High efficiency encapsulation of charged therapeutic agents in lipid vesicles

• US 20050255153A1
• Filed: 09/09/2003
• Published: 11/17/2005
• Est. Priority Date: 05/14/1997
• Status: Active Grant

• Current Assignee: University of British Columbia
• Original Assignee: University of British Columbia
• Inventors: Debeyer, Dan, Ansell, Steven M., Scherrer, Peter, Klimuk, Sandra K., Semple, Sean C., Harasym, Troy, Hope, Michael J., Cullis, Pieter
• Granted Patent: US 7,341,738 B2
• Time in Patent Office: Days
• 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: A61K 9/1277 Processes for preparing; Proliposomes
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: Y10T 428/2984 Microcapsule with fluid core [includes liposome]

Abstract

Methods for the preparation of a lipid-nucleic acid composition are provided. According to the methods, a mixture of lipids containing a protonatable or deprotonatable lipid, for example an amino lipid and a lipid such as a PEG- or Polyamide oligomer-modified lipid is combined with a buffered aqueous solution of a charged therapeutic agent, for example polyanionic nucleic acids, to produce particles in which the therapeutic agent is encapsulated in a lipid vesicle. Surface charges on the lipid particles are at least partially neutralized to provide surface-neutralized lipid-encapsulated compositions of the therapeutic agents. The method permits the preparation of compositions with high ratios of therapeutic agent to lipid and with encapsulation efficiencies in excess of 50%.

Claims

1-84. -84. (canceled)

85. A nucleic acid-lipid particle, said nucleic acid-lipid particle comprising:

a nucleic acid;

an amino lipid;

a neutral lipid;

a sterol; and

a polyethyleneglycol-diacylglycerol (PEG-DAG) conjugate.

86. The nucleic acid-lipid particle in accordance with claim 85, wherein said amino lipid is a member selected from the group consisting of:

(1,2-dioleyloxy-3-dimethylamino-propane (DODAP), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), and a mixture thereof.

87. The nucleic acid-lipid particle in accordance with claim 85, wherein said neutral lipid is a member selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), distearoylphosphatidylcholine (DSPC), sphingomyelin and a mixture thereof.

88. The nucleic acid-lipid particle in accordance with claim 85, wherein said sterol is cholesterol.

89. The nucleic acid-lipid particle in accordance with claim 88, wherein the cholesterol comprises from 35% to 55% of the total lipid present in said particle.

90. The nucleic acid-lipid particle in accordance with claim 85, wherein said amino lipid comprises from 10% to 40% of the total lipid present in said particle.

91. The nucleic acid-lipid particle in accordance with claim 85, wherein said amino lipid comprises from 10% to 35% of the total lipid present in said particle.

92. The nucleic acid-lipid particle in accordance with claim 85, wherein said neutral lipid comprises from 25% to 45% of the total lipid present in said particle.

93. The nucleic acid-lipid particle in accordance with claim 85, wherein said PEG-DAG conjugate comprises from 0.5% to 15% of the total lipid present in said particle.

94. The nucleic acid-lipid particle in accordance with claim 85, wherein said neutral lipid is distearoylphosphatidylcholine (DSPC).

95. The nucleic acid-lipid particle in accordance with claim 85, wherein the nucleic acid-lipid particle comprises:

N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA);

distearoylphosphatidylcholine (DSPC);

a sterol; and

a PEG-DAG conjugate.

96. The nucleic acid-lipid particle in accordance with claim 95, wherein the sterol is cholesterol.

97. The nucleic acid-lipid particle in accordance with claim 85, wherein said nucleic acid is DNA.

98. The nucleic acid-lipid particle in accordance with claim 85, wherein said nucleic acid is a plasmid.

99. The nucleic acid-lipid particle in accordance with claim 85, wherein said nucleic acid is an antisense oligonucleotide.

100. The nucleic acid-lipid particle in accordance with claim 85, wherein said nucleic acid is a ribozyme.

101. The nucleic acid-lipid particle in accordance with claim 85, wherein said nucleic acid encodes a therapeutic product of interest.

102. The nucleic acid-lipid particle in accordance with claim 101, wherein said therapeutic product of interest is a peptide or protein.

103. The nucleic acid-lipid particle in accordance with claim 85, wherein the nucleic acid in said nucleic acid-lipid particle is not substantially degraded after exposure of said particle to a nuclease at 37°

C. for 20 minutes.

104. The nucleic acid-lipid particle in accordance with claim 85, wherein the nucleic acid in said nucleic acid-lipid particle is not substantially degraded after incubation of said particle in serum at 37°

C. for 30 minutes.

105. The nucleic acid-lipid particle in accordance with claim 85, wherein the nucleic acid is fully encapsulated in said nucleic acid-lipid particle.

106. A pharmaceutical composition comprising a nucleic acid-lipid particle in accordance with claim 85 and a pharmaceutically acceptable carrier.

107. A pharmaceutical composition in accordance with claim 106 comprising a nucleic acid-lipid particle, wherein the nucleic acid-lipid particle comprises:

N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA);

distearoylphosphatidylcholine (DSPC);

cholesterol;

a PEG-DAG conjugate; and

a pharmaceutically acceptable carrier.

108. A method of introducing a nucleic acid into a cell, said method comprising contacting said cell with a nucleic acid-lipid particle comprising an amino lipid, a neutral lipid, a sterol, a PEG-DAG conjugate, and a nucleic acid.