EP1562963A1

EP1562963A1 - Novel lipophilic compounds and uses thereof

Info

Publication number: EP1562963A1
Application number: EP03786052A
Authority: EP
Inventors: Hervé Des Abbayes; Jean-Jacques Yaouanc; Jean-Claude Clement; Karine Le Ny; Claude Ferec; Tristan Montier; Pascal Delepine
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Centre Hospitalier Regional et Universitaire de Brest; Univerdite de Bretagne Occidentale
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Centre Hospitalier Regional et Universitaire de Brest; Univerdite de Bretagne Occidentale
Priority date: 2002-11-08
Filing date: 2003-11-07
Publication date: 2005-08-17
Also published as: AU2003295057A8; FR2846967A1; US20060165768A1; AU2003295057A1; FR2846967B1; US7667070B2; WO2004043970A1; CA2505414A1; CA2505414C

Abstract

The invention relates to a cationic lipophilic compound of general formula (I), wherein: a) R1 and R'1 independently represent an alkyl chain, alkenyl chain, or polyalkenyl chain of 10 to 24 carbon atoms, the polyalkenyl chain being provided with 2 to 4 double bonds; b) R2 represents a hydrogen atom or an alkyl chain with 1 to 4 carbon atoms; c) R3 represents a group of formula (IIa), i.e. -(CH2)n-, or formula (IIb), i.e. -C(=NH)-NH-(CH2)n-, n representing one of the whole numbers 0, 1, 2, 3, or 4; d) A+ represents an organic cation; and e) X- represents an anion.

Description

New lipophilic compounds and their uses

FIELD OF THE INVENTION

The present invention relates to new lipophilic compounds having an affinity for nucleic acids. These new compounds can be used as non-viral vectors to introduce a nucleic acid of interest into a chosen host cell or into a chosen host organism.

PRIOR ART

In recent years, many authors have been interested in the development of non-viral vectors intended to transport DNA of interest through the cell membrane and to the cell nucleus, in particular within the framework of the development of methods of genetical therapy.

In the review by A.D. MILLER entitled “Cationic liposomes for gene therapy” (Angewandte Chem. Int., Ed. Engl., 1998, Vol. 37: 1768-

1785), which constitutes a general review on cationic lipids, it can be noted that the positive charge of the cation is always carried by a nitrogen atom.

Among the lipophilic compounds used in the prior art as non-viral vectors, mention may be made of the halides of -1, 2-3-dioleyl trimethylammonium deoxyglycerol, commonly known as DOTAP, the -1, 2-3-dioleyl-trimethylammonium, commonly known as DOTMA , dimethylammonium ethyloxycarbonylcholesterol, commonly known as DC-chol.

Phosphonolipids have also been described, such as those described by

G. Le Bolc'h et al. (Tetrahedron Lett., 1995, 36, 6681) and V. Floch et al (Eur. J. Med. Chem., 1998, 33, 12.), phosphonolipids in the form of an ammonium cation salt (V. Floch et al., Eur. J. Med. Chem., 1998 /, Vol. 33: 923-934) or in the form of a phosphonium or arsonium cation salt ( E. Guénin et al., Anew. Chem Int. Ed., 2000, Vol. 39 (3); V. Floch et al., J. Med. Chem., 2000, Vol. 43 (24): 4617-4628 ).

However, non-viral cationic lipophilic vectors have a reduced capacity for transfection of DNA into cells and have cytotoxic properties with respect to these cells.

There is therefore a need, in the prior art, for non-viral vectors more efficiently transporting a nucleic acid through the cell membrane, to the cell nucleus, in order to obtain transfection yields higher than those observed. with known non-viral vectors.

Another objective sought is the obtaining of new non-viral vectors having an improved transfection capacity combined with a low cytotoxicity for the cells to be transfected.

SUMMARY OF THE INVENTION

The applicant has now synthesized new cationic lipophilic compounds, of the mono- or bis-phosphoramide type, and containing a cationic part of the “Onium” type, which have a high capacity for transfection of a nucleic acid into cells, greater than that known non-viral vectors, in particular known phosphonolipids. In addition, the new lipophilic compounds according to the invention have reduced cytotoxicity properties, compared with the non-viral vector compounds described in the state of the art.

The subject of the invention is therefore new cationic lipophilic compounds, of the mono- or bisphosphoramide type, and containing a cationic part of the “Onium” type, which are described later in the detailed description of the invention. The "Onium" part of the compounds cationic lipophiles of the invention can be an ammonium, a phosphonium or an arsonium, or an organic cation, such as the imidazolium, thiazolium or pyridinium cations.

The present invention also relates to lipid vesicles, unilamellar or multilamellar, comprising, or consisting mainly or almost exclusively of a lipophilic compound according to the invention, preferably in the form of a complex between said lipophilic compound and a nucleic acid of interest.

The invention also relates to a complex formed between a nucleic acid of interest and a lipophilic compound as defined above.

It also relates to methods for introducing, in vitro or in vivo, a nucleic acid of interest into a host cell or a host organism, using a complex formed between said nucleic acid of interest and a lipophilic compound of the invention, where appropriate presented in the form of unilamellar or multilamellar lipophilic vesicles.

The invention also relates to a pharmaceutical composition containing, as active principle, at least one complex formed between a nucleic acid of interest and a lipophilic compound as defined above, optionally in combination with one or more physiologically compatible excipients.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1 illustrates the comparative results of in vitro transfection of cells of the K562 cell line with lipophilic compounds of the prior art and lipophilic compounds according to the invention or with a mixture of a lipophilic compound according to the invention with a lipophilic compound of the state of the prior art. The transfection was carried out with DNA of interest encoding the marker protein luciferase. On the abscissa, the different compounds or mixtures of compounds used to form complexes with the DNA of interest before cell transfection. In parentheses are reported the mass ratios between the lipid compound (s) and the DNA.

On the ordinate, the results of transfection efficiency correspond to the luciferase activity found in the different samples of cells transfected in culture, these results being expressed in TRLU units (Total Relative Light Units), as described in the section "Materials and Methods From Example 5.

FIGURE 2 illustrates the comparative results of in vitro transfection of cells of the Jurkat cell line with lipophilic compounds of the prior art and lipophilic compounds according to the invention or also with a mixture of a lipophilic compound according to the invention with a lipophilic compound of the state of the prior art. The transfection was carried out with DNA of interest encoding the marker protein luciferase. On the abscissa, the different compounds or mixtures of compounds used to form complexes with the DNA of interest before transfection of the cells. In parentheses are reported the mass ratios between the lipid compound (s) and the DNA.

In the Figure, "DC-CHOL" means the commercial product (3β- [N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol.

On the ordinate, the results of transfection efficiency correspond to the luciferase activity found in the different samples of cells transfected in culture, these results being expressed in TRLU units, as described in the section "Materials and Methods" of Example 5 .

FIGURE 3 illustrates the comparative results of in vitro transfection of cells of the Daudi cell line with lipophilic compounds of the prior art and lipophilic compounds according to the invention or with a mixture of a lipophilic compound according to the invention with a lipophilic compound of the state of the prior art. The transfection has was carried out with a DNA of interest encoding the marker protein luciferase.

On the abscissa, the different compounds or mixtures of compounds used to form complexes with the DNA of interest before transfection of the cells. In parentheses are reported the mass ratios between the lipid compound (s) and the DNA.

On the ordinate, the results of transfection efficiency correspond to the luciferase activity found in the different samples of cells transfected in culture, these results being expressed in TRLU units, as described in the section "Materials and

Methods ”of Example 5.

FIGURE 4 illustrates the comparative results of in vitro transfection of mouse cardiomyocyte cells in primary culture with lipophilic compounds of the prior art and lipophilic compounds according to the invention or with a mixture of a lipophilic compound according to the invention with a lipophilic compound of the state of the prior art. The transfection was carried out with DNA of interest encoding the marker protein luciferase.

On the abscissa, the different compounds or mixtures of compounds used to form complexes with the DNA of interest before transfection of the cells, used according to three distinct charge + / charge - ratios. For example, in report 2, the composition tested contains twice as many positive charges (cationic lipophilic compound) as negative charges (provided by the DNA molecules).

Methods ”of Example 5. In Figure 4, "D" means

DOPE (dioleylphosphatidylethanolamine) and "DD" means DOPE + DOTAP.

FIGURE 5 illustrates comparative cytotoxicity results between different lipophilic compounds of the invention, lipophilic compounds of the prior art and mixtures of lipophilic compounds of the invention with lipophilic compounds of the prior art.

Methods ”of Example 5.

FIGURE 6 illustrates the comparative results of in vivo transfection efficiency of a DNA coding for luciferase, between different lipophilic compounds of the invention, different lipophilic compounds of the prior art and mixtures between lipophilic compounds of the invention and lipophilic compounds of the prior art.

On the abscissa, the different compounds or mixtures of compounds used to form complexes with the DNA of interest before transfection of the cells. In Figure 6, "D" means DOPE and "Chol" means cholesterol.

On the ordinate, the comparative results of transcription efficiency are visualized according to the amount of luciferase found in the lungs of the animals sacrificed, these results being expressed in RLU units, as described in the “Materials and Methods” section of Example 6. DETAILED DESCRIPTION OF THE INVENTION

The applicant has synthesized new cationic lipophilic compounds, of the mono-phosphoramide or bis-phosphoramide type, and having a cationic part of the “Onium” type, combining a higher capacity for transfection of cells with a nucleic acid of interest and properties of reduced cytotoxicity, compared to previously known non-viral lipophilic vectors.

The subject of the invention is a cationic lipophilic compound of general formula (I) below:

in which: a) R ¹ and R ' ¹ each represent, independently of one another, an alkyl chain, an alkenyl chain or a polyalkenyl chain of 10 to 24 carbon atoms, with the polyalkenyl chain having from 2 to 4 double bonds; b) R ² is a hydrogen atom or an alkyl chain having from 1 to 4 carbon atoms; and

R ³ is a group of the following formula (Ma): - (CH2) n- or (llb) following:

-C (= NH) -NH- (CH ₂ ) n- in which:

- n is an integer equal to 0, 1, 2, 3 or 4; and d) A ⁺ is an organic cation; e) X ^" is an anion. By "alkyl" is meant according to the invention an aliphatic hydrocarbon group, which can be linear or branched. The alkyl chain may be substituted, on one or more of the carbon atoms constituting it, by a group chosen from hydroxy, alkoxy and alkylthio groups. Preferably, a carbon atom of the hydrocarbon chain comprises at most a single substituent. Preferably, at most three carbon atoms of the hydrocarbon chain are substituted by at least one of the above groups.

By "alkenyl" is meant according to the invention an alkyl group containing a carbon-carbon double bond, which can be located at any point of the hydrocarbon chain.

By "polyalkenyl" is meant according to the invention an alkyl group containing from two to four carbon-carbon double bonds in the hydrocarbon chain, which can be located at any point of the hydrocarbon chain in relative "malonic" positions.

By “organic cation” is meant according to the invention any organic chemical group contained in a lipophilic compound of the invention and positively charged in solution. By "anion" is meant according to the invention any organic or mineral molecule which is negatively charged in solution.

Without wishing to be bound by any theory, the applicant believes that the improved properties of the above lipophilic compounds allowing efficient transfection of a nucleic acid of interest, both in vitro and in vivo, are due to the brittleness of the covalent bond between the lipophilic part and the cationic part of these compounds, said bond being cleaved after the passage of the lipophilic compound through the cell membrane, which releases the cationic part which retains the binding properties to the polyanionic nucleic acid d of interest, and which can be transported to the nucleus of cells. The free lipophilic part, after cleavage, can be degraded under the action of various cellular enzymes, especially various enzymes found in the cytoplasm.

The applicant has in fact observed, by measuring the ¹ H and ³¹ P NMR spectrum, that the bond between the phosphorus atom (P) and the nitrogen atom (N) of a cationic lipophilic compound of formula (I ) is rapidly hydrolyzed, in the absence of an enzyme, to a pH value of about 4-5, which is the pH found in the endosomes or intracytoploplasmic lysosomes of the cell. In particular, in the absence of any enzyme, the PN bond is completely hydrolyzed after an incubation of the compound of formula (I) for 6 hours at a pH of about 4-5, at a temperature of 20 ° C.

Preferably, X ^" is an anion chosen from CF3CO2 ^" , CF3SO ₃ ^" ,

HS04 ^" , and a halogen. Preferably, the halogen is chosen from

CI ^' , Bf and P. According to a first preferred embodiment, a lipophilic compound as defined above is characterized in that group A is a five or six-membered heterocyclic aromatic ring comprising a heteroatom consisting of a nitrogen quaternary linked by a covalent bond to the group R ³ . Preferably, the heterocyclic aromatic ring comprises a second heteroatom chosen from S or N. Preferably, when the second heteroatom is N, said second heteroatom is substituted by an alkyl chain of 1 to 4 carbon atoms.

According to a preferred aspect of this first embodiment of a lipophilic compound, group A is a thiazolium or an imidazolium, with the second nitrogen heteroatom optionally substituted by an alkyl chain of 1 to 4 carbon atoms.

Preferred compounds belonging to this first embodiment of the invention are the following: - ditetradecyl iodide 2- (1-methyl-1 H-imidazol-3-ium-3-yl) ethylamidophosphate [compound KLN27]; dioleyl iodide 2- (1-methyl-1H-imidazol-3-ium-3-yl) ethylamidophosphate [compound KLN28];

- ditetradecyl iodide 2- (1, 3-thiazol-3-ium-3yl) ethylamidophosphate [compound KLN37]. - ditetradecyl iodide 2- (1-methyl-1 H-imidazol-3-ium-3-yl) propylamidophosphate [compound KLN16a].

According to a second preferred embodiment, a lipophilic compound as defined above is characterized in that group A is a six-membered heterocyclic aromatic ring comprising a quaternary nitrogen heteroatom. Preferably, group A is a pyridinium ring.

According to a preferred aspect of this second embodiment, the lipophilic compound of the invention is characterized in that group A is represented by the following formula (III):

in which R ¹³ represents an alkyl chain of 1 to 4 carbon atoms.

A preferred compound belonging to the second embodiment of a lipophilic compound according to the invention is the compound: - ditetradecyl iodide 2- (1-methyl-1 - / - pyridin-1-ium-4-yl) ethylamidophosphate [compound KLN38]

According to a third preferred embodiment, a lipophilic compound according to the invention is characterized in that group A is a cation of formula (IV) below:

_D 6

in which : f) Z represents N, P or As; g) R ⁴ and R ⁵ each represent, independently of one another, an alkyl chain having from 1 to 4 carbon atoms; and h) R ⁶ represents an alkyl chain having from 1 to 4 carbon atoms.

It has been shown according to the invention that the compounds belonging to the third embodiment above and having a high level of capacity to transfect a nucleic acid in a target cell are preferably those for which the groups R ⁴ , R ⁵ and R ⁶ each represents a methyl group.

Preferably, the group R ² represents hydrogen, a methyl group or an ethyl group.

Preferably, the group R ³ of formula (IIa) [- (CH ₂ ) n-] or (IIb) [- C (= NH) -NH- (CH ₂ ) _n -] is such that n is an equal integer to 2, 3 or 4. Preferably, Z represents P or A _s , and very preferably, Z represents As.

Preferred compounds belonging to the third embodiment of a lipophilic compound according to the invention are chosen from the following compounds: - iodide of 3 - [[bis (tetradecyloxy) phosphoryl] (methyl) amino] -NNN- trimethylpropanaminium [compound KLN5 ]; 3 - [[bis (oleyloxy) phosphoryl] (methyl) amino] -N.N.N-trimethylpropanaminium iodide [compound KLN6];

- iodide of 3 - [[bis (tetradecyloxy) phosphoryl] (ethyl) amino] -N.N.N- trimethylethanaminium [compound KLN13]; 3 - [[bis (oleyloxy) phosphoryl] (ethyl) amino] -N.N.N-trimethylethanaminium iodide [compound KLN14]; ditetradecyl 2- (trimethylphosphonio) ethyl propylamidophosphate iodide [compound KLN19]; - dioleyl 3- (trimethylphosphonio) propylamidophosphate iodide

[compound KLN20]; - ditetradecyl 2- (trimethylarsonio) ethylamidophosphate iodide [compound KLN29];

- dioleyl iodide 2- (t methylarsonio) ethylamidophosphate [compound KLN30]; - ditetradecyl iodide 3- (trimethylarsonio) propylamidophosphate

[compound KLN31];

- dioleyl iodide 3- (trimethylarsonio) propylamidophosphate [compound KLN32];

The cationic lipophilic compounds belonging to the first, second and third embodiments above are all compounds of the mono-phosphoramide type.

According to a fourth embodiment of the invention, an active lipophilic compound is obtained by covalently linking two compounds of the mono-phosphoramide type, the two monophosphoramide compounds being linked to each other by an alkyl chain of 1 to 4 atoms of carbon, which replaces the R ⁶ group of each of the two mono-phosphoramide compounds, resulting in a bis-phosphoramide compound which also forms part of the invention.

Thus, the subject of the invention is also a lipophilic compound of formula (I) as defined above, said compound being characterized in that group A is a cation of formula (IV) below:

(IV) in which: i) Z represents N, P or As; j) R ⁴ and R ⁵ each represent, independently of one another, an alkyl chain having from 1 to 4 carbon atoms; k) R ⁶ represents the following group of formula (V): in which :

I) R ⁷ and R ⁸ each represent, independently of one another, an alkyl chain of 1 to 4 carbon atoms, and m) R ⁹ represents a group of formula (VI) below:

in which: n) R ¹⁰ represents - (CH ₂ ) n- where n is an integer equal to 0, 1, 2, 3 or 4; o) R ¹¹ represents H or an alkyl group of 1 to 4 carbon atoms; and p) R ¹² and R ′ ¹² each represent, independently of one another, an alkyl chain, an alkenyl chain or a polyalkenyl chain of 10 to 24 carbon atoms, with the polyalkenyl chain having from 2 to 4 double connections; Preferably, according to this fourth embodiment of a lipophilic compound according to the invention, the groups R ¹² and R ¹² are identical to the groups R ¹ and R ¹ , respectively. Preferably, the groups R ³ and R ¹⁰ are identical.

Preferably, the groups the groups R ⁴ and R ⁵ are identical to the groups R ⁷ and R ⁸ , respectively. A preferred bis-phosphoramide compound belonging to the fourth embodiment above is the following compound: diiodide of N ¹ , N ⁴ -bis (3-

^< {[bis (tetradecyloxy) phosphoryl] amino} propyl) -N ¹ , N ¹ , N, N ⁴ - tetramethyl-1, 4 butane diaminium [compound KLN39].

In general, for the lipophilic compounds of formula (I) of the monophosphoramide type according to the invention, the groups R ⁴ , R ⁵ and R ⁶ are preferably identical.

Likewise, for the lipophilic compounds of formula (I) of the bisphosphoramide type as defined above, the groups R ⁴ , R ⁵ , R ⁷ and R ⁸ are preferably identical.

Preferably, for bisophosphoramide lipophilic compounds as defined above, the groups R ² and R ¹¹ are identical. As already mentioned above, the lipid groups R ¹ and R ' ¹ , and, for bis-phosphoramide compounds, also the lipid groups R ¹² and R' ¹² , each represent, independently of one another, an alkyl chain from 10 to 24 carbon atoms, an alkenyl chain of 10 to 24 carbon atoms or a polyalkenyl chain of 10 to 24 carbon atoms and having from 2 to 4 double bonds per chain.

According to a first aspect, the groups R ¹ and R ′ ¹ are identical. According to a second aspect, the R ¹² and R ^'12 are identical. According to a third aspect, the groups R ¹ , R ' ¹ , R ¹² and R' ¹² are all identical.

When at least one group, among the groups R ¹ , R ' ¹ , R ¹² and R' ¹² , represents an alkyl chain of 10 to 24 carbon atoms, said alkyl chain is preferably chosen from chains of 14 to 20 atoms of carbon. A preferred lipophilic group having an alkyl chain of 10 to 24 carbon atoms is the tetradecyl or myristyl group having 14 carbon atoms.

When at least one group, among the groups R ¹ , R ' ¹ , R ¹² and R' ¹² , represents an alkenyl chain of 10 to 24 carbon atoms, said alkenyl chain is preferably chosen from chains of 14 to 20 atoms of carbon.

A preferred lipophilic group having an alkenyl chain of 18 carbon atoms and having an olefinic double bond is the oleyl group.

When at least one group, among the groups R ¹ , R ' ¹ , R ¹² and R' ¹² , represents a polyalkenyl chain, said polyalkenyl chain is preferably chosen from chains of 14 to 20 carbon atoms.

Preferably, the polyalkenyl chain is chosen from the groups Ci8 _{: 2} and Cι _8: 3, in which the first number represents the number of carbon atoms in the alkenyl chain and the second number represents the number of double bonds in the chain alkenyl.

In general, the technical problem of the invention can be solved with groups R ¹ , R ' ¹ , R ¹² and R' ¹² , each group representing, independently of one another, an alkyl chain of 10 to 24 carbon atoms, an alkenyl chain of 10 to 24 carbon atoms or a polyalkenyl chain of 10 to 24 carbon atoms and having 2 to 4 double bonds per chain, since these different alkyl, alkenyl or polyalkenyl chains are all sufficiently hydrophobic to allow a lipid compound of the invention to come into contact with the cell membrane, then to enter the cell by crossing the lipid bilayer of the cell membrane, then to reach the nucleus by passing through the membrane nuclear.

The synthesis of the various embodiments of the lipophilic mono-phosphoramide compounds is described in detail in the Examples. For example, to synthesize a lipophilic compound of formula (I), in which the group A is a cation of formula (IV) with the group R ⁶ representing an alkyl chain having from 1 to 4 carbon atoms, a compound is reacted phosphite of the following formula (VII):

a diamine of formula (VIII) below

R ²

in the presence of an appropriate phase transfer agent, such as benzyltriethylammonium chloride, then the compound of formula (I) is recovered as defined above by quaternizing the amine -N (R ⁴ R ⁵ ) of the compound of formula (VIII) above by an appropriate alkyl halide R ⁶ -X.

By way of illustration, to prepare a compound of formula (I) in which group A represents a five-membered heterocyclic aromatic ring comprising a heteroatom consisting of a quaternary nitrogen linked by a covalent bond to the group R ³ , such as a heterocycle imidazole or thiazole, the following synthesis process is used, exemplified for imidazole. a) A phosphite compound of the following formula (VII) is reacted:

with an amino compound of the following formula (IX) R ²

^ -R ³ Br (| X) in the presence of an appropriate phase transfer agent, such as triethyl benzylammonium chloride, in order to obtain the intermediate compound of formula (X) below:

b) then the compound of formula (X) above is reacted with N-methyl imidazole, in order to obtain the compound of formula (XI) below:

To prepare a compound of formula (I) in which the group A is a six-membered heterocyclic aromatic ring comprising a quaternary nitrogen heteroatom, such as pyridine, the procedure is carried out according to the reaction scheme defined above for the compounds of which group A consists of a six-membered heterocyclic aromatic ring comprising a quaternary nitrogen atom.

To prepare a lipophilic compound of the bis-phosphoramide type according to the invention, the process below can be used, for example. A phosphite compound of formula (VII) as described above is reacted with a diamine of formula (VIII) as described above, under the same conditions, then reacting 2 equivalents of the compound obtained with an equivalent of an appropriate alkyl dihalide X - R ¹⁰ - X.

In general, any other conventional synthesis method can be implemented by a person skilled in the art to prepare a lipophilic monophosphoramide or bisphosphoramide compound according to the invention.

Without wishing to be bound by any theory, the applicant believes that the cationic part of the lipophilic compound according to the invention carries the binding properties of said compound with the nucleic acid of interest, said nucleic acid being a polyanionic compound. The lipid part of the lipophilic compound, represented by the groups R ¹ and R ' ¹ and, for bisphosphoramide compounds, also by the groups R ¹² and R' ¹² , allows the lipophilic compound of the invention to bind to the cell membrane , to cross the lipid bilayer of the cell membrane then to reach the cytoplasm and / or the nucleus, at the level of which the nucleic acid of interest is transcribed, or even at the level of which the nucleic acid of interest s' hybrid with a target nucleic acid naturally present in the cell, either in the cytoplasm or in the nucleus.

For the purposes of the present description, the terms “nucleic acid”, polynucleotide ”and“ oligonucleotide ”include DNA, RNA molecules, DNA / RNA hybrid molecules of more than two nucleotides in length, either in the simple form strand or double strand.

Thus, any of the lipophilic compounds of the invention can be used as such, preferably in solution, to be complexed with a nucleic acid of interest, the introduction of which by transfection into a host cell is sought.

According to a fifth preferred embodiment of the invention, a lipophilic compound according to the invention is used in the form of lipid vesicles, which are then brought into contact with an acid nucleic acid of interest so as to form a complex between said nucleic acid and the lipid vesicles thus prepared.

According to a first preferred aspect, the lipid vesicles are prepared using exclusively a lipophilic monophosphoramide or bisphosphoramide compound of the invention. According to this first aspect, the lipid vesicles can be prepared from a mixture of several lipophilic compounds of the invention, preferably at most four, and very preferably two distinct lipophilic compounds according to the invention. Part of the invention thus forms lipid vesicles comprising a lipophilic monophosphoramide or bisphosphoramide compound chosen from the compounds defined above.

Also part of the invention are lipid vesicles essentially consisting of one or more lipophilic monophosphoramide or bisphosphoramide compounds as defined above. By "essentially" means lipid vesicles comprising at least 90% by weight of one or a plurality of lipophilic compounds of the invention.

Also part of the invention are lipid vesicles consisting exclusively of one or more lipophilic monophosphoramide or bisphosphoramide compounds as defined above.

According to a second preferred aspect, the lipid vesicles are prepared by using a mixture of at least one lipophilic compound of the invention with at least one lipophilic compound not forming part of the invention, very preferably a lipophilic compound binding to a nucleic acid and usable as a non-viral vector of a nucleic acid of interest. Such a lipophilic non-viral vector compound is for example a lipophilic compound of the state of the art, such as DOPE, DOTAP, DOTMA, or cholesterol. Such lipid vesicles comprise at most four, and preferably at most two, distinct lipophilic compounds according to the invention. Of similarly, such lipid vesicles comprise at most four, and preferably at most two, distinct lipophilic compounds not forming part of the invention. Most preferably, the lipid vesicles encompassed by the second preferred aspect of the fifth embodiment above respectively comprise a single lipophilic compound of the invention, in admixture with a single lipophilic compound not forming part of the invention . In these vesicles, the proportions of the various lipophilic compounds are variable. When the vesicles comprise a lipophilic compound of the invention and a lipophilic compound not forming part of the invention, a weight ratio of the compound of the invention is preferred:: other lipophilic compound of between 10:: 1 to 1:: 2, preferably between 6:: 1 and 1:: 1, and most preferably between 4:: 1 and 1:: 1.

According to this second preferred aspect of the fifth embodiment above, also form part of the invention lipid vesicles comprising at least one lipophilic monophosphoramide or bisphosphoramide compound of the invention and at least one other lipophilic compound.

According to a third aspect of the fifth embodiment above, the lipid vesicles are in the form of unilamellar lipid vesicles, specifically small unilamellar vesicles.

According to a fourth aspect, the lipid vesicles are in the form of multilamellar lipid vesicles.

The lipid vesicles of the invention can be prepared by any technique known to those skilled in the art, for example by prior dissolution of the lipophilic compound (s) in a solvent, such as an aqueous medium, for example pyrogen-free distilled water or a physiologically compatible saline solution, then sonication of the solution thus obtained. Once prepared, the lipid vesicles of the invention can be stored for a long time, for example in frozen form in liquid nitrogen. However, an extemporaneous preparation of these vesicles is preferred, at most a few hours, for example at most 4 hours, before they are brought into contact or incubation with a nucleic acid of interest. According to a sixth preferred embodiment of the invention, the nucleic acid of interest which must be introduced into a host cell encodes a protein or a peptide. The protein can be any protein useful for carrying out a method of gene therapy, preferably somatic gene therapy, and includes, but is not limited to, cytokines, structural proteins, hormones, antigens, immunogens, receptors etc.

According to a second preferred aspect of the sixth embodiment above, the nucleic acid of interest encodes a sense or antisense polynucleotide which hybridizes with a target nucleic acid encoding a protein whose inhibition of expression in a cell host is sought.

According to yet another preferred aspect, the nucleic acid of interest consists of a recombinant vector, preferably a recombinant expression vector, into which is inserted the nucleic acid of interest, the coding sequence of which is placed under the control regulatory sequences, in particular promoter or activator sequence (“enhancer”), necessary for the expression of said nucleic acid of interest in the transfected host cell.

According to the invention, the nucleic acid of interest, linear or circular, single-stranded, or double-stranded, is firstly complexed with a lipophilic monosphoramide or bisphosphoramide compound of the invention, before being introduced, under the form of the complex, in the host cell.

As described above, the complexes are preferably formed by incubation of the nucleic acid with the lipid vesicles defined above. However, in some cases, the complexes can be formed by incubating the nucleic acid of interest with a lipophilic compound of the invention, or a mixture of several lipophilic compounds of the invention, the complexes thus formed being subsequently used for the transfection of host cells. According to an alternative, lipid vesicles, unilamellar or multilamellar, are formed from the nucleic acid / lipophilic compound (s) complexes previously prepared, then the vesicles are used to transfect the host cells.

Preferably, the complexes formed between nucleic acid molecules and molecules of lipophilic compounds comprise a weight ratio of nucleic acid / lipophilic compounds of between 0.5 and 100, advantageously between 1 and 10, and preferably between 2 and 5.

Another subject of the invention is the use of a lipophilic compound or of a lipid vesicle as defined above, for introducing, in vitro or in vivo, a nucleic acid into a host cell or into a host organism.

The invention also relates to a method for introducing, in vitro or in vivo, a nucleic acid into a host cell or a host organism, characterized in that it comprises the following steps: a) bringing said nucleic acid into contact with a compound lipophilic or with a lipid vesicle as defined above, in order to obtain a complex between said nucleic acid, on the one hand, and said compound or said lipid vesicle, on the other hand; b) incubating the host cell with the complex formed in step a), or administering, preferably by injection, the complex formed in step a) to the host organism.

Preferably, said host cell is a non-human mammalian cell or a human cell

Preferably, the host organism is a man or a non-human mammal, although the application of the above method cannot be excluded in other higher organisms such as plants. The invention also relates to a complex formed between a nucleic acid and a lipophilic phosphoramide or bisphosphoramide compound or a lipid vesicle as defined above.

The invention also relates to a composition comprising a complex formed between a nucleic acid and a lipophilic phosphoramide or bisphosphoramide compound or a lipid vesicle as defined above.

As already mentioned previously, the complexes formed between a nucleic acid of interest and a lipophilic compound or a lipid vesicle of the invention can be administered by any suitable method allowing their introduction into the cells of a man or of an animal, such as by injection into interstitial tissue species (heart, muscle, skin, lung, liver, intestines, etc.). Preferably, the complexes are presented in the form of a composition also containing a physiologically compatible vehicle.

In a particular embodiment of administration of the complexes according to the invention, the composition containing these complexes is in a form suitable for administration by aerosol, for example for inhalation. For the injection of one between a lipophilic compound or a lipid vesicle of the invention and a nucleic acid of interest, the quantity of DNA, RNA or DNA / RNA of interest for a dose injectable is advantageously between 0.005 mg / kg and 50 mg / kg of the weight of the man or animal to be treated. Preferably, the amount of nucleic acid ranges from 0.005 mg / kg to 20 mg / kg, and most preferably from 0.05 mg / kg to 5 mg / kg.

Of course, those skilled in the art are able to adapt the quantity of nucleic acid in a dose for injection, depending in particular on the pathology to be treated and the injection site. The quantity of nucleic acid for an injectable dose is determined by a person skilled in the art. The invention also relates to a method for introducing in vivo a nucleic acid of interest into the cells of a host organism, said method comprising the following steps: a) a) bringing said nucleic acid into contact with a lipophilic compound or with a lipid vesicle as defined above in order to obtain a complex between said nucleic acid, on the one hand, and said compound or said lipid vesicle, on the other hand; b) administering the complexes formed in step a) to said host organism.

As already mentioned, the host organism is preferably a human or a non-human mammal, although it can also be a plant.

In general, the complexes formed between a nucleic acid of interest and a lipophilic compound or a lipid vesicle of the invention are present in a suitable liquid solution, such as sterile and pyrogen-free distilled water, in amounts of suitable complexes. . The solution can be used as it is, or also contain one or more stabilizing agents, such as Tween® (20, 40, 60 or 80), NaCl, or even DMPE-PEG 5000.

The present invention also relates to a pharmaceutical composition comprising a complex formed between a nucleic acid of interest and a lipophilic compound or a lipid vesicle of the invention, where appropriate in combination with one or more physiologically compatible vehicles or excipients.

The present invention is further illustrated, without however being limited, by the following examples.

EXAMPLES

In general, the structure of each of the new compounds described in the following examples was verified by nuclear magnetic resonance spectroscopy (NMR) ¹ H, ¹³ C and ³¹ P. EXAMPLE 1 Synthesis of iodide of 3-rrbis (tetradecyloxy) phosphoryl (methyl) amino1-N, N, N-trimethylpropanaminium (KLN5).

A solution of 2.37 g (5 mmol) of ditetradecylphosphite, 0.581 g (5 mmol) of NNN 'trimethyl-1, 3 propanediamine and 70 mg of triethylbenzylammonium chloride in 3 ml of dichloromethane were added to a two-phase mixture consisting of 3 ml of dichloromethane, 3 ml of carbon tetrachloride and 4 ml of a 20% aqueous sodium hydroxide solution, keeping the reaction temperature between 0 ° C and 5 ° C.

After stirring this mixture for one hour at a temperature between 0 and 5 ° C, the mixture is stirred for 1 hour at room temperature.

Then, the organic phase is washed twice with 5 ml of water, then dried over magnesium sulfate. Solvents are removed.

The structure of the final product is verified by ¹ H and ³¹ P NMR spectroscopy. Then, the intermediate tertiary amine is dissolved in 5 ml of dichloromethane, to which is added 0.4 ml of methyl iodide, which is three times in excess. with respect to the tertiary amine intermediate. After stirring overnight at room temperature, the solvent was removed and the 3- iodide compound

[[bis (tetradecyloxy) phosphoryl (methyl) amino] -N, N, N- trimethylpropanaminium (KLN5) is precipitated from diethyl ether, then dried under vacuum, until a white powder is obtained. The final reaction yield is 85%.

EXAMPLE 2 Synthesis of dioleyl 3- (trimethylphosphonio) propylamidophosphate iodide (KLN20).

A solution consisting of 2.91 g (5 mmol) of dioleylphosphite,

1.095 g (5 mmol) of bromopropylamine hydrobromide and 15 mg of benzyltriethylammonium chloride in dichloromethane (3 ml) is added slowly to a two-phase system consisting of carbon tetrachloride (3 ml), dichloromethane (3 ml) and 4 ml of a 20% aqueous solution of sodium hydroxide, keeping the reaction temperature between 0 ° C and 5 ° C.

Stirring is continued for one hour at a temperature between 0 ° C and 5 ° C, then stirring is continued for an additional period of one hour at room temperature.

The organic layer was washed twice with 5 ml of water and then dried over magnesium sulfate.

The solvents were removed in vacuo. The crude dioleylbromopropylphosphoramide was obtained in the form of an oil. The final reaction yield was 80%.

To 1 g (1.4 mmol) of the bromophosphoramide obtained above, dissolved in 5 ml of tetrahydrofuran (THF), 3 ml of a 1.0 M solution of trimethylphosphine in THF was added under an atmosphere of nitrogen.

The reaction mixture was stirred for one week under a nitrogen atmosphere at room temperature. Then the solvent was removed in vacuo and the crude phosphonium was crystallized twice from ethyl acetate. Dioleyl 3- (trimethylphosphonio) propylamidophosphate iodide (KLN20) was obtained, the purity of which was greater than 99%, was checked both by ¹ H, ¹³ C and ³¹ P NMR and by thin layer chromatography (TLC). (eluent CHCIa / MeOH 9: 1; Rf = 0.21).

The final reaction yield was 60%.

EXAMPLE 3 Synthesis of ditetradecyl iodide 2- (1-methyl-1H-imidazol-3-ium-3-yl.ethylamidophosphate (KLN 27). To a solution of ditetradecylphosphite (2.37 g, 5 mmol), 3-bromopropylamine hydrobromide (1, 946 g, 5 mmol) and approximately 70 mg of triethylbenzylammonium chloride in 6 ml of dichloromethane and 3 ml of tetrachloride of carbon, 4 ml of a 20% aqueous sodium hydroxide solution was added, while maintaining the reaction temperature between 0 ° C and 5 ° C.

After stirring for one hour at the temperature from 0 ° C to 5 ° C, stirring was continued for one hour at room temperature.

As in Example 1, the reaction leads to the synthesis of the bromophosphoramide intermediate of formula

(C ₁₄ H ₂₉ 0) ₂ P (0) NHCH ₂ CH ₂ Br.

The above bromophosphoramide intermediate was mixed with two equivalents of N-methyl imidazole. This mixture was heated to the temperature of 40 ° C-50 ° C with stirring for three days. Then, after cooling, the reaction mixture was diluted in 15 ml of dichloromethane and the excess of imidazole was removed by treatment with a sulfonic acid resin (DOWEX 50W x 8, 1 meq

H ⁺ / g).

Then the solvent was removed in vacuo to obtain the phosphoramide of ditetradecyl iodide 2- (1-methyl-1 H-imidazol-3-ium-3- yl) ethylamidophosphate (KLN 27), with a purity> 99% determined according to the same methods as in Example 2.

The final reaction yield was 84%.

EXAMPLE 4 Synthesis of 5-imino-N iodide, N.N-trimethyl- 7 (tetradecyloxy, -8-oxa-4,6-diaza-7-phosphadocosan-1 -aminium 7-oxide (KLN 67):

1.74 g are added to 4.74 g (10 mmol) of ditetradecylphosphite

(10 mmol) 2-ethyl-2-thiopseudourea hydrobromide, 1.93 mL (20 mmol) of carbon tetrachloride and 3.48 mL (20 mmol) of diisopropylethylamine while maintaining the temperature between 0 and 5 ° C. This is kept stirring at this temperature for one hour, then allowed to return to room temperature for another hour. 1.1 g (15 mmol) of N, N-dimethylethylenediamine and 20 ml of toluene are then added and the mixture is brought to reflux for 24 hours. Toluene and excess amines are removed in vacuo. After cold precipitation in a little diethyl ether and washing in the same solvent, 4 g of the aminophosphoguanidine (Ci ₄ H ₂₉ 0) ₂ P (0) NH-C (= NH) -NH-CH ₂ are obtained. -CH ₂ - N (CH ₃ ) ₂ which is dissolved in 50 ml of dichloromethane to which 4.26 g (30 mmol) of iodomethane are added. After 24 hours of stirring at room temperature, the solvent and the excess iodomethane are evaporated under vacuum. The residue is precipitated in a little cold diethyl ether and then dried under vacuum. 4.93 g (65% yield) of KLN 67 are obtained, of formula (Cι ₄ H ₂ 9θ) 2P (0) NH-C (= NH) -NH-CH ₂ -CH2-N ⁺ (CH ₃ ) 3 1 ^" in the form of a white powder whose purity is controlled by the methods described in the preceding examples.

EXAMPLE 5 Transfection in vitro of a nucleic acid of interest complexed with a lipophilic compound according to the invention. A. MATERIALS AND METHODS. A.1 Cell lines and plasmids

For the in vitro experiments, the cell lines K562, Jurkat, Daudi and HeLa were used.

The cells were cultured in RPMI-1640 medium or in MEM culture medium supplemented with 10% fetal calf serum (SVF), 0.2 mM glutamine, 100 U / L of penicillin, 100 U / ml of streptomycin and 1% fungizone. All the cells were maintained in an atmosphere at 5% CO ₂ at the temperature of 37 ° C. The plasmid used is the plasmid pTG11033 encoding the luciferase protein under the control of the cytomegalovirus promoter (pCMV), developed by the company TRANSGENE (Strasbourg, France).

A.2 Preparation of cationic phosphonolipid / DNA complexes

Each of the cationic phosphonolipids was prepared individually or in combination with the neutral lipid DOPE (sold by the company SIGMA CHEMICALS, Saint-Quentin Fallavier, France). The phosphonolipids were formulated by mixing solutions of the different lipids in chloroform in glass tubes, then removing the chloroform by evaporation on a rotary evaporator, in order to obtain dry films of lipids.

Sterile, pyrogen-free distilled water was then added to the glass tubes, before sealing the tubes, which were then placed at a temperature of 4 ° C overnight.

Small unilamellar vesicles (suv) were prepared by sonicating the compounds for ten minutes in a sonicator device (sold by the company PROLABO, Paris, France). To prepare the cationic phosphonolipid / DNA complexes, the plasmid pTG11033 was first diluted in sterile, pyrogen-free distilled water, then added to the lipid solution. The cationic phosphonolipid / DNA complexes were stored for 30 minutes at room temperature before being administered to animals, or used for in vitro transfections.

Two types of preparation were carried out. For the first type of preparation, 12.5 μg of cationic lipid compound was mixed with 4 μg of DNA (the plasmid pTG 11033). For the second type of preparation, 25 μg of cationic lipid compound are mixed with 8 μg of DNA (the plasmid pTG 11033). In addition, the complexes were formulated either (i) with a cationic lipid compound of the invention used alone or (ii) with a mixture of a lipid compound of the invention and DOPE (dioleylphosphatidyl ethanolamine).

A. 3. In vitro transfection and test using a marker gene.

The in vitro transfection activity of cationic phosphonolipid / DNA complexes was tested using the cell lines K562, Jurkat, Daudi and Hela.

Non-adherent cells (cell lines K562, Jurkat and Daudi) were cultured in 75 cm ² culture plates. For the transfection tests, the cells were seeded in 24-well tissue culture plates, at a rate of 500,000 cells per well. For the adherent cell line (HeLa), the cells were seeded in 24-well tissue culture plates at the rate of 150,000 cells per well.

The cells were incubated in the tissue culture plates for 24 hours before the transfection step, then incubated overnight in a culture incubator in a humid atmosphere at 5% CO ₂ , at a temperature of 37 ° C. .

The transfection of the cells was carried out according to the technique described by FELGNER et al. (Proc. Natl. Acad. Sci. USA, 1987, vol. 84: 7413), with the following modifications: The appropriate amounts of cationic lipids and of the plasmid vector PTG11033 were complexed in 100 μl of an OptiMEM solution (Ref. 31985-047, Gibco BRL / Life Technologies / Cergy- Pontoise, France) supplemented with L-glutamine, Sodium bicarbonate (2.4 g / l), HEPES buffer, sodium pyruvate, hypoxanthine, thymidine, growth factor and red phenol (1.1 mg / l). The preparation is identical to that described in Section A.2 above. 100 μl of the cationic phospholipid / DNA complex solution was added to each culture well.

After 2.5 hours at 37 ° C, the cells were added with 2 ml of the appropriate culture medium. After an additional 48 hour incubation at 37 ° C., the cells were tested for the expression of the gene coding for luciferase, using a chemiluminescent kit (sold by the company PROMEGA, Charbonnières, France). The tests were carried out according to the manufacturer's recommendations. The results are expressed in TRLU units (for “Total Relative

Light Units ”), for an average of 8 identical culture wells.

A.4. Determination of cell toxicity

The relative cytotoxicity of the different cationic phosphonolipid / DNA complexes was determined as a representation of the number of living cells after the transfection experiment, measured using a chemiluminescent test, the CYTOLITE® test (marketed by the company PACKARD, France), according to the manufacturer's recommendations.

The cells were transfected as described in Section A.3 above.

On the day of the transfection, the cells were seeded in the wells of a 24-well plate, at a rate of: - 500,000 cells per well for the non-adherent cell lines (K 562, Jurkat, Daudi); and

- 150,000 cells per well for adherent cell lines (HeLa, CFPAC, cells in primary culture).

The cells were treated for transfection as described above and incubated for an additional 48 hours. After incubation, the cytotoxicity test was carried out according to the manufacturer's recommendations.

The amount of Relative Light Units (RLU) formed is proportional to the number of living cells. Untransfected cells were used as a control.

Final results were expressed using a toxicity index

The toxicity index represents the ratio between the number of living cells in the control cultures (without cationic lipid / DNA complex) and the number of living cells in the culture wells containing the transfected cells.

Toxicity analysis of the 3- iodide compound

[[bis (tetradecyloxy) phosphoryl (methyl) amino] -N, N, N- trimethylpropanaminium [compound KLN 5] shows that there is no difference between the control cell cultures and the transfected cell cultures and thus the transfection shows no toxicity. The toxicity index increases with the cytotoxicity properties of the cationic lipid tested.

B. RESULTS In this example, the toxicity properties of cationic phosphonolipid / DNA complexes of the invention were compared with the cytotoxicity of lipid vector / DNA complexes known in the prior art, respectively the compounds EG 308, DOTAP and PEI (polyethyleneimine), the formulas of which are shown below.

DOTAP - [CH ₂ -CH ₂ -NH] _n - PEI

For a strict comparative evaluation of the efficacy of the cationic phosphonolipids of the invention, compared with the compounds known in the prior art, each preparation of lipid vector / DNA complexes described in section A.2. of the “Materials and methods” section above was used on each of the cell lines described in section A.1 of the “Materials and Methods” section above, at the same time, and under conditions identical to those described in section A.3 of the “Materials and Methods” section above.

The results presented in FIGS. 1, 2, 3 and 4 show that the cationic lipid compounds of the invention, which contain a phosphoramide bond are more effective and less cytotoxic than the compounds of the prior art such as the compound EG 308 , and that commonly marketed transfection agents, such as DOTAP and PEI.

EXAMPLE 6 Study of the in vivo transfection capacity of the cationic phosphonolipid DNA complexes of the invention.

A. MATERIALS AND METHODS

A.1 In vivo transfection by intravenous injection

Cationic lipid / DNA complexes are produced at a charge ratio (+/-) of 4 and are administered to mice of the Swiss line six weeks old, by a single injection into the tail vein. Each animal received a volume of 200 μl of a solution of the lipid / DNA complex tested, corresponding to 50 μg of plasmid DNA coding for the luciferase protein. The animals were killed 24 hours after the injection and each organ of interest was frozen in liquid nitrogen, then stored at the temperature of -70 ° C.

The frozen tissues were ground with steel balls in a 2 ml Epperdorf type tube with a round bottom using a device of the Mill MM300 mixer type (sold by the company QIAGEN; reference: 0030120-094).

Protein extraction from each tissue was carried out by incubating the ground tissue in a PLB lysis buffer (Promega) then centrifugation at 10,000 g, 10 min at 4 ° C.

The luciferase activity was tested on the lysis supernatants using the chemiluminescence test "Luciferase Assay System" sold by the company PROMEGA.

The results were expressed in relative light units (RLU for "Relative Light Unit").

In parallel, the total protein concentration was measured, using the “Coomassie Plus Reagent Assay Kit” kit (sold by the company Pierce). The RLU values were normalized by expressing the results per mg of total protein.

A.2. Transfection in vivo by aerosol.

The cationic lipid / plasmid DNA complexes were prepared as described in Example 5, with charge ratios (+/-) varying from 0 to 6. The lipid / DNA complexes were administered to mice of the Swiss line. six weeks of age, by inhalation of 150 μl of the complex solution, using an aerosol device. The inhalation was carried out using the “Microsprayer” aerosol device (sold by the company PENN-CENTURY, Inc.), which allows atomization to be carried out directly in the trachea. Each animal received a dose of 50 μg of plasmid DNA coding for the luciferase protein, in the form of the lipid / DNA complex.

The animals were sacrificed 72 hours after the injection. The lungs were collected, then frozen in liquid nitrogen and stored at -70 ° C. Protein extraction from frozen lungs and measurement of RLU units per mg protein were performed as described in section A.1 above.

B. RESULTS A comparative study of different cationic lipids was carried out by varying the binding part (Group R ³ ) between the hydrophobic domain and the hydrophilic domain, in mice. Each lipid was evaluated in isolation, or in admixture with the DOPE compound or cholesterol, used as co-lipids, as described in the “Materials and Methods” section.

The results are shown in FIG. 6. The results of FIG. 6 show the advantage of the lipophilic transfection compounds according to the invention, compared with the lipophilic compounds of the state of the art or even with DNA alone. Compounds with a C18: 1 hydrophobic chain are particularly effective at transfecting the lungs of mice, whether used alone or in combination with cholesterol. The KLN20 compound is the most effective.

Claims

1. Cationic lipophilic compound of general formula (I) below

in which: a) R ¹ and R ' ¹ each represent, independently of one another, an alkyl chain, an alkenyl chain or a polyalkenyl chain of 10 to 24 carbon atoms, with the polyalkenyl chain having from 2 to 4 double bonds; b) R ² is a hydrogen atom or an alkyl chain having from 1 to 4 carbon atoms; and c) R ³ is a group of the following formula (IIa): - (CH ₂ ) _n - or (IIb) following: -C (= NH) -NH- (CH ₂ ) _n - in which:

- n is an integer equal to 0, 1, 2, 3 or 4; and d) A ⁺ is an organic cation; e) X ^" is an anion.

2. Compound according to claim 1, characterized in that X- represents an anion chosen from CF ₃ C0 ₂ ^' , CF3SO3 ^" , HS04 ^" , and a halogen.

3. Compound according to claim 2, characterized in that the halogen is chosen from CI ^" , Br ^" and I ^" .

4. Compound according to one of claims 1 to 3, characterized in that the group A is a five-membered heterocyclic aromatic ring comprising a heteroatom consisting of a quaternary nitrogen linked by a covalent bond to the group R ³ .

5. Compound according to claim 4, characterized in that the heterocyclic aromatic cycle comprises a second heteroatom chosen from S or N.

6. Compound according to claim 5, characterized in that when the second heteroatom is N, the heteroatom is substituted by an alkyl chain of 1 to 4 carbon atoms.

7. A compound according to claim 4, characterized in that the group A is a thiazolium or an imidazolium, with the second nitrogen heteroatom optionally substituted by an alkyl chain of 1 to 4 carbon atoms.

8. Compound according to one of claims 5 to 7, characterized in that it is chosen from the following compounds: ditetradecyl iodide 2- (1 -methyl-1 H-imidazol-3-ium-3-yl) ethylamidophosphate dioleyl iodide 2- (1-methyl-1 H-imidazol-3-ium-3-yl) ethylamidophosphate - ditetradecyl iodide 2- (1, 3-thiazol-3-ium-3-yl) ethylamidophosphate ditetracetyl iodide 2 - (1-methyl-1 H-imidazol-3-ium-3-yl) propylamidophosphate.

9. Compound according to one of claims 1 to 3, characterized in that group A is a six-membered heterocyclic aromatic ring comprising a quaternary nitrogen heteroatom.

10. Compound according to claim 8, characterized in that group A is a pyridinium ring.

11. Compound according to claim 10, characterized in that group A is represented by the following formula (III):

in which R ¹³ represents an alkyl chain of 1 to 4 carbon atoms.

12. Compound according to claim 1 1, characterized in that it is the following compound: ditetradecyl iodide 2- (1-methyl-1 H-ρyridin-1-ium-4-yl) ethylamidophosphate.

13. Compound according to one of claims 1 to 3, characterized in that group A is a cation of formula (IV) below:

in which: f) Z represents N, P or As; g) R ⁴ and R ⁵ each represent, independently of one another, an alkyl chain having from 1 to 4 carbon atoms; h) R ⁶ represents an alkyl chain having from 1 to 4 carbon atoms.

14. Compound according to claim 13, characterized in that the group R ² represents hydrogen, a methyl group or an ethyl group.

15. Compound according to one of claims 13 or 14, characterized in that the group R ³ of formula (IIa) [- (CH ₂ ) _n -] or (llb) [-C (= NH) -NH- ( CH ₂ ) _n -] is such that n is an integer equal to 2, 3 or 4.

16. Compound according to one of claims 13 to 15, characterized in that it is chosen from the following compounds: - iodide of 3 - [[bis (tetradecyloxy) phosphoryl] (methyl) amino] -N.N.N- trimethylpropanaminium; 3 - [[bis (oleyloxy) phosphoryl] (methyl) amino] -N.N.N-trimethylpropanaminium iodide;

- iodide of 3 - [[bis (tetradecyloxy) phosphoryl] (ethyl) amino] -N.N.N- trimethylethanaminium; 3 - [[bis (oleyloxy) phosphoryl] (ethyl) amino] -N.N.N-trimethylethanaminium iodide; ditetradecyl 2- (trimethylphosphonio) ethyl propylamidophosphate iodide; - dioleyl iodide 3- (trimethylphosphonio) propylamidophosphate;

- ditetradecyl iodide 2- (trimethylarsonio) ethylamidophosphate;

- dioleyl 2- (trimethylarsonio) ethylamidophosphate iodide;

- ditetradecyl iodide 3- (trimethylarsonio) propylamidophosphate;

- dioleyl iodide 3- (trimethylarsonio) propylamidophosphate;

17. Compound according to one of claims 1 to 3, characterized in that group A is a cation of formula (IV) below:

wherein: i) Z represents N, P or As; j) R ⁴ and R ⁵ each represent, independently of one another, an alkyl chain having from 1 to 4 carbon atoms; k) R ⁶ represents the following group of formula (V):

in which :

in which: n) R ¹⁰ represents - (CH ₂ ) n- where n is an integer equal to 0, 1, 2, 3 or

4; o) R ¹¹ represents H or an alkyl group of 1 to 4 carbon atoms; and p) R ¹² and R ′ ¹² each represent, independently of one another, an alkyl chain, an alkenyl chain or a polyalkenyl chain of 10 to 24 carbon atoms, with the polyalkenyl chain having from 2 to 4 double connections;

18. Compound according to claim 17, characterized in that the groups R ¹² and R ¹² are identical to the groups R ¹ and R ¹ , respectively.

19. Compound according to one of claims 17 or 18, characterized in that the groups R ³ and R ¹⁰ are identical.

20. Compound according to one of claims 17 to 19, characterized in that the groups the groups R ⁴ and R ⁵ are identical to the groups R ⁷ and R ⁸ , respectively.

21. Compound according to one of claims 17 to 20, characterized in that it is the following compound: diiodide of N ¹ , N ⁴ -bis (3- ^< | [bis (tetradecyloxy) phosphoryl] amino} propyl ) - N ¹ , N ¹ , N, N ⁴ -tetramethyl-1, 4 butane diaminium.

22. Compound according to one of claims 1 to 21, characterized in that the groups R ¹ and R ' ¹ are chosen from:

- the tetradecyl group; - the oleyl group; and

- C _{18: 2} and Cιβ: ₃ polyalkenyl groups, in which the first number represents the number of carbon atoms in the alkenyl chain and the second number represents the number of double bonds in the alkenyl chain.

23. A compound according to one of claims 17 to 21, characterized in that the R ¹² and R ^'12 are chosen from:

- the tetradecyl group;

- the oleyl group; and - the polyalkenyl groups C _{18: 2} and C18.3, in which the first number represents the number of carbon atoms in the chain alkenyl and the second number represents the number of double bonds in the alkenyl chain.

24. Lipid vesicle comprising a compound according to one of claims 1 to 23.

25. Lipid vesicle essentially consisting of a compound according to one of claims 1 to 23.

26. Lipid vesicle comprising at least one compound according to one of claims 1 to 23 and at least one other lipophilic compound.

27. Lipid vesicle according to one of claims 24 to 26, consisting of a unilamellar vesicle.

28. Lipid vesicle according to one of claims 24 to 26, consisting of a multilamellar vesicle.

29. Use of a compound according to one of claims 1 to 23 or of a lipid vesicle according to one of claims 24 to 28, for introducing in nucleic acid into a host cell.

30. A method of introducing a nucleic acid into a host cell in vitro, characterized in that it comprises the following steps: * a) bringing said nucleic acid into contact with a compound according to one of claims 1 to 23 or with a lipid vesicle according to one of claims 24 to 28 in order to obtain a complex between said nucleic acid, on the one hand, and said compound or said lipid vesicle, on the other hand; b) incubating the host cell with the complex formed in step a).

31. The method according to claim 30, characterized in that said host cell is a non-human mammalian cell or a human cell.

32. Complex formed between a nucleic acid and a compound according to one of claims 1 to 23 or a lipid vesicle according to one of claims 24 to 28.

33. Composition comprising a complex according to claim 32.

34. Pharmaceutical composition comprising a complex according to claim 32.