Compositions for the delivery of negatively charged molecules

Details Compositions for the delivery of negatively charged molecules Compositions for the delivery of negatively charged molecules

1998-07-21

2002-05-28

Davenport, Avis M. (Department: 1632)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C424S450000, C424S422000, C424S423000, C424S449000, C554S105000, C564S159000, C564S157000, C564S511000, C435S242000, C435S173300, C435S173300, C560S158000, C560S159000, C560S180000, C560S190000

Reexamination Certificate

active

06395713

ABSTRACT:

BACKGROUND OF THE INVENTION
The following is a brief description of the delivery of biopolymers. This summary is not meant to be complete but is provided only for understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
Trafficking of large, charged molecules into living cells is highly restricted by the complex membrane systems of the cell. Specific tranporters allow the selective entry of nutrients or regulatory molecules, while excluding most exogenous molecules such as nucleic acids and proteins. The two major strategies for improving the transport of foreign nucleic acids into cells are the use of viral vectors or cationic lipids and related cytofectins. Viral vectors can be used to transfer genes efficiently into some cell types, but they cannot be used to introduce chemically synthesized molecules into cells. An alternative approach is to use delivery formulations incorporating cationic lipids, which interact with nucleic acids through one end and lipids or membrane systems through another (for a review see Felgner, 1990,
Advanced Drug Delivery Reviews,
5,162-187; Felgner 1993,
J. Liposome Res.,
3,3-16). Synthetic nucleic acids as well as plasmids may be delivered using the cytofectins, although their utility is often limited by cell-type specificity, requirement for low serum during transfection, and toxicity.
Since the first description of liposomes in 1965, by Bangham (
J. Mol. Biol.
13, 238-252), there has been a sustained interest and effort in the area of developing lipid-based carrier systems for the delivery of pharmaceutically active compounds. Liposomes are attractive drug carriers since they protect biological molecules from degradation while improving their cellular uptake.
One of the most commonly used classes of liposomes formulation for delivering polyanions (e.g., DNA) are those that contain cationic lipids. Lipid aggregates can be formed with macromolecules using cationic lipids alone or including other lipids and amphiphiles such as phosphatidylethanolamine. It is well known in the art that both the composition of the lipid formulation as well as its method of preparation have effect on the structure and size of the resultant anionic macromolecule-cationic lipid aggregate. These factors can be modulated to optimize delivery of polyanions to specific cell types in vitro and in vivo. The use of cationic lipids for cellular delivery of biopolymers have several advantages. The encapsulation of anionic compounds using cationic lipids is essentially quantitative due to electrostatic interaction. In addition, it is believed that the cationic lipids interact with the negatively charged cell membranes initiating cellular membrane transport (Akhtar et al., 1992,
Trends Cell Bio.,
2, 139; Xu et al., 1996,
Biochemistry
35, 5616).
The transmembrane movement of negatively charged molecules such as nucleic acids may therefore be markedly improved by coadministration with cationic lipids or other permeability enhancers (Bennett et al., 1992
Mol. Pharmacol.,
41, 1023-33; Capaccioli et al., 1993,
BBRC,
197,818-25; Ramila et al., 1993
J. Biol. Chem.,
268,16087-16090; Stewart et al., 1992,
Human Gene Therapy,
3, 267-275). Since the introduction of the cationic lipid DOTMA and its liposomal formulation Lipofectin™ (Felgner et al., 1987,
PNAS
84, 7413-7417; Eppstein et al., U.S. Pat. No. 4,897,355), a number of other lipid-based delivery agents have been described primarily for transfecting mammalian cells with plasmids or antisense molecules (Rose, U.S. Pat. No. 5,279,833; Eppand et al. U.S. Pat. No. 5,283,185; Gebeyehu et al., U.S. Pat. No. 5,334,761; Nantz et al., U.S. Pat. No. 5,527,928; Bailey et al., U.S. Pat. No. 5,552,155; Jesse, U.S. Pat. No. 5,578,475). However, each formulation is of limited utility because it can deliver plasmids into some but not all cell types, usually in the absence of serum (Bailey et al., 1997,
Biochemistry,
36, 1628). Concentrations (charge and/or mass ratios) that are suitable for plasmid delivery (~5,000 to 10,000 bases in size) are generally not effective for oligonucleotides such as synthetic ribozymes or antisense molecules (~10 to 50 bases). Also, recent studies indicate that optimal delivery conditions for antisense oligonucleotides and ribozymes are different, even in the same cell type. However, the number of available delivery vehicles that may be utilized in the screening procedure is highly limited, and there continues to be a need to develop transporters that can enhance nucleic acid entry into many types of cells.
Epstein et al., U.S. Pat. No. 5,208,036, disclose a liposome, LIPOFECTIN™, that contains an amphipathic molecule having a positively charged choline head group (water soluble) attached to a diacyl glycerol group (water insoluble). Promega (Wisconsin) markets another cationic lipid, TRANSFECTAM™, which can help introduce nucleic acid into a cell.
Wagner et al., 1991,
Proc. Nat. Acad. Sci. USA
88, 4255; Cotten et al., 1990,
Proc. Nat. Acad. Sci. USA
87, 4033; Zenke et al., 1990,
Proc. Nat. Acad. Sci. USA
87, 3655; and Wagner et al.,
Proc. Nat. Acad. Sci. USA
87, 3410, describe transferrin-polycation conjugates which may enhance uptake of DNA into cells. They also describe the feature of a receptor-mediated endocytosis of transferrin-polycation conjugates to introduce DNA into hematopoietic cells.
Wu et al.,
J. Biol. Chem.
266, 14338, describe in vivo receptor-mediated gene delivery in which an asialoglycoprotein-polycation conjugate consisting of asialoorosomucoid is coupled to poly-L-lysine. A soluble DNA complex was formed capable of specifically targeting hepatocytes via asialoglycoprotein receptors present on the cells.
Biospan Corporation International PCT Publication No. WO 91/18012, describe cell internalizable covalently bonded conjugates having an “intracellularly cleavable linkage” such as a “disulfide cleavable linkage” or an enzyme labile ester linkage.
Brigham, U.S. Pat. No. 5,676,954 describes a method for the expression of nucleic acid following transfection into a target organ consisting of mammalian cells.
The references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the delivery vehicles of the instant invention.
SUMMARY OF THE INVENTION
This invention features cationic lipid-based compositions to facilitate delivery of negatively charged molecules into a biological system such as animal cells. The present invention discloses the design, synthesis, and cellular testing of novel agents for the delivery of negatively charged molecules in vitro and in vivo. Also disclosed are screening procedures for identifying the optimal delivery vehicles for any given nucleic acid and cell type. In general, the transporters described here were designed to be used either individually or as part of a multicomponent system. The compounds of the invention generally shown in
FIG. 1
, are expected to improve delivery of negatively charged molecules into a number of cell types originating from different tissues, in the presence or absence of serum.
The “negatively charged molecules” are meant to include molecules such as nucleic acid molecules (e.g., RNA, DNA, oligonucleotides, mixed polymers, peptide nucleic acid, and the like), peptides (e.g., polyaminoacids, polypeptides, proteins and the like), nucleotides, pharmaceutical and biological compositions, that have negatively charged groups that can ion-pair with the positively charged head group of the cationic lipids of the invention
In a first aspect the invention features a cationic lipid having the formula I:
wherein, n is 1, 2 or 3 carbon atoms; n, is 2, 3, 4 or 5 carbon atoms; R and R
1
independently represent C12-C22 alkyl chain which are saturated or unsaturated, wherein the unsaturation is represented by 1-4 double bonds; and R
2
and R
3
are independently H, acyl, alkyl, carboxamidine, aryl, acyl, substituted carboxamidine, polyethylene glycol (PEG) or a combination thereo

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