Cationic cholesteryl derivatives containing cyclic polar groups

Details Cationic cholesteryl derivatives containing cyclic polar groups Cationic cholesteryl derivatives containing cyclic polar groups

2001-06-01

2003-06-24

Badio, Barbara P. (Department: 1616)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Cyclopentanohydrophenanthrene ring system doai

Reexamination Certificate

active

06583131

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to cationic lipids and their use in delivering biologically active substances to cells. In particular, the invention relates to novel cationic cholesteryl derivatives containing cyclic polar groups and the use of these derivatives to deliver biologically active substances to cells and to transfect nucleic acids into cells.
BACKGROUND OF THE INVENTION
The development of new forms of therapeutics which use macromolecules such as proteins or nucleic acids as therapeutic agents has created a need to develop new and effective means of delivering such macromolecules to their appropriate cellular targets. Therapeutics based on either the use of specific polypeptide growth factors or specific genes to replace or supplement absent or defective genes are examples of therapeutics which may require such new delivery systems. Clinical application of such therapies depends not only on efficacy of new delivery systems but also on their safety and on the ease with which the technologies underlying these systems can be adapted for large scale pharmaceutical production, storage, and distribution of the therapeutic formulations.
Gene therapy has become an increasingly important mode of treating various diseases. The potential for providing effective treatments, and even cures, has stimulated an intense effort to apply this technology to diseases for which there have been no effective treatments. Recent progress in this area has indicated that gene therapy may have a significant impact not only on the treatment of single gene disorders, but also on other more complex diseases such as cancer.
Success of a gene therapy protocol largely depends upon the vehicle used to deliver the gene. A variety of means exist to introduce a gene inside the cell including physical means such as microinjection (Capecchi, M. R.
Cell
(1980) 22:479-485), electroporation (Pacqereau, L. et al.
Anal. Biochem.
(1992) 204:147-151) and particle bombardment (Yang, N.-S. et al.
Proc. Natl. Acad. Sci. USA
(1990) 87:9568-9572)), biological means such as viruses (Ferry, N. et al.
Proc. Natl. Acad. Sci.
(1991) 88:8377-8381) and chemical means such as calcium phosphate (Wiegler, M. et al.
Cell
(1977) 11:223-232), DEAE dextran (Ishikawa, Y. et al.
Nucl. Acid Res.
(1992) 20:4367-4370), polylysine (Wu, G. Y. et al.
J. Biol. Chem.
(1988) 263:4429-4432) and cationic liposomes (Felgner, P. L. et al.
Proc. Natl. Acad. Sci.
(1987) 84:7413-7417)). Clinical application of such therapies depends not only on the efficacy of new delivery systems but also on their safety and on the ease with which the technologies underlying these systems can be adapted for large scale pharmaceutical production, storage, and distribution of the therapeutic formulations. Thus, an ideal vehicle for the delivery of exogenous genes into cells and tissues should be highly efficient in nucleic acid delivery, safe to use, easy to produce in large quantity and have sufficient stability to be practicable as a pharmaceutical.
Non-viral vehicles, which are represented mainly by cationic lipids, are one type of vehicle which have, for the following reasons, been considered for use in gene therapy. First, the plasmid DNA required for liposome-mediated gene therapy can be widely and routinely prepared on a large scale and is simpler and carries less risk than the use of viral vectors such as retroviruses. Second, cationic lipids are less toxic and less immunogenic than viral vectors and the DNA complexed with the lipids is better protected from degradation by nucleases. Third, liposome-mediated gene delivery, unlike retroviral-mediated gene delivery, can deliver either RNA or DNA. Thus, DNA, RNA or an oligonucleotide can be introduced directly into cells using cationic liposomes.
Among the numerous cationic amphiphiles which have been referred to as useful for transfecting nucleic acids into cells are cationic derivatives of cholesterol. For example, cholesterol (4′-trimethylammonio) butanoate (ChOTB) contains a trimethylammonium group connected to the 3′-hydroxyl group of cholesterol via a butanoyl spacer arm and cholesterol hemisuccinate choline ester (ChOSC) contains a choline moiety connected to the 3′-hydroxyl group via a succinyl spacer arm. However, the transfection activities of these amphiphiles are generally weak. (Leventis, R. et al. (1989)
Biochim. Biophys. Acta.,
1023:124-132).
Epand et al. (U.S. Pat. No. 5,283,185) describe cationic derivatives of cholesterol in which primary, secondary-tertiary or quaternary alkyl ammonium groups are linked to cholesterol via the 3-hydroxy group. These cationic cholesterol derivatives, including 3&bgr;[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), are disclosed to be useful in transfecting nucleic acids into cells.
SUMMARY OF THE INVENTION
This invention relates to novel compounds having the formula:
X—R
2
—R
1
—O-Cholesteryl,
or salts thereof, where
R1 is a linker bond
R
2
is a direct bond, a C
1
-C
10
linear or branched chain alkylene or alkenylene group, a C
3
-C
7
cyclolalkylene, preferably C
5
-C
6
, or a phenylene; and X is a 4-7-membered nitrogen-containing cyclic structure wherein said cyclic structure can optionally include further heteroatoms such as S, O or NR
3
. The X-moiety can be linked to the R
2
spacer either via a carbon atom on the nitrogen-containing cyclic structure or via a nitrogen atom of the cyclic structure. R
3
mentioned hereinabove is H, CH
3
, C
2
H
5
, CH
2
(CH
2
)
Z
SH or CH
2
(CH
2
)
z
OH where z=0−3.
The invention further relates to lipid dispersions which comprise at least one compound of the present invention where by “lipid dispersions” as used throughout the specification and claims is meant liposomes, micelles, emulsions or lipoproteins.
The present invention also relates to biologically active substance: lipid complexes formed by mixing a biologically active substance with lipid dispersions comprising at least one compound of the invention. The invention further relates to biologically active substance:lipid:polycation complexes formed by mixing a biologically active substance with polycation and with lipid dispersions comprising at least one compound of the invention.
The invention therefore provides methods for delivering biologically active substances to cells where such methods may be utilized to deliver substances to cells in vitro or in vivo by contacting cells with the complexes of this invention.
The invention also provides a method of transfecting cells comprising (a) mixing nucleic acid which encodes a protein or peptide or, which effects gene expression, with lipid dispersions comprising at least one compound of the invention and optionally, polycation, to form nucleic acid:lipid or nucleic acid:lipid:polycation complexes and (o) contacting the cells with the complex. It is contemplated that the methods of transfection may be utilized in vitro or in vivo.
The invention further provides pharmaceutical compositions comprising at least one compound of the invention; pharmaceutical compositions comprising a lipid dispersion containing at least one compound of the invention; and pharmaceutical compositions comprising at least one complex of the invention.
The invention further relates to a kit containing a compound of the invention and/or a lipid dispersion containing at least one such compound. The invention also provides kits containing the complexes of the invention.


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