Liposome having an exchangeable component

• US 6,673,364 B1
• Filed: 06/12/1998
• Issued: 01/06/2004
• Est. Priority Date: 06/07/1995
• Status: Expired due to Fees

• Current Assignee: University of British Columbia
• Original Assignee: University of British Columbia
• Inventors: Holland, John W., Cullis, Pieter R., Madden, Thomas D.
• Primary Examiner(s): Kishore, Gollamudi S.
• Application Number:
  US09/094,540
• Time in Patent Office: 2,034 Days
• Field of Search: 424/480, 424/1.21, 424/9.32, 424/9.51, 424/812, 424/417, 424/94.3, 436/829, 935/54, 428/402.2, 264/4.1, 264/4.3, 264/4.6
• US Class Current: 424/450
• 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: Y10T 428/2984 Microcapsule with fluid core [includes liposome]

Abstract

The present invention provides a fusogenic liposome comprising a lipid capable of adopting a non-lamellar phase, yet capable of assuming a bilayer structure in the presence of a bilayer stabilizing component; and a bilayer stabilizing component reversibly associated with the lipid to stabilize the lipid in a bilayer structure. Such fusogenic liposomes are extremely advantageous because the rate at which they become fusogenic can be not only predetermined, but varied as required over a time scale ranging from minutes to days. Control of liposome fusion can be achieved by modulating the chemical stability and/or exchangeability of the bilayer stabilizing component(s). The fusogenic liposomes of the present invention can be used to deliver drugs, peptide, proteins, RNA, DNA or other bioactive molecules to the target cells of interest.

Claims

1. A method for increasing the rate of exchange of a bilayer stabilizing component from a liposome formulation to improve multiple dosing, said method comprising:

(a) producing a first liposome composition having a lipid, a bioactive agent and a first bilayer stabilizing component, wherein said first bilayer stabilizing component is DSPE-PEG₂₀₀₀;

(b) determining the rate of exchange of DSPE-PEG₂₀₀₀ from said first liposome composition using a fluorescent lipid mixing assay; and

(c) preparing a second liposome formulation nearly identical to said first liposome formulation having said lipid, said bioactive agent, but substituting DSPE-PEG₂₀₀₀ from said first liposome composition with a second bilayer stabilizing component having a faster rate of exchanges wherein said second liposome composition results in improved dosing.

2. The method of claim 1, wherein said first and second liposome compositions further comprise cholesterol.

3. The method of claim 1, wherein said lipid is a cationic lipid.

4. The method of claim 3, wherein said cationic lipid is a member selected from the group consisting of 3β

-(N-(N′

,N′

-dimethylaminoethane)carbamoyl)cholesterol, N,N-distearyl-N,N-dimethylammonium bromide, N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide, N,N-dioleyl-N,N-dimethylammonium chloride, diheptadecylamidoglycyl spermidine, N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride, N-(1-(2,3dioleyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-bis(oleoyloxy)-3-dimethylaminopropane.

5. The method of claim 1, wherein said lipid is a member selected from the group consisting of dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine and distearoylphosphatidylethanolamine.

6. The method of claim 5, wherein said lipid is distearoylphosphatidylcholine.

7. The method of claim 1, wherein said second bilayer stabilizing component is polyethylene glycol conjugated to a lipid.

8. The method of claim 1, wherein said second bilayer stabilizing component is polyethylene glycol conjugated to a lipid which is a member selected from the group consisting of ceramides, phosphatidylethanolamines, phosphatidylcholines, sphingomyelins and cholesterol.

9. The method of claim 8, wherein said second bilayer stabilizing component is a hydrophilic polymer conjugated to a ceramide having a carbon chain length between C₈ to C₁₆.

10. The method of claim 9, wherein said second bilayer stabilizing component is a hydrophilic polymer conjugated to a ceramide having a carbon chain length between C₈ to C₁₆, wherein said carbon chain has at least one site of unsaturation.

11. The method of claim 10, wherein said second bilayer stabilizing component is polyethylene glycol conjugated to a C-₁₄-ceramide lipid.

12. The method of claim 11, wherein said second bilayer stabilizing component is polyethylene glycol conjugated to a C-₁₄-ceramide lipid, said C-₁₄-ceramide lipid having at least one site of unsaturation.

13. The method of claim 1, wherein said bioactive agent is a member selected from the group consisting of drugs, peptides, proteins, DNA and RNA.

14. The method of claim 1, wherein said faster rate of exchange varies from minutes to hours.

15. The method of claim 14, wherein said exchange rate is less than 4 hours.

16. The method of claim 14, wherein said exchange rate is less than about 1 hour.

17. The method of claim 1, wherein said lipid compositions further comprise cholesterol and a second lipid.

18. The method of claim 17, wherein said second lipid is a cationic lipid.

19. The method of claim 18, where said cationic lipid is a member selected from the group consisting of 3β

-(N-(N′

,N′

-dimethylaminoethane)carbamoyl)cholesterol, N,N-distearyl-N,N-dimethylammonium bromide, N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide, N,N-dioleyl-N,N-dimethylammonium chloride, diheptadecylamidoglycyl spermidine, N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride, N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-bis(oleoyloxy)-3 dimethylaminopropane.

20. The method of claim 1, wherein said second liposome composition comprises a lipid which is distearoylphosphatidylcholine, a second lipid which is 1,2-bis(oleoyloxy)-3-dimethylaminopropane, said bioactive agent is a nucleic acid, and said bilayer stabilizing component is a polyethylene glycol conjugated to a C-₁₄ ceramide lipid.

21. The method of claim 1, wherein said fluorescent lipid mixing assay is determined by the equation:

$\%<br><br>\text{\hspace{1em}<br><br>}<br><br>\mathrm{Fusion}=\frac{\frac{\left(F_{\left(t\right)}-L_{\left(t\right)}\right)}{\left(F_T-L_T\right)}-\frac{\left(F_O-L_O\right)}{\left(F_T-L_T\right)}}{\frac{\left(M_{\left(t\right)}-L_{\left(t\right)}\right)}{\left(M_T-L_T\right)}-\frac{\left(F_O-L_O\right)}{\left(F_T-L_T\right)}}\times <br><br>100$

wherein;

% fusion rate is relative to the % bilayer stabilizing component exchange rate,

F_(t) is fluorescence intensity at time t of said liposome composition;

F_O is fluorescence intensity at zero time of said liposome composition;

F_T is fluorescence intensity in the presence of a detergent of said liposome composition;

M_(t) is fluorescence intensity at time t for a fused control;

M_T is fluorescence intensity in the presence of a detergent of said fused control;

L_O is fluorescence intensity at zero time of a light scattering control;

L_T is fluorescence intensity in the presence of a detergent of said light scattering control; and

L_(t) is fluorescence intensity at time t for said light scattering control.