Lipid derivatives of polythiourea

Details Lipid derivatives of polythiourea Lipid derivatives of polythiourea

2002-05-14

2004-11-02

Ketter, James (Department: 1636)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C514S476000, C514S580000, C435S458000

Reexamination Certificate

active

06812218

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to novel compounds which make it possible to transfer nucleic acids into cells. More precisely, these novel compounds are lipid derivatives of polythiourea. They are useful for the in vitro, ex vivo or in vivo transfection of nucleic acids into various cell types.
With the development of biotechnology, the possibility of effectively transferring nucleic acids into cells has become a necessity. It may involve the transfer of nucleic acids into cells in vitro, for example, for the production of recombinant proteins, or in the laboratory for studying the regulation of the expression of genes, the cloning of genes, or any other manipulation involving DNA. It may also involve the transfer of nucleic acids into cells in vivo, for example for the creation of transgenic animals, the production of vaccines, labeling studies or also therapeutic approaches. It may also involve the transfer of nucleic acids into cells ex vivo, in approaches including bone marrow transplants, immunotherapy or other methods involving the transfer of genes into cells collected from an organism for the purpose of their subsequent readministration.
Several methods have been proposed for the intracellular delivery of exogenous genetic material. One of them, in particular, is based on the use of nonviral vectors which constitute a highly advantageous alternative to the viral methods which are not completely risk free. These synthetic vectors have two main functions: to complex and to compact the nucleic acid to be transfected, and to promote its passage across the plasma membrane and possibly across the nuclear envelope.
Several families of synthetic vectors have thus been developed, such as for example polymers or alternatively biochemical vectors (consisting of a cationic protein combined with a cellular receptor ligand), but a major advance has in particular been made with the development of lipofectants and more particularly of cationic lipids. It has thus been demonstrated that cationic lipids, because of their overall positive charge, spontaneously interfere with DNA which is globally negative, forming nucleolipid complexes capable both of protecting the DNA against nucleases and of binding to the cellular membranes for intracellular release of the DNA.
Various types of cationic lipids have been synthesized to date: lipids comprising a quaternal ammonium group (for example DOTMA, DOTAP, DMRIE, DLRIE, and the like), lipopolyamines such as for example DOGS, DC-Chol or alternatively the lipopolyamines disclosed in Patent Application WO 97/18185, lipids combining both a quaternary ammonium group and a polyamine such as DOSPA, or alternatively lipids comprising various other cationic entities, in particular amidinium groups (for example ADPDE, ADODE or the lipids of patent application WO 97/31935).
However, the use of these cationic lipids as transfection agent still poses numerous problems, and their efficiency remains to be improved. In particular, it has been observed that to obtain efficient and stable nucleolipid complexes, it is in general necessary for these complexes to be highly cationic. However, it would be desirable to be able to have available vectors which are not cationic so as to form, with the nucleic acid, particles which are globally neutral or negative. Indeed, it has been observed that the globally cationic complexes formed between the nucleic acid and the cationic lipids tend to be captured by the reticuloendothelial system, which induces their elimination. In addition, the plasma proteins tend to become adsorbed at their surface because of the overall positive charge of the complexes formed, and this results in a loss of the transfection power. Furthermore, in a context of local injection, the presence of a large overall positive charge prevents the diffusion of the nucleic acid complexes away from the site of administration because the complexes become adsorbed onto the extracellular matrices; the complexes can therefore no longer reach the target cells, which consequently causes, a decrease in the transfer efficiency in relation to the injected quantity of complexes. Finally, it has also been observed, in many instances, that cationic lipids have an inflammatory effect.
BRIEF SUMMARY OF THE INVENTION
The object of the present invention is precisely to provide novel transfecting compounds which are innovative by virtue of their polythiourea functional group and which are capable of being efficiently used for the in vitro, ex vivo or in vivo transfection of nucleic acids. These novel compounds are particularly advantageous because:
the absence of positive charges from their structure makes it possible to solve the many problems raised by the use of cationic vectors discussed above,
just like cationic lipids, they are capable of complexing and compacting nucleic acids and of promoting their transfection.
A first subject of the present invention is thus transfecting compounds characterized in that they consist of a polythiourea part linked to a lipid via a spacer.
In particular, the subject of the present invention is transfecting compounds of general formula (I):
in which:
l is an integer chosen from 0 and 1,
n is an integer chosen from 1, 2, 3, 4, 5 and 6,
m is an integer chosen from 2, 3 and 4, it being possible for m to take different values within the different groups —[NH—CS—NH—(CH)
m
]—,
R′ represents a group of general formula (II):
 in which q is an integer chosen from 1, 2, 3, 4, 5 and 6, and p is an integer chosen from 2, 3 and 4, it being possible for p to take different values within the different groups —[(CH
2
)
p
—NH—CS—NH]—,
R represents either a hydrogen atom or a group of general formula (II) as defined above, it being understood that when n is 1 and l is 0, then at least one group R is of formula (II),
X, in the formulae (I) and (II), represents a saturated or unsaturated, linear or cyclic aliphatic group, comprising 1 to 8 carbon atoms, a mercaptomethyl (—CH
2
SH) group, or alternatively a hydrophilic chain chosen from the groups:
—(CH
2
)
x
—(CHOH)
u
—H with x an integer chosen from 1 to 10 and u an integer chosen from 1, 2, 3, 4, 5 and 6, or alternatively,
—(OCH
2
CH
2
O)
v
—H with v an integer chosen from 1, 2 and 3, it being understood that no more than one substituent X, both in the formulae (I) and (II), represents a hydrophilic chain,
Y represents a spacer,
and L represents:
either a group —N(R
1
)R
2
with R
1
and R
2
which represent, independently of each other, a hydrogen atom or alternatively a fatty aliphatic chain, or alternatively a group of formula —(CH
2
)
t
—OZ with t representing an integer chosen from 11, 12, 13, 14 or 15 and Z represents a sugar, a polyol or a PEG, it being understood that at least one of R
1
and R
2
is different from hydrogen,
or a group —OR
3
, with R
3
which represents a steroid derivative.
According to the present invention, the term “spacer” is understood to mean any chemical group which makes it possible both to provide the linkage between the polythiourea part and the lipid part of the molecule, and to keep these two parts apart so as to attenuate any undesirable interruption between them. Preferred spacers may for example consist of one or more chemical functional groups chosen from alkyls having 1 to 6 carbon atoms, ketone, ester, ether, amide, amidine, carbamate or thiocarbamate functional groups, glycerol, urea, thiourea, or else aromatic rings. For example, the spacer may be chosen from the groups of formula:
—NH—C(O)—CH
2
—CH
2
—
or:
—(CH
2
—)
i
—W—(CH
2
)
j
—
in which i and j are integers chosen between 1 and 6 inclusive and W is a group chosen from ketone, ester, ether, amide, amidine, carbamate or thiocarbamate functional groups, glycerol, urea, thiourea, or alternatively aromatic rings.
For the purposes of the present invention, the expression “fatty aliphatic chains” is understood to mean alkyl groups containing 10 to 22 carbon atoms which are saturated or unsaturated and optionally containing one or more heteroatoms, pr

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