Cationic reagents of transfection

Details Cationic reagents of transfection Cationic reagents of transfection

1999-05-04

2004-05-11

Nguyen, Dave T. (Department: 1633)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C514S04400A, C435S320100, C435S455000, C435S458000

Reexamination Certificate

active

06733777

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to cationic reagents for transfection, useful for delivery of exogenous compounds into cells, in vitro and in vivo.
BACKGROUND OF THE INVENTION
Currently four main methods for introducing nucleic acids into eukaryotic cells are in use: (1) electroporation; (2) calcium-phosphate-based transfection; (3) DEAE-dextran-based transfection; and (4) liposome-mediated transfection.
Compared to other methods, liposome-mediated transfection is characterized by high reproducibility, low cytotoxicity and simple procedures. However, many cationic compounds useful for liposome-mediated transfection are based on ester-linkages and are rapidly degraded by hydrolysis. Compared to infectious agents, cationic liposomes often show low overall efficiencies. Moreover, the commercially available cationic liposomes cannot be used or adapted for transfection of specific subpopulations of cells either in vitro or in vivo.
Advantages of the Invention Over Existing Technologies
The compounds of the present invention are easily preparable from inexpensive reagents, and therefore highly suitable for the preparation of liposomes for large-scale use. The compounds of Formula (I) are not based on ester-linkages, therefore, they are not degraded by hydrolysis. Transfection using the compounds of the present invention results in a high overall transfection efficiency. Adaption for transfection of specific cells is easily possible by structural changes of the compounds of the present invention and by choice of the accompanying counter ion. The compounds of the present invention provide an easy and reproducible procedure for liposome preparation, preferably without the need for sonication.
SUMMARY OF THE INVENTION
The present invention relates to compounds of Formula (I) useful for delivery of exogenous compounds into cells, in vitro and in vivo.
The present invention further provides liposomes comprising (a) a neutral lipid such as dioleoylphosphatidylethanolamine (DOPE) or similar lipid like compounds such as 1,2-dioleoyloxiphosphatidylethanolamine or other lipid-like structures and (b) one or more of the compounds of Formula (I). The present invention also relates to methods of delivery of exogenous compounds, for example macromolecules and pharmaceutical compositions, into cells in vitro and in vivo using the compounds of the present invention.
Also within the scope of this invention are transfection kits comprising the compounds of the present invention.
According to the present invention, the delivery of desired exogenous compounds to target cells may be modulated by, among other things, varying the following: (1) the structure of the compounds of Formula (I), (2) the ratio of neutral lipids to the compounds of Formula (I), (3) the method of preparing liposomes, or (4) the counter ion being prepared with the compounds of the present invention.
DETAILED DESCRIPTION
The present invention provides compounds of Formula (I):
wherein
A denotes an anion selected from the group of chloride, bromide, iodide, hydrogenphosphate (HPO
4
2−
), dihydrogenphosphate (H
2
PO
4
−
), sulphate, thiosulphate, hydroxy and/or oxalate.
k denotes an integer 1, 2, 3, 4 or 5;
B denotes an alkandiyl bridge (CH
2
)
n
wherein
n denotes an integer 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
R
1
, R
3
and R
4
, which may be identical to one another or different, denote hydrogen, straight-chained or branched C
1
-C
6
-alkyl, C
1
-C
6
-alkenyl, C
1
-C
6
-alkynyl;
R
2
denotes straight-chained or branched C
8
-C
20
-alkyl, C
8
-C
20
-alkenyl, C
8
-C
20
-alkynyl;
R
5
denotes for k=1
straight-chained or branched C
8
-C
20
-alkyl, C
8
-C
20
-alkenyl, C
8
-C
20
-alkynyl;
denotes for k>1
hydrogen, straight -chained or branched C
1
-C
6
-alkyl, C
1
-C
6
-alkenyl, C
1
-C
6
-alkynyl;
R
6
denotes for k=1
hydrogen, straight -chained or branched C
1
-C
6
-alkyl, C
1
-C
6
-alkenyl, C
1
-C
6
-alkynyl;
denotes for k>1
a straight-chained or branched C
8
-C
20
-alkyl, C
8
-C
20
-alkenyl, C
8
-C
20
-alkynyl and the repeating unit —B—NR
4
R
6
may be identical to one another or different.
Preferred are compounds of general Formula (I) wherein
A denotes an anion selected from the group of chloride, bromide, iodide, hydrogenphosphate(HPO
4
2−
), dihydrogenphosphate (H
2
PO
4
−
), sulphate, thiosulphate, hydroxy and/or oxalate.
k denotes an integer 1, 2 or 3;
B denotes an alkandiyl bridge (—CH
2
)
n
— and
n denotes an integer 1, 2, 3, 4, 5 or 6;
R
1
, R
3
and R
4
, which may be identical to one another or different, denote hydrogen or straight-chained or branched C
1
-C
6
-alkyl;
R
2
denotes straight-chained or branched C
8
-C
20
-alkyl, C
8
-C
20
-alkenyl, C
8
-C
20
-alkynyl;
R
5
denotes for k=1
a straight-chained or branched C
8
-C
20
-alkyl, C
8
-C
20
-alkenyl, C
8
-C
20
-alkynyl;
denotes for k>1
hydrogen, straight-chained or branched C
1
-C
6
-alkyl;
R
6
denotes for k=1
hydrogen, straight-chained or branched C
1
-C
6
-alkyl, C
1
-C
6
-alkenyl, C
1
-C
6
-alkynyl;
denotes for k>1
a straight-chained or branched C
8
-C
20
-alkyl, C
8
-C
20
-alkenyl, C
8
-C
20
-alkynyl and the repeating unit —B—NR
4
R
6
is preferably identical to one another.
Specifically preferred are compounds of general Formula (I) wherein
A denotes an anion selected from the group of bromide, iodide, dihydrogenphosphate (H
2
PO
4
−
) and/or thiosulphate;
k denotes an integer 1 or 2;
B denotes for k=1
an alkandiyl bridge —(CH
2
)
n
wherein
n represents an integer 2, 3 or 4;
B denotes for k=2
an ethylenebridge —(CH
2
—CH
2
)—;
R
1
, R
3
and R
4
which are identical to one another denote CH
3
;
R
2
denotes straight-chained C
10
-C
20
-alkyl;
R
5
denotes for k=1
straight-chained C
10
-C
20
-alkyl and is identical to R
2
;
denotes for k=2
CH
3
;
R
6
denotes for k=1
CH
3
denotes for k=2
straight-chained C
10
-C
20
-alkyl and is identical to R
2
.
A pharmaceutically acceptable ion is a mono-, di- or multi-valent, preferably non cytotoxic, ion. The different salts can be synthesized by methods which are known per se from the state of the art, in particular using ion exchange methods.
C
1
-C
6
-alkyl generally represents a straight-chained or branched hydrocarbon radical having 1 to 6 carbon atoms which may optionally be substituted by one or several halogen atoms—preferably fluorine—which may be identical to one another or different. The following radicals may be mentioned by way of example:
methyl, ethyl, propyl, 1-methylethyl (isopropyl), butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl and 1-ethyl-2methyl-propyl.
The same definition applies accordingly to alkandiyl radicals.
C
8
-C
20
-alkyl refers specifically to a straight-chained or branched hydrocarbon radical having 8 or 20 carbon atoms—for example octyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, dodecadecyl, nonadecyl and eicosyl.
Unless otherwise stated aklyl groups having 1 to 3 carbon atoms such as methyl, ethyl, n-propyl or isopropyl are preferred. The same definition applies to alkandiyl radicals.
Alkenyl in general represents a straight-chained or branched hydrocarbon radical having 3 to 6 carbon atoms and one or more double bonds, preferably one double bond, which may optionally be substituted by one or several halogen atoms—preferably fluorine—which may be identical to another or different. C
8
-C
20
-alkenyl refers specifically to a straight-chained or branched hydrocarbon radical having 8 or 20 carbon atoms and one or more double bonds.
Examples include:
2-propenyl (allyl), 2-butenyl, 3-butenyl, 1-methyl

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