FR2797188A1 - CATIONIC LIPID COMPOUNDS AND THEIR USE FOR THE TRANSFER OF NEGATIVELY CHARGED SUBSTANCES OF THERAPEUTIC INTEREST - Google Patents

Abstract

The invention concerns the use of a monocationic lipid compound of general formula I(A): R1O-CO-NH-(CH2)n-N<+>(C2H5)3, I<-> wherein: R1 represents the cholesteryl radical; n represents an integer equal to 1, 2 or 3 and in particular 3- beta [N-(N',N',N'-triethylaminopropane iodide)-carbamoyl] cholesterol (TEAPC-Chol) and/or a monocationic lipid compound of general formula I(B): R2-CO-NH-(CH2)n-N<+>(R)3,X<-> wherein: R2 represents in particular the abietic acid radical, or the radical of a derivative of cholanic (cholic, deoxycholic, dehydrocholic, lithocholic) acid; R represents in particular a methyl or ethyl radical; n represents an integer equal to 2 or 3; X represents in particular a chlorine or iodine atom; and/or a dicationic lipid compound of general formula II(A): R1O-CO-N[CH2- CH2-N<+>(R)3,X<->]2; and/or a dicationic lipid compound of general formula II(B): R1O-CO-CH2-CH2-CO-N[CH2-CH2-N<+>(R)3, X<->]2; and/or a dicationic lipid compound of formula II(C): R2-CO-N[CH2-CH2-N<+>(R)3,X<->]2 wherein R1 represents the cholesteryl radical; R2 represents in particular the abietic acid radical or the radical of a derivative of a cholanic (cholic, deoxycholic, dehydrocholic, lithocholic) acid; R represents in particular a hydrogen atom, a methyl or ethyl radical; X represents in particular a chlorine or iodine atom. Said compounds are useful for transfecting living organisms <i>in vivo</i>, organs <i>in vivo</i>, tumours <i>in vivo</i>, or cells in <i>vitro</i> or <i>ex vivo</i>.

Description

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CATIONIC LIPID COMPOUNDS AND THEIR USE FOR THE TRANSFER OF NEGATIVELY CHARGED SUBSTANCES OF THERAPEUTIC INTEREST
The subject of the present invention is cationic lipid compounds, and their use for the transfer of negatively charged substances of therapeutic interest, and in particular nucleic acids.

Apart from a few very special cases, nucleic acids alone do not enter cells, organs or organisms, and thus require the use of a vector. To date, various techniques have been described for the transfer of nucleic acids and in particular DNA, the most important and promising of which involve viral vectors or lipid vectors.

Viral vectors are effective but present certain risks, including in particular pathogenicity, immunogenicity, transmission, recombination, replication, transformation etc ...; there is therefore a security problem in the use of the latter.

Lipid vectors, in particular cationic lipids, are experiencing increasing development. In fact, cationic lipids can, through their positive charges, easily form complexes with negatively charged nucleic acids and help them cross the lipid cell barrier, which is also negatively charged; the nucleic acids can then pass into the nucleus. Efforts are being made to develop cationic lipids having a high level of transfection. This is only achieved if the lipid vector forms, in high yield, complexes with the nucleic acid to be transported, helps to protect said nucleic acid from extra and intracellular enzymatic degradation, and exhibits acceptable cytotoxicity.

( 1-(2,3-dioléyloxy)propyl)-N,N,N-triméthylammonium), - Lipofectamine , dont le lipide de base est le DOSPA (trifluoroacétate de 2,3-dioléyloxy-N-(2(spermine carboxamido)éthyl)-N,N-diméthyl-1- propanaminium), The cationic lipid formulations currently on the market have an average price of 1500 F / mg, and are marketed: # by Gibco (USA) under the names of: - Lipofectin, whose base lipid is DOTMA (N chloride -

(1- (2,3-dioleyloxy) propyl) -N, N, N-trimethylammonium), - Lipofectamine, the base lipid of which is DOSPA (2,3-dioleyloxy-N- (2 (spermine carboxamido) trifluoroacetate) ethyl) -N, N-dimethyl-1-propanaminium),

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- Lmvllr, , UUl11 lu yuuv uc Ui:1::i1:; I:;M lu <\ mvmct orurnure ue 1,L.- dimyristyloxypropyl-3-N,N-diméthyl hydroxy ammonium), # par Boehringer (Allemagne) sous le nom de DOTAP , dont le lipide de base est le DOTAP (N-(1-(2,3-dioléyloxy)propyl-N,N,N-triméthylammonium sulfate de méthyle)), # par Proméga (USA) sous le nom de Transfectam , dont le lipide de base est le DOGS (dioctadécylamidoglycyl spermine) # par Eurogentec (Belgique) sous le nom de DAC 30 , dont le lipide de base est le DAC-Chol (3-p-(N-(N,N'-diméthylaminoéthane)-carbamoyle) cholestérol).

- Lmvllr,, UUl11 lu yuuv uc Ui: 1 :: i1 :; I:; M lu <\ mvmct orurnure ue 1, L.- dimyristyloxypropyl-3-N, N-dimethyl hydroxy ammonium), # by Boehringer (Germany) under the name DOTAP, whose base lipid is DOTAP (N - (1- (2,3-dioleyloxy) propyl-N, N, N-trimethylammonium methyl sulfate)), # by Proméga (USA) under the name Transfectam, whose base lipid is DOGS (dioctadecylamidoglycyl spermine) # by Eurogentec (Belgium) under the name DAC 30, the base lipid of which is DAC-Chol (3-p- (N- (N, N'-dimethylaminoethane) -carbamoyl) cholesterol).

(3[N-(N,N'-diméthylaminoéthane)-carbamoyle] cholestérol [5]. Regarding non-marketed products, two main families of cationic lipids have been reported to date. All of these compounds have a hydrophobic tail linked to an amino polar head bearing one or more positively charged nitrogen atoms. These families are distinguished by the hydrophobic part which can be a double alkyl chain or a derivative of cholesterol. These compounds form complexes with nucleic acids (plasmids or oligonucleotides). Encouraging results on the level of transfection of cells in culture have been published [1-4, 13,14]. Among the monoaminated cholesterol derivatives, the best known derivative is DC-Chol, corresponding to 3-P [N- (N ', N'-dimethylaminoethane) -carbamoyl] cholesterol [3,4] and, the commercial derivative is the DAC-Chol, corresponding to 3-

(3 [N- (N, N'-dimethylaminoethane) -carbamoyl] cholesterol [5].

Furthermore, it would appear that compounds with a polyamine head, such as dioctadecylamidoglycylspermine (DOGS), could have a greater capacity to form complexes with nucleic acids [6,7] than compounds with a single amine function in their polar head. These results have led to the synthesis of a whole series of compounds derived from cholesterol having a polar head of the polyamine type (spermine, spermidine, guanidinium, etc.) [8,9].

However, in all these experiments, the medium used must be practically serum-free: for example, the maximum percentage of serum used does not exceed 20% for a formulation of DC-Chol in tests on A431 cells, and a formulation of DAC-Chol in assays on HepG2 and COS-1 cells.

Thus, the use of these vectors to deliver nucleic acids in intravenous in vivo assays is still problematic.

Recently, another type of cationic amphiphile has been synthesized by the attachment of polyamines to the polar sites of cholic acid [10]. For COS-7 cells,

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levels of transection of b a IL) higher than that of lipotectin were obtained with molecules carrying a spermine, pentamine or hexamine group.

These molecules differ from those of the present invention because they do not contain a spacer arm between the polar sites and the hydrophobic part.

One of the aims of the invention is to provide cationic lipids capable of transporting in particular negatively charged substances, in particular nucleic acids, including in a medium containing a high concentration of serum, for the purposes of gene therapy or vaccination. , biology, physiology, genetics and biotechnology.

One of the other aims of the invention is to provide cationic lipids having an improved transfecting power compared to the cationic lipids known to date.

Another aspect of the present invention is to provide simple methods for the synthesis of cationic lipids, so as to synthesize the cationic lipids of the invention on an industrial scale, while having high yields.

Rz - CO - NH - (CHz) - N+ (R)3 , X (I) dans laquelle R2 représente le radical de l'acide abiétique, le radical d'un dérivé de l'acide cholanique, notamment celui choisi dans le groupe constitué par le radical de

l'acide 3a,7a,12a-trihydroxy-53-cholanique, l'acide 3a,12a-dihydroxy-53-cholanique, l'acide 3a-hydroxy-5p-cholanique ou l'acide 3,7,12-trioxo-5p-cholanique, ou le groupe R1-O dans lequel Ri représente le radical cholestéryle, R représente un atome d'hydrogène, un radical alkyle et notamment un méthyle (-CH3) ou un éthyle (-C2H5). n représente un nombre entier égal à 2 ou 3, X représente un atome d'halogène tel que le chlore ou l'iode, et/ou d'un composé lipidique dicationique de formule générale (II)

R'z - CO - N[CHz - CHZ - N+ (R)3 , X ]2 (II) dans laquelle R'2 représente le radical de l'acide abiétique, le radical d'un dérivé de l'acide cholanique, notamment celui choisi dans le groupe constitué par le radical de

l'acide 3a,7a,12a-trihydroxy-53-cholanique, l'acide 3a,12a-dihydroxy-5p-cholanique, l'acide 3a-hydroxy-5p-cholanique ou l'acide 3,7,12-trioxo-5P-choIanique, ou le groupe R1-O ou R1-O-CO-(CH2)2, dans lequel RI représente le radical cholestéryle, R représente un atome d'hydrogène, un radical alkyle, et notamment un méthyle (-CH3) ou un éthyle (-C2Hs), X représente un atome d'halogène tel que l'iode ou le chlore, The present invention relates to the use of a monocationic lipid compound of general formula (I)

Rz - CO - NH - (CHz) - N + (R) 3, X (I) in which R2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group constituted by the radical of

3a, 7a, 12a-trihydroxy-53-cholanic acid, 3a, 12a-dihydroxy-53-cholanic acid, 3a-hydroxy-5p-cholanic acid or 3,7,12-trioxo- 5p-cholanic, or the group R1-O in which R1 represents the cholesteryl radical, R represents a hydrogen atom, an alkyl radical and in particular a methyl (-CH3) or an ethyl (-C2H5). n represents an integer equal to 2 or 3, X represents a halogen atom such as chlorine or iodine, and / or a dicationic lipid compound of general formula (II)

R'z - CO - N [CHz - CHZ - N + (R) 3, X] 2 (II) in which R'2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group consisting of the radical of

3a, 7a, 12a-trihydroxy-53-cholanic acid, 3a, 12a-dihydroxy-5p-cholanic acid, 3a-hydroxy-5p-cholanic acid or 3,7,12-trioxo- 5P-choIanique, or the group R1-O or R1-O-CO- (CH2) 2, in which RI represents the cholesteryl radical, R represents a hydrogen atom, an alkyl radical, and in particular a methyl (-CH3) or an ethyl (-C2Hs), X represents a halogen atom such as iodine or chlorine,

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for transrection to living organisms in vivo, to organs in vivo, of tumors in vivo, or of cells in vitro or ex vivo.

Likewise, a subject of the invention is the cationic lipid compounds of general formula (I) and (II) defined above, as well as the processes for preparing said compounds.

l'acide 3a,7cc,12a-trihydroxy-5p-cholanique, l'acide 3a,12a-dihydroxy-5p-cholanique, l'acide 3a-hydroxy-5p-cholanique ou l'acide 3,7,12-trioxo-5p-cholanique, ou le groupe R1-O dans lequel R1 représente le radical cholestéryle, Z1 représente le chlore, ou le groupement O-CO-O-R3 dans lequel R3 représente un radical alkyle et notamment un méthyle ou un éthyle, à l'action d'un composé de formule générale (IV) H2N-(CH2)n-N(R)2 (IV) dans laquelle R représente un atome d'hydrogène, un radical alkyle et notamment un méthyle ou un éthyle, n représente un nombre entier égal à 2 ou 3, pour former le composé de formule générale (V)
R2 - CO - HN - (CH2)n -N (R)2 (V) dans laquelle R2, R et n ont les significations mentionnées ci-dessus, que l'on soumet à l'action du composé de formule générale (VI) R-X (VI) dans laquelle R a la signification mentionnée ci-dessus, et X représente un atome d'halogène tel que l'iode ou le chlore, pour obtenir le composé lipidique monocationique de formule générale (I) :

R2 - CO - NH - (CHz)n - N+ (R)3 X (I) dans laquelle R2 , R, X et n ont les significations mentionnées ci-dessus. By way of example, a process for preparing a monocationic lipid compound of general formula (I) according to the invention is characterized in that a compound of general formula (IIIa) is subjected
R2-CO-Z1 (IIIa) in which R2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group consisting of the radical of

3a, 7cc, 12a-trihydroxy-5p-cholanic acid, 3a, 12a-dihydroxy-5p-cholanic acid, 3a-hydroxy-5p-cholanic acid or 3,7,12-trioxo- 5p-cholanic, or the R1-O group in which R1 represents the cholesteryl radical, Z1 represents chlorine, or the O-CO-O-R3 group in which R3 represents an alkyl radical and in particular a methyl or an ethyl, to l 'action of a compound of general formula (IV) H2N- (CH2) nN (R) 2 (IV) in which R represents a hydrogen atom, an alkyl radical and in particular a methyl or an ethyl, n represents a number integer equal to 2 or 3, to form the compound of general formula (V)
R2 - CO - HN - (CH2) n -N (R) 2 (V) in which R2, R and n have the meanings mentioned above, which is subjected to the action of the compound of general formula (VI ) RX (VI) in which R has the meaning mentioned above, and X represents a halogen atom such as iodine or chlorine, to obtain the monocationic lipid compound of general formula (I):

R2 - CO - NH - (CHz) n - N + (R) 3 X (I) in which R2, R, X and n have the meanings mentioned above.

R, O - CO - NH - (CHz)" - N'+ (R)3 , X - I(A) According to the process for preparing the compounds of general formula (I) described above, it is possible more particularly to obtain the compounds of general formula I (A) and I (B):

R, O - CO - NH - (CHz) "- N '+ (R) 3, X - I (A)

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R2- CO - NH - (CH2) - N+ (R)3 , X I (B) dans laquelle R2 représente le radical de l'acide abiétique, le radical d'un dérivé de l'acide cholanique, notamment celui choisi dans le groupe constitué par le radical de

l'acide 3a,7a,12a-trihydroxy-53-cholanique, l'acide 3a,12a-dihydroxy-5(3-cholanique, l'acide 3a-hydroxy-5p-cholanique ou l'acide 3,7,12-trioxo-5p-cholanique, R représente un radical méthyle ou éthyle, n représente un nombre entier égal à 2 ou 3, X représente un atome d'halogène tel que le chlore ou l'iode,
Le procédé de préparation dudit composé lipidique monocationique de formule générale (I) est encore caractérisé en ce que le composé de formule générale (IIIa)
R2 - CO - Z1 (nia) dans laquelle R2 représente le radical de l'acide abiétique, le radical d'un dérivé de l'acide cholanique, notamment celui choisi dans le groupe constitué par le radical de

l'acide 3a,7a,12a-trihydroxy-5|3-cholanique, l'acide 3a,12a-dihydroxy-5p-cholanique, l'acide 3a-hydroxy-5p-cholanique ou l'acide 3,7,12-trioxo-5p-cholanique, ou le groupe R1-O dans lequel Ri représente le radical cholestéryle, Z1 représente le groupement 0CO-O-R3 dans lequel R3 représente un radical alkyle et notamment un méthyle ou un éthyle, est préparé en soumettant le composé de formule générale (VIII)
R2 - CO - OH (VIII) dans laquelle R2 a la signification mentionnée ci-dessus, à l'action d'un composé de formule générale (IX) N(R4)3 (IX) dans laquelle R4 représente un radical alkyle et notamment un éthyle, pour obtenir le composé de formule générale (X)

R2 - CO - O - NIi(Rn)3 (X) dans laquelle R2, R4 ont les significations mentionnées ci-dessus, que l'on soumet à l'action du composé de formule générale (XI)
R3-O-CO-X (XI) dans laquelle R3 a la signification mentionnée ci-dessus, X représente un atome de chlore, pour obtenir le composé de formule générale (IIIc)

Rz-CO-O-CO-O-R3 (IIIc) in which R1 represents the cholesteryl radical, K represents a methyl or ethyl radical, n represents an integer equal to 2 or 3, X represents a halogen atom such as chlorine or iodine,

R2- CO - NH - (CH2) - N + (R) 3, XI (B) in which R2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group constituted by the radical of

3a, 7a, 12a-trihydroxy-53-cholanic acid, 3a, 12a-dihydroxy-5 (3-cholanic acid, 3a-hydroxy-5p-cholanic acid or 3,7,12- trioxo-5p-cholanic, R represents a methyl or ethyl radical, n represents an integer equal to 2 or 3, X represents a halogen atom such as chlorine or iodine,
The process for preparing said monocationic lipid compound of general formula (I) is further characterized in that the compound of general formula (IIIa)
R2 - CO - Z1 (nia) in which R2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group consisting of the radical of

3a, 7a, 12a-trihydroxy-5 | 3-cholanic acid, 3a, 12a-dihydroxy-5p-cholanic acid, 3a-hydroxy-5p-cholanic acid or 3,7,12- trioxo-5p-cholanic, or the R1-O group in which Ri represents the cholesteryl radical, Z1 represents the OCO-O-R3 group in which R3 represents an alkyl radical and in particular a methyl or an ethyl, is prepared by subjecting the compound of general formula (VIII)
R2 - CO - OH (VIII) in which R2 has the meaning mentioned above, to the action of a compound of general formula (IX) N (R4) 3 (IX) in which R4 represents an alkyl radical and in particular an ethyl, to obtain the compound of general formula (X)

R2 - CO - O - NIi (Rn) 3 (X) in which R2, R4 have the meanings mentioned above, which is subjected to the action of the compound of general formula (XI)
R3-O-CO-X (XI) in which R3 has the meaning mentioned above, X represents a chlorine atom, to obtain the compound of general formula (IIIc)

Rz-CO-O-CO-O-R3 (IIIc)

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corresponding to the compound of general formula (IIIa) in which Z1 represents the O-CO-O-R3 group.

l'acide 3a,7a,12a-trihydroxy-5p-cholanique, l'acide 3a,12a-dihydroxy-5p-cholanique, l'acide 3a-hydroxy-5(3-cholanique ou l'acide 3,7,12-trioxo-5p-cholanique, ou le groupe R1-O ou R1-O-CO-(CH2)2 dans lequel R1 représente le radical cholestéryle, Z2 représente un halogène tel que le chlore, à l'action d'un composé de formule générale (VII)
HN[CH2 - CH2 - N (R)3+, X-]2 (VII) dans laquelle R représente un atome d'hydrogène, un radical alkyle et notamment un radical méthyle ou éthyle, X représente un atome d'halogène tel l'iode ou le chlore, pour obtenir le composé lipidique dicationique de formule générale (II)

R'z - CO - N[CHz - CHz - N+ (R)3 , X ]z (il) dans laquelle R'2, R et X ont les significations mentionnées ci-dessus. The process for preparing a dicationic lipid compound of general formula (II) according to the invention is characterized in that a compound of general formula (IIIb) is subjected
R'2 - CO - Z2 (IIIb) in which R'2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group consisting of the radical of

3a, 7a, 12a-trihydroxy-5p-cholanic acid, 3a, 12a-dihydroxy-5p-cholanic acid, 3a-hydroxy-5 (3-cholanic acid or 3,7,12- trioxo-5p-cholanic, or the group R1-O or R1-O-CO- (CH2) 2 in which R1 represents the cholesteryl radical, Z2 represents a halogen such as chlorine, to the action of a compound of formula general (VII)
HN [CH2 - CH2 - N (R) 3+, X-] 2 (VII) in which R represents a hydrogen atom, an alkyl radical and in particular a methyl or ethyl radical, X represents a halogen atom such as 'iodine or chlorine, to obtain the dicationic lipid compound of general formula (II)

R'z - CO - N [CHz - CHz - N + (R) 3, X] z (il) in which R'2, R and X have the meanings mentioned above.

Rz - CO - N[CHz - CHz - N+ (R)3 , X ']z II(C) dans laquelle R2 représente le radical de l'acide abiétique, le radical d'un dérivé de l'acide cholanique, notamment celui choisi dans le groupe constitué par le radical de

l'acide 3a,7a,12a-trihydroxy-53-cholanique, l'acide 3a,12a-dihydroxy-5p-cholanique, l'acide 3[alpha]-hydroxy-5ss-cholanique ou l'acide 3,7,12-trioxo-5p-cholanique, R représente un atome d'hydrogène, X représente un atome d'halogène tel que le chlore ou l'iode. According to the process for preparing the compounds of general formula (II) described above, it is possible more particularly to obtain the dicationic lipid compounds of general formulas II (A), II (B) and II (C): R, 0 - CO - N [CH2 - CH2 - N + (R) 3, X -] 2 II (A)
Ri O- CO - CH2 - CH2 - CO - N [CH2 - CH2 - N + (R) 3, X -] 2 II (B) in which R1 represents the cholesteryl radical, R represents a hydrogen atom, X represents a halogen atom such as chlorine or iodine,

Rz - CO - N [CHz - CHz - N + (R) 3, X '] z II (C) in which R2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group consisting of the radical of

3a, 7a, 12a-trihydroxy-53-cholanic acid, 3a, 12a-dihydroxy-5p-cholanic acid, 3 [alpha] -hydroxy-5ss-cholanic acid or 3,7,12 acid -trioxo-5p-cholanic, R represents a hydrogen atom, X represents a halogen atom such as chlorine or iodine.

A more particular subject of the present invention is the use of a monocationic lipid compound of general formula I (A)

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R2 - CO - NH - (CH2)n - N (R)3 X I (B) dans laquelle R2 représente le radical de l'acide abiétique, le radical d'un dérivé de l'acide cholanique, notamment celui choisi dans le groupe constitué par le radical de

l'acide 3a,7a,12a-trihydroxy-53-cholanique, l'acide 3a,12a-dihydroxy-53-cholanique, l'acide 3(x-hydroxy-50-cholanique ou l'acide 3,7,12-trioxo-5p-cholanique, R représente un radical méthyle ou éthyle, n représente un nombre entier égal à 2 ou 3, X représente un atome d'halogène tel que le chlore ou l'iode, et/ou d'un composé lipidique dicationique de formule générale II(A)
R1 O- CO - N[CH2 - CH2 - N+ (R)3 , X ]2 II(A) dans laquelle R1 représente le radical cholestéryle, R représente un atome d'hydrogène, un radical méthyle ou éthyle, X représente un atome d'halogène tel que le chlore ou l'iode, et/ou d'un composé lipidique dicationique de formule générale II(B)
R1 0 - CO - CH2 - CH2 - CO - N[CH2 - CH2 - N+ (R)3 , X -]2 II (B) dans laquelle R1 représente le radical cholestéryle, R représente un atome d'hydrogène, un radical méthyle ou éthyle, X représente un atome d'halogène tel que le chlore ou l'iode, et/ou d'un composé lipidique dicationique de formule générale II(C)

R2 - CO - N[CHZ - CH2 - N+ (R)3 , X ]2 n(C) dans laquelle R2 représente le radical de l'acide abiétique, le radical d'un dérivé de l'acide cholanique, notamment celui choisi dans le groupe constitué par le radical de

l'acide 3a,7a,12a-trihydroxy-5P-choIanique, l'acide 3a,12a-dihydroxy-5P-cholanique, l'acide 3[alpha]-hydroxy-5ss-cholanique ou l'acide 3,7,12-trioxo-5p-cholanique, R représente un atome d'hydrogène, un radical méthyle ou éthyle, X représente un atome d'halogène tel que le chlore ou l'iode, pour la transfection d'organismes vivants in vivo, d'organes in vivo, de tumeurs in vivo, ou de cellules in vitro ou ex vivo. R1 O-CO-NH- (CH2) n - N '(R) 3, X- I (A) in which R1 represents the cholesteryl radical, R represents a methyl or ethyl radical, n represents an integer equal to 2 or 3, X represents a halogen atom such as chlorine or iodine, and / or a monocationic lipid compound of general formula I (B)

R2 - CO - NH - (CH2) n - N (R) 3 XI (B) in which R2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group constituted by the radical of

3a, 7a, 12a-trihydroxy-53-cholanic acid, 3a, 12a-dihydroxy-53-cholanic acid, 3 (x-hydroxy-50-cholanic acid or 3,7,12- trioxo-5p-cholanic, R represents a methyl or ethyl radical, n represents an integer equal to 2 or 3, X represents a halogen atom such as chlorine or iodine, and / or a dicationic lipid compound of general formula II (A)
R1 O- CO - N [CH2 - CH2 - N + (R) 3, X] 2 II (A) in which R1 represents the cholesteryl radical, R represents a hydrogen atom, a methyl or ethyl radical, X represents an atom halogen such as chlorine or iodine, and / or a dicationic lipid compound of general formula II (B)
R1 0 - CO - CH2 - CH2 - CO - N [CH2 - CH2 - N + (R) 3, X -] 2 II (B) in which R1 represents the cholesteryl radical, R represents a hydrogen atom or a methyl radical or ethyl, X represents a halogen atom such as chlorine or iodine, and / or a dicationic lipid compound of general formula II (C)

R2 - CO - N [CHZ - CH2 - N + (R) 3, X] 2 n (C) in which R2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular the one chosen in the group consisting of the radical of

3a, 7a, 12a-trihydroxy-5P-cholanic acid, 3a, 12a-dihydroxy-5P-cholanic acid, 3 [alpha] -hydroxy-5ss-cholanic acid or 3,7,12 acid -trioxo-5p-cholanic, R represents a hydrogen atom, a methyl or ethyl radical, X represents a halogen atom such as chlorine or iodine, for the transfection of living organisms in vivo, of organs in vivo, tumors in vivo, or cells in vitro or ex vivo.

The compounds of general formula I (A), II (A), II (B) and II (C) exhibit an improved level of transfection compared to the cationic lipid compounds known to date. To determine the level of transfection, a reporter gene is used. The

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The protein produced from this gene is detected by chemilummescence. The intensity is expressed in relative units (RLU) and related to the total quantity of proteins expressed by the transfected cells.

R1 O - CO - NH - (CHz)" - N+ (R)3 , X I(A) dans laquelle R1 représente le radical cholestéryle, R représente un radical méthyle ou éthyle, n représente un nombre entier égal à 2 ou 3, X représente un atome d'halogène tel que le chlore ou l'iode, - le composé lipidique monocationique de formule générale I(B)

R2 - CO - NH - (CH2)n - N+ (R)3, X 1 (IB) dans laquelle R2 représente le radical de l'acide abiétique, le radical d'un dérivé de l'acide cholanique, notamment celui choisi dans le groupe constitué par le radical de

l'acide 3a,7a,12a-trihydroxy-53-cholanique, l'acide 3a,12a-dihydroxy-5p-cholanique, l'acide 3a-hydroxy-5p-cholanique ou l'acide 3,7,12-trioxo-5{3-cholanique, R représente un radical méthyle ou éthyle, n représente un nombre entier égal à 2 ou 3, X représente un atome d'halogène tel que le chlore ou l'iode. A more particular subject of the invention is also the monocationic lipid compound, characterized in that it is chosen from the group consisting of: - the monocationic lipid compound of general formula I (A)

R1 O - CO - NH - (CHz) "- N + (R) 3, XI (A) in which R1 represents the cholesteryl radical, R represents a methyl or ethyl radical, n represents an integer equal to 2 or 3, X represents a halogen atom such as chlorine or iodine, - the monocationic lipid compound of general formula I (B)

R2 - CO - NH - (CH2) n - N + (R) 3, X 1 (IB) in which R2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group consisting of the radical of

3a, 7a, 12a-trihydroxy-53-cholanic acid, 3a, 12a-dihydroxy-5p-cholanic acid, 3a-hydroxy-5p-cholanic acid or 3,7,12-trioxo- 5 {3-cholanic, R represents a methyl or ethyl radical, n represents an integer equal to 2 or 3, X represents a halogen atom such as chlorine or iodine.

- le 3i[N-(N',N',N'-triméthylaminoéthane iodure)-carbamoyle] cholestérol (TMAEC-Chol) représenté par la formule générale I (A) danslaquelle R1 représente le radical cholestéryle, R représente le radical méthyle, n est égal à 2 et X représente l'iode, - le composé N-(N', N', N'-triméthylaminoéthane iodure) abiétamide représenté par la formule générale I (B) danslaquelle R2 représente le radical de l'acide abiétique, R représente le radical méthyle, n est égal à 2 et X représente l'iode, - le composé N-(N', N', N'-triméthylaminopropane iodure) abiétamide représenté par la formule générale I (B) laquelle R2 représente le radical de l'acide abiétique, R représente le radical méthyle, n est égal à 3 et X représente l'iode,

- le composé 3a,7a,12a-trihydroxy-5p-[N-(N',N',N'-triméthylaminoéthane iodure) ] cholanamide, dénommé Triméthylaminoéthane Cholamide (TAE- Cholamide) , représenté par la formule générale I (B) laquelle R2 représente le As such, the invention is more particularly aimed at the monocationic lipid compound as defined above, characterized in that it is chosen from the group consisting of:

- 3i [N- (N ', N', N'-trimethylaminoethane iodide) -carbamoyl] cholesterol (TMAEC-Chol) represented by general formula I (A) in which R1 represents the cholesteryl radical, R represents the methyl radical, n is equal to 2 and X represents iodine, - the compound N- (N ', N', N'-trimethylaminoethane iodide) abietamide represented by the general formula I (B) in which R2 represents the radical of abietic acid , R represents the methyl radical, n is equal to 2 and X represents iodine, - the compound N- (N ', N', N'-trimethylaminopropane iodide) abietamide represented by the general formula I (B) in which R2 represents the radical of abietic acid, R represents the methyl radical, n is equal to 3 and X represents iodine,

- the compound 3a, 7a, 12a-trihydroxy-5p- [N- (N ', N', N'-trimethylaminoethane iodide)] cholanamide, called Trimethylaminoethane Cholamide (TAE-Cholamide), represented by the general formula I (B) which R2 represents the

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radical de l'acide 3a,7a,12a-trihydroxy-5p-cholanique, R représente le radical méthyle, n est égal à 2 et X représente l'iode, - le composé 3a,7a,12a-trihydroxy-5P-[N-(N',N',N'-triméthylaminopropane iodure) ] cholanamide, dénommé Triméthylaminopropane Cholamide (TAP- Cholamide) , représenté par la formule générale I (B) danslaquelle R2 représente le radical de l'acide 3a,7a,12a-trihydroxy-5p-cholanique, R représente le radical méthyle, n est égal à 3 et X représente l'iode, - le composé 3a,12a-dihydroxy-5(3-[N-(N',N',N'-triméthylaminoéthane iodure) ] cholanamide, dénommé Triméthylaminoéthane Déoxycholamide (TAE- Déoxycholamide) représenté par la formule générale I (B) laquelle R2 représente

le radical de l'acide 3a,12a-dihydroxy-5p-cholanique, R représente le radical méthyle, n est égal à 2 et X représente l'iode, - le composé 3a,12a-dihydroxy-5P-[N-(N',N',N'-triméthylaminopropane iodure) ] cholanamide, dénommé Triméthylaminopropane Déoxycholamide (TAP- Déoxycholamide) , représenté par la formule générale I (B) laquelle R2 représente le radical de l'acide 3a,12a-dihydroxy-5p-cholanique, R représente le radical méthyle, n est égal à 3 et X représente l'iode,

- le composé 3a-hydroxy-5P-[N-(N',N',N'-triméthylaminoéthane iodure)] cholanamide, dénommé Triméthylaminoéthane Lithocholamide (TAE- Lithocholamide) , représenté par la formule générale I (B) laquelle R2 représente

le radical de l'acide 3a-hydroxy-5P-cholanique, R représente le radical méthyle, n est égal à 2 et X représente l'iode, - le composé 3a-hydroxy-5P-[N-(N',N',N'-triméthylaminopropane iodure)] cholanamide, dénommé Triméthylaminopropane Lithocholamide (TAP- Lithocholamide) , représenté par la formule générale I (B) laquelle R2 représente le radical de l'acide 3a-hydroxy-5p-cholanique, R représente le radical méthyle, n est égal à 3 et X représente l'iode,

- le composé 3, 7, 12-trioxo-5P-[N-(N',N',N'-triméthylaminoéthane iodure)] cholanamide, dénommé Triméthylaminoéthane Déhydroxycholamide (TAE- Déhydrocholamide) , représenté par la formule générale I (B) laquelle R2

représente le radical de l'acide 3,7,12-trioxo-5(3-cholanique, R représente le radical méthyle, n est égal à 2 et X représente l'iode, - le composé 3, 7, 12-trioxo-5P-[N-(N',N',N' -triméthylaminopropane iodure)] cholanamide, dénommé Triméthylaminopropane Déhydroxycholamide (TAP-

radical of 3a, 7a, 12a-trihydroxy-5p-cholanic acid, R represents the methyl radical, n is equal to 2 and X represents iodine, - compound 3a, 7a, 12a-trihydroxy-5P- [N - (N ', N', N'-trimethylaminopropane iodide)] cholanamide, called Trimethylaminopropane Cholamide (TAP-Cholamide), represented by the general formula I (B) in which R2 represents the radical of the acid 3a, 7a, 12a- trihydroxy-5p-cholanic, R represents the methyl radical, n is equal to 3 and X represents iodine, - compound 3a, 12a-dihydroxy-5 (3- [N- (N ', N', N'- trimethylaminoethane iodide)] cholanamide, referred to as Trimethylaminoethane Deoxycholamide (TAE- Deoxycholamide) represented by the general formula I (B) in which R2 represents

the radical of 3a, 12a-dihydroxy-5p-cholanic acid, R represents the methyl radical, n is equal to 2 and X represents iodine, - the compound 3a, 12a-dihydroxy-5P- [N- (N ', N', N'-trimethylaminopropane iodide)] cholanamide, referred to as Trimethylaminopropane Deoxycholamide (TAP-Deoxycholamide), represented by the general formula I (B) in which R2 represents the 3a, 12a-dihydroxy-5p-cholanic acid radical , R represents the methyl radical, n is equal to 3 and X represents iodine,

- the compound 3a-hydroxy-5P- [N- (N ', N', N'-trimethylaminoethane iodide)] cholanamide, called Trimethylaminoethane Lithocholamide (TAE-Lithocholamide), represented by the general formula I (B) in which R2 represents

the radical of 3a-hydroxy-5P-cholanic acid, R represents the methyl radical, n is equal to 2 and X represents iodine, - the compound 3a-hydroxy-5P- [N- (N ', N' , N'-trimethylaminopropane iodide)] cholanamide, referred to as Trimethylaminopropane Lithocholamide (TAP-Lithocholamide), represented by the general formula I (B) in which R2 represents the radical of 3a-hydroxy-5p-cholanic acid, R represents the methyl radical , n is equal to 3 and X represents iodine,

- the compound 3, 7, 12-trioxo-5P- [N- (N ', N', N'-trimethylaminoethane iodide)] cholanamide, called trimethylaminoethane dehydroxycholamide (TAE- Dehydrocholamide), represented by the general formula I (B) which R2

represents the 3,7,12-trioxo-5 (3-cholanic acid radical, R represents the methyl radical, n is equal to 2 and X represents iodine, - compound 3, 7, 12-trioxo- 5P- [N- (N ', N', N '-trimethylaminopropane iodide)] cholanamide, referred to as Trimethylaminopropane Dehydroxycholamide (TAP-

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Dehydrocholamide), represented by the general formula I (B) in which R2 represents the radical of 3,7,12-trioxo-5p-cholanic acid, R represents the methyl radical, n is equal to 3 and X represents iodine .

l'acide 3a,7a,12a-trihydroxy-5p-cholanique, l'acide 3a,12a-dihydroxy-5p-cholanique, l'acide 3a-hydroxy-5p-cholanique ou l'acide 3,7,12-trioxo-5p-cholanique, R représente un atome d'hydrogène, un radical méthyle ou éthyle, X représente un atome d'halogène tel que le chlore ou l'iode,
A ce titre, l'invention vise plus particulièrement le composé lipidique dicationique tel que défini ci-dessus, caractérisé en ce qu'il est choisi dans le groupe constitué par : - le 3P[N,N-(Bis(aminoéthane chlorhydrate))-carbamoyle] cholestérol (BAECChol) représenté par la formule générale II(A), dans laquelle R1 représente le radical cholestéryle, R représente un atome d'hydrogène et X représente un atome de chlore, - le 3P[N,N-(Bis(aminoéthane chlorhydrate))-succinamide cholestérol (BAES- Chol) représenté par la formule générale II (B), laquelle R1 représente le radical cholestéryle, R représente un atome d'hydrogène et X représente un atome de chlore, - le composé N,N-(Bis(aminoéthane chlorhydrate))-abiétamide représenté par la formule générale II(C), dans laquelle R2 représente le radical de l'acide abiétique, R représente le radical méthyle et X représente un atome de chlore, A subject of the invention is also more particularly the dicationic lipid compound, characterized in that it is chosen from the group consisting of: - the dicationic lipid compound of general formula II (A)
Ri O- CO - N [CH2 - CH2 - N + (R) 3, X -] 2 II (A) in which Ri represents the cholesteryl radical, R represents a hydrogen atom, a methyl or ethyl radical, X represents a halogen atom such as chlorine or iodine, - the dicationic lipid compound of general formula II (B)
R1 0 - CO - CH2 - CH2 - CO - N [CH2 - CH2 - N + (R) 3, X -] 2 II (B) in which R1 represents the cholesteryl radical, R represents a hydrogen atom or a methyl radical or ethyl, X represents a halogen atom such as chlorine or iodine, - the dicationic lipid compound of general formula II (C)
R2 - CO - N [CH2 - CH2 - N + (R) 3, X -] 2 II (C) in which R2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group consisting of the radical of

3a, 7a, 12a-trihydroxy-5p-cholanic acid, 3a, 12a-dihydroxy-5p-cholanic acid, 3a-hydroxy-5p-cholanic acid or 3,7,12-trioxo- 5p-cholanic, R represents a hydrogen atom, a methyl or ethyl radical, X represents a halogen atom such as chlorine or iodine,
As such, the invention relates more particularly to the dicationic lipid compound as defined above, characterized in that it is chosen from the group consisting of: - 3P [N, N- (Bis (aminoethane hydrochloride)) -carbamoyl] cholesterol (BAECChol) represented by the general formula II (A), in which R1 represents the cholesteryl radical, R represents a hydrogen atom and X represents a chlorine atom, - the 3P [N, N- (Bis (aminoethane hydrochloride)) - succinamide cholesterol (BAES- Chol) represented by the general formula II (B), in which R1 represents the cholesteryl radical, R represents a hydrogen atom and X represents a chlorine atom, - the compound N, N- (Bis (aminoethane hydrochloride)) - abietamide represented by the general formula II (C), in which R2 represents the radical of abietic acid, R represents the methyl radical and X represents a chlorine atom,

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radical de l'acide 3a,7a,12a-trihydroxy-53-cholanique, R représente le radical méthyle et X représente un atome de chlore, - le composé N,N-(Bis(aminoéthane chlorhydrate))- 3a,12a-dihydroxy-5(3- cholanamide représenté par la formule générale II(C), dans laquelle R2 représente le

radical de l'acide 3a,12a-dihydroxy-53-cholanique, R représente le radical méthyle et X représente un atome de chlore, - le composé N,N-(Bis(aminoéthane chlorhydrate))-3a-hydroxy-5p-cholanamide représenté par la formule générale II(C), dans laquelle R2 représente le radical de l'acide

3a-hydroxy-5(3-cholanique, R représente le radical méthyle et X représente un atome de chlore, - le composé N,N-(Bis(aminoéthane chlorhydrate))-3,7,12-trioxo-5(3-cholanamide représenté par la formule générale II(C), dans laquelle R2 représente le radical de l'acide 3,7,12-trioxo-5p-cholanique, R représente le radical méthyle et X représente un atome de chlore. - the compound N, N- (Bis (aminoethane hydrochloride)) - 3 [alpha], 7 [alpha], 12 [alpha] -trihydroxy-5ss-cholanamide represented by the general formula II (C), in which R2 represents the

3a, 7a, 12a-trihydroxy-53-cholanic acid radical, R represents the methyl radical and X represents a chlorine atom, - the compound N, N- (Bis (aminoethane hydrochloride)) - 3a, 12a-dihydroxy -5 (3-cholanamide represented by general formula II (C), wherein R2 represents

radical of 3a, 12a-dihydroxy-53-cholanic acid, R represents the methyl radical and X represents a chlorine atom, - the compound N, N- (Bis (aminoethane hydrochloride)) - 3a-hydroxy-5p-cholanamide represented by the general formula II (C), in which R2 represents the radical of the acid

3a-hydroxy-5 (3-cholanic, R represents the methyl radical and X represents a chlorine atom, - the compound N, N- (Bis (aminoethane hydrochloride)) - 3,7,12-trioxo-5 (3- cholanamide represented by the general formula II (C), wherein R2 represents the 3,7,12-trioxo-5p-cholanic acid radical, R represents the methyl radical and X represents a chlorine atom.

The invention also relates to a composition characterized in that it contains a cationic lipid compound as defined above.

l'acide 3a,7a,12a-trihydroxy-5p-cholanique, l'acide 3a,12a-dihydroxy-5p-cholanique, l'acide [alpha]-hydroxy-5ss-cholanique ou l'acide 3,7,12-trioxo-5p-cholanique, et un solvant tel que l'éthanol. As such, the invention relates to a composition characterized in that it is in the form of a cationic lipid solution containing a cationic lipid compound as defined above, in which R2 represents the radical of a derivative of cholanic acid, in particular that chosen from the group consisting of the radical of

3a, 7a, 12a-trihydroxy-5p-cholanic acid, 3a, 12a-dihydroxy-5p-cholanic acid, [alpha] -hydroxy-5ss-cholanic acid or 3,7,12- trioxo-5p-cholanic, and a solvent such as ethanol.

hydroxy-5p-cholanique ou l'acide 3,7,12 -tri oxo- 5 P-cholanique, et un solvant tel que le chlorure de méthylène pour la préparation de liposomes. The invention also relates to a composition characterized in that it is in the form of a cationic lipid solution containing a cationic lipid compound as defined above, in which R1 represents the cholesteryl radical, R2 represents the acid radical. abietic, the radical of a derivative of cholanic acid, in particular that chosen from the group consisting of the radical of 3a, 7a, 12a-trihydroxy-5p-cholanic acid, 3a, 12a-dihydroxy-5p -cholanic acid, 3a-

5p-hydroxy-cholanic or 3,7,12-tri oxo-P-cholanic acid, and a solvent such as methylene chloride for the preparation of liposomes.

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a composition as defined above, and a neutral lipid (or colipid), in particular dioleoylphosphatidylethanolamine (DOPE), and optionally a lipid conjugated (in particular with a polyethylene glycol, or with an antibody fragment), and / or a surfactant, in particular chosen from the group consisting of simulsol 59, simulsol 165 or simulsol 989.

The invention is also aimed at a complex characterized in that it comprises a cationic lipid compound as defined above, or a composition as defined above, and a negatively charged compound, in particular a nucleic acid (deoxyribonucleic acid, ribonucleic acid, gene, plasmid, ribozyme or oligonucleotide, which may or may not be modified covalently) having therapeutic or vaccination effects, or applications in genetics or biotechnology.

The invention also relates to a complex characterized in that it comprises a liposome as defined above, and a negatively charged compound, in particular a nucleic acid (deoxyribonucleic acid, ribonucleic acid, gene, plasmid, ribozyme or oligonucleotide, which can be modified (covalently or not) having therapeutic or vaccination effects, or applications in genetics or biotechnology.

Likewise, a subject of the invention is also a pharmaceutical composition characterized in that it comprises, as active principle, a complex as defined above, in combination with a pharmaceutically acceptable vehicle or excipient.

The pharmaceutical composition according to the invention is suitable for any mode of administration, in particular parenterally, topically or by inhalation.

The pharmaceutical composition according to the invention is further characterized in that the amount of complex varies from 0.5 to 30 mg / kg of body weight for a unit dose.

Description of figures
FIGS. 1 to 4 represent the synthesis diagrams of monocationic and dicationic lipid compounds according to the invention.

FIG. 1 represents the synthesis diagram of the monocationic lipid TMAECChol. Step (a) consists in adding to the starting product (1) (cholesteryl chloroformate), 2 equivalents of N, N-dimethylethylenediamine and anhydrous ether to

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a temperature of 0 C, to obtain the carbamate of formula (2). Step (b) consists in adding an excess of methyl iodide on said carbamate (2) to obtain the numbered compound (3): TMAEC-Chol corresponding to the general formula I (A).

Figure 2 represents the diagram of the synthesis of the dicationic lipid BAEC-Chol.

Step (a) consists in carrying out the synthesis of diethylene triamine dihydrochloride by reacting diethylene triamine with 2 equivalents of hydrochloric acid. Step (b) consists in adding 2.25 equivalents of diethylene triamine dihydrochloride thus obtained to cholesteryl chloroformate (1), to obtain the compound numbered (2): BAECChol corresponding to general formula II (A).

Figure 3 shows the synthesis scheme of the dicationic lipid BAES-Chol.

Step (a) consists in carrying out the synthesis of diethylene triamine dihydrochloride by reacting diethylene triamine with 2 equivalents of hydrochloric acid.

Step (b) consists in adding oxalyl chloride (COCl) 2 to cholesteryl hemisuccinate (1) in the presence of methylene chloride (CH2Cl2), to obtain the corresponding succinoyl chloride (2). Step (c) consists in reacting the succinoyl chloride (2) with 2.25 equivalents of diethylene triamine dihydrochloride, to obtain the numbered compound (3): BAES-Chol corresponding to general formula II (B) .

FIG. 4 represents the synthesis diagram of the monocationic lipid TAPLithocholamide.

Step (a) consists in adding to the starting compound (1) (lithocholic acid also represented by R2COOH), triethylamine in THF, to obtain the intermediate compound (2) to which is added the compound C2H5-O -CO-Cl (step (b)) to obtain the mixed anhydride (3). Step (c) consists in adding to the mixed anhydride (3), the N, N-dimethylpropylene diamine to obtain the tertiary amine (4). Step (d) consists in taking the tertiary amine (4) obtained in step (c) above in tetrahydrofuran (THF) and methyl iodide (ICH3), to obtain the numbered compound (5) : TAP-Lithocholamide corresponding to the general formula I (B).

FIGS. 5 to 13 represent the results obtained during the use of the cationic lipid compounds according to the invention for the transfer of nucleic acids.

FIG. 5 represents the percentage of oligonucleotides not bound to liposomes, as a function of the concentration of cationic lipids (expressed in µM).

FIG. 6 represents the activity levels of β-galactosidase (103 RLU relative light unit / mg) expressed respectively (from left to right) in the following cells: MCF7,3T3, A549 and U373MG, said cells being transfected with using TMAEC-Chol liposomes according to the invention.

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FIG. 7 represents the relative levels of transfection (%) in MCF-7 cells obtained respectively (from left to right) with the following liposomes: Lipofectin, TMAEC-Chol according to the invention, DC-Chol and DAC-30.

FIG. 8 represents the levels of transfection in CEM cells obtained respectively (from left to right) with the following liposomes: Lipofectin, TAPlithocholamide according to the invention and TMAEC-Chol according to the invention. The level of transfection is measured by the activity of β-galactosidase (RLU).

FIG. 9 represents the activity levels of β-galactosidase (103 RLU / mg) expressed in the MCF7 cells transfected using the TMAEC-Chol (Lip +) liposomes according to the invention as a function of the ratio r = DOPE / lipid cationic (Lip (+)) used.

FIG. 10 represents the relative transfection levels (%) in MCF7 tumor cells by PCMVss-TMAEC-Chol liposomes complexes according to the invention as a function of the content of fetal calf serum (%).

FIG. 11 represents the activity levels of β-galactosidase (103 RLU / mg) expressed in the MCF7 tumor cells transfected using the complexes formed between pCMVp and the liposomes prepared respectively from the cationic lipids TMAEC-Chol according to l invention (hatched histogram), cationic lipids DMAEC-Chol (checkered histogram), TMAPC-Chol (white histogram) and DCChol (black histogram) as a function of the calf serum content f # tal (%).

Figure 12 represents the percentage of viable MCF7 cells as a function of the concentration (expressed in M) of cationic liposomes prepared from the cationic lipids according to the invention TMAEC-Chol (see curve comprising the black rectangle) and BAEC-Chol (see curve comprising the black circle), and from the known DC-Chol lipid (see curve comprising the black triangle).

Figure 13 represents the relative levels of transfection (%) of MCF7 tumor cells by pCMVp-liposome BAEC-Chol complexes according to the invention, in the presence of fetal calf serum, as a function of the molar charge ratio X = Lip (+) / nucleotide. The hatched histogram represents 0% f # tal calf serum (FCS), the checkered histogram represents 25% FCS, the white histogram 50% FCS, and the black histogram 10% f # calf serum tal.

The cationic lipid compounds according to the invention exhibit numerous advantages which are in particular illustrated in the examples given below.

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The examples below illustrate the present invention and do not limit it in any way.

A. PROPERTIES OF CATIONIC LIPID COMPOUNDS ACCORDING TO THE INVENTION Example 1: monocationic cholesterol (TMAEC-Chol).

R,-O-CO-NH-(CHZ)2-N+(CH3)3 1-1 dans laquelle R1 est le groupement cholestéryle
Les composés lipidiques de l'art antérieur donnés à titre de comparaison dans cet exemple sont des dérivés monoaminés du cholestérol, à savoir le DC-Chol , correspondant au 3-(3[N-(N',N'-diméthylaminoéthane)-carbamoyle] cholestérol [3,4],

et le DAC-Chol , correspondant au 3-[i[N-(N,N'-diméthylaminoéthane)-carbamoyle] cholestérol [5]. The monocationic lipid compound according to the invention studied in this example is designated by the abbreviation TMAEC-Chol, and corresponds to 3 (3 [N- (N ', N', N'-trimethylamino ethane iodide) -carbamoyl] cholesterol , of molar mass M = 642.75 g / mol, represented by the general formula I (A) below:

R, -O-CO-NH- (CHZ) 2-N + (CH3) 3 1-1 where R1 is the cholesteryl group
The lipid compounds of the prior art given by way of comparison in this example are monoamine derivatives of cholesterol, namely DC-Chol, corresponding to 3- (3 [N- (N ', N'-dimethylaminoethane) -carbamoyl ] cholesterol [3,4],

and DAC-Chol, corresponding to 3- [i [N- (N, N'-dimethylaminoethane) -carbamoyl] cholesterol [5].

1) Comparison between TMAEC-Chol according to the invention, DC-Chol and DAC-Chol.

The advantages regarding the chemical originality of TMAEC-Chol compared to DCChol and DAC-Chol are listed below.

(a) The cationic lipid TMAEC-Chol is in the form of an ion consisting of a quaternary ammonium bearing 3 methyl groups. It is different from DC-chol which is a tertiary amine.

In the publications of the prior art [3, 4], a positive charge has been attributed to DC-Chol which has a neutral chemical structure! This error has also been noted by other authors [11,12].

Thus, the cationic lipid compound according to the invention can easily fix negatively charged substances, unlike neutral DC-Chol which tends rather to repel them due to the free doublet on nitrogen.

(b) The yield obtained by the synthesis process according to the invention of TMAEC-Chol is 65% while that obtained for DC-Chol [3] is only 21.8%.

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(c) Le lipide 1MA;C:-C:rio1 selon l'invention est clntérent, par rapport au DAC- Chol (lipide de base du DAC30 ), par le bras de liaison ( linker ) et par la tête polaire. Le bras de liaison de TMAEC-Chol est un amide secondaire alors que celui de DAC-Chol est un amide tertiaire. Le lipide TMAEC-Chol ne présente pas de risque de formation de produits de dégradation, éventuellement toxiques, propres aux amines.

(c) The 1MA; C: -C: rio1 lipid according to the invention is clntérent, with respect to DAC-Chol (base lipid of DAC30), by the linker arm and by the polar head. The TMAEC-Chol linker is a secondary amide while that of DAC-Chol is a tertiary amide. TMAEC-Chol lipid does not present any risk of formation of degradation products, possibly toxic, specific to amines.

(d) The polar head of the DAC-Chol lipid is a secondary amine hydrochloride, it is therefore an acidic compound, unlike TMAEC-Chol which is neutral and therefore more stable with respect to the variation in pH.

(e) Due to the fact that it is a quaternary ammonium, TMAEC-Chol is not very sensitive to the variation in pH in media whose pH is close to neutral pH. This is the case with culture media and intracellular media. It is such as to protect the negatively charged and complexed entities against any degradation due to variations in pH.

(f) In the synthesis of the DAC-Chol lipid, the product N, N'dimethylethylenediamine used is a symmetrical molecule. The yield of the reaction giving rise to the molecule having an amine head cannot exceed 50%.

(g) TMAEC-Chol lipid is soluble in methylene chloride (CH2Cl2).

Liposomes based on TMAEC-Chol can therefore be prepared using CH2Cl2 which must be evaporated after the first liposome preparation step which consists of producing a thin film. Since CH2C12 (Eb. 40 C) is eliminated much more easily than the chloroform used hitherto (Eb. 60 C), the liposomes prepared from the TMAEC-Chol lipid dissolved in CH2Cl2 do not exhibit toxicity due to the solvent. .

(h) The materials used in the process for preparing the cationic lipid compounds, and the liposomes according to the invention make it possible to reduce the manufacturing cost, estimated at 10 cents / mg or at 40 F (labor included), to laboratory scale.

2) Comparison between TMAEC-Chol according to the invention, DC-Chol and Lipofectin.

The advantages regarding the originality of the applications of TMAEC-Chol compared to DC-Chol [3,4] and lipofectin [1] are listed below.

(a) Using dioleoylphosphatidylethanolamine (DOPE) as colipid or neutral lipid in a molar or weight ratio of between 0.5 and 3, the TMAEC-Chol lipid gives liposomes of size between 110 to 180 nm. These

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liposomes are very stable over time, since these values only change by less than 5% after 1 year.

(b) These liposomes instantly form complexes with nucleic acids. For phosphodiester or phosphorothioate oligonucleotides with a length of 10 to 28 m, provided that a suitable molar charge ratio [cationic lipid (Lip +) / nucleotide] is taken, the complexation yield can reach 100%. This ratio is 2 for the phosphodiesters.

(c) The oligonucleotide-liposome complexes formed enter cells in culture, adherent cells or cells in suspension. The oligonucleotides are thus internalized in the cytoplasm or in the nucleus, depending on the sequence and the molecular structure chosen for the oligonucleotide.

(d) Intracellular residence times as long as 72 h and even 8 days could be observed for these oligonucleotides.

(e) For the plasmids, the reaction is instantaneous and the yield depends on the DOPE / Lip + ratio. Formulation 2: of DOPE / Lip + appears to be the best for several plasmids.

(f) The liposome / plasmid complex penetrates cells in culture, adherent or in suspension, in 3-D culture in nodules and in solid tumors. The level of expression of the reporter gene (luciferase or P-galactosidase) is high, and in all the tests carried out higher than that of lipofectin and DC-Chol.

(g) Liposome / plasmid complexes are resistant to serum. Tests on cells in culture media containing up to 50% serum have been possible.

(h) The liposome-plasmids or liposomes-oligonucleotides complexes are not very toxic for several cells up to a molar concentration of 100 M in cationic lipid TMAEC-Chol.

Example 2: cholesterol dicationics (BAEC-Chol and BAES-Chol).

The dicationic compound designated by the abbreviation BAEC-Chol, corresponds to 3p [N, N- (Bis (aminoethane hydrochloride)) - carbamoyl] cholesterol, of molar mass M = 588.74 g / mol, represented by the general formula II (A) below:
R1-O-CO- [CH2-CH2-NH3 +, Cl-] 2, wherein R1 represents the cholesteryl group.

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groupement cholestéryle : R-O-CO-(CH2)z-CO-N[CH2-CHz-NH/ ' CI-] 2
Les avantages quant à l'originalité des lipides dicationiques BAEC-Chol et BAES-Chol sont énumérés ci-dessous. The dicationic compound denotes by i aoreviation BASE-Chol, corresponds to 3p [N, N- (Bis (aminoethane hydrochloride)) - succinamide] cholesterol, of molar mass M = 644.81 g / mol, represented by general formula II ( B) in which R] represents the

cholesteryl group: RO-CO- (CH2) z-CO-N [CH2-CHz-NH / 'CI-] 2
The originality advantages of BAEC-Chol and BAES-Chol dicationic lipids are listed below.

(a) With a head having 2 positively charged nitrogen atoms, the power of complex formation with nucleic acids is enhanced compared to a monocationic head.

(b) The charged groups are hydrochlorides of primary amines.

It should be remembered that triaminocholesterol [8] is an entity not electrically charged.

(c) The spacer is a biodegradable tertiary amide.

(d) The dicationic lipids according to the invention are products soluble in THF, which avoids the use of chlorinated solvents in the preparation of the liposomes.

(e) BAEC-Chol and BAES-Chol are recrystallizable from THFmethanol mixture (50/50), thus avoiding the use of very heavy chromatographic purification techniques.

(f) The synthesis of BAEC-Chol takes place in a single, very rapid step after the preparation of the diethylene triamine dihydrochloride, without resorting to the blocking of the —NH2 functions. The yield of the synthesis is high (77%).

Thus, compared to the synthesis in several steps of the product of the prior art [8] which is an amine, the synthesis of BAEC-Chol in a single step constitutes a fundamental improvement. The cost excluding labor is less than 5 cents / mg.

These particularities make it possible to envisage a synthesis on an industrial scale.

Example 3: monocationics of cholanic acid derivatives (TAP-cholamide, TAP-deoxycholamide, TAP-lithocholamide and TAP-dehydrocholamide).

The monocationic compounds designated by the abbreviations TAP-cholamide, TAP-deoxycholamide, TAP-lithocholamide and TAP-dehydrocholamide, are represented by the following formula I (B): R2-CO-NH- (CH2) 3-N + (CH3) 3 , I-, in which R2 is a group derived from cholanic acid

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Les avantages quant a i ongmame aes iipiaes monocauoniques 1 Al'-Cn01amlGe, TAP-déoxycholamide, TAP-lithocholamide et TAP-déhydrocholamide sont énumérés ci-dessous.

The advantages in relation to the monocauonic lipiaes 1 Al'-Cn01amlGe, TAP-deoxycholamide, TAP-lithocholamide and TAP-dehydrocholamide are listed below.

(a) The linker arm is a biodegradable amide.

(b) The head is quaternary ammonium.

B. PROCESS FOR PREPARING CATIONIC LIPID COMPOUNDS ACCORDING TO THE INVENTION
Example 4 Synthesis of the TMAEC-Chol monocationic lipid.

TMAEC-Chol was synthesized according to the scheme of Figure 1. The new lipid was characterized by IR spectroscopy.

Cholesteryl chloroformate (98% purity), N, N-dimethylethylenediamine (99% purity), anhydrous ether (+ 99% purity) and iodomethane (99.5% purity) are products marketed by Aldrich, used without further purification.

A solution of cholesteryl chloroformate (2.7g; 6 mmol in 50 cm3 of anhydrous ether) is added dropwise in a solution of N, Ndimethylethylenediamine (1.06g; 12 mmol in 50 cm3 of anhydrous ether maintained at 0 C (ice bath) The hydrochloride formed is removed by filtration and the solvent is removed on a rotary evaporator The residual carbamate is precipitated by adding 20 cm3 of absolute methanol at 0 C, filtered and then dried under vacuum. synthesis of the quaternary ammonium salt is carried out by reaction at reflux (4 hours) of excess methyl iodide (18 mmol) on said carbamate in solution in anhydrous ether. After removal of the solvent, of the excess of methyl iodide and recrystallization from absolute methanol, 2.44 g (ie 65%) of white powder corresponding to the pure cationic salt TMAEC-Chol are collected.

Example 5: Synthesis of the dicationic lipid BAEC-Chol.

The synthesis of BAEC-Chol was carried out according to the scheme of FIG. 2. Cholesteryl chloroformate (98%), diethylene triamine (99%), tetrahydrofuran (99 +%) are Aldrich products used without further purification. The other reagents are common laboratory chemicals.

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The first step consists in carrying out the synthesis of diethylene triamine dihydrochloride of formula HN [CH2-CH2-NH3 +, Cl-] 2 by reacting diethylene triamine with two equivalents of hydrochloric acid (HCl) in aqueous solution (1M). The formation of this dihydrochloride is possible by this method, since the pKa of the two NH 2 functions of diethylene triamine is 10, while that of its NH is only 3.7. It should be noted that this result, which constitutes a very simple blocking of the two NH2, is not applicable to amines such as spermine or spermidine widely used in the synthesis of cationic lipids, since all their primary and secondary amine functions are dosed at a single equivalent point. The dihydrochloride is obtained with a yield of 77% after two recrystallizations from an ethanolmethanol mixture (50/50), and its melting point is 176 C.

The dihydrochloride in slight excess (2.25 equivalents) is placed in a porcelain crucible and melted on an electric plate. The chloroformate (5mmol, 1 eq., F = 125 C) is added in small portions. The pasty mixture is stirred with the agitator until the end of the evolution of HCl. After cooling, the straw-yellow solid obtained is crushed and then taken up for 2 hours at reflux with 50 cm3 of tetrahydrofuran (THF). The excess dihydrochloride and the diethylene triamine trihydrochloride formed, insoluble in THF, are removed by filtration. After concentrating the filtrate to approximately 5 cm3, the addition of absolute CH3OH releases the BAEC-Chol, which, after filtration, is taken up successively with a new portion of THF and then of alcohol. Finally, 1.51 g (yield 52%) of BAEC-Chol are collected, the melting point of which is 211 C. The IR band of the C = O group (KBr pellet) is at 1690.8 cm-1.

Example 6: Synthesis of the dicationic lipid BAES-Chol.

The synthesis of BAES-Chol was carried out according to the scheme of Figure 3.

Cholesteryl hemisuccinate (98%) is a product marketed by Lancaster; oxalyl chloride (99%), diethylene triamine (99%), tetrahydrofuran (THF) (99 +%) are products marketed by Aldrich, all used without further purification. The other reagents are common laboratory chemicals.

The first step consists in carrying out the synthesis of diethylene triamine dihydrochloride of formula HN [CH2-CH2-NH3 +, Cl-] 2 by reacting diethylene triamine with two equivalents of HCl in aqueous solution (1M) as described above.

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In a second step, 10-2 mol of cholesteryl hemisuccinate in 50 cm3 of CH2Cl2 at room temperature and with stirring for 20 hours, are treated with 3 # 10-2 mol of oxalyl chloride. This leads to the corresponding succinoyl chloride with a yield of 96%, after removal of the methylene chloride and then of excess (COCl) 2 under vacuum.

The dihydrochloride in slight excess (2.25 equivalents) is placed in a porcelain crucible and melted on an electric plate. Succinoyl chloride (1 eq.) Is added in small portions. The pasty mixture is stirred with the agitator until the end of the evolution of HCl. After cooling, the straw-yellow solid obtained is crushed and then taken up for 2 hours at reflux with 50 cm3 of THF. The excess dihydrochloride and the diethylene triamine trihydrochloride formed, insoluble in THF, are removed by filtration. After concentrating the filtrate to approximately 5 cm3, the addition of absolute CH3OH releases the BAES-Chol which, after filtration is taken up successively with a new portion of THF and then of alcohol. Finally, 0.70 g (yield 55%) of BAEC-Chol is collected from initially 1 g of succinoyl chloride. The IR band of the C = O group (KBr pellet) is at 1651.8 cm-1 for the amide function and at 1734.1 cm-1 for the ester function.

Example 7: Synthesis of the TAP-lithocholamide monocationic lipid.

The synthesis of TAP-lithocholamide was carried out according to the scheme of Figure 4. At 9.5 g (2.5 # 10-2 mol) of lithocholic acid (> 99%, Fluka) in 100 cm3 of anhydrous THF ( 99.9%, Aldrich), 2.55 g of triethylamine (99 +%, Aldrich) in 20 cm3 of THF, then 2.72 g of ethyl chloroformate (> 98%, Fluka). The triethylamine hydrochloride is separated from the ethereal solution of mixed anhydride by filtration. After adding 2.55 g of N, Ndimethylpropylene diamine (99 +%, Aldrich) to the preceding filtrate, the mixture is stirred for 24 hours at room temperature. After cooling (0 ° C.), the lipid is isolated by filtration in its tertiary amine form. The solid obtained is taken up in 100 cm3 of THF and 10.5 g (7.5 # 10-2 mol) of ICH3 (99.5%, Aldrich), and maintained for 3 hours at 50 C. Finally, 13 is collected, 7 g (91%) of TAPlithocholamide. Solid phase Fourier transform infrared spectroscopy (FTIR) analysis with KBr gives (-CO-N) at 1658.9 cm-1 and (-NH-) at 3378 cm-1.

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- . - ....- 1 --- "-''-''.1''''''''' '-""-'..&....tU ..&....t...... A.Ì.&.",V..L..IU '-'..(""1......."""J.",.a.'lu..a.:..t.J SELON L'INVENTION POUR LE TRANSFERT D'ACIDES NUCLEIQUES.

-. - ....- 1 --- "-''- ''. 1 ''''''''''-""-'..& .... tU .. & .... t ...... A.Ì. &. ", V..L..IU '-' .. (""1.......""" J. ",. a.'lu. .a.: .. tJ ACCORDING TO THE INVENTION FOR THE TRANSFER OF NUCLEIC ACIDS.

Example 8: Use of the monocationic derivative TMAEC-Chol.

The transfection tests described according to the following are carried out with TMAECChol prepared according to the protocol described in Example 1. a) Preparation of the liposomes:
A mixture of TMAEC-Chol cationic lipid and neutral lipid (or colipid): dioleolylphosphatidylethanolamine (DOPE), in a 1: 1 weight ratio, is dissolved in chloroform and then evaporated under vacuum in a rotary evaporator. The resulting thin film is dried for a further at least 5 hours before being rehydrated with sterile water. Methylene chloride can be used in place of chloroform.

The final cationic lipid concentration is 1 mg / mL, or 1.55 mM.

The mixture is vortexed for 2 minutes and then sonicated intermittently for 30 minutes using a Bransonic, model 1210 sonicator. A clear solution is obtained. The solution is centrifuged for 15 min with a speed of 12000 g. The supernatant is taken and, if necessary, filtered through a Millipore filter of 0.22 μm pore diameter and the filtrate is stored at 4 C. The liposomes are characterized in size by a dynamic light scattering apparatus. The number distribution indicates a single mode with an average diameter of 124 nm.

The same preparation protocol is applicable to BAEC-Chol. b) Oligonucleotides and plasmids:
The oligonucleotides used, of variable length from 12 to 28 mers, are supplied in lyophilized form by Genosys (Great Britain) or Eurogentec (Belgium). In order to visualize their internalization, certain oligonucleotides are labeled with fluorescein in the 5 ′ position. The oligonucleotides are dissolved in sterile water at a concentration of 3 nM in nucleotide.

The plasmids used are pCMV-P plasmids carrying a reporter gene encoding β-galactosidase (Clonetech), or pGL2-luc carrying a reporter gene encoding luciferase (Promega). The plasmids were amplified according to the standard protocol. They are stored in a Tris-EDTA (ethylenediamine tetraacetic acid) buffer, pH = 8 at a concentration of 1 mg / mL.

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c) Cell cultures:
The cells used are adherent cells, of cancerous origin (MCF7, A549, U373MG) or not (3T3), or cells in suspension (CEM, U937). The cells are cultured according to the standard conditions for each type. The DMEM medium (Dulbecco's Modified Eagle Medium) is used for the adherent cells MCF7, A549, U373MG, 3T3 and the RPMI 1640 medium (Roswell Park Memorial Institute) for the cells in suspension CEM and U937. The media contain 10% fetal calf serum (FCS) and penicillin-streptomycin antibiotics. d) In vitro transfection protocol:
For the adherent cells, the day before the day of transfection, the cells are inoculated (5 × 10 cells per well in 6-well Falcon plates or 5 × 10 cells per well of a 4-well Labtek (Nunc) slide). Thus on the day of transfection the cells are 60-70% confluent.

A quantity of DNA (plasmid or oligonucleotide) of 1 g and the selected quantity of cationic TMAEC-Chol liposomes were separately diluted in 10 L of sterile water and slightly vortexed then mixed.

After 15 minutes, the mixture is diluted in 1 ml of OptiMEM medium (Gibco) without serum. The cells are washed with the serum-free medium and then the mixture in OptiMEM is added to the cells. After 6 hours of incubation at 37 ° C., in an atmosphere containing 5% C02, the OptiMEM is removed, replaced by 1 mL of culture medium containing 10% serum. The cells continue to be incubated for the desired time. In assays to test the transfection capacity in the presence of serum, 1 mL of medium containing the desired serum content is used in place of OptiMEM.

tri cyclo(3.3. 1. I)décane)-4-yl)phényl- P-D -galactopyranoside (Tropix). En présence de P-galactosidase, l'AMPGD à pH > 9, conduit à l'adamantanone et à l'anion méthyl The same protocol is used for control transfections using DC-Chol synthesized according to the method of Gao and Huang (1991) [3]. Concerning the transfections using lipofectin, the complexes are obtained according to the protocol recommended by the manufacturer. e) Evaluation of the level of transfection:
The method of evaluating the level of transfection consists in detecting ssgalactosidase by the reagent AMPGD (3- (4-methoxyspiro (1,2-dioxetane-3,2'-

tri cyclo (3.3. 1. I) decane) -4-yl) phenyl-PD -galactopyranoside (Tropix). In the presence of P-galactosidase, AMPGD at pH> 9, leads to adamantanone and methyl anion

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allows to perform the measurement [15].

The β-galactosidase or luciferase activity is measured 48 hours after transfection using the Galactolight Plus detection kit (Tropix) following the protocol recommended by the manufacturer. The cells are washed twice, in 1 ml of phosphate buffer solution (PBS) each time, then lysed with 200 μl of lysis buffer containing 1 mM of freshly prepared dithiotreiol. The extracts are harvested, centrifuged at 12,000 g for 5 min.

An aliquot (20 L) of the supernatant is diluted in 200 μl of Galacton reagent (based on AMPGD) and left at room temperature. After 1 hour, the accelerator is added and the chemiluminescence is immediately measured by a BCL luminometer (Gouteyron, France) in integration mode of 10 seconds. The intensities are expressed in RLU (relative light unit).

The total protein concentration is measured using the Bio-Rad kit for this purpose. The activity results are expressed by normalized values RLU / mg relative to 1 mg of total protein. f) Observation of internalization of oligonucleotides:
Internalization of oligonucleotides is observed using light microscopy. For this, after the incubation periods of 24, 48 or 72 hours, the cells transfected in wells of the Labtek slide (Nunc) are washed twice with PBS, fixed with paraformaldehyde (4%) for 15 minutes and mounted. with Mowiol (Calbiochem). g) Possibility of strengthening transfection:
The transfection rate can be further enhanced by using a plasmid condensing agent such as spermine, prior to complexation with TMAECChol liposomes.

On the other hand, in the formulation of liposomes it is possible to add a surfactant such as simulsol 59, simulsol 165 or simulsol 989 (SEPIC), or a lipid conjugated with a polyethylene glycol (PEG) or with a group such as a antibody fragment making it possible to orient the liposomes towards specific receptors.

Example 9: Results

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a) Kenaemeni ue compiexauon liposomes 11vlL-Lnol/o11gonuCleOtIaes.

a) Kenaemeni ue compiexauon liposomes 11vlL-Lnol / o11gonuCleOtIaes.

The 1 g aliquots of the oligonucleotide (ATC5G6) (d (ATCCCCCGGGGGG)) are mixed with the liposomes 1: 1, according to varying charge ratios X. The mixtures are filtered through Millipore filters with a limiting mass of 30 kDa so as to pass only free oligonucleotides, not bound to the liposomes. The oligonucleotides are assayed by UV spectroscopy and the percentage of unbound oligonucleotides (relative to those not mixed with the liposomes) is represented as a function of X (FIG. 5). The variation is practically linear. From the value of X = 2, there are no unbound oligonucleotides. This makes it possible to determine the optimal complexation ratio for which all the oligonucleotides are linked. b) Level of transfection of different cell lines by TMAEC-Chol / pCMVss complexes.

FIG. 6 represents the levels of β-galactosidase activity expressed respectively in cells of MCF7,3T3, A549 and U373MG, transfected with the aid of TMAEC-Chol liposomes. The best results are observed for 3T3 cells. c) Comparison of the level of transfection of MCF7 cells by TMAEC-Chol / pCMVss complexes, with those obtained from lipofectin, DC-Chol and DAC-30.

In Figure 7, the levels of transfection in MCF-7 cells obtained with TMAEC-Chol liposomes are compared with those of known vectors.

Figure 7 indicates that TMAEC-Chol liposomes have a transfection level 2 to 3 times higher than that of lipofectin or DAC-30. d) Comparison of the level of transfection of CEM cells by TAP-lithocholamide-Chol / pCMVP complexes with those obtained from lipofectin.

In Figure 8, the levels of transfection in CEM cells obtained with TAP-lithocholamide liposomes are compared with those of lipofectin.

It can be seen that the TAP-lithocholamide liposomes have a transfecting power 30 times greater than that of lipofectin. e) Effect of the DOPE / Lip + ratio on the level of transfection of MCF7 tumor cells by TMAEC-Chol / pCMVss complexes.

The content of the neutral dioleolylphosphatidylethanolamine lipid in the formulation of TMAEC-Chol liposomes, represented by the ratio r = DOPE / Lip + affects their level of cell transfection. It seems that this depends on the type of cells. In the case of MCF7 cells shown in Figure 9, the highest level is observed

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l'VU! !'" ICZIJIJ%Jlt 1 - 1. w.rw.ttuam Yvut uauuw w.uutw, ta mtututauvtt avcv. 1 - 4 palan optimale. f) Niveau de transfection des cellules tumorales MCF7 par des complexes TMAEC-Chol/pCMVP en présence de sérum.

the VU! ! '"ICZIJIJ% Jlt 1 - 1. w.rw.ttuam Yvut uauuw w.uutw, ta mtututauvtt avcv. 1 - 4 optimum hoist. F) Level of transfection of MCF7 tumor cells by TMAEC-Chol / pCMVP complexes in the presence of serum.

FIG. 10 represents the levels of transfection of MCF7 tumor cells by pCMVp-TMAEC-Chol liposome complexes in the presence of fetal calf serum. Even at 50% serum, a level equivalent to the level obtained when the transfection medium is devoid of serum is still observed. g) Comparison of the levels of transfection of MCF7 tumor cells by TMAEC-Chol / pCMVss complexes and by complexes obtained from TMAEC-Chol homologues in the presence of serum.

The originality of the TMAEC-Chol lipid lies in the resistance of the complexes formed from the TMAEC-Chol liposomes in the presence of serum. The polar head of TMAEC-Chol is a quaternary ammonium containing 3 methyl groups.

The bulk should allow the molecule to be less sensitive to pH.

The cationic lipids DMAEC-Chol, TMAPC-Chol and DC-Chol were synthesized and the levels of transfection obtained were compared with that of TMAEC-Chol.

DC-Chol and DMAEC-Chol have different pole heads than TMAECChol. That of DC-Chol is a tertiary amine, while that of DMAEC-Chol is a tertiary ammonium. TMAPC-Chol contains a quaternary ammonium like TMAEC-Chol, but the spacer arm of TMAPC-Chol is longer than that of TMAEC-Chol and has an additional CH2 group.

FIG. 11 indicates the levels of transfection of the MCF7 cells by complexes formed by 1 g of pCMVss and 6 nmoles of liposomes prepared respectively from the cationic lipids TMAEC-Chol, DMAEC-Chol, TMAPCChol or DC-Chol. It is observed that only TMAEC-Chol is resistant to serum. h) Comparison of the cytotoxicity of the lipids TMAEC-Chol, BAEC-Chol and that of DC-Chol.

The viability of the cells is detected by the MTT test. The MTT test consists of detecting viable cells using the MTT reagent (3- (4,5-dimethylthiazol-2-yl) - 2,5-diphenyl tetrazolium bromide (Sigma). For living cells, the dehydrogenases of the mitochondria react with MTT and reduce it to formazan, which is a blue product, the amount of which can be determined by absorption measurements at 570 nm [16].

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The MCF7 cells deposited the day before transfection in the wells of a 96-well Falcon plate (5 # 104 cells per well) are transfected with the complexes formed by mixing 1 g of oligonucleotides and variable amounts of TMAEC-Chol liposomes. or BAEC-Chol according to the invention, according to the protocol described above.

After 48 h of incubation at 37 ° C., the cells are washed twice with PBS, then 100 l of MTT reagent (1 mg / ml of DMEM medium containing serum) are added and then the cells are incubated again. After 3 h, the reagent is removed, replaced with 100 L of DMSO (dimethylsulfoxide) for 20 min. The "viable" cells give a blue coloration, the optical density of which at the wavelength 570 nm varies in proportion to the number of these cells.

FIG. 12 represents the variations in the percentage of viable MCF7 cells as a function of the concentration (expressed in M) of the cationic liposomes used, prepared from TMAEC-Chol, BAEC-Chol or DC-Chol. The percentage is calculated relative to the optical density obtained from untransfected cells.

The curves in this figure clearly show that the cationic lipids TMAEC-Chol and BAEC-Chol are markedly less toxic than DC-Chol, itself less cytotoxic than lipofectin. i) Level of transfection of MCF7 tumor cells by BAEC-pCMVp complexes in the presence of serum.

FIG. 13 represents the levels of transfection of MCF7 tumor cells by BAEC-Chol / pCMVp liposome complexes in the presence of fetal calf serum.

The levels are normalized to that obtained with the molar charge ratio (Lip (+) / nucleotide) X = 2 in the absence of serum. A variation in the level of transfection is observed as a function of the ratio X. Thus, in the absence of serum, the maximum is observed for X = 1 while for 50% of serum, the maximum is observed for X = 4.

By varying the ratio X, these cells can be transfect in the presence of varying levels of serum.

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Acad. Sci. USA, 84: 7413-7417.

[2] FELGNER J.H., KUMAR R., SRIDHAR C. N., WHEELER C. J., TSAI Y.J., BORDER R., RAMSEY P., MARTIN M., FELGNER P. (1994). Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J.

Biol. Chem., 269,1550-2561.

[3] GAO X. and HUANG L. (1991) A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochem. Biophys. Res. Comm., 179, 280-285.

[4] LEE R.J. and HUANG L. (1997). Lipidic vector systems for gene transfer.

Critical Review in "Ther. Drug Car. Systems", 14, 173-206.

[5] RESZKA R., (1996). New cholesterol derivative for liposomal gene transfer.

International Patent No. WO 96/20208.

[6] BEHR J.P., DEMENEIX B., LOEFFLERJ. P. and PEREZ-MUTUL J. (1989) Efficient gene transfer into mammalian primary endocrine cells with lipopolyaminecoated DNA. Proc. Natl. Acad. Sci. USA, 86, 6982-6986.

[7] SCHWARTZ B., BENOIST C., ABDALLAH B., SCHERMAN D., BEHR J. P., DEMENEIX, B. (1995). Lipospermine-based gene transfer into the newborn mouse brain is optimized by a low lipospermine / DNA charge ratio. Human Gene Therapy, 6, 1515-1524.

[8] SULLIVAN S., MENG X.Y. (1997). Cationic polymer / lipid nucleic acid delivery vehicles. International patent WO 97/45069.

[9] VIGNERON J.P., OUDDRHIRI N., FAUQUET M., VERGELY L., BRADLEY J.C., BASSEVILLE M., LEHN P., LEHN J.M. (1996). Guanidiniumcholesterol cationic lipids: efficient vectors for the transfection of eukaryotic cells.

Proc. Natl. Acad. Sci. USA, 93,9682-9686.

[10] WALKER S., SOFIA M.J., KAKARLA R., KOGAN N. A., WIERICHS L., LONGLEY C. B., BRUCKER K., AXELROD H. R., MIDHA S., BABU S., KAHNE D.

(1996), Cationic facial amphiphiles: promising class of transfection agents. Proc.

Natl. Acad. Sci. USA, 93, 1585-1590.

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[Il] WY5t J., W AKNr;K C. ( 1 yyb), uatiomc l1pld acid salt os !jeta (N-(N ,N - dimethylaminoethane)-carbamoyl)cholestérol, Brevet international n WO 96/40067, p. 2.

[II] WY5t J., W AKNr; K C. (1 yyb), uatiomc l1pld acid salt os! Jeta (N- (N, N - dimethylaminoethane) -carbamoyl) cholesterol, International Patent No. WO 96/40067, p. 2.

[12] MILLER A. D., COOPER R., ETHERIDGE C.J. (1997), Polycationic sterol derivatives as transfection agents, International Patent No. WO 97/45442.

[13] FARHOOD H., BOTTEGA R., EPAND R.M. and HUANG L. (1992) Effect of cationic cholesterol derivatives on gene transfer and protein kinase C activity.

Biochim. Biophys. Acta, 1111, 239-246.

[14] EPAND R., BOTTEGA R., HUANG L., (1993). Method for delivering nucleic acids into cells, International Patent, 1993.

[15] JAIN V.J. and MAGRATH I.T. (1991), A chemiluminescent assay for quantification of P-galactosidase in the femtogram range, Anal. Biochem., 199, 119-124.

[16] LAPPALAINEN K., URTI A., JAASKELAINEN L, SYRJANEN K. and SYRJANEN S. (1994), Cationic liposomes mediated delivery of antisense oligonucleotides targeted to HPV 16E7 mRNA in CaSki cells, Antiviral Res., 23, 119-130 .

Claims (1)

CLAIMS 1. Use of a monocationic lipid compound of general formula I (A) RO - CO - NH - (CHz) n - N (R) 3, X 'I (A) in which Ri represents the cholesteryl radical, R represents a methyl or ethyl radical, n represents an integer equal to 2 or 3, X represents a halogen atom such as chlorine or iodine, and / or a monocationic lipid compound of general formula I (B) R2 - CO - NH - (CHz) n - lr (R) 3 XI (B) in which R2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group consisting by the 3a, 7a, 12a-trihydroxy-5p-cholanic acid radical, 3a, 12a-dihydroxy-S (3-cholanic acid, 3 [alpha] -hydroxy-5ss-cholanic acid or acid 3,7, 12-trioxo-5p-cholanic, R represents a methyl or ethyl radical, n represents an integer equal to 2 or 3, X represents a halogen atom such as chlorine or iodine, and / or a compound dicationic lipid of general formula II (A)

RO - CO - N [CHz - CHz - N (R) 3, X -] z II (A) in which R1 represents the cholesteryl radical, R represents a hydrogen atom, a methyl or ethyl radical, X represents an atom halogen such as chlorine or iodine, and / or a dicationic lipid compound of general formula II (B) Ri O- CO - CH2 - CH2 - CO - N [CH2 - CH2 - N + (R) 3, X -] 2 II (B) in which R1 represents the cholesteryl radical, R represents a hydrogen atom, a methyl radical or ethyl, X represents a halogen atom such as chlorine or iodine, <Desc / Clms Page number 31> and / or a dicationic lipid compound of general formula II (C)

R2 - CO - N [CHZ - CHz - N + (R) 3, X] zn (C) in which R2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group consisting of the radical of

3a, 7a, 12a-trihydroxy-53-cholanic acid, 3a, 12a-dihydroxy-53-cholanic acid, [alpha] -hydroxy-5ss-cholanic acid or 3,7,12- trioxo-5p-cholanic, R represents a hydrogen atom, a methyl or ethyl radical, X represents a halogen atom such as chlorine or iodine, for the transfection of living organisms in vivo, of organs in vivo, tumors in vivo, or cells in vitro or ex vivo. 2. Monocationic lipid compound characterized in that it is chosen from the group consisting of: - the monocationic lipid compound of general formula I (A)

R, O - CO - NH - (CHz) "- N + (R) 3, X - I (A) in which Ri represents the cholesteryl radical, R represents a methyl or ethyl radical, n represents an integer equal to 2 or 3, X represents a halogen atom such as chlorine or iodine, - the monocationic lipid compound of general formula I (B)

R2- CO - NH - (CH2) n - IV (R3, X 1 (B) in which R2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group constituted by the radical of

3a, 7a, 12a-trihydroxy-53-cholanic acid, 3a, 12a-dihydroxy-5P-choIanic acid, 3a-hydroxy-5p-cholanic acid or 3,7,12-trioxo- 5p-cholanic, R represents a methyl or ethyl radical, n represents an integer equal to 2 or 3, X represents a halogen atom such as chlorine or iodine. <Desc / Clms Page number 32> 3. Monocationic lipid compound according to claim 2, characterized in that it is chosen from the group consisting of:

- 3 | 3 [N- (N ', N', N'-trimethylaminoethane iodide) -carbamoyl] cholesterol (TMAEC-Chol) represented by the general formula I (A) in which R1 represents the cholesteryl radical, R represents the radical methyl, n is equal to 2 and X represents iodine, - the compound N- (N ', N', N'-trimethylaminoethane iodide) abietamide represented by the general formula I (B) in which R2 represents the radical of abietic acid, R represents the methyl radical, n is equal to 2 and X represents iodine, - the compound N- (N ', N', N'-trimethylaminopropane iodide) abietamide represented by the general formula I (B) which R2 represents the radical of abietic acid, R represents the methyl radical, n is equal to 3 and X represents iodine,

- the compound 3a, 7a, 12a-trihydroxy-53- [N- (N ', N', N'-trimethylaminoethane iodide)] cholanamide, called Trimethylaminoethane Cholamide (TAE-Cholamide), represented by the general formula I (B) which R2 represents the

radical of 3a, 7a, 12a-trihydroxy-53-cholanic acid, R represents the methyl radical, n is equal to 2 and X represents iodine, - compound 3a, 7a, 12a-trihydroxy-Si- [N - (N ', N', N'-trimethylaminopropane iodide)] cholanamide, called Trimethylaminopropane Cholamide (TAP-Cholamide), represented by the general formula I (B) in which R2 represents the

radical of 3a, 7a, 12a-trihydroxy-53-cholanic acid, R represents the methyl radical, n is equal to 3 and X represents iodine, - compound 3a, 12a-dihydroxy-5p- [N- ( N ', N', N'-trimethylaminoethane iodide)] cholanamide, referred to as Trimethylaminoethane Deoxycholamide (TAE- Deoxycholamide) represented by the general formula I (B) in which R2 represents

the 3 (x, 12 (x-dihydroxy-50-cholanic acid radical, R represents the methyl radical, n is equal to 2 and X represents iodine, - compound 3a, 12a-dihydroxy-5/3 - [N- (N ', N', N'-trimethylaminopropane iodide)] cholanamide, referred to as Trimethylaminopropane Deoxycholamide (TAP-Deoxycholamide), represented by the general formula I (B) in which R2 represents

the radical of 3a, 12a-dihydroxy-5p-cholanic acid, R represents the methyl radical, n is equal to 3 and X represents iodine, <Desc / Clms Page number 33> - the compound 3a-hydroxy-5p- [N- (N ', N', N'-trimethylaminoethane iodide)] cholanamide, called Trimethylaminoethane Lithocholamide (TAE-Lithocholamide), represented by the general formula I (B) in which R2 represents

the radical of 3a-hydroxy-5p-cholanic acid, R represents the methyl radical, n is equal to 2 and X represents iodine, - the compound 3a-hydroxy-53- [N- (N ', N' , N'-Trimethylaminopropane iodide)] cholanamide, referred to as Trimethylaminopropane Lithocholamide (TAP-Lithocholamide), represented by the general formula I (B) wherein R2 is

the radical of 3a-hydroxy-5p-cholanic acid, R represents the methyl radical, n is equal to 3 and X represents iodine, - the compound 3, 7, 12-trioxo-5P- [N- (N ', N', N'-trimethylaminoethane iodide)] cholanamide, referred to as Trimethylaminoethane Dehydroxycholamide (TAE-Dehydrocholamide), represented by the general formula I (B) in which R2 represents the radical of 3,7,12-trioxo-5p acid -cholanic, R represents the methyl radical, n is equal to 2 and X represents iodine,

- the compound 3, 7, 12-trioxo-5p- [N- (N ', N', N'-trimethylaminopropane iodide)] cholanamide, called trimethylaminopropane dehydroxycholamide (TAP-Dehydrocholamide), represented by the general formula I (B) in which R2 represents the 3,7,12-trioxo-5p-cholanic acid radical, R represents the methyl radical, n is equal to 3 and X represents iodine. 4. Dicationic lipid compound characterized in that it is chosen from the group consisting of: - the dicationic lipid compound of general formula II (A)

R1 O - CO - N [CHz - CHz - N + (R) 3, X -] z II (A) in which Ri represents the cholesteryl radical, R represents a hydrogen atom, a methyl or ethyl radical, X represents a halogen atom such as chlorine or iodine, - the dicationic lipid compound of general formula II (B) R, O- CO - CH2 - CH2 - CO - N [CH2 - CH2 - N + (R) 3, X -] 2 II (B) <Desc / Clms Page number 34> in which R1 represents the cholesteryl radical, R represents a hydrogen atom, a methyl or ethyl radical, X represents a halogen atom such as chlorine or iodine, - the dicationic lipid compound of general formula II (C) R2 - CO - N [CH2 - CH2 - N + (R) 3, X -] 2 II (C) in which R2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group consisting of the radical of

3a, 7a, 12a-trihydroxy-5p-cholanic acid, 3a, 12a-dihydroxy-53-cholanic acid, 3a-hydroxy-5p-cholanic acid or 3,7,12-trioxo- 5p-cholanic, R represents a hydrogen atom, a methyl or ethyl radical, X represents a halogen atom such as chlorine or iodine, 5. Dicationic lipid compound according to claim 4, characterized in that it is chosen from the group consisting of: - 3p [N, N- (Bis (aminoethane hydrochloride)) - carbamoyl] cholesterol (BAECChol) represented by the formula general II (A), in which R1 represents the cholesteryl radical, R represents a hydrogen atom and X represents a chlorine atom, - 3P [N, N- (Bis (aminoethane hydrochloride)) - succinamide cholesterol (BAESChol) represented by the general formula II (B), in which R1 represents the cholesteryl radical, R represents a hydrogen atom and X represents a chlorine atom, - the compound N, N- (Bis (aminoethane hydrochloride)) - abietamide represented by the general formula II (C), in which R2 represents the radical of abietic acid, R represents the methyl radical and X represents a chlorine atom, - the compound N, N- (Bis (aminoethane hydrochloride)) - 3 [alpha], 7 [alpha], 12 [alpha] -trihydroxy-5ss-cholanamide represented by general formula II (C), in which R2 represents the

3a, 7a, 12a-trihydroxy-5p-cholanic acid radical, R represents the methyl radical and X represents a chlorine atom, - the compound N, N- (Bis (aminoethane hydrochloride)) - 3a, 12a-dihydroxy -5p-cholanamide represented by general formula II (C), wherein R2 represents

3a, 12a-dihydroxy-53-cholanic acid radical, R represents the methyl radical and X represents a chlorine atom, <Desc / Clms Page number 35>

- the compound N, N- (Bis (aminoethane hydrochloride)) - 3a-hydroxy-5p-cholanamide represented by the general formula II (C), in which R2 represents the radical of the acid

3a-hydroxy-5p-cholanic, R represents the methyl radical and X represents a chlorine atom, - the compound N, N- (Bis (aminoethane hydrochloride)) - 3,7,12-trioxo-5 (3-cholanamide shown by general formula II (C), in which R2 represents the radical of 3,7,12-trioxo-5p-cholanic acid, R represents the methyl radical and X represents a chlorine atom. 6. Composition characterized in that it contains a cationic lipid compound according to any one of claims 2 to 5. 7. Composition according to claim 6, characterized in that it is in the form of a cationic lipid solution containing a cationic lipid compound according to any one of claims 2 to 5 in which R2 represents the radical of a derivative of cholanic acid, in particular that chosen from the group consisting of

3a, 7a, 12a-trihydroxy-S13-cholanic acid radical, 3 (X, 12 (X-dihydroxy-50-cholanic acid, 3a-hydroxy-5p-cholanic acid or 3, 7,12-trioxo-5p-cholanic, and a solvent such as ethanol. 8. Composition according to claim 6, characterized in that it is in the form of a cationic lipid solution containing a cationic lipid compound according to any one of claims 2 to 5 in which R1 represents the cholesteryl radical, R2 represents the radical of abietic acid, the radical of a derivative of cholanic acid, in particular that chosen from the group consisting of the radical of

3a, 7a, 12a-trihydroxy-5p-cholanic acid, 3a, 12a-dihydroxy-S (3-cholanic acid, 3 [alpha] -hydroxy-5ss-cholanic acid or 3.7 , 12-trioxo-5p-cholanic, and a solvent such as methylene chloride for the preparation of liposomes. 9. Liposome characterized in that it contains a composition according to any one of claims 6 to 8, and a neutral lipid (or colipid), in particular dioleoylphosphatidylethanolamine (DOPE), and optionally a conjugated lipid (in particular with a polyethylene glycol , or an antibody fragment), and / or a <Desc / Clms Page number 36> surfactant, in particular chosen from the group consisting of simulsol # 59, simulsol 165 or simulsol 989. 10. Complex characterized in that it comprises a cationic lipid compound according to any one of claims 2 to 5 or a composition according to any one of claims 6 to 8, and a negatively charged compound, in particular a nucleic acid (acid deoxyribonucleic, ribonucleic acid, gene, plasmid, ribozyme or oligonucleotide, which can be modified covalently or not) having therapeutic or vaccination effects, or applications in genetics or biotechnology. 11. Complex characterized in that it comprises a liposome according to claim 9, and a negatively charged compound, in particular a nucleic acid (deoxyribonucleic acid, ribonucleic acid, gene, plasmid, ribozyme or oligonucleotide, which can be modified covalently or not. ) having therapeutic or vaccination effects, or applications in genetics or biotechnology. 12. Pharmaceutical composition characterized in that it comprises, as active principle, a complex according to claim 10 or claim 11, in association with a pharmaceutically acceptable vehicle or excipient. 13. Pharmaceutical composition according to claim 12, characterized in that it is suitable for any mode of administration, in particular parenterally, topically or by inhalation. 14. Pharmaceutical composition according to claim 12 or claim 13, characterized in that the amount of complex varies from 0.5 to 30 mg / kg of body weight for a unit dose.