MXPA97003928A - New transfection agents and their pharmaceutical applications - Google Patents

Abstract

The present invention relates to cationic lipids of general formula I. This also refers to the pharmaceutical compositions containing them and their applications for the transfection of nucleic acids, in vitro or in vivo in the cells.

Description

NEW TRANSFER AGENTS AND THEIR PHARMACEUTICAL APPLICATIONS DESCRIPTION OF THE INVENTION The present invention relates to new compositions related to the family of lipopolyamines, to the pharmaceutical compositions containing them and to their applications for the transfection in vi and / or in vi tro of nucleic acids. Numerous genetic diseases are. associated to a failure of the expression and / or to an abnormal expression, that is to say deficient or excessive, of one or several nucleic acids. Gene therapy has the main objective of correcting this type of genetic anomalies by means of the bias of cell expression in vi tro or in vi vo of the cloned genes. Nowadays, several methods for the intracellular distribution of this type of genetic information are proposed. One of them, in particular, lies in the use of chemical or biochemical vectors. Synthetic vectors have two main functions, to compact the DNA to be transfected, and to promote its cellular fixation as well as its passage, through the plasmidic membrane and, as the case may be, to cross the two nuclear membranes. REF: 24530 An important progress has been achieved in this mode of transfection with the development of a technology based on the use of a cationic lipid. It has been shown that a positively charged cationic lipid, N- [l- (2,3-dioleyloxy) propyl-N, N, N-dimethylammonium chloride (DOTMA), will interfere, in the form of liposomes or from small vesicles, spontaneously with the DNA, which is negatively charged, to form lipid-DNA complexes, capable of fusing with the membranes, cells, and thus allow the intracellular distribution of DNA. However, although this molecule is effective at the level of transfection, it has the disadvantage of being non-biodegradable and of having a toxic nature with respect to cells. From the DOTMA, other cationic lipids have been developed on this structure model: the lipophilic group associated with an amino group via an arm called "spacer". Among these, there may be mentioned more particularly, those comprising as a lipophilic group two fatty acids or a cholesterol derivative, and which also includes, as the case may be, as an amino group, a quaternary ammonium group. DOTAP, DOBT or ChOTB may be cited mainly as representatives of this category of cationic lipids. The other compounds, such as DOSC and ChOSC, are characterized by the presence of a choline group instead of a quaternary ammonium group. In general, the transfectant activity of these compounds is nevertheless very weak. Another category of cationic lipids, lipopolyamines, has also been described. In this type of compound, the cationic group is represented by the radical L-5carboxypermine which contains four ammonium groups, two primary and secondary dosi. The DOGS and DPPES are mainly part. These lipopolyamines are more particularly effective for the transfection of primary endocrine cells. In fact, an ideal synthetic transfection agent should present a high level of transfection, and this for a large spectrum of cells, have a zero toxicity or, if not, a very minimized toxicity at the doses of use, and finally, be biodegradable to get rid of all side effects at the level of the treated cells. The object of the present invention is precisely to propose novel compounds capable of being used efficiently in the transfection in and / or in vi ve of cells mainly for the vectorization of nucleic acids. This has as its first objective the lipopolyamines, under the form D, L or LD and their salts, represented by the general formula I: H2N- ((CH) ra-NHd-H I R wherein m is an integer comprised between 2 and 6 inclusive, n is an integer comprised between 1 and 9 inclusive and more preferably between 1 and 5 with a single R group different from hydrogen present in the general formula, and the values of variables or identical, within different groups - (CH) n- and - (CH2) m-, I R represents a hydrogen atom or a radical of the general formula II: II in which X and X 'represent, independently of one another, an oxygen atom, a methylene group: - (CH1) q- with q equal to 0, 1, 2, or 3, or an amino group -NH - or -NR '- with R' represents an alkylene group of 1 to 4 carbon atoms. Y and Y 'independently represent one of the other a methylene group, a carbonyl group or a C = S group. R3, R4 and R5 represent independently of one another a hydrogen atom or an alkyl radical, substituted or not, of 1 to 4 carbon atoms, with p which may vary between ü and 5, R6 represents a cholesterol derivative or a group alkylamino -NR [beta] R2 with Ri and R2 independently representing an aliphatic radical, saturated or not, linear or branched from 12 to 22 carbon atoms.

Of particular interest are the compounds in which R is represented by the general formula II ' wherein R3, R4, R5, Re and p correspond to the preceding definitions and X represents an oxygen atom or a group - (CH2) q- with q that is equal to zero. This family of compounds is characterized mainly by the presence of an internal ester bond, interesting on the plane of biodegradation. As preferred lipopolyamines according to the invention, the following compounds can be mentioned more particularly: HH alfacolßstьrilo HH under the form D, L, DL or one of its salts. The present invention also aims at any therapeutic application of the lipopolyamines according to the invention, either directly or in pharmaceutical compositions.

As explained above, the compounds of the general formula I are found to be very particularly interesting for transfection in < vi tro and in vi vo of nucleic acids. These effectively compact the DNA and advantageously have a very low toxicity if not with respect to the treated cells. In addition, these are biodegradable mainly by hydrolysis of their ester bond.

To obtain a maximum effect of the compositions of the invention, the respective proportions of the compounds of the general formula I of the nucleic acid are preferably determined such that the ratio R, positive charges of the lipopolyamine considered by negative charges of the nucleic acid is optimum This optimum ratio varies in particular according to the mode of use namely in vi or in vi tro, and according to the cell type to be transfected, this is optimized according to the case. The optimization responds to the competence of the person skilled in the art.

In the compositions of the present invention, the polynucleotide can also be a deoxyribonucleic acid as well as a ribonucleic acid. It can be sequences of natural or artificial origin, and mainly of DNA. genomic, cDNA, mRNA, tRNA, dARNr, hybrid sequence or synthetic or semi-synthetic sequence. These nucleic acids may be of human, animal, plant, bacterial, viral, etc. origin. These can be obtained by any technique known to the person skilled in the art, mainly by selection of banks, by chemical synthesis, or even by mixed methods that include the chemical or enzymatic modification of sequences obtained by selection of banks. These can be otherwise incorporated into vectors, such as plasmid vectors.

More particularly, deoxyribonucleic acids can be single-stranded or double-stranded. These deoxyribonucleic acids can carry therapeutic genes, transcriptional or replication regulatory sequences, modified or non-modified antisense sequences, regions of attachment to other cellular compounds, etc.

In the sense of the invention, the therapeutic gene is understood as meaning any gene which. codifies for a protein product that has a therapeutic effect. The protein product encoded in this way can be a protein, a peptide, etc. This protein product can be homologous to the target cell (ie a product that is mainly expressed in the target cell when it does not present any pathology). In this case, the expression of a protein makes it possible, for example, to shovel an insufficient expression in the cell or the expression of an inactive or weakly active protein, by virtue of a modification, or even to overexpress said protein. The therapeutic gene may also code for a mutant of a cellular protein, which has an increased stowage, a modified activity, etc. The protein product can also be heterologous to the target cell. In this case, an expressed protein can be, for example, completing or contributing a deficient activity in the cell, allowing it to fight against a pathology, or stimulate an immune response.

Among the therapeutic products in the sense of the present invention, mention may be made more particularly of enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, etc. (French Patent FR 9203120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / pleiotrophin, etc. , dystrophin or a minidistrofin (French Patent FR 9111947), CFTR protein associated with mucoviscidosis, tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev, etc. (French Patent FR 93 04745), the genes that code for the factors involved in coagulation: Factors VII, VIII, IX, genes involved in DNA repair, suicide genes (thymidine kinase, cytosine deaminase), the genes of hemoglobin or other protein transporters, the genes that correspond to the proteins involved in the lipid metabolism, of the apolipoprotein type chosen among the apolipoproteins AI, A-II, A-IV, B, CI, C-II , C-III, D, E, F, G, H, J and apo (a), enzymes of metabolism such as lipoprotein-lipase, hepatic lipase, lecithin-cholesterol acyltransferase, 7-alpha- cholesterol-hydroxylase, phosphatidic acid phosphatase, or even lipid transfer proteins such as cholesterol ester transfer protein and transfer protein, phospholipids, an HDL binding protein or even a receptor chosen for example between LDL receptors, receptors e the chylomicrons-remnants and the purifying receptors, etc.

The therapeutic nucleic acid can also be a gene or an antisense sequence, where the expression of the target cell allows to control the expression of the genes or the transcription of the cellular mRNAs. Such sequences can, for example, be transcribed in the target cell in the complementary RNAs of the cellular mRNAs and thus block its translation into protein, according to the technique described in European Patent EP 140 308. The therapeutic genes also comprise the sequences coding for the ribozymes, which are capable of selectively destroying the target RNAs (European Patent EP 321 201).

As indicated above, the nucleic acid may also possess one or more genes encoding an antigenic peptide, capable of generating an immune response in man or animal. In this particular embodiment, the invention thus allows the realization of either vaccines or immunotherapeutic treatment applied to man or animal, mainly against microorganisms, viruses or cancers. It can be mainly antigenic peptides specific to Epstein Barr virus, HIV virus, hepatitis B virus (European Patent No. EP 185 573), pseudo-rabies virus, "syncytium formation virus". , of other viruses and even tumor-specific ones (European Patent No. EP 259 212).

Preferably, the nucleic acid also comprises sequences allowing the sequences of the therapeutic gene and / or the gene encoding the antigenic peptide in the desired cell or organ. These may be sequences that are naturally responsible for the expression of the gene considered when these sequences are capable of functioning in the infected cell. It can also be sequences of different origin (responsible for the expression of other proteins, or even synthetic). Primarily, it can be promoter sequences of eukaryotic or viral genes. For example, it can be promoter sequences from the genome of the cell to be infected. In the same way it can be promoter sequences, outputs of the genome of a virus. In this regard, mention may be made, for example, of the promoters of the genes E1A, MLP, CMV, RSV, etc. In addition, these expression sequences can be modified by the addition of activation, regulation, etc. sequences. It can also be promoter, inducible or repressible.

On the other hand, the nucleic acid can also possess, in particular, upstream of the therapeutic gene, a signal sequence that directs the therapeutic product synthesized in the secretion pathways of the target cell. This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence. The nucleic acid may also possess a signal sequence which directs the synthesized therapeutic product into a particular compartment of the cell.

In another embodiment, the present invention relates to compositions comprising a nucleic acid, a lipopolyamine as claimed and an adjuvant capable of associating, the lipopolyamine / nucleic acid complex and improving the transfectant power. The applicant has indeed shown that the transfectant power of the lipopolyamines can be unexpectedly increased in the presence of certain adjuvants (lipids or proteins for example), capable of associating with the lipopolyamine / nucleic acid complex.

More preferably, the compositions of the invention comprise, as an adjuvant, one or more neutral lipids. Such compositions are particularly advantageous, especially when the ratio R is small. The applicant has indeed shown that the addition of a neutral lipid makes it possible to improve the formation of nucleolipid particles and, surprisingly, to favor the penetration of the particle in the cell, destabilizing the membrane.

More preferably, the neutral lipids used in the context of the present invention are lipids of 2 fatty chains.

In a particularly advantageous manner, the natural or synthetic, amphoteric or non-ionic charged lipids are used under physiological conditions. These can be chosen more particularly between dioleylphosphatidylethanolamine (DOPE), oleoyl-palmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -myristoyl-phosphaitidylethanolamines, as well as their N-methylated derivatives 1 to 3 times; the phosphatidylglycerols, the diacylglycerols, the glycosyldiacylglycerols, the cerebrosides (mainly such as galactocerebrate acids), the sphingolipids (mainly such as sphingomyelins) or even asialogangliosides (mainly such as asialoGMl and GM2).

These different lipids can be obtained either by synthesis, or by extraction from organs (eg, the brain) or from eggs, by the classical techniques well known to the person skilled in the art. In particular, the extraction of natural lipids can be carried out by means of organic solvents (see also Lehninger, Biochemistry). Preferably, the compositions of the invention comprise from 0.1 to 20 equivalents of adjuvant per one equivalent of compound of the general formula I and, more preferably from 1 to 5. The compositions according to the invention can be formulated with a view to administrations via via tó. ica, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intramuscular, transdermal, etc. Preferably, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable carrier for an injectable formulation, primarily for direct injection at the level of the desired organ, or for administration via the topical route (on the skin and / or mucosa). It can be, in particular, sterile, isotonic, or dry, mainly lyophilised, compositions which, by addition according to the case of sterilized water or physiological saline, allow the constitution of injectable solutes. The doses of nucleic acid used for the injection, as well as the number of administrations, can be adapted according to different parameters, and mainly depending on the mode of administration used, of the pathology in question, of the gene to express, or even of the duration of the treatment sought. As regards more particularly the mode of administration, it can be treated either by a direct injection into the tissues, or by a treatment of cells in the culture followed by its reimplantation in vi, by injection or grafting. The present invention thus provides a particularly advantageous method for the treatment of diseases, comprising the in vi ve or in vi tro administration of a nucleic acid capable of correcting the disease associated with a compound of the general formula I under the conditions defined previously. More particularly, this method is applicable to diseases resulting from a deficiency in a protein or nucleic product, and the administered nucleic acid encodes the protein product or contains the nucleic product.

This extends to any use of a lipopolyamine according to the invention for the transfection into a cell or cell. The present invention will be more fully described with the help of the following examples, which should be considered as illustrative and not as limiting.

ABBREVIATIONS OF SYMBOLS AcOEt: Ethyl acetate BOP: Benzotriazole-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate DOCC: Dicyclohexylcarbodiimide DCU: Dicyclohexylurea DMAP: 4-Dimethylaminopyridine DMF: Dimethylformamide DMSO: Dimethyl sulfoxide DODA: Dioctadecylamine EP: Petroleum ether EtOH: Ethanol NEt3: Triethylamine Rf: Coefficient of frontal retention TFA: Trifluoroacetic acid THF: Tetrahydrofuran TMS: Tetramethylsilane UV: Ultra-Violet MATERIAL AND METHODS A. Products used The DODA, the NEt3, the TFA, the L-ornithine, the tetramethylammonium hydroxide, the Raney nickel, the deglycolic anhydride come from Fluka; the BOP, from, Propeptide France; DMAP, isobutyl chloroformate and N-methylmorpholine from Aldrich. THF comes from Merck; all the other solvents used are RP Rolabo products. The solutions of sodium chloride, of sodium hydrogen carbonate are saturated; the potassium acid sulfate solution is at 0.5 M.

B. Physical measures The proton nuclear magnetic resonance spectra (1H NMR) have been recorded on a Bruker spectrometer. Chemical shifts are expressed in ppm relative to TMS.

C. Chromatographies on silica. Thin layer chromatography (CCD) has been carried out on 0.2 mm thick Merck silica gel plates. Disclosures: At UV (254 nm) With ninhydrin, vaporizing (lightly sprayed) an ethanolic solution of ninhydrin (40 mg / 100 ml EtOH) to reveal amines or amides heating at 150 ° C. , With fluorescamine, vaporizing a solution (40 mg / 100 ml of acetone) to reveal the primary amines. With iodine, coating the iodine powder plate. Column chromatographies are carried out on 60 Merck silica gel granules of 0.063-0.200 mm.

EXAMPLE 1.

Synthesis of 2,5-bis- (3-amino-propylamino) -pentyl (Dioctadecyl-carbamoylmethoxy) -acetate. 1. a Preparation of 2, 5-Bis- (2-cyanoethylamino) -pentanoate tetramethylammonium (l.a) The monohydrochloride of (s) -2,5-diaminopentanoic acid (L-ornithine) (6.74 g, 40 mmol) and tetramethylammonium hydroxide pentahydrate (14.48 g, 80 mmol) are dissolved in methanol (20 ml). The water formed and the methanol evaporate under reduced pressure (with the help of an oil pump) in order to obtain a dry residue.

N, N-dimethylformamide (30 ml) is added to the previously obtained salts. The mixture is degassed with nitrogen, then stirred vigorously for 10 minutes, which makes it possible to solubilize tetramethylammonium 2,5-diaminopentanoate (tetramethylammonium chloride remains in suspension in dimethylformamide (DMF)).

Acrylonitrile (6 ml, 85.6 mol) is added dropwise; The ball flask heats slightly again. The reaction sample is left for 1 h at room temperature, under a nitrogen atmosphere. It is then filtered to remove the tetramethylammonium chloride. The dimethylformamide is evaporated under reduced pressure. The obtained residue is an oily liquid; the rotary evaporator is removed, solidifies. Acetonitrile (100 ml) is added and the ball flask is heated slightly until a translucent solution is obtained. Tetrahydrofuran is then added until a cloudy solid is obtained. The ball flask is stored for ten days in the freezer; the tetramethylammonium salt of L-2, 5-bis- (2-cyanoethylamino) -pentanoic acid is recovered by filtration in the form of a white solid. This is rinsed with tetrahydrofuran on a frit, then dried over P205. 6.06 g or a yield of 49% (crude) are obtained. l.b. Preparation of the tetramethylammonium salts of 2,5-bis- (3-amino-propylamino) -pentanoic acid (l.b) The l.a product is solubilized in a mixture of ethanol (18 ml), water (2 ml) and 2M potash. (5 ml). The solution is purged with argon. Raney nickel (2 ml of the Fluka suspension) is added. The hydrogenation is carried out in an autoclave, purged with nitrogen, at 26 ° C. In three hours the pressure goes from 50.6 bar to 44.7 bar, then it is. Stabilizes in more than an hour. The autoclave produces 250 ml and the reaction mixture, 30 ml. The suspension obtained is filtered, rinsed with water and passed through the rotary evaporator. A yellow oil is obtained. The fluorescamine test is positive. 1. c. Preparation of 2,5-bis- [tert -butoxycarbonyl- [3 (tert-butoxycarbonylamino) -propyl-amino] -pentanoic acid l.c) The product l.b is dissolved in dioxane (30 ml). Ditherbutyl dicarbonate (17.46 g, 80 mmol) is added dropwise. The mixture is stirred overnight. The dioxane evaporates. Potassium acid sulfate is added. The product is extracted with chloroform (3 x 100 ml). The organic phase is then washed successively with sodium hydrogen carbonate (pH = 7.5), sodium chloride, then dried over magnesium sulphate and evaporated under reduced pressure. 10 g of the product are obtained (15.4 mmol, or a redeeming of 39% in relation to ornithine). High resolution liquid chromatography (CLAP): MeCN / H20: 0-3 min 50% MeCN; 3-20 in 50-100% MeCN; 20-40 min 100% MeCN, K '= 7.96 * (Column RP-18, expense = l, ml / min) Thin layer chromatography (CCD): Rf (CHC13: AcOEt; 95! 5 (v: v) ) = 0.28 NMR (Nuclear Magnetic Resonance) (ppm): 1.3 (m, 8H, N + CH2-CH2-CH2-CHN + COO / 2xN + CH2CH2CH2N +); 2.7-3.0 (m, 10 H 5x N + CH2); 3.8 (t, 1H, N + CHCOO) Mass Spectrum (MS): MH * - 647, (MW = 646) 1 - . 1 -d. Preparation of 2, 5-bis-. { tert-butoxycarbonyl- [3- (er-butoxycarbonylamino) propyl] -amino} -pentan-l-ol (l.d) The L-5-carboxitetrater-butoxycarbonylpermine (1.94 g, 3 mmol) is dissolved in 30 ml of tetrahydrofuran. 370 ml (3.1 mmol) of N-methylmorpholine are introduced with a micropipette. The reaction mixture, placed under a nitrogen atmosphere, is cooled to 15 ° C (in a dry ice bath and ethylene glycol): 390 ml (3.1 mmol) of isobutyl chloroformate are then added. After three minutes, the reaction mixture is poured into a vessel containing NaBH 4 (2 g) dissolved in 20 ml of water at 4 ° C. The tetrahydrofuran is evaporated, potassium acid sulfate is added (until pH = 7). The product is extracted with ethyl acetate, rinsed with sodium hydrogen carbonate, with sodium chloride, dried over magnesium sulfate, filtered and then evaporated. 1.15 g are obtained, or a yield of 61%. The CCD leads to two spots: a separation on a column of silica, with the same eluent, is then carried out and leads to 0.97 g of the product (yellow oil). The yield of the separation is 84%. The yield of the reduction is 51%. CCD: Rf (EP: AcOEt; 1: 1 (v: v)) = 0.24 NMR (Nuclear Magnetic Resonance) (ppm): 1.42 (s, 4xB? C); 1.5-1.7 (m, 8H N + CH2-CH2.CH2-CHN / 2x N + CH2CH2CH2N); 2.85-3.2 (m, 10H 5x NCH2), 3.4 (m, 1H, NCHCH2OH), 3.7 (d, 2H, CH2OH), 6.8 (m, 2H, NH) Mass Spectrum (MS): MH + = 633, MW = 632 1. e Preparation of 2,5-bis- [3- (tert-butoxycarbonylamino) -propyl] - acid. { tert-butoxycarbonylamino) -penenyl icarbonylmethoxy-acetic acid (1. e) 0. 51 g (0.806 mmol) of product l.d are dissolved in 10 ml of methylene chloride; Two equivalents of diglycolic anhydride (1,535 moi, 0.178 g), 2.2 equivalents of triethylamine are then added (1687 mmol: 235 μl) as well as 5 mg of DMAP. After an hour, the CCD shows that alcohol has? After reaction, methylene chloride is added to the mixture, then washed three times with 50 ml of potassium hydrogen sulfate, 3 times with 50 ml of sodium chloride, dried over magnesium sulfate, filtered and then evaporated. 0.26 g of a solid or a yield of 43% CCD are obtained: Rf (CHC13: MeOH: AcOH; 90: 8: 2 (v: v: v) = 0.75) Mass Spectrum (MS): MH * = 749 , PM- 748 1. F. Preparation of 2-5-bis (Dioctadecyl-carbamoyl ethoxy) -acetate. { ter-butoxycarbonyl- [3- (terbutoxycarbonyl-amino) -propyl] -amino} -pentyl (l.f) 0. 123 g of the preceding product (0.164 mmol), 1 equivalent (0.0856 g) of dioctadecylamine, 3 equivalents of tritylamine (68.4 ml), 1.1 equivalent (0.0798 g) of BOP are dissolved in chloroform. The reaction mixture is stirred at room temperature. After two hours, 100 ml of methylene chloride are added to the mixture, then washed with 3 portions of 100 ml of potassium acid sulfate, sodium hydrogen carbonate, sodium chloride (up to pH = 7), dry, then evaporate. 0.13 g of the product are obtained, or a yield of 63%.

CCD: Rf (EP: AcOEt; 1: 1 (v: v)) = 9.62; disclosure of iodine, ninhydrin and fluorescamine. NMR (Nuclear Magnetic Resonance) (ppm): 0.90 (t, J = 7.5 Hz, 6H: CH3, 1.20-1.75 (m, 108H: CH2 / C (CH3) 3), 3.05-3.35 (m, 14 H: CH2N ), 4.15-4.35 (m, 3H: NCHCH2OCO), 4.23-4.27 (2s, 2x 2H: OCH2CO), 4.5-5.5 (m, extended, 2 H: NH) Mass Spectrum (MS): MH + = 1252, P.M. = 1251 Elemental analysis: Crude formula: C7? H137N50? 2% theoretical: C 68.06 H 11.02 N 5.59% obtained 67.14 H 11.93 N 5.86 l. Di-di-carbamoylmethoxy diol) - 2-5-bis- (3-amino-propylamino) -pentyl (l.q) 1 ml of trifluoroacetic acid is added to or from. cro uc l.f0.02 mmol) in a tube • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1.5 ml, and left for one hour at room temperature. Biological Mass Spectrum (MS): MH * = 852, MW = 851 EXAMPLE 2 Preparation of (Dioctadecyl-carbamoylmethoxy) -acety 1,3-bis- (3-aminopropylamino) -2-propyl (2.f): N HH 2 to . Preparation of 1,3-bis- (2-cyano-ethylamino) -propan-2-or l (2. A) 3. 6 g (40 mmol) of 1,3-diaminopropan-2-ol are dissolved in 75 ml of methanol. 5.76 ml (80 mmol) of acrylonitrile are added in 15 min. The fluorescamine test is positive. The reaction mixture is colorless. This is left under stirring at room temperature, overnight. The fluorescamine test is then negative. The methanol is evaporated. 7.9 g of product are collected (100% yield for the crude product). 2. b Preparation of 1,3-bis- (3-amino-propylamino) -propan-2-ol (2.b) The hydrogenation is carried out in an autoclave, purged with nitrogen at 27 ° C. The product 2.a is solubilized in a mixture of 15 ml of methanol, 10 ml of ethanol, 5 ml of KOH (2 M). The solution is purged with argon and 4 ml of the Raney nickel suspension are added to the autoclave. In 3 h and 30 minutes, the pressure goes from 51.6 bar to 37.6 bar, then stabilizes for more than an hour. The autoclave produces 250 ml and the reaction mixture, 30 ml. The obtained suspension is filtered, rinsed with water and passed to the rotary evaporator. A yellow oil is obtained. The test with fluoroescamine is positive. 2. c. Preparation of 1, 3-bis-. { 3-tert-butoxycarbonyl-amino- (tert-butoxycarbonyl-aminopropyl) -2-propan-2-ol (2.c) The product 2.b is protected from the same », so that l.c. 100 ml of methylene chloride are added. 39.2 g (179 mmol) of di-tert-butyl dicarbonate dissolved in 100 ml of dioxane are added dropwise. The solution is left under stirring for 72 h. The solvent is evaporated, potassium acid sulfate is added. The product is extracted with ethyl acetate (3 x 100 ml); the phases are combined and rinsed with potassium hydrogen sulfate, with sodium hydrogen carbonate, with sodium chloride, dried over magnesium sulphate and evaporated. 20 g are obtained (83% yield in relation to the initial product) The product is crystallized in the EP. CCD: Rf (EP: AcOEt: 1: 1 (v: v)) = 0.23 Rf (EP: AcOEt; 1: 2 (v: v)) = 0.63 NMR (Nuclear Magnetic Resonance) (ppm): 1.4 (2s, 36H: 4x (CH3) 3)); 1.65 (qt, 4H: 2 x NCH; CH; CH2NH); 2.95 (c, 4H: 2 X NHCH2CH2); 3.1 (2d, 4H: NCH: CHCHN); 3.2 (t, 4H: NCH2CH2); 3.85 (m, 1H: 0H); 5.9 (s, 2H: NH) Mass Spectrum (MS): MH * = 605, MW = 604 2. d Preparation of 1,3-bis- acid. { 3-tert-butoxycarbonyl-amino- (tert-butoxycarbonyl-aminopropyl) -2-propy1-2-ethoxycarbonylmethoxy-acetic acid (2.d) 604 mg (1 mmol) of product 2.c are dissolved in 20 ml of methylene chloride. 2 equivalents of anhydride (2 mmol, 0.232 g), 2.2 equivalents of NEt3 (307 μl, and then 100 μl), 6.5 g of DMAP are introduced. After 24 h of stirring at room temperature, methylene chloride is added to the mixture; it is washed with potassium hydrogen sulfate, with sodium chloride, dried over magnesium sulfate, filtered and then evaporated. 0.156 g of the product are obtained (22% yield). CCD: Rf (CHC13: MeOH: AcOH; 90: 8: 2 { V: v: v.}.) = 0.71 NMR (Nuclear Magnetic Resonance) (ppm): 1.2-1.4 (2s, 36H: 4 x ( CH3) 3); 1.5 (, 4H: 2 x NHCH2CH2CH2N); 2.85 (c, 4H: 2 x NHCH 2 CH 2); 3.1 (m, 8H: 4 x NCH2); 3.9- 4. 2 (2 s, 4h: 2 x OCH2COO); 5 . 25 (m, 1H: CH2CHCH2); 6 7 (2H: NH) Mass Spectrum (MS): MH + = 721, MW = 720 2. e Preparation of 1,3-bis- (Dioctadecyl-carbamoylmethoxy) -acetate. { 3-tert-butoxycarbonyl-amino- (tert-butoxycarbonyl-1-aminopropyl) -2-propyl (2.e) 0. 216 mmoles of 2.d acid are dissolved in 3 ml of chloroform. One equivalent of DODA is added (0.113 g), 3 equivalents of NEt3 (100 μl) and 1.1 equivalent of BOP (0.11 g). The reaction mixture is stirred at room temperature for 24 h. The chloroform evaporates. 100 ml of ethyl acetate are added. The organic phase is washed with HCl (0.5 M), with sodium hydrogen carbonate, with sodium chloride until pH = 7, dried over magnesium sulfate and then evaporated. 1,185 g are obtained (70% yield). A column is made on silica. 110 mg of product are collected (yield 42%). CCD: Rf (CHC13: MeOH: AcOH; 90: 8: 2 (v: v: v)) = 0.81 Rf (EP: AcOEt; 1: 1 (v: v)) = 0.67 NMR (Nuclear Magnetic Resonance) (ppm ): 0.85 (t, 6H: 2 x CH3); 1.20-1.55 (m, 100H: CH2 / C (CH3) 3 = 1.6 (m, 4H, 2 x NHCH2CH2CH2N), 3.05-3.4 (m, 16H: 2 x NHCH2CH2CH2N / NCH2CHCH2N / 2 x CH2N); 4.1 (s) , 2H: NCOCH: 0), 4.15 (s, 2H: OCH2COO), 5.25 (m, 1H: CH2CHCH;), 6.15 (, 2H: NH) Mass Spectrum (MS): MHF = 1224, MW = 1223 2. f Preparation of 1,3-bis- (Dioctadecyl-carbamoylmethoxy) -acety. { 3-tert-butoxycarbonyl-amino- (tert-butoxycarbonyl-aminopropyl) -2-propyl (2.f) Add 1 ml of TFA to 0.0247 g of product. 2. e (0.021 mmol) in a 1.5 ml "Eppendorf" ® tube and leave for 1 hour at room temperature. The TFA evaporates. 505 μl of ethanol is added in order to obtain a solution at 40 mM, necessary for biological tests. Mass Spectrum (MS): MH + = 824, P.M. = 823 EXAMPLE 3: Preparation of 2- (3-amino-propylamino) -1- (3-amino-propylamino-methyl-ethyl Jt) (N, N-Dioctadecyl) -succinate H H The compound is prepared according to the protocol described above in Example 2, substituting the diglycolic acid, for succinic acid. The mass spectrometry analysis of the product obtained in this way indicates an MH1 fragment "of 808, which is in accordance with the expected mass.

USE OF LIPOPOLLAMINS ACCORDING TO THE INVENTION FOR THE JN VITRO TRANSFECTION OF GENETIC MATERIAL Airprod-PUCD. Plasmids used for gene transfer in vi tro Plasmid pCMV-LUC is used. This is a construction that includes the transporter or reporter gene of the "luciferase", derived either from the plasmid pGL2-Basic Vector (Promega) or from the plasmid pGL2-Control Vector (Promega) by inserting an Mlu I-HindIII fragment containing the human cytomegalovirus (CMV) promoter, extracted from the plasmid vector pcDNA3 (Invitrogen).

B. Protocol for preparation of solutions. used for transfection The lipopolyamines prepared according to the preceding examples are put into solution in 40 mM in ethanol, then diluted in an ethanol / water mixture maintained in an ethanolic concentration of less than 10%.

The nucleic acid solutions to be transfected are diluted in physiological saline (NaCl 0.15M) then added to the lipopolyamine solutions, in a ratio 1/1 (v / v). After homogenization by vortexing and incubation for 15 minutes at room temperature, the DNA / lipopolyamine solutions are distributed at the final 9% (v / v) in the wells where the cells have been washed with culture medium devoid of proteins ( serum).

EXAMPLE 4: Influence of the charge ratio (amines / phosphates) on the efficiency of transfection.

Samples of 1 x 105 NIH 3T3 cells in exponential growth phase on 2 cm2, are treated with. lipopolyamine / pCMV-LUC solutions, which have variable loading ratios, for 4 hours at 37 ° C with 5% C02; each sample receives 1 μg of nucleic acid. The search for the expression or reporter is made after the vision of final fetal calf serum at 8%, followed by a 40-hour incubation in a C02 stove.

The luciferase activity is dosed by the emission of light [RLU = unit of relative light] in the presence of luciferin, coenzyme A and ATP for 20 seconds, and reported in μg of proteins in the supernatant, after lysis of the cell. The results obtained are reported in table I below.

TABLE I Each value corresponds to the average of three independent tests. This experiment clearly shows that the lipopolyamines according to the invention allow the transfer of genes under the conditions required by their expression.

EXAMPLE 5: Influence of nucleic acid concentration in DNA / lipopolyamine mixtures.

According to the same protocol as that described in the previous example, NIH 3T3 cells are treated with mixtures of DNA / lipopolyamines under the conditions where different concentrations of DNA are used for the same charge ratio. In this case, luciferase activity is measured for 20 seconds, and it is reported at 2.5 x 103 treated cells. The results are presented in Table II and III below.

Lipopolyamine of Example 1 TABLE II Lipopolyamine of Example 2 TABLE III Each value corresponds to the average of three independent tests. The values reported in parentheses correspond to the coefficient of variation expressed in%. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (22)

ION REVIVIRE

1. a lipopolyamine under the form D, L or DL or one of its salts characterized in that it is represented by the general formula I H2N - ((CH) m-NH) n-H R where m is an integer between 2 'and 6 inclusive, n is an integer between 1 and 9 inclusive and more preferably between 1 and 5, when n is between 2 and 9, a single R group different from hydrogen is presented in the general formula, and variable or identical m values, within different groups - ( CH) m- or - (CH2) m-, R represents a hydrogen atom or a radical of the general formula II: II wherein - X and X 'represent, independently of one another, an oxygen atom, a methylene group - (CH2) q- with q equal to O, 1, 2, or 3, or an amino group -NH- or -NR '- with R' represents an alkylene group of 1 to 4 carbon atoms. - Y and Y 'independently represent one of the other a methylene group, a carbonyl group or a C = S group. R3, R4 and R5 represent independently of one another a hydrogen atom or an alkyl radical, substituted or not, of 1 to 4 carbon atoms, with p that can vary between 0 and 5, Rβ represents a cholesterol derivative or a group alkylamino -NRR2 with Ri and R2 representing independently of one another an aliphatic radical, saturated or not, linear or branched from 12 to 22 carbon atoms.

2. The lipopolyamine according to claim 1, characterized in that R is preferably represented by general formula II ' ? J wherein R, R6 and p correspond to the definitions proposed in claim 1 and X represents an oxygen atom, or a group - (CH2) q- with q that is equal to zero.

3. The lipopolyamine according to claim 1 or 2, characterized in that it is under the form D.L.DL or one of its salts.

4. The lipopolyamine according to claim 1 or 2, characterized in that it is HH under the form D, L, DL or one of its salts.

5. The lipopolyamine according to claim 1 or 2, characterized in that it is H H under the form D, L, DL or one of its salts

6. The lipopolyamine according to claim 1 or 2, characterized in that it is alfacolestßrilo under the form D, L, DL or one of its salts.

7. The pharmaceutical composition, characterized in that it contains at least one lipopolyamine according to any of claims 1 to 6, and at least one nucleic acid.

3. The composition according to claim 7, characterized in that the nucleic acid is a deoxyribonucleic acid.

9. The composition according to claim 1, characterized in that the nucleic acid is a ribonucleic acid.

10. The composition according to claim 7, 8 or 9, characterized in that the nucleic acid is chemically modified.

11. The composition according to any of claims 7 to 10, characterized * because the nucleic acid is an antisense.

12. The composition according to any of claims 7 to 10, characterized in that the nucleic acid includes a therapeutic gene.

13. The composition according to claim 12, characterized in that the therapeutic gene codes for a protein involved in lipid metabolism, such as the apolipoprotein chosen among the apolipoproteins AI, A-II, A-IV, B, CI , C-II, C-III, D, E, F, G, H, J and apo (a), an enzyme such as lipoprotein-lipase, hepatic lipase or other lipases, lecithin-cholesterol-acyltransferase, 7-alpha-cholesterol-hydroxylase, the phosphated phosphatidic acid, a lipid transfer protein such as the transfer protein of cholesterol esters, and the transfer protein of phospholipids, an HDL binding protein or even a chosen receptor, for example LDL receptors, clilomicron-remnant receptors and purifying receptors.

14. The pharmaceutical composition, characterized in that it comprises a nucleic acid, a lipopolyamine according to any of claims 1 to 6, and an adjuvant capable of associating with the lipopolyamine / nucleic acid complex and improving its transfectant power.

15. The composition according to claim 14, characterized in that the adjuvant is one or several neutral lipids.

16. The composition according to claim 15, characterized in that the neutral lipid or lipids are chosen from synthetic or natural lipids, amphoteric or devoid of ionic charge under physiological conditions.

17. The composition according to claim 16, characterized in that the neutral lipid (s) are lipids with 2 fatty chains.

18. The composition according to claim 17, characterized in that the neutral lipid (s) are chosen from dioleylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -myristoyl-phosphatidylethanolamine as well as their N-methylated derivatives. 1 to 3 times; phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (mainly such as galactocerebrosides), sphingolipids (mainly such as sphingomyelins) and asialogangliosides (mainly such as asialoGMl and GM2).

19. The composition according to any of claims 14 to 18, characterized in that it comprises from 0.1 to 20 equivalents of adjuvants per 1 equivalent of lipopolyamine, and more preferably, from 1 to 5.

20. The composition according to any of claims 7 to 19, characterized in that it comprises a pharmaceutically acceptable carrier for an injectable formulation.

21. The composition according to any of claims 7 to 19, characterized in that it comprises a pharmaceutically acceptable carrier for an application on the skin and / or mucous membranes.

22. The use of a lipopolyamine according to any of claims 1 to 6, characterized in that it is for transfection in vivo or in vitro cell.