Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
  • C07D233/04—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
  • C07D233/28—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D233/44—Nitrogen atoms not forming part of a nitro radical
  • C07D233/48—Nitrogen atoms not forming part of a nitro radical with acyclic hydrocarbon or substituted acyclic hydrocarbon radicals, attached to said nitrogen atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4164—1,3-Diazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
  • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
  • C07D239/06—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
  • C07D239/08—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms directly attached in position 2
  • C07D239/12—Nitrogen atoms not forming part of a nitro radical
  • C07D239/14—Nitrogen atoms not forming part of a nitro radical with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, attached to said nitrogen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

• the present invention relates to novel compounds useful as nucleic acid transfer agents in cells. These new compounds are more particularly related to the lipopolyamine family, and comprise at least one cyclic amidine function. They are useful for transfection of nucleic acids into different cell types, both in vitro and ex vivo or in vivo.
• nucleic acids into cells may be the transfer of nucleic acids into cells in vitro, for example for the production of recombinant proteins, or in the laboratory, for the study of the regulation of gene expression, the cloning of genes, or any other manipulation involving DNA. It can also involve the transfer of nucleic acids into cells in vivo, for example for the creation of transgenic animals, the production of vaccines, labeling studies or also therapeutic approaches. It may also involve the transfer of nucleic acids into cells ex vivo, in approaches to bone marrow transplants, immunotherapy or other methods involving the transfer of genes into cells taken from an organism, in particular view of their subsequent re-administration.
• cationic lipids have interesting properties. These vectors consist of a polar, cationic part, interacting with nucleic acids, and a lipid, hydrophobic part, promoting cell penetration. Particular examples of cationic lipids are in particular monocationic lipids (DOTMA: Lipofectin®), certain cationic detergents (DDAB), lipopolyamines and in particular dioctadecylamidoglycyl spermine (DOGS) or 5-carboxyspermylamide of palmitoylphosphatidylethanolamine (DPPES) preparation has been described for example in patent application EP 394 1 1 1. Another family of lipopolyamines is represented by the compounds as described in patent application WO 97/18185 incorporated herein by reference, and are illustrated in FIG. 1.
• DOTMA monocationic lipids
• DDAB certain cationic detergents
• DOGS dioctadecylamidoglycyl spermine
• DPES 5-carboxyspermy
• the compounds according to the invention have the unexpected advantage of having a level of transfection in vivo in the muscle at least equivalent to that obtained with unformulated DNA and in any event a very good level of transfection in other tissues.
• the association with a compound according to the invention protects the DNA from degradation by nucleases and / or from deterioration during lyophilization, which contributes to significantly improving the stability of the nucleolipid formulations.
• such an association allows a slow controlled release of nucleic acids.
• the compounds according to the present invention belong to the family of cationic lipids and carry an original cationic region which gives said compounds improved properties, in particular a reduced cytotoxicity compared to the cationic vectors of the prior art.
• This cationic part is in fact more precisely represented by one or more particular polyamine (s), carrying one or more cyclic amidine functions which very probably have the effect of "delocalizing" the positive charges, making the compound overall less cationic, with the effects resulting benefits in terms of toxicity.
• a first subject of the invention relates to new compounds in D, L or DL form, of general formula (I):
• CD CA represents a cycloamidine group and its mesomeric forms of general formula (II):
• n are integers independent of each other between 0 and 3 inclusive and such that m + n is greater than or equal to 1,
• Y represents a carbonyl, amino, methylamino or else methylene group, Y possibly having different meanings within the different groups [( CH 2 ) P -Y], and (*) represents either a hydrogen atom or is the site of bond to the Rep group, it being understood that Ri can be linked to any atom of the general formula (II) , including Z, and that there is a single group Ri in formula (II),
• X represents a group NR 2 or CHR 2 , R 2 being either a hydrogen atom or the bond to the group Ri as defined above,
• r is an integer between 0 and 10 inclusive, r can have different meanings within the different groupings -NR4- (CH) r -,
• R 3 which can have different meanings within the different groups NR 4 - (CH) r R 3 , represents a hydrogen atom, a methyl group, or a group of general formula (VII): - ⁇ iCH ⁇ r- N ⁇ H (VU)
• R4 is defined in the same way as R 3 or else represents a CA group as defined above, it being understood that the CA groups are independent of each other and may be different, and
• R is linked to the carbonyl function of the group Rep of general formula (VI), or else if Rep is absent R is linked directly to the group CA, and represents:
• R ⁇ and R independently of one another represent a hydrogen atom or an aliphatic radical saturated or not, linear or branched, optionally fluorinated, containing 1 to 22 carbon atoms , with at least one of the two substituents R ⁇ or R 7 different from hydrogen and the other containing between 10 and 22 carbon atoms,
• R 9 is a saturated or unsaturated aliphatic radical, optionally fluorinated, containing 8 to 22 carbon atoms, or a steroid derivative, and the two substituents R ⁇ are, independently one of the other, defined as above, or else Rs represents a group -OR 9 for which R 9 is defined as above.
• the group Ri is linked either to Z or to V on the one hand and to the group Rep on the other hand via Y.
• the cycloamidine group CA of formula (II) has 5, 6, 7 or 8 members.
• Rep is a distributor with 1, 2, or 3 "arms".
• distributors 1, 2, or 3 "arms”.
• R 3 represents a hydrogen atom or a methyl and R4 is as defined above, or else R 3 and R 4 present in formula (VI) represent hydrogen atoms, or well R 4 is a hydrogen atom and R 3 is a group of formula (VII) in which R 5 represents a group CA
• p and q are chosen independently of one another from 2, 3 or 4.
• the group R contains at least one hydrophobic segment.
• hydrophobic segment is intended to mean any group of lipid type, promoting cell penetration.
• the group R contains at least one aliphatic chain or at least one steroid derivative.
• the group R represents a group of formula NRôR-7, R O and R 7 being defined as above, or represents a group of general formula (VIII) wherein Q represents a group C (O) NReR 7 , R ⁇ and R 7 being defined as above.
• Re and / or R 7 independently of one another represent a linear saturated or unsaturated aliphatic chain containing 10 to 22 carbon atoms, preferably in 12, 14, 16, 17, 18, or 19 carbon atoms .
• These are, for example, groups (CH 2 ) ⁇ CH 3 , (CH 2 ), 3 CH 3 , (CH 2 ) ⁇ 5 CH 3 , (CH 2 ) ⁇ 7 CH 3 , oleyl, etc.
• the groups R ⁇ and R are identical or different and each represents an aliphatic chain, saturated or not, 8
• R represents a steroid derivative
• this is advantageously chosen from cholesterol, cholestanol, 3- ⁇ -5-cyclo-5- ⁇ -cholestan-6- ⁇ -ol, cholic acid, cholesteryl formiate, chotestanylformiate, 3 ⁇ , 5-cyclo-5 ⁇ -cholestan- 6 ⁇ -yl formiate, cholesterylamine, 6- (1,5-dimethylhexyl) -3a, 5a-dimethyl-hexadecahydrocyclopenta [a] cyclopropa [2,3] cyclopenta [1, 2-f] naphtha-len-10-ylamine, or cholestanylamine.
• These new compounds of general formula (I) can be in the form of non-toxic and pharmaceutically acceptable salts.
• These non-toxic salts include the salts with mineral acids (hydrochloric, sulfuric, hydrobromic, phosphoric, nitric) or with organic acids (acetic, propionic, succinic, maleic, hydroxymaleic, benzoic, fumaric, methanesulfonic or oxalic acids) or with mineral bases (soda, potash, lithine, lime) or even with organic bases (tertiary amines such as triethylamine, piperidine, benzylamine).
• the compounds of the invention can be prepared in various ways. According to a first method, the compounds of the invention can be obtained by synthesis of analogous lipopolyamines (that is to say the same structure but without cycloamidine group), the cyclization into cycloamidine groups being carried out in a second step. Analogous lipopolyamines can be obtained by any method known to those skilled in the art, and in particular according to the methods described in application WO 97/18185 or by analogous methods. The cyclization of the amidine heads can for example be carried out by reaction between one and / or more primary amines of the lipopolyamine and reagents such as O-methylisourea sulfate hydrogen sulfate [J. Med.
• the operation is carried out in an aqueous medium in the presence of a hot base [J. Med. Chem., 1985, pp. 694-698 and J. Med. Chem., 1996, pp. 669-672].
• a hot base mention may be made of water / alcohol mixtures or dimethylformamide.
• a base triethylamine, N-ethyldiisopropylamine, sodium hydroxide, potassium hydroxide, etc. may be used.
• the temperature is preferably between 40 ° C. and 60 ° C., and even more preferably, the reaction is carried out. at 50 ° C.
• Another method consists in carrying out a synthesis of bricks carrying the cycloamidine function which are then grafted on lipids equipped with distributors. This method has the advantage of accessing a large number of products.
• the term "bricks" means any functional segment of the molecule.
• the cycloamidine group CA as defined in the general formula (II), Rep or even R constitute bricks which are distinct from one another within the meaning of the invention. 1 1
• R represents -NP sR- ?
• R represents -NP sR- ?
• R represents -NP sR- ?
• it is commercially available or it can be synthesized according to one of the following methods: • by alkylative reduction between an amine carrying the group R ⁇ s and an aldehyde carrying the group R. It is preferably carried out in a chlorinated solvent (for example dichloromethane, chloroform, dichloro-1,2-ethane, etc. [J. Org. Chem., 1996, pp.
• a leaving group there may be mentioned the halogen atoms (Br, Cl, I) or the substituents tosyl, mesyl, etc.
• the operation is preferably carried out in the presence of a basic reagent, for example carbonate sodium, potassium, sodium hydroxide triethylamine etc., in an alcohol (eg ethanol) at reflux [J. Am. Chem. Soc, 1996, pp. 8524-8530]
• reaction can be carried out in the presence of a non-nucleophilic base in suitable aprotic solvents (such as chloroform, dimethylformamide, methylpyrrolidone, acetonitrile, 12
• Q is either commercially available, or when Q represents a group C (O) R8 with R8 of formula (IX), it can be synthesized by reaction between a commercial chloroformate (for example cholesteryl chloroformate) or obtained according to the conventional methods known to those skilled in the art from a commercial chloroformate, and a commercial diamine (for example N-ethylenediamine) or obtained according to conventional methods known to those skilled in the art.
• a commercial chloroformate for example cholesteryl chloroformate
• a commercial diamine for example N-ethylenediamine
• the procedure is carried out in a chlorinated solvent (for example dichloromethane, chloroform, dichloro-1,2-ethane etc.) or in any other organic solvent compatible with the reaction such as for example dimethylformamide, dimethylsulfoxide, acetonitrile etc.
• a chlorinated solvent for example dichloromethane, chloroform, dichloro-1,2-ethane etc.
• any other organic solvent compatible with the reaction such as for example dimethylformamide, dimethylsulfoxide, acetonitrile etc.
• the group H- [NH- (CH2) x] y-COOH is a commercial amino acid when y is equal to 1, or is obtained by one or more cyanoethylation reactions according to the methods described below in the synthesis of Rep when y is greater than 1.
• the Rep group is obtained by cyanoethylation or by dicyanoethylation (depending on whether one wishes to obtain a linear or branched Rep structure) of an amino acid of formula HOOC- (CH 2 ) r -NH 2 then by reduction of the nitrile functions to amines, a) mono- or di-cyanoethylation:
• the operation is carried out in a basic aqueous medium.
• the reaction is carried out in solvents such as water, alcohols (for example methanol, ethanol etc.) in the presence of a base such as sodium hydroxide, potassium hydroxide, triethylamine etc.
• solvents such as water, alcohols (for example methanol, ethanol etc.)
• a base such as sodium hydroxide, potassium hydroxide, triethylamine etc.
• monocyanoethylation work is preferably carried out cold [J. Am. Chem. Soc, 1950, pp. 2599-2603].
• dicyanoethylation it is preferably carried out hot and with an excess of acrylonitrile [J. Am. Chem. Soc, 1951, pp.
• b) The reduction of the nitrile functions to amines is carried out by catalytic hydrogenation in basic medium or by any other method known to those skilled in the art.
• catalytic hydrogenation in basic medium or by any other method known to those skilled in the art.
• platinum oxide or Raney nickel as a catalyst.
• the solvent chosen is an alcohol (for example methanol, ethanol etc.) in the presence of a base, for example soda, potash etc.
• the Rep-R brick is obtained by peptide coupling between the Rep acid and the R amine obtained in the preceding steps.
• Peptide coupling is carried out according to conventional methods known to those skilled in the art (Bodanski M., Principles and Practices of peptide Synthesis, Ed. Springe-Verlag) or by any known analogous method.
• the reaction can be carried out in the presence of a non-nucleophilic base in suitable aprotic solvents (such as chloroform, dimethylformamide, methylpyrrolidone, acetonitrile, dichloromethane etc.), at a temperature between 0 and 100 ° C, the pH being adjusted between 9 and 11.
• suitable aprotic solvents such as chloroform, dimethylformamide, methylpyrrolidone, acetonitrile, dichloromethane etc.
• CA-Rep-R The compounds according to the invention are obtained according to several possible methods: a) by coupling in basic medium between the terminal amine present on Rep-R obtained in step above, and CA-S-CH 3 , according to the conventional methods known to those skilled in the art. It is preferably carried out in a chlorinated solvent (for example dichloromethane, chloroform etc.) or in other organic solvents compatible with the reaction such as for example water, alcohols, 14
• dimethylformamide etc. in the presence of a base (for example triethylamine, soda, potash, N-ethyldiisopropylamine etc.). and at room temperature (around 20 ° C).
• a base for example triethylamine, soda, potash, N-ethyldiisopropylamine etc.
• the CA-S-CH 3 brick is either commercially available (this is the case for example of 2-methylthio-2-imidazoline hydroiodide), or it can be obtained by the action of a carbon disulfide on a diamine appropriate (that is to say chosen as a function of the cycloamidine group which it is desired to obtain), followed by methylation.
• a carbon disulfide on a diamine appropriate that is to say chosen as a function of the cycloamidine group which it is desired to obtain
• the reaction process is carried out in an alcohol (for example ethanol).
• the methylation step is carried out by the action of a halogenomethyl, the halogen atom possibly being, for example, an iodine atom [J. Am. Cem. Soc, 1956, pp. 1618-1620 and Bioorg. Med. Chem. Lett., 1994, pp. 351-354].
• CA-COOH brick can be obtained in different ways:
• CA-S-CH 3 brick is obtained in the same way as above, and the amino or polyamine acid is chosen according to the compound according to the desired invention, or else
• the operation is carried out in an ethanolic medium in the presence of a base (for example sodium hydroxide) and at the reflux temperature of the mixture.
• a base for example sodium hydroxide
• the amino substituents present in the different groups can interfere with the reactions used, it is preferable to protect them beforehand with compatible radicals which can be put in place and eliminated without touching the rest of the molecule .
• the protective radicals can be chosen from the radicals described by T.W. GREENE, Protective Groups in Organic Synthesis, J. Wiley-Interscience Publication (1991) or by McOmie, Protective Groups in Organic Chemistry, Plenum Press (1973).
• Another subject of the invention relates to a composition comprising at least one compound of formula (I) as defined above.
• another object according to the present invention comprises a compound of formula (I) as defined above and a nucleic acid.
• a compound according to the invention and a nucleic acid are brought into contact, they form a complex by interaction between the positive charges present at physiological pH on the compound according to the invention and the negative charges of the nucleic acid.
• This complex is called “nucleolipid complex” throughout.
• the compound according to the invention and the nucleic acid are present in amounts such that the ratio of the positive charges of the compound to the negative charges of the nucleic acid is between 0.1 and 50, preferably between 0.1 and 20. This ratio can be easily adjusted by a person skilled in the art depending on the compound used, the nucleic acid, and the applications sought.
• nucleic acid is understood to mean both a deoxyribonucleic acid and a ribonucleic acid. They can be natural or artificial sequences, and in particular genomic DNA (gDNA), complementary DNA (cDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (RRNA), hybrid sequences or synthetic or semi-synthetic sequences, of modified or unmodified oligonucleotides. These nucleic acids can be of human, animal, plant, bacterial, viral, etc. origin. They can be obtained by any technique known to those skilled in the art, and in particular by screening of banks, by chemical synthesis, or also by mixed methods including modification 17
• deoxyribonucleic acids can be single or double stranded as well as short oligonuleotides or longer sequences.
• the nucleic acids advantageously consist of plasmids, vectors, episomes, expression cassettes, etc.
• deoxyribonucleic acids can carry an origin of functional or non-functional replication in the target cell, one or more marker genes, transcription or replication regulatory sequences, genes of therapeutic interest, modified or unmodified antisense sequences, regions to other cellular components, etc.
• the nucleic acid comprises an expression cassette consisting of one or more genes of therapeutic interest under the control of one or more promoters and a transcriptional terminator active in the target cells.
• the expression “gene expression cassette” means a DNA fragment which can be inserted into a vector at specific restriction sites.
• the DNA fragment comprises a nucleic acid sequence coding for an RNA or a polypeptide of interest and further comprises the sequences necessary for expression (enhancer (s), promoter (s), polyadenylation sequences etc.) of said sequence.
• the cassette and restriction sites are designed to ensure insertion of the expression cassette into a reading frame suitable for transcription and translation.
• plasmid or an episome carrying one or more genes of therapeutic interest.
• the term “gene of therapeutic interest” in particular means any gene coding for a protein product having a therapeutic effect.
• protein thus encoded can be in particular a protein or a peptide.
• This protein product can be exogenous homologous or endogenous with respect to the target cell, that is to say a product which is normally expressed in the target cell when the latter presents no pathology.
• the expression of a protein makes it possible for example to compensate for an insufficient expression in the cell or the expression of an inactive or weakly active protein due to a modification, or else to overexpress said protein.
• the gene of therapeutic interest can also code for a mutant of a cellular protein, having increased stability, modified activity, etc.
• the protein product can also be heterologous towards the target cell.
• an expressed protein can, for example, supplement or bring about a deficient activity in the cell, allowing it to fight against a pathology, or stimulate an immune response.
• therapeutic products within the meaning of the present invention, there may be mentioned more particularly enzymes, blood derivatives, hormones, lymphokines: interieukins, interferons, TNF, etc. (FR 92/03120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / pleiotrophin, etc.
• BDNF trophic factors
• CNTF NGF
• IGF IGF
• GMF aFGF
• bFGF thelial growth factor
• NT3, NT5 HARP / pleiotrophin
• apolipoproteins (ApoAI, ApoAIV, ApoE, etc., FR 93/05125), dystrophin or a minidystrophin (FR 91/1 1947), the CFTR protein associated with cystic fibrosis, tumor suppressor genes (p53, Rb, RaplA, DCC, k-rev, etc., FR 93/04745), genes coding for factors involved in coagulation (Factors VII, VIII, IX), genes involved in DNA repair, suicide genes (thymidine kinase, cytosine deaminase), genes of hemoglobin or other protein transporters, enzymes of metabolism, catabolism etc.
• the nucleic acid of therapeutic interest can also be an antisense gene or sequence, the expression of which in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs.
• Such sequences can, for example, be transcribed in the target cell into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique. 19
• the therapeutic genes also include the sequences coding for ribozymes, which are capable of selectively destroying target RNAs (EP 321 201).
• the nucleic acid can also contain one or more genes coding for an antigenic peptide, capable of generating in humans or animals an immune response.
• the invention allows the production of either vaccines or immunotherapeutic treatments applied to humans or animals, in particular against microorganisms, viruses or cancers. These may in particular be antigenic peptides specific for the Epstein Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudo-rabies virus, the "syncitia forming virus", d other viruses or tumor-specific antigenic peptides (EP 259,212).
• the nucleic acid also comprises sequences allowing the expression of the gene of therapeutic interest and / or of the gene coding for the antigenic peptide in the desired cell or organ.
• sequences which 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).
• they may be promoter sequences of eukaryotic or viral genes.
• they may be promoter sequences originating from the genome of the cell which it is desired to infect.
• they may be promoter sequences originating from the genome of a virus.
• promoters of the El A, MLP, CMV, RSV, etc. genes can be modified by adding activation, regulation sequences, etc. It can also be a promoter, inducible or repressible.
• the nucleic acid can also comprise, in particular upstream of the gene of therapeutic interest, a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell.
• This signal sequence may be the natural signal sequence of the therapeutic product, but it may 20
• the nucleic acid may also include a signal sequence directing the synthesized therapeutic product to a particular compartment of the cell.
• compositions according to the invention may also comprise one or more adjuvants capable of associating with the complexes formed between the compound according to the invention and the nucleic acid, and of improving their transfecting power.
• the present invention therefore relates to compositions comprising a nucleic acid, a compound of formula (I) as defined above and one or more adjuvants capable of associating with the nucleolipid complexes compound (I ) / nucleic acid and improve its transfecting power.
• This type of adjuvants lipids, peptides or proteins for example
• compositions of the invention can comprise, as an adjuvant, one or more neutral lipids.
• Such compositions are particularly advantageous, especially when the charge ratio R is low.
• the Applicant has indeed shown that the addition of a neutral lipid makes it possible to improve the formation of nucleolipid particles and to promote the penetration of the particle into the cell by destabilizing its membrane.
• the neutral lipids used in the context of the present invention are lipids with two fatty chains.
• natural or synthetic lipids are used, zwitterionic or devoid of ionic charge under physiological conditions. They can be chosen more particularly from dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, - mirystoyl phosphatidylethanolamines as well as their N-methyl derivatives 1 to 3 times, phospol glycols, glycol glycols glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins) or alternatively asialogangliosides (such as in particular asialoGMl and GM2). 21
• DOPE dioleoylphosphatidylethanolamine
• lipids can be obtained either by synthesis or by extraction from organs (example: the brain) or eggs, by conventional techniques well known to those skilled in the art.
• extraction of natural lipids can be carried out using organic solvents (see also Lehninger, Biochemistry).
• a product which may or may not intervene directly in the condensation of the nucleic acid (WO 96/25508).
• product involved in the condensation of nucleic acid is meant to define a compacting product, directly or indirectly, nucleic acid. More precisely, this product can either act directly at the level of the nucleic acid to be transfected or intervene at the level of an additional product which is directly involved in the condensation of this nucleic acid.
• the precompacting product can be any polycation, for example polylysine.
• this product involved in the condensation of the nucleic acid is derived in whole or in part from a protamine, a histone, or a nucleoline and / or one of their derivatives.
• Such a product can also consist, in whole or in part, of peptide units (KTPKKAKKP) and / or (ATPAKKAA), the number of units can vary between 2 and 10.
• these units can be repeated continuously or not. Thus they can be separated by links of a biochemical nature, for example by one or more amino acids, or of a chemical nature.
• compositions of the invention comprise from 0.01 to 20 equivalents of adjuvant (s) for an equivalent of nucleic acids in mol / mol and, more preferably, from 0.5 to 5. 22
• compositions of the present invention further comprise a targeting element making it possible to direct the transfer of the nucleic acid.
• This targeting element can be an extracellular targeting element making it possible to direct the transfer of DNA to certain, cell types or certain desired tissues (tumor cells, hepatic cells, hematopoietic cells, etc.). It can also be an intracellular targeting element making it possible to direct the transfer of the nucleic acid towards certain privileged cellular compartments (mitochondria, nucleus etc.).
• the targeting element can be linked to the compound according to the invention or also to the nucleic acid as has been specified previously.
• sugars peptides, proteins, oligonucleotides, lipids, neuromediators, hormones, vitamins or their derivatives.
• these are sugars of peptides or proteins such as antibodies or fragments of antibodies, ligands of cellular receptors or fragments thereof, receptors or fragments of receptors, etc.
• they may be ligands for growth factor receptors, cytokine receptors, cell lectin receptors, or ligands with RGD sequence with an affinity for adhesion protein receptors such as integrins. Mention may also be made of the transferrin, HDL and LDL receptors, or the folate transporter.
• the targeting element can also be a sugar making it possible to target lectins such as receptors for asialoglycoproteins or for syalydes such as sialyde Lewis X, or alternatively an Fab fragment of antibodies, or a single chain antibody (ScFv).
• lectins such as receptors for asialoglycoproteins or for syalydes such as sialyde Lewis X, or alternatively an Fab fragment of antibodies, or a single chain antibody (ScFv).
• the association of the targeting elements with the nucleolipid complexes of the invention can be carried out by any technique known to those skilled in the art, for example by coupling to a hydrophobic part or to a part which interacts with the nucleic acid of the compound of general formula (I) according to the invention, or to a group which interacts with the compound of general formula (I) according to the invention or with the nucleic acid.
• the interactions in question can be, according to a preferred mode, of ionic or covalent nature.
• compositions of the invention can also incorporate at least one nonionic surfactant in an amount sufficient to stabilize the size of the particles of nucleolipid complexes composed of general formula (I) / nucleic acid.
• non-ionic surfactants prevents the formation of aggregates, which makes the composition more particularly suitable for administration in vivo.
• the compositions according to the invention incorporating such surfactants have an advantage in terms of safety. They also have an additional advantage in that they reduce the risk of interference with other proteins given the reduction in the overall charge of the compositions of nucleolipid complexes.
• the surfactants advantageously consist of at least one hydrophobic segment, and at least one hydrophilic segment.
• the hydrophobic segment is chosen from aliphatic chains, polyoxyalkylene, alkylidene polyester, polyethylene glycol with a benzyl polyether head and cholesterol
• the hydrophilic segment is advantageously chosen from polyoxyalkylene, polyvinyl alccols, polyvinylpyrrolidones or saccharides.
• nonionic surfactants have been described in application WO 98/34648.
• a subject of the invention is also the use of the compounds of general formula (I) as defined above for manufacturing a medicament intended to treat diseases by transfer of nucleic acids (and more generally of polyanions) in primary cells or in the established lines. It can be in particular fibroblastic, muscular, nervous cells (neurons, astrcytes, glyal cells), hepatic, hematopoietic lineage (lymphocytes, CD34, dendritics etc.), epithelial, etc., in differentiated or pluripotent forms ( precursors).
• compositions according to the invention can be formulated for topical, cutaneous, oral administration. , rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal, intraperitoneal, etc.
• the compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for a direct injection into the desired organ, or for topical administration (on the skin and / or mucosa).
• nucleic acid used for the injection may in particular be sterile, isotonic solutions, or dry compositions, in particular lyophilized, which, by addition as appropriate 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 in particular according to the mode of administration used, the pathology concerned, the gene to be expressed, or even the duration of the treatment sought.
• the mode of administration it may be either a direct injection into the tissues, for example at the level of tumors, or the circulatory tract, or a treatment of cells in culture followed by their reimplantation in vivo, by injection or graft.
• the tissues concerned in the context of the present invention are for example the muscles, the skin, the brain, the lungs, the liver, the spleen, the bone marrow, the thymus, the heart, the lymph, the blood, the bones, cartilage, pancreas, kidneys, bladder, stomach, intestines, testes, ovaries, rectum, nervous system, eyes, glands, connective tissue, etc.
• the transfected tissues are the muscles and the lungs.
• the invention further relates to a method for transferring nucleic acids into cells, comprising the following steps:
• nucleic acid bringing the nucleic acid into contact with a compound of general formula (I) as defined above, to form a nucleolipid complex, and 25
• the contacting of the cells with the nucleolipid complex can be carried out by incubation of the cells with the said complex (for uses in vitro or ex vivo), or by injection of the complex into an organism (for uses in vivo).
• the incubation is preferably carried out in the presence of, for example, from 0.01 to 1000 ⁇ g of nucleic acid per 10 6 cells.
• doses of nucleic acid of between 0.01 and 10 mg can for example be used.
• compositions of the invention additionally contain one or more adjuvants as defined above
• the adjuvant (s) are previously mixed with the compound of general formula (I) according to the invention or else with the nucleic acid.
• the present invention thus provides a particularly advantageous method for the treatment of diseases by administration of a nucleic acid coding for a protein or which can be transcribed into a nucleic acid capable of correcting said disease, said nucleic acid being associated with a compound of general formula (I) as defined above, under the conditions defined above. More particularly, this method is applicable to diseases resulting from a deficiency in a protein or nucleic product, the administered nucleic acid coding for said protein product or being transcribed into a nucleic product or even constituting said nucleic product.
• the invention extends to any use of a compound of formula (I) according to the invention for the in vivo, ex vivo, or in vitro transfection of cells.
• the present invention also includes other characteristics and advantages which will emerge from the examples and figures which follow, and which should be considered as illustrating the invention without limiting its scope.
• the Applicant proposes, without limitation, various operating protocols as well as reaction intermediates capable of being used 26
• Figure 1 Structure of synthetic vectors called lipid A, lipid B, lipid c and lipid D in the present invention and described in patent application WO 97/18185 incorporated herein by reference.
• Figure 2 Schematic representation of the plasmid pXL2774.
• FIGS. 3 Phase diagram of the nucleolipid complex compounds (1YADN.
• the binding of compound (1) to DNA was determined by following the decrease in fluorescence (in%, 100% being the fluorescence of naked DNA) of the ethidium bromide (EtBr) (symbol •, solid line), as described along the y-axis on the right.
• the size of the complex particles (in nm) is indicated on the y-axis on the left.
• x represents the transfer agent / DNA charge ratio.
• the size of the nucleolipid complexes without co-lipid is represented by the symbol * in solid line.
• the size of the nucleolipid complexes containing 25% of cholesterol is represented by the symbol D in broken line.
• the size of the nucleolipid complexes containing 40% of DOPE is represented by the broken line symbol. The method does not allow the size of the particles to be determined beyond 3 ⁇ m.
• Figure 4 In vitro gene transfer activity in HeLa cells of the nucleolipid complexes containing the compound (1) according to the present invention without co-lipid (middle bar in dark gray), with 25% cholesterol (left bar in medium gray), and with 40% molar of DOPE (light gray line bar), compared to naked DNA. Only the nucleolipid complexes in which the DNA is completely saturated with the compound according to the invention and whose size is between 100 nm and 300 nm were used. 27
• Figure 5 In vitro gene transfer activity of nucleolipid complexes formed from compound (3), in HeLa cells. On the ordinate is the expression of luciferase, expressed in pg per well transfected. The abscissa shows the charge ratio between the compound (3) and the DNA in nmol / ⁇ g. The expression was measured each time for formulations without co-lipid (micelles), with DOPE and with cholesterol.
• Figure 6 In vitro gene transfer activity of nucleolipid complexes formed from compound (5), in HeLa cells. On the ordinate is the expression of luciferase, expressed in pg per well transfected. The abscissa shows the charge ratio between the compound (5) and the DNA in nmol / ⁇ g. The expression was measured each time for formulations without co-lipid (micelles), with DOPE and with cholesterol.
• Figure 7 In vitro gene transfer activity of nucleolipid complexes formed from compound (6), in HeLa cells. On the ordinate is the expression of luciferase, expressed in pg per well transfected. The abscissa shows the charge ratio between the compound (6) and the DNA in nmol / ⁇ g. The expression was measured each time for formulations without co-lipid (micelles), with DOPE and with cholesterol.
• Figure 8 In vivo gene transfer activity after direct injection into the muscle of the complexes containing the compound (1) according to the present invention or the compound of formula H 2 N (CH 2 ) 3 NH (CH 2 ) 4 NH (CH 2 ) 3 NHCH 2 COArgN [(CH 2 ) 17 CH 3 ] 2 (called “lipid A” below) without co-lipid (bar in dark gray), with 25% cholesterol (bar in medium gray), and with 40 mol% of DOPE (light gray bar), compared to naked DNA. Only the complexes in which the DNA is completely saturated in lipid and whose size is between 100 nm and 300 nm were used.
• Figure 9 The importance of the invention is illustrated by comparing the gene transfer activity of two different lipids, the compound (1) according to the invention and lipid A, and naked DNA via two routes d administration: intravenously (iv) and 28
• intramuscular Only the complexes in which the DNA is completely saturated in lipid and whose size is between 100 nm and 300 nm were used.
• Figure 10 In vivo gene transfer activity 48 hours after i.m. injection nucleolipid complexes containing the compounds (5) or (6) according to the present invention without co-lipid and at charge ratio 0.25 / 1, compared to naked DNA. The expression is expressed in pg of luciferase per ml.
• the bars represent: (a) negative control; (b) naked DNA; (c) Compound (5) and (d) compound (6).
• the starting amino acids, polyamines (or their derivatives) are commercially available. This is the case for example with N- (3-aminopropyl) glycine, N- (2-cyanoethyl) glycine, or even 2,4-diaminobutyric acid, or can be synthesized by conventional methods known in the art. skilled in the art.
• Cyclic isothioureas are also commercial products, such as for example 2-methylthio-2-imidazoline hydroiodide, or can be synthesized by conventional methods known to those skilled in the art.
• Products such as triethylamine, trifluoroacetic acid, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (dimethylamino) phosphonium (BOP), dimethylaminopyridine (DMAP), benzyl chloroformate, di-tert-butyl dicarbonate are commercial products.
• the sodium chloride and sodium carnbonate solutions are saturated.
• the potassium sulphate solution is concentrated to 0.5 M. 29
• TLC thin layer chromatographies
• the apparatus is an assembly for liquid phase chromatography in gradient mode allowing UV detection.
• This preparative chain is composed of the following elements: Pump A: GILSON model 305 equipped with a 50 SC head. Pump B: GILSON model 303 fitted with a 50 SC head. Injection loop. : 5 ml. Pressure module: GILSON model 806. 30
• GILSON model 811 C equipped with a 23 ml head.
• UV detector GILSON model 119 equipped with a preparatory cell.
• Fraction collector GILSON model 202 fitted with racks n ° 21 and 10 ml glass tube.
• Integrator SHIMADZU model C-R6A.
• the solution of product to be purified is loaded onto the column via the injection loop, the eluate is collected in fractions of a tube in 30 seconds.
• the detector is set to the wavelengths of 220 nm and 254 nm. the mobile phases are defined as follows:
• the columns for the analytical separations are Browlee stainless steel columns, 3 cm long and 0.46 cm in diameter, sold by APPLIED BIOSYSTEM. 31
• the stationary phase consists of 7 micron Butyl Aquapore.
• the mobile phases are water (with trifluoroacetic acid) and acetonitrile (with trifluoroacetic acid).
• the injections are 20 ⁇ l of a solution of approximately 1 mg / cm 3 in a loop valve of 0.1 cm 3 .
• the flow rate for the analyzes is adjusted between 1 cmVmin and 4 cm 3 / min.
• the pressure is around 180 bars.
• the separation conditions are summarized below:
• Solvent A Solvent B Demineralized water 2500 cm 3 Acetonitrile for HPLC 2500 cm 3
• the Gly group the amines of which are protected by Boc groups (10 mmol) and the ditetradecylamine (10 mmol) are introduced into a 250 ml flask, and 100 cm J of dichloromethane are added. The mixture is stirred until complete dissolution. 30 mmol of N-ethyldiisopropylamine (DIE A) and 11 mmol of phosphonium benzotriazol-1-yloxytrisdimethylamine (BOP) are then added. The pH is maintained at 10 thanks to the DIEA, and the mixture is stirred for 2 hours. When the reaction is complete, (followed by TLC and / or HPLC), the dichloromethane is evaporated and 35
• the solid obtained above is dissolved in IN sodium hydroxide (200 cm 3 / amine) and dioxane (200 cm 3 ).
• the solution is stirred in an ice bath, then a solution of (t-butoxycarbonyl) 2 O or p-chlorobenzyloxycarbonyl (0.14 mol / amine) in 200 cm 3 of dioxane is added dropwise.
• the pH is maintained at a value greater than 9.
• the mixture is stirred at room temperature (20 ° C) overnight.
• the dioxane is evaporated in vacuo, then the mixture is acidified to pH 3 using a potassium sulphate solution.
• the product (f) whose amines are protected is introduced into a flask equipped with a magnetic bar and dissolved in 10 cm J of methanol per gram of product.
• Palladium on carbon (10%, lg / g of product) and ammonium formate (1 g / g of product) are added at room temperature.
• the hydrogenolysis is followed by HPLC. After two hours, the reaction is complete, the mixture is filtered, and the filter washed with 3 times 10 cm " of methanol per gram of product.
• NCH 2 of propyls and NCH 2 of fatty chains 3.68 (s, 8H: 2 NCH 2 CH 2 N); 3.72 (broad s, 2H: NCH 2 CON); 4.06 (s, 2H: CONCH 2 CON of glycyl).
• the solvent is evaporated under vacuum, then the mixture is acidified to pH 3 with a solution of potassium sulfate. What is insoluble is extracted with ethyl acetate (3 times 200 cm 3 ), then washed with a solution of sodium chloride (2 times 100 cm 3 ). The organic phase is dried over magnesium sulfate, then filtered and evaporated in vacuo. The product obtained is optionally purified on a silica column.
• the product (e) (50 mmol) is introduced into a 1 liter stainless steel autoclave.
• a solution of 10 cm 3 of ethanol (95%) and 3.3 g of sodium hydroxide is prepared at the same time. 80 mol)
• this solution is introduced into the autoclave
• a stream of nitrogen is passed through the autoclave and 2 cm "of Raney nickel on carbon are introduced
• the autoclave is closed
• the initial pressure of hydrogenation is about 52 bar, and it drops to about 48.5 bar overnight at room temperature (20 ° C)
• the suspension is filtered on paper, the filter is washed with ethanol (4 times 25 cm 3 ) , and the filtrates are concentrated to dryness under vacuum.
• the product obtained (0.1 mol amine) is dissolved in IN sodium hydroxide (200 cm 3 / amine) and dioxane (200 cm 3 ) The solution is stirred in an ice bath, then a solution is added dropwise p-chlorobenzyloxycarbonyl (0.14 mol amine) in 200 cm 3 of dioxane The pH is maintained at a value greater than 9 Then the mixture is stirred at room temperature (20 ° C) overnight The dioxane is evaporated in vacuo, then the mixture is acidified to pH 3 using a potassium sulphate solution. What is insoluble is extracted with 42
• the product (g) is introduced into a flask equipped with a magnetic bar and dissolved in 10 cm 'of methanol / g of product.
• Palladium on carbon (10%, lg / g of product) and ammonium formate (1 g / g of product) are added at room temperature (20 ° C).
• the hydrogenolysis is followed by HPLC. After two hours, the reaction is complete, the mixture is filtered, and the filter washed with 3 times 10 cm 3 of methanol / g of product.
• Bi-distilled water is added, and the solution is frozen and lyophilized, or the filtrate is concentrated to dryness and the solid is taken up in ethyl acetate (300 cm 3 ).
• Example 5 The procedure is the same as previously in Example 5. A white solid is obtained which is used without further purification after a TLC analysis.
• the product obtained is used in the same way as above in order to protect the terminal amine with a benzyloxycarbonyl group.
• This example illustrates the preparation of nucleolipid complexes according to the invention.
• the compound used in this example is compound (1) in solution in chloroform. Amounts of 10 nmol of compound (1) (i.e. 11.8 ⁇ g) per ⁇ g of DNA were used. In some cases, a neutral co-lipid, Cholesterol or DOPE, is previously mixed with the compound. A thin lipid film forms when the chloroform is evaporated using a light stream of argon, then it is rehydrated in a mixture of 5% dextrose and 20 mM sodium chloride, overnight, 4 ° C. The samples are then treated with ultrasound for 5 minutes, heated at 65 ° C for 30 minutes, and finally treated again with ultrasound for 5 minutes. Lipid suspensions are thus obtained which are stored at 4 ° C. until they are used.
• the DNA used is the plasmid pXL2774 (FIG. 2) in solution in a mixture of 5% dextrose and 20 mM sodium chloride at a concentration of 0.5 mg / ml or 1.0 mg / ml.
• the plasmid pXL2774 has the following characteristics: - level of endotoxins less than 50 EU / mg,
• RNA content that is to say of mRNA, tRNA and ribosomal RNA, (determined by HPLC) less than 5%
• nucleolipid complexes according to the invention are prepared by rapidly mixing equal volumes of DNA solution and lipid suspension such as 48
• the amount of compound complexed with DNA varies from 0.5 nmol / ⁇ g of DNA to 12 nmol / ⁇ g of DNA.
• Example 8 behavior of complexes formed with different charee ratio
• the size of the complexes was first analyzed by measuring the hydrodynamic diameter by dynamic light scattering (Dynamic Laser Light Scattering) using a Coulter N4plus device the samples are diluted 20 times in a solution containing 5% dextrose and 20 mM sodium chloride to avoid multiple diffusions
• the effect of the cycloamidine group, of the lipid composition, and of the charge ratio on the size of the nucleolipid complexes according to the invention has thus been studied
• FIG. 3 illustrates these 3 phases for the compound ( 1) The same behavior can be observed for other compounds according to the invention
• phase B the DNA is completely saturated with the compound (1), and the complexes are generally neutral or slightly positive.
• This phase is unstable because the ion repulsions are minimal and a phenomenon of “crosslinking” can occur.
• the particle size is well above the detection limit by dynamic light scattering (much greater than 3 ⁇ m). This unstable phase is called "phase B".
• Such a size of complex is not suitable for uses in injection. However, this does not mean that the complexes are inactive in phase B, but they are only in a formulation which is not suitable for their injection for pharmaceutical purposes.
• phase C the nucleolipid complexes are in a form such that DNA is very well protected against enzymes, and their generally positive charge facilitates the passage of the cell membrane of an anionic nature.
• the complexes of phase C are therefore particularly suitable for use for the transfer of nucleic acids into cells.
• the use of a neutral co-lipid has a strong impact on the stability of the complexes, as illustrated in FIG. 3.
• the addition of the neutral co-lipid increases the instability of the complexes, which leads to an increase in the amount of compound required to reach phase C. This is very clearly illustrated in FIG. 3 when comparing the charge ratio at which phase C is reached in the presence and in the absence of co-lipid.
• This example illustrates the capacity of the compound (1) according to the invention to transfect DNA in cells in vitro, compared to unformulated DNA.
• 24-well microplates are seeded with 60,000 HeLa cells (ATCC) per well, and transfected 24 hours later.
• Complexes containing 1 ⁇ g of DNA are diluted in 0.5 ml of DMEM culture medium (Gibco / BRL) in the absence of serum, then added to each well.
• the cells are incubated at 37 ° C for 4 hours.
• the medium containing the complexes is then removed and replaced with a mixture of DMEM and 10% fetal calf serum. Then, the cells are again cultured for 24 hours. Finally, the cells are lysed and tested using a luciferase test kit (Promega) and a Dynex MLX luminometer.
• This example illustrates the capacity of the compounds (3), (5) and (6) according to the invention to transfect DNA in cells in vitro, compared to unformulated DNA.
• This example illustrates the capacity of the compound (1) according to the invention to transfect DNA in cells in vivo, compared to unformulated DNA and to lipid A of condensed formula NH 2 (CH 2 ) 3 NH (CH 2 ) 4 NH (CH 2 ) 3 NHCH 2 COArgN [(CH 2 ), 7 CH 3 ] 2 described in application WO 97/18185 and the structure of which is shown represented in FIG. 1.
• each mouse received 30 ⁇ l of formulation containing 15 ⁇ g of DNA in the anterior tibia muscle.
• the tissues are recovered 7 days after the injection, they are frozen and stored at -80 ° C while waiting to carry out the luciferase activity tests.
• the luciferase activity measurements are made as in Example 8.
• each mouse received 200 ⁇ l of formulation containing 50 ⁇ g of DNA.
• the tissues are recovered in this case 24 hours after the injection, then frozen and stored in the same way as above. 52
• the results of gene transfer in vivo are presented in FIG. 8 and in FIG. 9.
• the ratio between the compound (1) and the DNA is 10 nmol / ⁇ g of DNA.
• the ratio between lipid A and DNA is 4 nmol / ⁇ g of DNA.
• FIG. 8 illustrates the in vivo activity in the muscle of the compound (1) according to the invention compared with naked DNA and lipid A. It can be seen that the levels of luciferase activity are equivalent between naked DNA and compound (1), the latter also having a very improved activity compared to lipid A.
• the transfer mechanisms involved seem different between naked DNA and the use of compound (1) according to the present invention. In fact, the complexes according to the invention used do not contain free DNA (phase C) and, moreover, their in vitro results are much higher than those of naked DNA.
• FIG. 9 compares the activities of compound (1) according to the invention, naked DNA and lipid A, intravenously and intramuscularly.
• the transfection efficiency is roughly equivalent intravenously for lipid A and for compound (1).
• the transfection efficiency of the compound (1) according to the invention is very much higher than that of the liquid A.
• the compound (1) Compared to naked DNA, the compound (1) has an intravenous transfection, in addition to the at least equivalent intramuscular transfection. It therefore appears that the transfer efficiency of the nucleic acid in vivo with the compound (1) according to the invention is generally greater than that with lipid A which is a known cationic lipid and that of unformulated DNA. .
• the complexes according to the invention have the advantage, compared to the transfection of naked DNA, of protecting DNA from degradation by nucleases, thus contributing to a significant improvement in the stability of the formulations.
• the compounds of the present invention can also be used to protect DNA from deterioration during lyophilization, again improving the stability of the formulations. 53
• This example illustrates the capacity of compounds (5) and (6) to efficiently transfect m vivo nucleic acid.
• FIG. 10 shows that the compound (5) and the compound (6), formulated in a charge ratio 0.25 1 with the DNA without co-lipid, have a level of transfection in vivo greater than or equal to naked DNA 48 hours after injection in im

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• 1999
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