FR2744453A1 - LIPOPOLYAMINE COMPOUNDS, PHARMACEUTICAL COMPOSITIONS COMPRISING NUCLEIC ACID TRANSFER VECTORS AND PROCESS FOR PREPARING THE SAME - Google Patents

Abstract

A compound of formula (II), wherein R1 is hydrogen and R2 is hydrogen or, for only one of its n substituents, a deoxy residue in the 3 position of the cholesterol or a mono- or polyvalent residue of a cholesterol derivative having one or more reactive groupings, respectively, said groupings being capable of reacting with a secondary amine group of a polyamine molecule; n is an integer from 2 to 6; and p is an integer from 2 to 6 and identical or different in the n (a) fragments.

Description

LIPOPOLYAMINE COMPOUND, PHARMACEUTICAL COMPOSITION AND
NUCLEIC ACID TRANSFER VECTOR COMPRISING, AND
PROCESS OF PREPARATION
The present invention relates to a lipopolyamine compound and pharmaceutical compositions comprising it. More particularly, the present invention relates to the use of this composition for the transfer of a nucleic acid into a host cell. Finally, the present invention relates to a process for preparing a compound according to the invention, and to a process for purifying an isomer of said compound.

 Gene transfer in a given cell is at the very heart of gene therapy. However, one of the major problems is to introduce a sufficient amount of therapeutic nucleic acid into the host cell to be treated. The most effective vectors in this respect are viral vectors, in particular retroviral and adenoviral vectors. Indeed, viruses have developed sophisticated mechanisms to cross cell membranes, escape degradation at the level of lysosomes and penetrate their genome into the nucleus.However, the viral approach has its limitations, including a cloning capacity in the cell. restricted viral genome, production of infectious viral particles susceptible to dissemination in the host organism and the environment, risk of insertional mutagenesis in the case of retroviral vectors, and induction of immune and inflammatory responses in the host. These important drawbacks in the context of human use justify the search for alternative systems for transferring nucleic acids.

 Many methods are currently available, including calcium phosphate coprecipitation, electroporation, microinjection or lipofection. The latter uses cationic lipids that interact spontaneously with the nucleic acid by interaction of charges to form positively charged complexes capable of fusing with the anionic cell membranes and penetrating the nucleic acid they carry.

 The use of cationic lipids as a transfer vector for various molecules of therapeutic interest has already been described in the literature. It has been shown that cationic lipids of various chemical structures allow the transfection of mammalian cells in culture with very different efficiencies depending on the cell type and the lipid-DNA complex formulation. However, the efficiencies are generally variable and weak in the animal, in particular as regards a muscular administration, and the results obtained with the synthetic molecules currently available, are not satisfactory. As a whole, these studies did not allow to identify factors influencing the efficiency of the gene transfer mechanism and optimization tests according to structural parameters such as particle size, charge, DNA content etc ... did not bring any clear answers.

 EP 0 349 111 and WO95 / 18863 (Behr et al.) Relate to the lipid composition comprising a 5-carboxyspermylglycine dioctadecylamide compound (also referred to as DOGS) which results from the combination of spermine with a linear-chain lipid. WO95 / 18863 discloses successful in vivo transfection experiments with a +/- charge ratio of less than 2 only in newborn mice by intracerebral injections. No transfection by intramuscular or intravenous administration is obtained, in particular in mice other than newborns.

 The article by Gao and Huang, 1991 in BBRC 179, 280-285 and the international application WO93 / 05162 describe a lipopolyamine, DC-CHOL resulting from the reaction of cholesteryl chloroformate and a tertiary amine (dimethylethylene diamine). DC-CHOL or 3 ([N - (N ', N'-dimethylaminoethane) carbamoyl] cholesterol has only a positive charge and therefore has a low transfecting power.

 The object of the present invention is to provide new, more efficient cationic lipids, in particular for application as a DNA transfer vector, in particular for intramuscular, intratracheal or intrapulmonary administration.

The subject of the present invention is therefore a lipopolyamine compound of formula C - (S) m (1) in which
C represents a deoxy residue at position 3 of cholesterol or a monovalent or polyvalent residue of a cholesterol derivative carrying one or more reactive groups capable of reacting with a primary or secondary amine group of a polyamine molecule;
S represents an amine residue of said amine group of a polyamine molecule, and
m represents an integer of 1 to 3.

 The term "cholesterol derivative" means a compound having the formula the backbone of cholesterol linked to one or more substituent (s) of which at least one comprises a reactive group capable of reacting with a primary or secondary amine group of a polyamine molecule.

As an illustration, we cite more particularly
compounds where m = 1, the compounds in which C represents a
remains monovalent deoxy in position 3 of cholesterol
compounds where m = 2, the compounds in which C represents a
divalent didesoxy residue of 5-cholesten-3B, 4B-diol, or 5-cholesten-3B,
25-diol, and,
compounds where m = 3, the compounds in which C represents a
trivalent tridesoxy-residue of cholesten - 3ss, 5B, 6ss-triol.

dans laquelle: - R1 et R2 représentent l'hydrogène ou un reste monovalent C; - le symbole R2 peut être identique ou différent dans les n fragments

- l'un seulement de R1 et des n substituants R2 représente undit reste monovalent C; - n est un nombre entier de 2 à 6 ; et - p est un nombre entier de 2 à 6 identique ou différent dans les n fragments

In a preferred embodiment S is a residue of a polyamine molecule of formula:
H2N - [- (CH2) p - NH] n- H (IX) and the lipopolyamine compound according to the invention corresponds to the formula

in which: R1 and R2 represent hydrogen or a monovalent residue C; the symbol R2 may be identical or different in the n fragments

only one of R 1 and n substituents R 2 represents one monovalent residue C; n is an integer from 2 to 6; and - p is an integer from 2 to 6 identical or different in the n fragments

 Preferably the compounds n = 2 or 3 and p = 3 or 4.

 More preferably, S represents a residue of natural spermine or spermidine compound corresponding to the compounds of formula (IX) in which n = 2 and p = 3 and 4 (spermidine) and n = 3 and p = 3, 4 and 3 respectively in different links (spermine).

 In a particular embodiment, the compound according to the invention is characterized in that the monovalent residue C is a monovalent residue deoxy in position 3 of cholesterol.

 The coupling of the amine S can then be carried out, for example, by nucleophilic substitution of a tosylate group at the 3-position of a cholesterol tosylate compound by an amine function of S, thus creating a direct amine bond. This has the advantage of preserving the number of positive charges of spermidine (3 charges) and spermine (4 charges).

But the coupling of the amine S can also be done by coupling a reactive group of C with an amine function of the compound of formula (IX). This is the case where C represents a residue of a cholesterol derivative. More specifically, the residue C can represent a residue of a 3-position cholesterol derivative of formula LR in which - L represents an aliphatic radical containing a residue of a group that can react with a primary or secondary amine group and - R represents the stays in position 3 of formula

In particular, the compounds in which the cholesterol derivative residue at the 3-position has the formula:

 This residue (V) is a residue of chloroformate derivative at the 3-position of cholesterol.

Mention may also be made of the compounds of formula (III) in which
L represents a hemisuccinate-type linkage of formula

que l'on peut obtenir en greffant un radical succinite anhydre sur le cholestérol. La réaction s'accompagne d'une ouverture du cycle succinite qui peut ensuite réagir avec la polyamine. Ce mode de réalisation introduit un bras entre cette dernière et le cholestérol.This remainder is then a remainder of a derivative

that can be obtained by grafting an anhydrous succinite radical on cholesterol. The reaction is accompanied by an opening of the succinite ring which can then react with the polyamine. This embodiment introduces an arm between the latter and cholesterol.

dans laquelle R1 et R2i, avec i = 1 et 2 ont les significations de R1 et R2 données précédement et des composés répondant à la formule:

dans laquelle R1 et R2i avec i = 1, 2 et 3 ont les significations de R1 et R2 données précédemment.The present invention also provides monosubstituted isomers of purified cholesterol having the formula

wherein R1 and R2i, with i = 1 and 2 have the meanings of R1 and R2 given previously and compounds of the formula:

wherein R1 and R2i with i = 1, 2 and 3 have the meanings of R1 and R2 given previously.

In particular, the compounds of formula (VI) for which
R2 represents one monovalent residue of cholesterol or a
cholesterol derivative in position 3 and,
- R1 and R22 represent H.

as well as the compounds for which
R1 and R21 represent H and
R22 represents a residue of cholesterol or a derivative of
cholesterol in position 3 and, the compounds for which
R1 represents a monovalent monovalent cholesterol residue of a derivative
cholesterol in position 3, and
- R21 and R22 represent H.

In the same way, a compound according to the invention is more particularly characterized in that in formula (VII)
R21 and R22 represent H, and
One of R1 and R23 represents said monovalent residue of a cholesterol derivative at the 3-position and the other represents H.

as well as the compounds according to the invention, characterized in that in the formula (VIl)
R1 and R23 represent H, and
One of R21 and R22 represents said cholesterol residue or a
cholesterol derivative at position 3 and the other represents H.

Those compounds in which the cholesterol or cholesterol derivative is substituted on a terminal amine of the compounds (VI) or (VII) are preferred.

 The experimental data described below show that the transfecting power of these isomers is greater when the remainder of a cholesterol derivative at the 3-position is grafted onto a primary terminal amine of the compound of formula (IX), in particular on the long arm. [(CH2) 4] of spermidine.

 The compounds according to the invention are advantageously used for their transfecting capacity for the transfer into a cell of a negatively charged therapeutically active substance.

 The present invention therefore also relates to a pharmaceutical composition comprising a compound according to the invention and a therapeutically active substance negatively charged.

 In particular, a composition is mentioned in which said active substance is an acid.

 Preferably, said active substance is a nucleic acid.

 The said nucleic acid may be a sense or antisense DNA or RNA.

Sense and antisense applications are well known to those skilled in the art.

In short, we mean by:
- meaning meaning "a nucleic acid having a sequence
complementary to a target sequence, for example a sequence
mRNA whose expression is sought to be blocked by hybridization
on the target sequence; and
- sense "a nucleic acid having a homologous sequence or
identical to a target sequence, for example a sequence that binds
to a protein transcription factor and involved in
the expression of a given gene.

 According to a preferred embodiment, the nucleic acid comprises a gene of interest and elements allowing the expression of said gene of interest. In this embodiment, said nucleic acid fragment is advantageously in plasmid form.

In the context of the present invention, the nucleic acid may be homologous or heterologous to the target cell. It may be advantageous to use a nucleic acid coding for a cytokine (interleukin including IL-2, interferon, colony stimulating factor, etc.), a cellular or nuclear receptor, a ligand or a coagulation factor (Factor VII ,
Factor VIII, Factor IX ...), CFTR protein (Cystic Fibrosis
Transmembrane Conductance Regulator), insulin, dystrophin, a growth hormone, an enzyme (urease renin, thrombin, etc.), an enzyme inhibitor (inhibitor of a viral protease, cx 1-antitrypsin). .), an anti-tumor effect polypeptide (tumor suppressor gene product, immune system-stimulating polypeptide, etc.), a polypeptide capable of inhibiting or slowing down the development of a bacterial, viral or parasitic infection (variant antigenic polypeptide, trans-dominant ...), an antibody, a toxin, an immunotoxin and finally a marker (luciferase, β-galactosidase, product conferring resistance to an antibiotic.). Of course, this list is not limiting and other genes can also be used.

Advantageously, the nucleic acid is placed under the control of the elements necessary for its expression in the host cell. By "necessary elements" is meant all the elements allowing the transcription of said DNA fragment into RNA (antisense RNA or mRNA) and the translation of the mRNA into a polypeptide. These elements include a regulatable or constitutive promoter, which may be heterologous or otherwise homologous to the parental virus. Mention may be made, by way of examples, of the promoter of the human or murine PGK gene (Phospho Glycerate Kinase), the early promoter of the SV40 virus (Simian Virus), the LTR of RSV (Rous
Sarcoma Virus), the TK promoter (Thymidine Kinase) of the HSV-1 virus (Herpes Simplex virus) and the adenoviral promoters E1A and MLP. The necessary elements may furthermore include additional elements (intronic sequence, secretion signal sequence, nuclear localization sequence, translation initiation site, poly A transcription termination signal, etc.).

 The transfection experiments show that advantageously the ratio by weight of the lipid compound according to the invention to said DNA fragment is from 0.01 to 100. The optimum ratio is from 0.1 to 10.

 The present invention also relates to the use of a pharmaceutical composition according to the invention for preparing a transfer vector of said nucleic acid in cells in vitro, ex vivo or in vivo.

 In general, a composition according to the invention can be used in various types of host cells. It is preferably a mammalian cell and in particular a human cell. Said cell may be a primary or tumoral cell of a hematopoietic origin (totipotent stem cell, leucocyte, lymphocyte, monocyte or macrophage ...) hepatic, epithelial, fibroblast ... and especially a muscle cell (myoblast, cardiomyocyte .. .), a tracheal or pulmonary cell. Furthermore, a composition according to the invention may comprise a targeting element to a particular cell, for example a ligand to a cellular receptor. Such targeting elements are known.

 A composition according to the invention can be administered intragastrically, subcutaneously, intravenously, intraperitoneally, intratumorally or nasally. Intra-cardiac, intramuscular, intrapulmonary or intratracheal routes of administration are particularly preferred. The administration may take place in single or repeated doses one or more times after a certain interval interval. The appropriate route of administration and dosage vary depending on various parameters, for example, the individual or the disease to be treated or the nucleic acid to be transferred.

 A composition according to the invention is intended for the preparation of a medicinal product intended for the treatment of the human or animal body and, preferably, by gene therapy. According to a first possibility, the medicament may be administered directly in vivo (for example in a muscle, in the lungs by aerosol, etc.). It is also possible to adopt the ex vivo approach which consists of taking patient cells (bone marrow stem cells, peripheral blood lymphocytes, muscle cells, etc.) and transferring them in vitro according to the techniques of the art. to re-administer them to the patient.

 The subject of the present invention is therefore also a vector for transferring nucleic acids into cells of microorganisms, plants, humans or animals, in particular mammals, characterized in that it comprises a pharmaceutical composition according to the invention comprising a nucleic acid and an adjuvant capable of improving the transfecting power of said composition.

 In one embodiment, said adjuvant is a neutral co-lipid.

 Examples include phospholipids such as phosphatidyl ethanolamine, phosphatidylcholine, phosphatidyl serine, phosphatidylglycerol and pharmaceutically acceptable detergents.

 It is also possible to combine the lipid / colipid mixture with a third substance to further improve the transfection efficiency or the stability of the complexes.

 The molar ratio of the lipopolyamine compound according to the invention to the adjuvant neutral co-lipid is preferably from 0.1 to 5, more preferably from 0.25 to 2.

dans laquelle R représente un reste désoxy en position 3 de cholestérol, avec un groupe amine du composé de formule:
H2Nt(CH2)p- NHinH (IX) dans laquelle p et n ont les significations données ci-dessus.The subject of the present invention is also a process for preparing a lipopolyamine compound according to the invention, characterized in that the coupling of a chloroformate compound of formula

in which R represents a deoxy residue at the 3-position of cholesterol, with an amine group of the compound of formula:
H2Nt (CH2) p-NHinH (IX) in which p and n have the meanings given above.

 To promote the production of mono substituted cholesterol derivatives, the process conditions had to be adapted.

 The reaction is carried out in the presence of an excess of amine (IX) 10 to 20 times, at a temperature of -20 ° C., and in an atmosphere of inert gas, In addition, the mono-substituted compound of formula (II) is advantageously obtained by extraction in a water / dichloromethane mixture followed by two crystallization steps in methanol of the dichloromethane fraction: a crystallization solvent (methanol) adapted to the hydrophobicity of the molecules according to the invention had to be selected , able to crystallize in the first place the most hydrophobic molecules (polysubstituted derivatives), then the monosubstituted (compounds of formula (II)).

 To obtain an isomer of a lipopolyamine compound according to the invention, a compound of formula (X) is prepared which corresponds to a compound of formula (IX) in which at least one amine function is not protected, but whose other functions are amines are protected. Compound (X) is then coupled with a chloroformate compound of formula (VIII). A compound is thus obtained whose free amine functions are protected and the said amine functions are deprotected to obtain a compound of formula (II).

 Preferably, to prepare the compound of formula (VI) or (VII) in which p = 3 and 4 and n = 2 or 3, the coupling of one mole of 1,4 diaminobutane and 1 or 2 moles of acrylonitrile, then protect the amine groups, then reduce the group (s) nitrile (s) group (s) amine (s). There is thus obtained a compound of formula (VI) or respectively (VIl) in which the residue C is substituted on a terminal primary amine of the compound of formula (IX).

 Finally, the subject of the present invention is also a process for purifying an isomer of a compound according to the invention from a mixture of isomers of said compounds, characterized in that a high performance liquid chromatography is carried out. reverse phase with a supelcosil LC-ABZ column.

 The method for preparing the isomers in substantially pure form by reverse phase HPLC has proved particularly difficult to implement because of their hydrophobic but nonetheless polar characteristics. In this respect, the choice of the column proved to be decisive: it was necessary to employ a silica-based column strongly derivatized in C 18 groups and deactivated at the surface, such as the supelcosilS LC-ABZ intended for the purification of compounds. basic. The gradient conditions applied are also important for optimizing the purification of isomers whose hydrophobicity is very close.

The results of transfection of cell lines in culture show a capacity for transfection of cells of tissue origin and of different species. In a first series of experiments carried out in parallel with DC Chol (3 ss [N, N '- dimethylaminomethane) carbamoyl] cholesterol), which differs from the compounds of the invention by the presence of
N, N '- dimethylethylenediamine (1 single positive charge) instead of spermine or spermidine (2 or 3 positive charges), there is a significant increase in gene transfer in muscle cells which are usually refractory to transfection . Therefore, the cationic lipids of the invention are particularly suitable for the treatment of muscular diseases, including Duchenne and Becker myopathies. In addition, they are also effective in transfecting lung cells and are suitable for the treatment of lung diseases, including cystic fibrosis, lung cancer and asthma.

 Another advantage of the compounds according to the invention, such as sperminecholesterol and spermidine-cholesterol, is that they result from the association of molecules naturally present in the human body. Cholesterol forms cell membranes and spermine and spermidine are ubiquitously present molecules in cells. This characteristic, which is not shared by the cationic lipids of the state of the art, is an additional advantage in the context of human use and limits the risks of immunogenicity and toxicity in the patient. In this respect, the compounds of the invention are in fact capable of being metabolized by enzymes into natural products, which are therefore not very toxic.

Therefore, they present security guarantees essential for human use.

 Other advantages and features of the present invention will become apparent in light of the detailed description which follows.

The following experimental data are illustrated with reference to the following Figures:
Figure 1 illustrates the analytical HPLC profile obtained with the spermidine-cholesterol compound after synthesis and 6 consecutive extractions water / dichloromethane.

 Figure 2 illustrates the profile obtained with the spermidine-cholesterol preparation in semipresparative HPLC and visualizes the three monoecholesterol isomers.

 Figure 3 illustrates the profile obtained with the spermine-cholesterol preparation in semipresparative HPLC and visualizes the two mono-cholesterol isomers.

 Figure 4 shows schematically the steps of synthesis of the Nl-spermidine cholesterol isomer through protecting groups.

 Figure 5 shows a thin-layer chromatography of spermine-cholesterol and spermidine-cholesterol isomers produced synthetically or purified from the lipid preparation (ninhydrin revealing).

 Figure 6 shows schematically the steps of synthesis of the isomer Ni-spermine cholesterol through protective groups.

 Figure 7 illustrates the transfectional efficacy of the spermidine-cholesterol / DOPE mixture (molar ratio of 1: 0.74). The Y axis represents the absorbance read at 405 nm, the X axis the amount of lipid / colipid (clg) per well and the Z axis the amount of DNA (blood) per well.

 Figure 8 illustrates the transfection efficiency of the C2C12 muscle line with cationic lipids (A) Transfectam, (B) DC-Chol, (C) spermidine-cholesterol and (D) spermine cholesterol.

 Figure 9 illustrates the transfection efficiency of the 3 isomers of spermidine-cholesterol (peak 1 to 3 of Figure 2). The X axis represents the absorbance read at 405 nm and the Y axis the amount of DNA (cil) per well.

1. Synthesis and purification of the spermidine-cholesterol compound
The synthesis is carried out under conditions favoring the production of monosubstituted derivatives. To do this, one places oneself in excess of amines (preferably 10 to 20 times of molar excess), at low temperature (-20 ° C) to slow down the rate of reaction and in an inert gas atmosphere (for example argon ) to meet the anhydrous conditions It is stated that the products spermidine, cholesteryl chloroformate and
N-Hydroxysuccinimide (NHS) are commercially available (eg Sigma, reference S-2626, C-6633 and H-7377 respectively).

We introduce into a first flask under argon:
- 1.6 g of cholesteryl chloroformate (ie 3.4 mmol) taken up in 15
ml of anhydrous dichloromethane (SDS, Peypin, France, reference
2910E16), and
0.5 g of NHS (4.1 mmol) dissolved in 5 ml of dioxane (SDS,
Peypin, France, reference 03610) made anhydrous by passage on
molecular sieve that traps water molecules.

 The mixture A thus formed is left stirring for 15 h at 4 ° C.

In parallel, in a second flask is introduced 10 g of spermidine (69 mmol). After drying under vacuum for 13 to 15 hours, to form mixture B is added:
5 ml of anhydrous dichloromethane, and
5 ml of diisopropylethylamine (40 mmol) (DIPEA, Applied)
Biosystem, St Quentin, France; reference 400136).

 Mixture B is also placed under an argon atmosphere.

 Mixture A is slowly added to mixture B through a 100 μm diameter capillary at a rate of about 1 ml per minute at -20 ° C. with stirring. connected by the same type of capillary to a related flask containing 20 ml of anhydrous dichloromethane placed at a given height which defines the flow rate from A to B.

 The preparation thus obtained is first lyophilized at -80 ° C. to evaporate the solvent and DIPEA. The lyophilisate is taken up in approximately 50 ml of dichloromethane and is then extracted with the same volume of water. After separation of the phases, the dichloromethane fraction is recovered which is subjected to several additional extractions (generally 5) under the above conditions. The free amines which are extremely hydrophilic pass into the aqueous phase. The dichloromethane fraction is collected and then dried.

 Finally, the quality of the product can be improved by two successive stages of crystallization in methanol. The first made in a volume of 120 ml, can crystallize the most hydrophobic products such as cholesterol and polysubstituted amine derivatives. The second crystallization is carried out on the supernatant of the first previously concentrated to 10 ml by evaporation at 65 ° C. The monosubstituted amines are recovered in the recrystallizate at -20 ° C. As an indication, 1 to 1.5 g of spermidine are obtained. cholesterol under these conditions (1.8 to 2.7 mmol).

 It is of course possible to carry out subsequent crystallizations to additional extractions in the water / dichloromethane (volume to volume) mixture to further improve the purity of the product.

2. Synthesis and purification of the spermine-cholesterol compound
The method used is similar to that described above except for the following modifications:
The mixture A is formed by adding 2.09 g of cholesteryl chloroformate (4.65 mmol) placed in 15 ml of anhydrous dichloromethane and 0.6 g of NHS (5.58 mmol) taken up in 5 ml of dioxane.

Mixture B results from the reaction of:
17.7 g of spermine (87.5 mmol, Sigma, reference S-3256)
previously dried under vacuum, and
5 ml of anhydrous dichloromethane, and
5 ml of DIPEA (40 mmol).

 As regards the yields, this process makes it possible to generate 0.5 to 1 g of product (0.8 to 1.6 mmol).

 3. Characterization of lipid preparations spermidine-cholesterol and spermine-cholesterol.

3.1 By thin layer chromatography
Thin layer chromatography analysis is performed after each key step of the purification protocol to rapidly determine the contamination of the synthetic product with the basic constituents. Conventional techniques are applied (E. Stahl, Dunnschicht
Chromatography, ein Laboratoriumshandbuch, 2 ed, Spinger-Verlag
Berlin, Germany, 1967).

 Briefly, an aliquot is deposited on a silica-on-glass plate, which is placed in the N-propanol / ammonia 32% (in water) / water migration solvent (6/3/1 by volume). The various constituents are visualized by the sulfuric acid which reveals all the molecules containing cholesterol or by the ninhydrin 0,1% in ethanol which reveals only the amino products.

 The separation is a function of the hydrophobicity, the most hydrophobic products migrating the fastest. Free spermine and spermidine are detected in the aqueous phases harvested during the water / dichloromethane extractions while the last dichloromethane phase contains the majority of the amine substituted by cholesterol. The characteristic frontal retention (Rf) of each product is 0.71 and 0.68 for mono-cholesterol spermidine and spermine monocholesterol.

3.2 By reverse phase high performance liquid chromatography
The quality of the lipid preparation can also be followed by HPLC (for High Performance Liquid Chromatography in English) under very particular analytical conditions. A water / isopropanol gradient is applied in the presence of 0.1% trifluoroacetic acid (TFA) under the following experimental conditions
column: supelcosil LC-ABZ 15 cm x 4.6 mm (Supelco; 5-9140)
duration of analysis: 35 minutes
flow rate: 0.5 ml / minute
injection: 10 to 100 mg in 100 ul of solvent at initial conditions (80 20% B) /:
gradient A = water + 0.1% trifluoroacetic acid
gradient B = isopropanol (HPLC 205 grade) + 0.1% trifluoroacetic acid
- initial conditions: 80% to 20% B
- 6 minutes: 60 Q / O to 40% B
- 20 minutes: 0% to 100% B - 30 minutes: 096 100% B
- 32 minutes: 80% to 20% B
detection: absorbance at 205 nm.

 In general, the various constituents are separated according to their hydrophobicity: free spermine and spermidine are not retained while the hydrophobic molecules are. The monosubstituted derivatives are eluted first before the polysubstituted derivatives.

 Figure 1 shows the typical HPLC profile obtained on the dichloromethane phase generated during the synthesis of the spermidine-cholesterol compound. The results show that the test preparation contains, for the most part, the desired spermidine mono-cholesterol (retention time 15 to 18 min). The polysubstituted derivatives are also present (22 to 28 min) but will be removed during the subsequent crystallization steps. A technique of indirect analysis of the non-retained fraction makes it possible to estimate the amount of free spermidine contaminating the preparation. To do this, the first fractions harvested are deposited on a thin layer chromatography plate, revealed by ninhydrin and compared to a concentration range established from commercial spermidine.If the contamination is greater than 1%, one or more Additional steps of dichloromethane extraction are introduced into the purification protocol.

 The same procedure is used to analyze the spermine-cholesterol preparations.

 4. Purification and characterization of monocholesterol isomers from lipid preparations.

4.1 Purification of isomers by semi-preparative HPLC
The different monosubstituted isomers have a very close hydrophobicity and have a similar retention time under the previously applied conditions. We must therefore define new conditions that favor their separation in order to be able to characterize them and study their respective transfection efficiency. For this purpose, several hundred g of lipid preparations were deposited on an HPLC supelcosil LC-ABZ column 25 cm × 10 mm (Supelco, reference 5-9170) and eluted in a water / acetonitrile gradient in the presence of 0.1% TFA. Experimental modalities for carrying out the chromatograms of Figures 2 and 3 which illustrate the isolation of the isomers of spermine and spermidine mono-cholesterol respectively are as follows
Figure 2 (Spermidine - monocholesterol)
column: supelcosil LC-ABZ 25 cm x 10 mm (Supelco; 5-9170);
duration of analysis: 12 minutes
flow rate: 5 ml / minute;
injection: - 100 μg in 100 cil of solvent at initial conditions
(70% A30% B):
gradient A = water + 0.1% trifluoroacetic acid
gradient B = acetonitrile (HPLC grade 205) + 0.1% trifluoroacetic acid
- initial conditions: 70% to 30% B
- 1.5 minutes: 65% to 35% B
- 2 minutes: 33 Q / O to 67% B
- 11 minutes: 28% to 72% B
- 11.5 minutes: 70% to 30% B
detection: absorbance at 205 nm
Figure 3 (Spermine - monocholesterol)
column: supelcosil LC-ABZ 25 cm x 10 mm (Supelco: 5-9170);
duration of analysis: 9 minutes
flow rate: 5 ml / minute;
injection: - 500 g in 100 FI of solvent with initial conditions
(65% A35% B):
gradient A = water + 0.1% trifluoroacetic acid
gradient B = acetonitrile (HPLC grade 205) + 0.1% trifluoroacetic acid
- initial conditions: 65% to 35% B - 0.2 minutes: 65% A35% B
- 1 minute: 39% to 61% B
- 8.5 minutes: 34% A66% B - 8.8 minutes: 65% AA 35 ab
detection: absorbance at 205 nm
The chromatogram (Figure 2) shows 3 spermidine-cholesterol peaks: a well-isolated first peak (peak 1 migrating at 7.2 min) and two less proximal peaks (peaks 2 and 3 migrating at 9.6 min and 10.6, respectively). min). Optionally, a reinjection of the fractions covering the latter reduces the contamination of one by the other.

 HPLC analysis of the spermine-cholesterol preparation (Figure 3) produced two well separated peaks exiting at 4.9 min and 7 min.

4.2 Characterization of isomer mass by ionization-electrospray mass spectrometry (ESI-MS)
The HPLC fractions spanning each of the peaks were pooled and analyzed by mass spectrometry (Edmonds and Smith, Electrospray ionization mass spectrometry, in Methods in Enzymology, Vol 193, pp 412-432, McCloskey ed., Academic Press, San Diego, USA , 1990).

 The 3 peaks of spermidine-cholesterol contain products with a molecular mass of about 558 Da which is consistent with that expected for a monosubstituted product (calculated mass: 557.84 Da). These data confirm the production during the chemical synthesis of the three isomers by attachment of the cholesterol group to one of the three amines of spermidine.

 Similarly, the HPLC fractions collected from the lipid spermine-cholesterol preparation contain products with a molecular weight of about 615 Da corresponding to that expected for spermine substituted by a cholesterol molecule (mass calculated 614.96 Da). . Due to the symmetry of spermine, only two isomers can be produced depending on the binding of cholesterol to a primary or secondary amine.

4.3 Characterization of isomers by 13C NMR
The semi-preparative HPLC fractions are extracted into a 20 mM sodium dichloromethane mixture in order to deprotonate the amines before being subjected to 13 C NMR (Gunther, NMR Spectroscopy, Wiley, Chickester, UK, 1995; and Goldstein, 1981, Anal Chem.

53, 789-793; Dagnall et al., 1984, J. Chem. Soc. Perkin Trans. II, 435-440).

This analysis makes it possible to determine which type of amine, primary or secondary, the cholesterol group is attached to. It is performed in parallel with spermine, spermidine and cholesterol standards and the spectra obtained with the test molecules and the standards are compared.

Displacement of signals relative to standards indicates a change in the chemical environment depending on the cholesterol binding site.

 With regard to the spermine-cholesterol compound, the peak 1 visualized on the HPLC profile reported in FIG. 3 contains the isomer in which the cholesterol is bound to the secondary amine and the peak 2 to the primary amine. For spermidine-cholesterol, the substituted isomer on the secondary amine is detected in the fractions covering the peak leaving the most rapidly (peak 1 of Figure 2) and the modified isomers on the primary amines correspond to the peaks. 2 and 3. These have been differentiated by comparison with a standard consisting of the isomer of spermidine-cholesterol obtained in pure form by chemical synthesis (see 5.1). Peak 2 corresponds to the synthetic isomer in which cholesterol is attached to the primary amine of the short arm (CH2) 3 of spermidine and peak 3 to that modified on the long arm (CH2) 4.

Example 5 Synthetic Isomer Preparation
5.1 Synthesis of the substituted spermidine-cholesterol isomer at the primary amine level of the short arm.

The main steps are shown in Figure 4:
1) synthesis of an intermediate product with two intermediates
possible depending on the addition site of the acrylonitrile part
2) protection of amines by a Boc protective group
(Boc = tert-butyloxycarbonyl)
3) Catalytic reduction of the terminal nitrile
4) reaction with cholesterylchloroformate, and
5) deprotection of amines with TFA.

 The protocol applied to obtain 1,4-Diboc-spermidine is published by Goodnow et al. (1990, Tetrahedron 46, 3267-3286) with the difference that the intermediate purification step is omitted. 50 mg (177 μmol) are taken up in 3 ml of anhydrous dichloromethane and 40Zl (417 fumoles) of diisopropylethylamine and then cooled to 0 ° C. The reaction is started by slowly adding 90 mg (200 μmol) of cholesterylchloroformate placed in 3 ml of anhydrous dichloromethane.

After one hour, the reaction is checked by thin layer chromatography. The deprotection of the amines is carried out by addition of 300 μl TFA (final concentration of 4.7%). The reaction mixture is left at room temperature overnight.

 The synthetic product is purified by chromatography on silica gel (5 g of gel for a column 2 cm in diameter and 3.5 cm in height) using a mixture of dichloromethane / ethanol (77/23) to charge the column and dichloromethane / methanol / triethylamine (15/5/1) for elution. The isomer is characterized by reverse phase HPLC and thin layer chromatography. As shown in Figure 5, the synthetic product has a Rf of 0.72 characteristic of spermidine-cholesterol (migration identical to the isomer of peak 2).

 5.2 Synthesis of the spermine-cholesterol isomer substituted at the primary amine level.

The synthesis protocol is schematically represented in the
Figure 6. NN 'dipropionitrile 1,4 diamino butane intermediate is formed by reaction of 2 moles of acrylonitrile and 1 mole of 1,4 diaminobutane. In practice, with stirring (i) 75.2 mmol of acrylonitrile (Fluka, reference 01710) is added at 0 ° C. to (ii) 37.6 mmol of 1,4 diaminobutane (Fluka, reference 32790) taken up in 2 ml of methanol. and 3.8 ml of dichloromethane at 0 ° C. After returning to ambient temperature, 99 mmol of Boc-On (2-Boc-oxyimino) -2-phenylacetonitrile (Fluka, reference 15475) suspended in a mixture are added to the mixture. dichloromethane / methanol (1/3). The Boc group binds to free amines, in this case secondary amines. The "protected" product (dipropionitrile di-Boc 1,4 diamino butane) is subjected to different cycles of evaporation and extractions to remove solvents and contaminants. . Then, the nitrile reduction step is carried out in the presence of 140 mmol of LiAIH4 (Sigma, LO260) taken up in ether while controlling the temperature (0 C).

When it is judged that the reduction is complete (by thin layer chromatography), the reaction is stopped by adding sodium hydroxide. The mixture is filtered, the ether fraction is recovered and the reaction by-products are removed by several extractions with 20% aqueous NaCl solution.

 The product is purified by chromatography on silica gel (5 g of silica per 1 g of product in 10 ml of equilibration solvent). The silica containing the adsorbed product is applied to a column 2 cm in diameter and 23 cm high loaded with 25 g of gel. The equilibration solvent consists of the dichloromethane / ethanol mixture (2/1) and the eluvium buffer with dichloromethane / methanol / triethylamine (15/5/1). The fractions containing spermine-diboc on which the cholesterylchloroformate (1.48 m moles of spermine diboc per 492 11 moles of cholesteryl chloroformate) are determined by thin layer chromatography. The reaction mixture is lyophilized, suspended in dichloromethane, then deprotected in the presence of 5% TFA and finally purified on silica gel chromatography under the same conditions as above except that the column used is more resolutive. The NI spermine-cholesterol isomer is characterized by analytical HPLC and thin layer chromatography. The frontal retention of the synthetic product is characteristic of spermine-cholesterol (FIG. 5) constituted here of the isomer of peak 2.

Example 6: In Vitro Nucleic Acid Transfer
6.1 Plasmids
The reporter plasmid pCH110N is used to evaluate the transfection efficiency of the compounds of the invention. It comprises the early promoter / enhancer block of the SV40 virus (Simien Virus) directing the expression of the LacZ gene coding for Escherichia coli β-galactosidase endowed with a nuclear localization signal from SV40 at the N-terminal.

But any other plasmid can also be used. For example, a plasmid expressing (i) an antibiotic resistance gene, (ii) a luciferase gene or (iii) an easily detectable gene may be mentioned. Such plasmids are known in the art. All experiments are performed in parallel with DC-Chol as a transfection control and, where indicated, transfectam and Upofectin are also included. DC-Chol is obtained by chemical synthesis on a scale of 20 g according to the method described in Gao et al. (1991,
BBRC 179, 280-285). Transfectam and Lipofectin are commercially available (Sepracor, MA, USA; 26395 1 and Gibco BRL, MD, USA; 18292.011 respectively). In order to increase transfection efficiencies, cationic lipids can be associated with a colipid. The selected colipid is DOPE (Sigma, P5078) which is widely used in the state of the art.

6.2 Determination of the optimal lipid to colipid ratio (DOPE)
Transfections are performed in 96-well plates (Falcon) each seeded with 2x104 to 4x104 A549 cells (ATCC CCL 185) derived from human lung carcinoma. The culture is left overnight at 37 ° C in a conventional medium supplemented with fetal calf serum (FCS) at a final concentration of 10%.

 In a first step, the cationic lipid is mixed with a variable amount of DOPE to evaluate the effect of this on the transfection efficiencies. 500 μl of spermine-cholesterol or spermidine-cholesterol are dissolved in 50 to 100 μl of dichloromethane, placed in a sterile glass tube and placed in the presence of a variable amount of DOPE taken up in 50 to 100 μl of chloroform (see table). below).

spermine-cholesterol spermidine-cholesterol
Ratio DOPE lipid Ratio DOPE lipid (M) (crude) ('in) (M) (clg) (crude) 1: 0.5 290 500 1: 0.5 334 500 1: 0.86 500 500 1: 0, 74 500 500 1: 1 580 500 1: 1 668 500 1: 2 1160 500 1: 2 1336 500 1: 4 2320 500 1: 4 2672 500
The mixture is dried under a stream of nitrogen. 1 ml of 0.9% NaCl is added and the mixture is left overnight at 4 ° C. before being sonicated for 3 to 5 min until a milky to translucent solution is obtained. The lipid mixture is stored at 4 C until used. Serial (2-to-2) dilutions of the plasmid solution and the lipid mixture are prepared. A wide range of lipid / DNA complexes are formed by transferring a given volume of DNA dilutions into an identical volume of dilutions. lipid.

 After having aspirated the culture medium, the cells are placed in the presence of 100 μl of the above complexes and incubated in a humid atmosphere at 37 ° C., 5 to 10% CO 2. The medium is added in two stages: 50 μl of medium comprising 30% of FCS 4 at 6 h after the transfection and then 100 l at 10% FCS 24 h after. The last two columns are usually reserved for witnesses and the standard range.

The β-galactosidase activity is revealed, 48 hours post-transfection, after lysis of the cells by adding 50 μl of lysis buffer (0.1% Triton X-100, 250 mM Tris-HCl pH8). 50 μl of PBS buffer comprising 0.5% of
BSA (Bovine Serum Albumin). The standard range consists of serial dilutions of a standard R-galactosidase (Boehringer
Mannheim; reference 567 779) prepared in the same PBS-BSA buffer.

As an indication, the range extends from 50 to 0.39 ng / ml. Finally, 150 μl of ONPG substrate (OrthoNitroPhenol Galactopyranoside Sigma reference N1127) is added at a concentration of 2 mg / ml in PBS, 0.5% BSA.

The absorbance is measured at 405 nm in an Elisa reader after 30 minutes, 4 hours or 24 hours of incubation.

 The best results are obtained when the cells are transfected in approximately equimolar condition (lipid / colipid 1: 1 to 1: 074). Thus, in the case of the spermidine-cholesterol / DOPE mixture at a ratio of 1: 0.74 (FIG. 7), the β-galactosidase activity is high (absorbance reaching 3 to 3.5) for a wide range of concentrations of lipids and DNA. The transfecting power of the mixture in a 1: 2 ratio is effective, especially in the high lipid concentration range. as for the extreme ratios (1: 0.5 or even 1: 4), the transfection activity, although lower, is still acceptable.

 For the spermine-cholesterol / DOPE mixture, a ratio of between 1: 0.86 to 1: 2 is preferred, for which the transfection efficiency is highest for a range of dilutions of the lipid mixture and 1 '. Rather large DNA.

 6.3 Transfection of Established Lines of Muscle Cells.

This series of experiments is conducted in parallel with the two compositions of the invention, DC-Chol and Transfectam on line C2C 12 (ATCC CRL-1772) derived from mouse myoblasts. The cells are distributed in 6-well culture plates due to 5 × 10 5 cells per well, cultured overnight under the usual conditions and transfected the following day by different dilutions of lipid / colipid / DNA complexes (cationic lipid / DOPE ratio approximately equimolar except in the case of the transfectant which is used as such). After 48 h of culture, the cells are fixed and the enzymatic activity is revealed by X-Gal. The nuclei of the transfected cells (which produce a nuclear β-galactosidase) appear in blue and the proportion of stained cells is estimated. The
Figure 8 illustrates the results obtained for each lipid composition.

The number of blue cells is significantly higher with the spermine-cholesterol or spermidine-cholesterol complexes (transfection rate roughly equivalent in both cases) than with those consisting of DC-Chol. In comparison, the transfection rate of Transfectam-based complexes is very low.

 6.4 Transfection of primary myoblasts.

Myoblasts are taken from adult C57 diaphragms
Black 10 or adult mdx mouse thighs (4 weeks). The muscle samples are dissociated in a mixture of dispase and collagenase and the cells are cultured in 24-well plates and in DMEM or F14 medium supplemented with growth factors (insulin, FGF,
EGF, glutamine) and calf serum at a concentration of 10%.

 The cells are transfected in serum-free medium with 1.875 Fg of plasmid pCH11ON complexed with various lipids (spermine-cholesterol, spermidine-cholesterol, DC-Chol and Transfectam) in the presence of DOPE (ratio lipid / DOPE 1: 1 and ratio DNA / mixture lipid 1: 4). For comparison, a naked DNA control is also constituted. 4 h after transfection, the culture is continued in a medium supplemented with serum. Myoblasts are able to proliferate and then merge into plurinuclear structures constituting muscle fibers or myotubes.

The cells are fixed after 48 hours, treated with X-Gal and the estimated number of colored cells. The best transfection rates are obtained with the spermine-cholesterol, spermidine-cholesterol and Transfectam complexes. Blue cells are much weaker in the case of DC-Chol. The naked DNA transfects even less (less than 1% of R-galactosidase positive cells).

 Another series of experiments is conducted on primary cardiomyococci of cardiac muscles of newborn C57 Black 10 pups, with the same lipid mixtures and also lipofectin / DOPE. The transfection is carried out after 4 days of culture in a conventional medium and the transfection efficiency determined 2 days later. Under these conditions, the Transfectam / DOPE mixture gives a percentage of blue cells slightly higher than those based on spermine-cholesterol and spermidine-cholesterol, but much higher than the DC-Chol and Lipofectin / DOPE mixtures. As before, the naked DNA transfects very badly.

6.5 Transfection efficiency of HPLC-purified isomers
The applied protocol is similar to that described in section 6.2 with the difference that 500 μg of isomers purified by semi-preparative HPLC are used in place of the lipid preparation. In the case of spermidine-cholesterol, the spermidine long arm primary amine substituted isomer (corresponding to peak 3 of Figure 2) has the best transfection activity in A549 cells compared to the other two isomers (Figure 9) and the mixture, (not shown).

Claims (33)

 1. Compound of formula:  C- (S) rn (I) in which  C represents a deoxy residue at position 3 of cholesterol or a monovalent or polyvalent residue of a cholesterol derivative carrying one or more reactive groups capable of reacting with a primary or secondary amine group of a polyamine molecule;  S represents an amine residue of said amine group of a polyamine molecule, and  m represents an integer of 1 to 3.  2. Compound of formula:

 in which - R1 and R2 represent hydrogen or a monovalent residue C; the symbol R2 may be identical or different in the n fragments

 Only one of R 1 and n substituents R 2 represents one monovalent residue C; n is an integer from 2 to 6; and - p is an integer from 2 to 6 identical or different in the n fragments

 3. Compound according to claim 2 characterized in that n = 2 or 3 and p = 3 or 4.  4. Compound according to one of claims 1 to 3, characterized in that the C residue is a monovalent residue deoxy position 3 of cholesterol.  5. Compound according to one of claims 1 to 3, characterized in that said residue of the cholesterol derivative is a monovalent residue in position 3 which has the formula:  -L-R (III) in which - L represents an aliphatic radical containing a residue of a group which can react with a primary or secondary amine group and - R represents the residue in the 3-position of the formula

 6. Compound according to claim 5, characterized in that said residue of cholesterol derivative in position 3 has the formula:

 7. Compound according to one of claims 2 to 6, characterized in that it corresponds to the formula:

 wherein R1 and R14, with i = 1 and 2 have the meanings of R1 and Rz respectively given in claim 2.  8. Compound according to one of claims 2 to 6, characterized in that it corresponds to the formula:

 wherein R1 and R12 with i = 1, 2 and 3 have the meanings of R1 and R2 respectively given in claim 2.  9. Compound according to claim 7, characterized in that in formula (VI)  R21 represents a monovalent monovalent residue of cholesterol or a  cholesterol derivative in position 3 and,  - R1 and R22 represent H.  10. Compound according to claim 7, characterized in that in formula (VI)  R1 and R21 represent H and  R22 represents a monovalent residue of cholesterol or a  cholesterol derivative in position 3.  11. Compound according to claim 10, characterized in that in formula (VI)  R1 represents a monovalent monovalent cholesterol residue of a derivative  cholesterol in position 3, and  - R21 and R22 represent H.  12. Compound according to claim 8, characterized in that in formula (VII)  R21 and R22 represent H, and  one of R1 and R23 represents one monovalent residue of a cholesterol derivative in position 3 and the other represents H.  13. Compound according to claim 8, characterized in that in formula (VII)  R1 and R23 represent H, and  one of R2 1 and R22 represents a monovalent residue of  cholesterol or a cholesterol derivative in position 3 and the other  represents H.  14. A pharmaceutical composition comprising a compound according to one of claims 1 to 13 and a therapeutically active substance negatively charged.  15. Composition according to claim 14, characterized in that said active substance is a nucleic acid.  16. Composition according to claim 15, characterized in that said nucleic acid fragment is a DNA or a sense or antisense RNA.  17. Composition according to claim 16, characterized in that said nucleic acid comprises a gene of interest and expression elements of said gene of interest.  18. Composition according to one of claims 15 to 17, characterized in that said nucleic acid is in plasmid form.  19. Composition according to one of claims 15 to 18, characterized in that the weight ratio of the compound of claims 1 to 12 said nucleic acid is from 0.01 to 100.  20. Composition according to one of claims 15 to 19, characterized in that said weight ratio is from 0.1 to 10.  21. Use of a composition according to one of claims 13 to 20 for preparing a transfer vector of said nucleic acid in cells in vitro, ex vivo or in vivo.  22. Nucleic acid transfer vector in cells of microorganisms, plants, humans or animals, especially mammals, characterized in that it comprises a composition according to one of claims 15 to 21 and an adjuvant capable of to improve the transfecting power of said composition.  23. Vector according to claim 22, characterized in that said adjuvant is a neutral co-lipid.  24. Vector according to claim 23, characterized in that said neutral colipid is a phospholipid.  25. Vector according to claim 24, characterized in that said phospholipid is dioleyl phosphatidyl ethanolamine (DOPE).  26. Vector according to one of claims 23 to 25, characterized in that the molar ratio of the compound according to one of claims 1 to 12 to adjuvant neutral co-lipid is from 0.1 to 5, preferably 0.25. to 2.  27. Use of a composition according to one of claims 14 to 20 for the preparation of undit vector of nucleic acid transfer in mammalian muscle cells.  28. Use of a composition according to one of claims 14 to 20 for the preparation of a nucleic acid transfer vector in mammalian lung cells.  29. Process for the preparation of a compound according to one of Claims 2 to 13, characterized in that the coupling of a chloroformate compound of formula

 in which R represents a deoxy residue at the 3-position of cholesterol, with an amine group of the compound of formula:  H2N + (CH2) p - NH H (IX) wherein n and p have the meanings given in claim 2.  30. The process as claimed in claim 29, characterized in that the reaction is carried out in the presence of an excess of amine (IX) of 10 to 20 times, at a temperature of -20 ° C., and in an atmosphere of inert gas.  31. Process for the preparation of a compound according to one of Claims 2 to 13, characterized in that:  a) a compound of formula (X) is prepared which corresponds to a compound  of formula (IX),  H2Ni (CH2) p- NH H  at least one amine function is not protected, but whose  other amino functions are protected  b) the coupling of the compound (X) with a compound  chloroformate of formula (VIII), and  (c) the protected amine functions are deprotected to obtain a  compound of formula (II).  32. Preparation process according to claim 31 for preparing compounds of formula (VI) or (VII) according to claims 10 to 12, characterized in that said compound of formula (X) is prepared by coupling a mole of 1,4 diaminobutane and 1 or 2 moles of acrylonitrile respectively, then the amine groups are protected, then the nitrile group (s) is (are) reduced in amine group (s) and thus a compound of formula (VI) or respectively (VII) in which the C residue is substituted on a terminal primary amine of the compound of formula (Ix) according to claims 10 or 11 or respectively 12.  33. Process for the purification of an isomer of a compound according to one of claims 1 to 13 from a mixture of isomers of said compounds, characterized in that a high performance liquid chromatography is performed in reverse phase with a supelcosil column LC-ABZ.