2
Application number	US-201314440834-A
Country	US
Kind code	B2
Filing date	Nov 19, 2013
Priority date	Nov 19, 2012
Publication date	May 23, 2017
Grant date	May 23, 2017

Abstract

The present invention provides a liposome based composition wherein the liposome includes a peptide conjugated thereto via a peptide bond, wherein the peptide includes a spacer amino acid and a short Apolipoprotein E recognition sequence or a short Amyloid beta recognition sequence. This invention further provides a process for making the liposome and methods of utilizing the liposome based composition for therapeutic and diagnostic purposes.

First claim

The invention claimed is:

1. A composition comprising a liposome and a liposome-anchored blood-brain-barrier (BBB) recognition peptide covalently conjugated to said liposome, said covalently conjugated is conjugated via a peptide bond between a carboxyl end of a succinate group within said liposome lipid bilayer and an amino group of said peptide, wherein said BBB recognition peptide comprises the amino acid sequence AHRERMS (SEQ ID NO: 6) or ARERMS (SEQ ID NO: 4).

2. The composition of claim 1 , wherein said liposome further comprises a second peptide comprising the amino acid sequence of SEQ ID NO: 2.

3. The composition of claim 2 , wherein said second peptide consists the amino acid sequence of ALRKLRKRLLR (SEQ ID NO: 3).

4. The composition of claim 1 , wherein said conjugated is conjugated via a peptide bond between a carboxyl end of a succinate group within the liposomal lipid bilayer and the amino group of said at least one spacer amino acid.

5. The composition of claim 1 , wherein said peptide consists the amino acid sequence AHRERMS (SEQ ID NO: 6).

6. The composition of claim 1 , wherein said liposome further comprises a brain therapeutic compound.

7. The composition of claim 1 , wherein said liposome further comprises a brain probing agent.

8. The composition of claim 1 , wherein said liposome comprises cholesterol (Ch), 1,2-dioleoyl-sft-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE).

9. The composition of claim 8 , wherein the weight ratio of: Ch, DOPC, and DOPE is 1:1:1.

10. The composition of claim 1 , wherein said liposome comprises cholesterol (Ch), 1,2-5 dioleoyl-sft-glycero-3-[phosphor-L-serine] (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

11. The composition of claim 10 , wherein the weight ratio of: Ch, DOPS, and DOPE is 10 1:1:1.

12. The composition of claim 1 , wherein said composition is lyophilized.

13. The composition of claim 1 , wherein the liposome's diameter is between 10 nm to 200 nm.

14. A method for delivering a liposome into a subject's brain, comprising administering to said subject the composition of claim 1 , thereby delivering a liposome into a subject's brain.

15. The method of claim 14 , wherein said delivering comprises systemically administering.

16. A process for the preparation of a liposome conjugated to a peptide comprising at least one spacer amino acid followed by an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, or 6, comprising the steps:
(a) dissolving in ethanol or chloroform: (1) Cholesterol (Ch), 1,2-dioleoyl-5nglycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); or (2) Cholesterol (Ch), 1,2-dioleoyl-5n-glycero-3-[phosphor-L-serine] (DOPS), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE);
(b) adding to the mixture of step (a) a peptide comprising at least one spacer amino acid followed by an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, or 6 conjugated to either 1,2-dioleoyl-5n-glycero-3-succinate or 1,2-dioleoyl-5n-glycero-3-succinate at a concentration of 0.1-1 mol %;
(c) removing said chloroform and obtaining a dried lipid film; and
(d) hydrating said dried lipid film in an isotonic buffer;
thereby obtaining a liposome conjugated to a peptide comprising at least one spacer amino acid followed by an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, or 6.

17. The process of claim 16 , wherein the weight ratio of: Ch, DOPC, and DOPE or the weight ratio of: Ch, DOPS, and DOPE is 1:1:1.

18. The process of claim 16 , wherein the liposome's diameter is between 10 nm to 200 nm.

19. The process of claim 16 , further comprising extruding the liposome through a 50 nm to 100 nm pore filter.

20. The process of claim 16 , further comprising lyophilizing said liposome.

21. The process of claim 16 , further comprising loading a hydrophilic drug onto said 5 liposome in an isotonic buffer in an amount equal to w/w ratio in the range from 2:1 to 100:1 drug to dry liposome.

22. The process of claim 16 , further comprising loading a lipid soluble drug onto said liposome comprising:
(a) heating a loading composition comprising said lipid soluble drug and said 10 liposome to a temperature that is 0.01° C. to 5° C. above the phase-inversion temperature thus obtaining a water in oil emulsion;
(b) cooling said loading composition to a temperature that is 0.01° C. to 10° C. below the phase-inversion temperature thus obtaining an oil in water emulsion.

23. The process of claim 22 , wherein steps (a) and (b) form a cycle that is repeated at least 15 twice.

24. The process of claim 22 , wherein said heating and said cooling are performed at a rate of 1° C. to 10° C./min.

25. The process of claim 22 , wherein said heating is heating to a temperature between 75° C. to 95° C.

26. The process of claim 22 , wherein said cooling is cooling to a temperature between 50° C. to 70° C.

27. A liposome obtained by the process of claim 16 .

Assignees

• Technion Res & Dev Foundation
• Technion Res & Dev Foundation

Inventors

• Allon Nahum
• Gavish Moshe

Classifications

• C12N15/88

  using microencapsulation, e.g. using {amphiphile} liposome vesicle · CPC title

• C12Q2563/161

  Vesicles, e.g. liposome · CPC title

• A61K9/1272 Primary

  comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers (lipids as modifying agents {A61K47/543}) · CPC title

• C12N2320/32

  Special delivery means, e.g. tissue-specific · CPC title

• A61K9/1277

  Preparation processes; Proliposomes · CPC title