Cationic peg-lipids and methods of use

• US 6,852,334 B1
• Filed: 04/20/2000
• Issued: 02/08/2005
• Est. Priority Date: 04/20/1999
• Status: Expired due to Fees

• Current Assignee: University of British Columbia
• Original Assignee: University of British Columbia
• Inventors: Wong, Kim, Fenske, David B., Chen, Tao, Palmer, Lorne R., Cullis, Pieter R.
• Primary Examiner(s): Nguyen, Dave T.
• Application Number:
  US09/553,639
• Time in Patent Office: 1,755 Days
• Field of Search: 435/320.1, 435/325, 435/455, 435/458, 424/450, 514/44
• US Class Current: 424/450
• CPC Class Codes: A61K 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 47/6911 the form being a liposome
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 49/0021 the fluorescent group being a small organic molecule
• CPC Class Codes: A61K 49/0041 Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal in vivo G01N
• CPC Class Codes: A61K 49/0054 Macromolecular compounds, i.e. oligomers, polymers, dendrimers
• CPC Class Codes: A61K 9/1272 with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle

Abstract

The present invention provides cationic-polymer-lipid conjugates (CPLs) such as distal cationic-poly(ethylene glycol)-lipid conjugates which can be incorporated into conventional and stealth liposomes or other lipid-based formulation for enhancing cellular uptake. The CPLs of the present invention comprise a lipid moiety; a hydrophilic polymer; and a polycationic moiety. Method of increasing intracellular delivery of nucleic acids are also provided.

Claims

1. A vesicle comprising:

(a) a cationic-polymer-lipid conjugate having the general structure of Formula I;

A—

W—

Y

wherein;

A is a lipid moiety which acts as a lipid anchor;

W is a hydrophilic polymer; and

Y is a non-specific targeting polycationic moiety, said polycationic moiety having between about 2 to about 15 positive charges, wherein said positive charges are located away from the surface of the vesicle;

(b) a bioactive agent; and

(c) a second lipid.

2. The vesicle according to claim 1, wherein Y is biocompatible.

3. The vesicle according to claim 1, wherein

W is a polymer selected from the group consisting of PEG, polyamide, polylactic acid, polyglycolic acid, polylactic acid/polyglycolic acid copolymers and combinations thereof, said polymer having a molecular weight of about 250 to about 7000 daltons.

4. The vesicle according to claim 1, wherein W is PEG.

5. The vesicle according to claim 4, wherein said PEG has a molecular weight of about 250 to about 3000.

6. The vesicle according to claim 4, wherein said PEG has a molecular weight of about 250 to about 1000.

7. The vesicle according to claim 4, wherein said second lipid is a PEG-lipid and W has a lower molecular weight than the PEG of said PEG-lipid.

8. The vesicle according to claim 4, wherein said second lipid is a PEG₃₄₀₀-lipid and said compound of Formula I has a formula of

A-PEG₁₀₀₀-Y.

9. The vesicle according to claim 4, wherein said second lipid is a PEG₂₀₀₀-lipid and said compound of Formula I has a formula of

A-PEG₁₀₀₀-Y.

10. The vesicle according to claim 4, wherein said second lipid is a PEG-lipid and W has a greater molecular weight that the PEG of said PEG-lipid.

11. The vesicle according to claim 4, wherein said second lipid is PEG₁₀₀₀-lipid and said compound of Formula I has a formula of

A-PEG₃₄₀₀-Y.

12. The vesicle according to claim 4, wherein said second lipid is PEG₁₀₀₀-lipid and said compound of Formula I has a formula of

A-PEG₂₀₀₀-Y.

13. The vesicle according to claim 1, wherein said bioactive agent comprises an anti-tumor drug.

14. The vesicle according to claim 13, wherein said antitumor drug is a member selected from the group consisting of actinomycin D, vincristine, vinblastine, cystine arabinoside, an anthracycline, an alkylative agent, a platinum compound, an antimetabolite, a nucleoside analog, methotrexate, a purine analog and a pyrimidine analog.

15. The vesicle according to claim 1, wherein said bioactive agent comprises a nucleic acid.

16. The vesicle according to claim 15, wherein said bioactive agent is a gene construct or an oligonucleotide.

17. The vesicle according to claim 1, further comprising a bilayer-stabilizing component.

18. The vesicle according to claim 17, wherein said bilayer stabilizing component is a PEG-lipid, wherein said PEG of said PEG-lipid has a greater molecular weight than said polymer W.

19. The vesicle according to claim 17, wherein said bilayer stabilizing component is a ATTA-lipid, wherein said ATTA of said ATTA-lipid has a greater molecular weight than said hydrophilic polymer.

20. The vesicle according to claim 3, wherein W is a PEG.

21. The vesicle according to claim 20, said compound having Formula II:

wherein;

X is a member selected from the group consisting of single bond and a functional group covalently attaching said hydrophobic lipid to at least one ethylene oxide unit or a single bond;

Z is a member selected from the group consisting of a single bond and a functional group covalently attaching said at least one ethylene oxide unit to a cationic head group or a single bond; and

n is an integer ranging from about 6 to about 50.

22. The vesicle according to claim 21, wherein

A is a member selected from the group consisting of a diacylglycerolyl moiety, a dialkylglycerolyl moiety, a N-N-dialkylamino moiety, 1,2-diacyloxy-3-aminopropane moiety and a 1,2-dialkyl-3-aminopropane moiety.

23. The vesicle according to claim 21, wherein

X is a member selected from the group consisting of a single bond, phosphatidylethanolamino, phosphatidylethanolamido, phosphoro, phospho, phosphoethanolamino, phosphoethanolamido, carbonyl, carbamate, carboxyl, carbonate, amido, thioamido, oxygen, sulfur and NR, wherein R is a hydrogen or alkyl group.

24. The vesicle according to claim 21, wherein

Z is a member selected from the group consisting of a single bond, phospho, phosphoethanolamino, phosphoethanolamido, carbonyl, carbamate, carboxyl, amido, thioamido, an amino group, and NR, wherein R is a member selected from the group consisting of hydrogen atom and alkyl group.

25. The vesicle according to claim 1, wherein

said formulation is in the form of a member selected from the group consisting of a liposome, a micelle, a virosome, a lipid-nucleic acid particle, a nucleic acid complex and mixtures thereof.

26. The vesicle according to claim 25, wherein said lipid-based drug formulation is a liposome.

27. The vesicle according to claim 26, wherein said lipid-based drug formulation is a liposome having an average size in the range of about 0.05 to about 0.5 microns wherein said bioactive agent is a gene construct or an oligonucleotide.

28. A method of increasing an intracellular delivery of a bioactive agent, said method comprising administering to the cell the cationic lipid forming vesicle of claim 1, thereby increasing the intracellular delivery of the bioactive agent.

29. The method of claim 28, wherein said delivery is in vivo.

30. The method of claim 28, wherein said increasing is at least 10-fold compared to a cationic lipid forming vesicle without said cationic-polymer-lipid conjugate.

31. A method of increasing delivery to a target cell of a drug which is part of a parenterally administered lipid-based drug formulation, said method comprising:

preparing a suspension of a lipid-based drug formulation comprising the cationic lipid forming vesicle of claim 1, wherein between 0.1 to 20 mole percent of said cationic-polymer-lipid conjugated is incorporated.

32. A method of administering a bioactive agent to a mammal, said method comprising:

preparing a suspension of a lipid-based drug formulation comprising the cationic lipid forming vesicle of claim 1, wherein between 0.1 to 20 mole percent of said cationic-polymer-lipid conjugated is prepared, and a pharmaceutically acceptable amount of a bioactive agent, and parenterally administering said lipid-based drug formulation to said mammal.

33. A method for transfection of a cell with a lipid-based drug formulation, said method comprising:

contacting said cell with a lipid-based drug formulation, which comprises the cationic lipid forming vesicle of claim 1, wherein between 0.1 to 20 mole percent of said cationic-polymer-lipid conjugate is incorporated.

34. A method for increasing transfection of a cell with a lipid-based drug formulation, said method comprising:

contacting said cell with a lipid-based drug formulation, which comprises the cationic lipid forming vesicle of claim 1, wherein between 0.1 to 20 mole percent of said cationic-polymer-lipid conjugate is incorporated, whereby the transfection efficiency of said lipid-based formulation is increased compared to a lipid-based drug formulation without the incorporated cationic-polymer-lipid conjugate.

35. The vesicle according to claim 1, wherein Y comprises at least one basic amino acid or derivative thereof.

36. The vesicle according to claim 1, wherein Y has at least 8 positive charges at a selected pH.

37. The vesicle according to claim 1, wherein

Y is a member selected from the group consisting of lysine, arginine, asparagine, glutamine, derivatives thereof and combinations thereof.

38. The vesicle according to claim 1, wherein W is a polyamide polymer.

39. The vesicle according to claim 38, wherein W has a molecular weight of about 250 to about 2000 daltons.

40. The vesicle according to claim 21, wherein

A is a diacylglycerolyl moiety;

X is phosphoethanolamido;

Z is NR, wherein R is a hydrogen atom; and

Y is a member selected from the group consisting of about 1 to about 10 basic amino acids or derivatives thereof.

41. The vesicle according to claim 21, wherein

A is a diacylglycerolyl moiety having 2 fatty acyl chains, wherein each acyl chain is independently between 2 and 30 carbons in length and is either saturated or has varying degrees of saturation.

42. The vesicle according to claim 21, wherein

Y is a member selected from the group consisting of lysine, arginine, asparagine, glutamine, derivatives thereof and combinations thereof.

43. The vesicle according to claim 21, wherein

A is a diacylglycerolyl moiety having 2 fatty acyl chains, wherein each acyl chain is a saturated C-18 carbon chain;

and

Y is a cationic group having 4 lysine residues or derivatives thereof.