Cationic liposomes for gene transfer

Details Cationic liposomes for gene transfer Cationic liposomes for gene transfer

1999-11-23

2003-12-02

Nguyen, Dave T. (Department: 1635)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C435S458000

Reexamination Certificate

active

06656498

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to synthetic cationic lipid compounds, liposome formulations and the use of such compounds via lipofection to introduce functional bioactive agents/biologically active substances to cultured cells.
BACKGROUND OF THE INVENTION
Lipofection (or transfection), a process of introducing functional nucleic acids into cultured cells by using positively charged liposomes, was first described by Philip Felgner et al. a decade ago, and later shown, by K. L. Brigham, as applicable in vivo to experimental animals. See, Felgner et al.,
Proc. Natl. Acad. Sci. USA
, 84, 74113-7417 (1987) and K. L. Brigham et al.,
Am. J. Med. Sci
., 298, 278-281 (1989). Cationic lipids have become an increasingly important tool for many in vitro gene transfer applications, including several recent human gene therapy trials. As non-viral, synthetic DNA carriers, cationic lipids are particularly attractive because they are non-immunogenic, simple to use, can deliver DNA of a broad range size, and can be manufactured in large quantity. Although lipofection is quite efficient in vitro under serum-free conditions, its use in vivo when delivered as DNA/lipid complex, lipoples, via intravenous or airway routes, was limited to the presence of proteins and polysaccharides in the body fluids and mucus that strongly inhibit the transfection efficiency. Recent efforts to search for more efficient lipids and/or improved DNA/liposome formulations have resulted in dramatic increases of in vivo transfection efficiency. See, Solodin, I. et al.,
Biochemistry
, 34, 13537-13544 (1995); Templeton, N. S., et al.,
Nat. Biotech
., 15(7), 647-652 (1997); Thierry, A. R. et al.,
Proc. Natl. Acad. Sci. USA
, 92, 9742-9746 (1995); Li, S. et al.,
Gene Ther
., 4, 891-900 (1997); Liu, Y., et al.,
Nat. Biotechnol
., 15(2), 167-173 (1997); Liu, F. et al.,
Gene Ther
., 4(6), 517-523 (1997); and Song Y. K., et al.,
Hum. Gene Ther
., 8, 1585-1594 (1997).
Using a few well known cationic lipids, researchers have carefully studied key parameters that affect transfection efficiency of intravenously administered lipoplex in mouse. See, also Hong K., et al.,
FEBS Letters
, 400, 233-237 (1997). These studies have shown that a 2-16 fold excess of cationic liposome over DNA were necessary for high level gene expression in lungs and other organs, liposomes composed of 1,2, -dioleyl-3-N,N,N-trimethyl amino propane chloride (DOTMA) were significantly more active than that composed other cationic lipids. See, Song et al. Liposomes prepared from pure cationic lipid, such as DOTMA or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or mixtures of a cationic lipid and cholesterol or Tween 80 could mediate more efficient gene transfer than those formulations composed of a cationic lipid and a neutral phospholipid 1,2-dioleoyl-sn-glycero-3-phosphotidylethanolamine (DOPE) and the lipoplex prepared from extruded multilamellar liposomes were more active than those prepared from sonicated small unilamellar liposomes. See, Hong et al., Liu, Y et al., Liu, F. et al, Song et al., and Li, S. et al. With these improvements, the level of reporter gene expression in lungs after intravenous administration of optimized lipoplex were estimated 100 to 1000 fold more efficient than those prepared according to previously reported studies using DOTMA/DOPE liposomes and less lipid to DNA ratios. However, the improvements were also associated with noticeable toxicity of varying degrees of lipid/DNA treated animals due to toxic effect of large excess of cationic lipids or lipoplexes themselves. Therefore, searching for cationic lipids with less toxicity and formulations that function at reduced lipid to DNA ratios seem to be reasonable approaches to the problem of lipofection-related toxicity. We report the synthesis of a novel series of cationic lipids and test of their in vivo transfection activity in mice.
High level transfection in vivo in lungs and several other organs using lipoplex have been reported by a number of groups, as cited in the text above. One of the conclusions drawn from these studies is that relatively high lipid to DNA ratios is required to achieve high levels of transfection in vivo (Hong, K., et al., Liu, Y., et al., Liu, F., et al., and Li, S., et al.) Besides the high charge ratios, other factors such as the use of cholesterol, instead of DOPE as helper lipid (Hong, K., et al., Templeton, N. S., et al., Liu, Y., et al, Song, Y. K., et al. and Li, S., et al); multilamellar liposomes of about 200 nm in size rather than small unilamellar liposomes (Liu, Y., et al.) and the use of polycations and polymers in the cationic lipid-DNA complexes also contribute to the high level transfection (Hong, K., et al. and Li, S., et al.). The least mentioned factor in these studies was the side effects that are associated with these high level transfections in vivo.
It is therefore an object of the present invention to provide cationic lipids which are less toxic in pharmaceutical formulations and function at reduced lipid to DNA ratios than existing cationic lipids.
It is also an object of the present invention to provide a liposome, with or without a helper lipid, which is less toxic than prior art liposomes.
It is also an object of the present invention to provide cationic liposome pharmaceutical formulations which enhance intracellular delivery of DNA to a less toxic extent than previously described lipophilic compounds.
It is also an object of this invention to provide a lipoplex that has a transfection activity which is higher than transfection activity of the prior art liposome.
It is also and object of this invention to provide improved lipid and liposome formulation for treating a disease in a mammal via transfection.
It is a further object of the present invention to provide cationic liposome formulations which demonstrate superior efficacy.
SUMMARY OF THE INVENTION
In accordance with the purpose(s) of this invention, as embodied and described herein, this invention, in one aspect, relates to novel cationic lipids, and their use in pharmaceutical formulations for the intra cellularly delivery of bioactive agent.
One aspect of the invention relates to a compound of the general formula I:
or a pharmaceutically acceptable salt or ester thereof; wherein:
R
1
and R
2
are the same or different and are from C
6
to C
24
alkyl or aryl;
Y and Z are both —O—C(O)— or —O—;
or —C(O)—O—; and
n=1-6.
Another aspect of the invention relates to a compound of formula I(a):
or a pharmaceutically acceptable salt or ester thereof, wherein:
R
1
and R
2
are independently from C
6
to C
24
alkyl or alkenyl or one of R
1
or R
2
is C
6
to C
24
alkyl or alkenyl and the other is absent, or R
1
and R
2
are independently aryl;
Y and Z are the same and are —O—C(O)—, or one of Y and Z is —O—C(O)— and the other is —OH;
R
3
is C
1-6
alkyl, aryl, aryloxy, alkene, or a protecting group; and
n=1-6.
Another aspect of the invention relates to a compound of formula I(b):
or a pharmaceutically aceptable salt or ester thereof, wherein:
R
1
and R
2
are the same or different and are from C
6
to C
24
alkyl or alkenyl or aryl;
Y and Z are both —O—C(O)—;
A is —C(O)—O—;
R
4
is C
1-6
alkyl;
a=0 or 1;
n=1-3;
q=0-3;
X
−
is a halogen anion or is absent; and
the S and N atoms each independently have a positive charge or the positive charge is absent.
The novel N-[(2,3-dioleoyloxy)propyl]-L-lysinamide (LADOP) cationic lipid has lysine as a head group and two long fatty chains as lipid anchors linked through biodegradable diester and amide bonds. Liposome/DNA complexes prepared with a plasmid containing a luciferase reporter gene delivered either intravenously or intratracheally to mice demonstrated high level expression of the transgene. The magnitude of transgene expression was related to the ratio of lipid to DNA; high level of transgene expression occurred when lipid/DNA charge ratios reached 1:8 to 1:24. The duration of the transgene expression in lungs of mice treated with liposome/D

Affiliated with

Also associated with

Rating

Cationic liposomes for gene transfer does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Cationic liposomes for gene transfer, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Cationic liposomes for gene transfer will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3102700