Pentaerythritol lipid derivatives and nuleic-acid complexes

Details Pentaerythritol lipid derivatives and nuleic-acid complexes Pentaerythritol lipid derivatives and nuleic-acid complexes

2000-03-27

2001-11-13

Wilson, James O. (Department: 1623)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C536S022100, C536S023100, C560S156000, C560S169000, C560S171000, C560S224000

Reexamination Certificate

active

06316421

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to pentaerythritol lipid derivatives which are useful for the intracellular delivery of polynucleotides. These cationic lipids are useful in the preparation of liposomes and other lipid vesicles for the delivery of nucleic acids into mammalian cells.
BACKGROUND OF THE INVENTION
The introduction of foreign nucleic acids and other molecules is a valuable method for manipulating cells and has great potential both in molecular biology and in clinical medicine. Many methods have been used for insertion of endogenous nucleic acids into eukaryotic cells. Genetic material can be introduced into cells to express an encoded protein which is deficient or defective. The use of such technology allows for the treatment of genetic based diseases. Gene transfer entails distributing nucleic acids to target cells and then transferring the nucleic acid across a target cell membrane in a form that can function in a therapeutic manner. Of the many methods used to facilitate entry of DNA into eukaryotic cells, cationic liposomes are among the most efficacious and have found extensive use as DNA carriers in transfection experiments. Cationic lipids themselves are known to bind to polynucleotides and to facilitate their intracellular delivery into mammalian cells. Nucleic acid is negatively charged and when combined with a positively charged lipid forms a complex that is suitable for formulation and cellular delivery. The use of cationic lipid carriers for transfection is well established. However, their ability to mediate transfection is not well understood.
The precise way in which nucleic acids and cationic lipids interact and the structure formed before and during the transfection process are not well known. It is commonly believed that the nucleic acids are entrapped within a lipid bilayer, which is the classic definition of a “liposome.” There is also a belief, however, that the nucleic acid does not become entrapped, but forms some other sort of aggregate with the cationic lipids. It has also been reported that liposome-DNA aggregate size and shape are a function of the ratio of the amount of DNA to that of cationic lipid. It has been concluded that DNA binds to the outer surface of liposomes, which then cluster into irregular spherical aggregates. Plasmid length had no effect on binding to liposomes and the structure of the liposome-DNA complex is believed to change at charge neutrality, while the DNA becomes organized into a very compact structure that is evidently quite different from a liposome. It has been concluded that the liposome probably uses at least two pathways to introduce DNA into cells: fusion with the plasma membrane and endocytosis.
The delivery and expression of a transfected gene constitute a complex process that includes steps involving transfection complex (lipoplex) formulation, cellular internalization, endosomal escape, and nuclear localization. Incorporation of cationic lipid in the cytoplasmic membrane can occur by cytoplasmic fusion or translocation after lipoplex uptake. Incorporation of the cationic lipid in the cytoplasmic membrane can occur by cytoplasmic fusion or translocation after lipoplex uptake. Cellular processes can be inhibited by the incorporation of positively charged lipids into the plasma membrane. This incorporation can lead to cell dysfunction and possibly cell death. Thus, although there are benefits to cationic lipid facilitated gene transfer, there are also deleterious effects of lipidic salts on cellular processes. The long-term administration of cationic lipoplexes has been shown to elicit inflammatory responses and cytotoxicity.
Lipid-associated cytotoxicity has been attributed to the inhibition of protein kinase C activity by cationic lipids after internalization of the lipoplex. This is presumably a consequence of cationic lipid incorporation into the plasma membrane. In addition, transfection is attributed to the formation of transmembrane pores. There are also resultant disruptions of signal transduction and gene regulation processes which impair cellular function. It is possible that enhanced clearance of the cationic lipids might alleviate the cytotoxicity.
There exists a need to design lipids which are effective in facilitating intracellular delivery of genetic material, but that will reduce the associated cellular toxicity. The present invention fulfills this and other needs.
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to an amphiphilic lipid for the intracellular delivery of polynucleotides having Formula I
In Formula I, R
1
is a functional group including, but not limited to, C
8
-C
24
alkyl and C
8
-C
24
alkenyl. The alkenyl groups may have more than one site of unsaturation and the double bonds may be cis or trans. R
2
, in Formula I, is a functional group including, but not limited to, C
8
-C
24
alkyl and C
8
-C
24
alkenyl. The alkenyl groups may have more than one site of unsaturation and the double bonds may be cis or trans. R
3
, in Formula I, is a functional group including, but not limited to, hydrogen, optionally substituted C
1
-C
8
alkyl, optionally substituted arylalkyl, C
1
-C
8
alkylthioalkyl, guanidinoalkyl, carboxyalkyl, aminoalkyl, carbamoyl C
1
-C
8
alkyl and heteroarylalkyl. R
4
, in Formula I, is a functional group including, but not limited to, hydrogen, optionally substituted C
1
-C
8
alkyl, optionally substituted arylalkyl, C
1
-C
8
alkylthioalkyl, guanidinoalkyl, carboxyalkyl, aminoalkyl, carbamoyl C
1
-C
8
alkyl and heteroarylalkyl. Y
1
, in Formula I, is a functional group including, but not limited to, hydrogen and C
1
-C
6
alkyl. Y
2
, in Formula I, is a functional group including, but not limited to, hydrogen and C
1
-C
6
alkyl. Y
3
, in Formula I, is a functional group including, but not limited to, hydrogen and C
1
-C
6
alkyl. Y
4
, in Formula I, is a functional group including, but not limited to, hydrogen and C
1
-C
6
alkyl. Y
5
, in Formula I, is a functional group including, but not limited to, hydrogen and C
1
-C
6
alkyl. Y
6
, in Formula I, is a functional group including, but not limited to, hydrogen and C
1
-C
6
alkyl. X, in Formula I, is an anion, such as a halogen, including chloride, iodide, fluoride and bromide or an oxyanion. In an alternative embodiment, R
3
, Y
1
and the atoms to which they are bound, join to form an optionally substituted 5- or 6-membered heterocyclic ring. In an alternative embodiment, R
4
, Y
6
and the atoms to which they are bound, join to form an optionally substituted 5- or 6-membered heterocyclic ring.
The cationic lipids of Formula I are attractive for a number of reasons. These novel cationic lipids are derived from pentaerythritol which provides a unique “linchpin” framework that differs from current diacylpropanaminium motifs commonly used in gene transfer. Furthermore, the lipids of Formula I are less toxic than known cationic lipids, due in part to their amino acid metabolic by-products. In addition, the liposomes and lipid complexes whose lipids include the cationic lipids of Formula I have been shown to have comparable transfection efficiencies to the cationic lipids of the prior art.
In another aspect, this invention relates to a lipid-nucleic acid complex, the lipid portion of which contains an amphiphilic cationic lipid of Formula I.
In yet another aspect, this invention relates to a method for transfecting a nucleic acid into a cell. In this method, the cell is contacted with a lipid-nucleic acid complex, or liposome, the lipid portion of which contains an amphiphilic cationic lipid of Formula I. Using standard techniques, the lipids of Formula I can facilitate the transfection of nucleic acids into cells, in vivo and in vitro, with high efficiency.
In still yet another aspect, this invention relates to a pharmaceutical composition or other drug delivery composition for administering a nucleic acid particle to a cell. This composition includes a lipid-nuclei acid complex, the lipid portion of which contains an amphiphilic cationic lipid of Formula I, and a pha

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