Title:  Stabilization of lipid:DNA formulations during nebulization

United States Patent:  6,375,980

Assignee:  Research Development Foundation (Carson City, NV)

Filed:  July 19, 1999

The present invention provides a liposomal aerosol composition, comprising a pharmaceutical compound, a cationic lipid, a neutral co-lipid; and tryptone. Also provided is a nebulized cationic lipid: neutral co-lipid: DNA suspension useful for lipid-DNA transfections, wherein the cationic lipid is bis(guanidinium)-tren-cholesterol and the neutral co-lipid is dioleoylphosphatidylethanolamine (DOPE).

DETAILED DESCRIPTION OF THE INVENTION

The present invention demonstrates the effects of cationic liposome-DNA formulation on both transfection efficiency (in vitro and in vivo) and jet nebulizer stability. The effects of nebulization and sonication on liposome-DNA particle size characteristics were also examined. Electron microscopy of promising formulations was performed using several fixation methods.

The cationic lipid bis-guanidinium-tren-cholesterol (BGTC), in combination with the neutral co-lipid dioleoylphosphatidylethanolamine (DOPE), was found to have a degree of stability adequate to permit effective gene delivery by the aerosol route. Optimal ratios of lipids and plasmid DNA were identified. Particle size analysis and ultrastructural studies revealed a remarkably homogeneous population of distinctly liposomal structures correlating with the highest levels of transfection efficiency and nebulizer stability.

Optimizing gene delivery vectors for pulmonary aerosol delivery to respiratory sites must take into account factors other than transfection efficiency in vitro. Effects of liposome-DNA formulation on liposomal morphology (i.e., particle size, multilamellar structure) appear to be relevant to stability during aerosolization. The present studies make it possible to identify formulations that hold promise for successful clinical application of aerosol gene delivery.

The present invention is directed to a liposomal aerosol composition, comprising a pharmaceutical compound, a cationic lipid, a neutral co-lipid, and tryptone. In one embodiment of this liposome aerosol composition, the pharmaceutical compound is a gene in the form of plasmid DNA. Representative examples of useful lipids include egg yolk phosphatidylcholine, hydrogenated soybean phosphatidylcholine, dimyristoylphosphatidylcholine, dilauroyl-phosphatidylcholine, dioleoylphosphatidylcholine, bis(guanidinium)-tren-cholesterol and dipalmitoyl phosphatidylcholine. The co-lipid is generally a neutral phospholipid. Representative examples of suitable co-lipids include dioleoylphosphatidylethanolamine (DOPE), analogs of dioleoylphosphatidylethanolamine and cholesterol. Typically, the tryptone is found in this composition in an amount sufficient to enhance transfection of a DNA:lipid suspension following nebulization. Generally, such concentrations are from about 0.1% to about 5%.

The present invention is also directed to a liposomal aerosol composition, comprising a pharmaceutical compound, a cationic lipid, a neutral co-lipid, and glutamic acid. In one embodiment of this liposome aerosol composition, the pharmaceutical compound is a gene. Representative examples of useful lipids include egg yolk phosphatidylcholine, hydrogenated soybean phosphatidylcholine, dimyristoylphosphatidylcholine, dilauroyl-phosphatidylcholine, dioleoylphosphatidylcholine, phosphatidylcholine, bis (guanidinium)-tren-cholesterol and dipalmitoyl phosphatidylcholine. Typically, the glutamic acid is found in this composition in an amount sufficient to enhance transfection of a DNA:lipid suspension following nebulization. Generally, such concentrations are from about 0.1% to about 5%.

The present invention is also directed to a nebulized cationic lipid:DNA suspension useful for lipid-DNA transfections, wherein said cationic lipid is bis(guanidinium)-tren-cholesterol and the neutral co-lipid is DOPE. Preferably, the bis(guanidinium)-tren-cholesterol/DOPE is contained in said suspension in a concentration relative to the plasmid DNA concentration determined to be optimal for transfection. The concentraton of the stable formulation depends on the relative concentrations of the three components, i.e., the plasmid DNA, the cationic lipid bis(guanidinium)-tren-cholesterol, and the neutral co-lipid DOPE. A preferred range of the plasmid DNA is from about 1 .mu.g/ml to about 1000 .mu.g/ml.

The optimal ratio of plasmid DNA to combined lipid (lipid formulation includes a 1:1 ratio of cationic lipid to neutral co-lipid) is approximately from 1:3 to 1:5. However, slightly different ratios often prove optimal for different applications or different plasmids, e.g., larger plasmids have a greater net charge and therefore require more lipid to neutralize this charge. Therefore, for 400 .mu.g/ml DNA, one would use approximately 600 .mu.g/ml of bis(guanidinium)-tren-cholesterol and 600 .mu.g/ml of DOPE. Concentrations of bis(guanidinium)-tren-cholesterol up to about 1.5 mg/ml (when present in a 1:1 ratio of DOPE) should be stable in a formulation with plasmid DNA. In one embodiment of the nebulized cationic lipid:DNA suspension of the present invention, the ratio of DNA concentration to combined cationic lipid and neutral co-lipid concentration is from about 1:1 to about 1:4.

Claim 1 of 9 ClaimsWhat is claimed is:

1. A liposomal aerosol composition with protection against nebulizer-induced loss of cell transfection activity comprising:

(a) a plasmid DNA containing a gene;

(b) a cationic lipid;

(c) a neutral co-lipid; and

(d) tryptone.

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