MXPA97008111A - Cationic lipids for gen therapy - Google Patents

Abstract

This invention provides novel cationic lipids, particularly guanidino lipids, and methods for their preparation. Polyanion and lipid complexes are also provided which include the lipids of the invention, their preparation and use for transporting biologically active substances, in particular nucleic acids to cellulose.

Description

DESCRIPTION OF THE INVENTION This invention relates to cationic lipid guanidine derivatives, to their preparation and use, and to pharmaceutical compositions in which said derivatives are used. Various lipid-based materials, such as, for example, liposomes, have been used effectively as transporters in various pharmaceutical and biological situations to introduce biologically active substances, such as drugs, radiotherapeutic agents, enzymes, viruses, nucleic acids (for example, constructions). of DNA and RNA, transcription factors and other cellular vectors) in cell lines in culture and in animals. For example, some drugs encapsulated in lipids, such as daunorubicin (DaunoXome ™) and amphotericin B (Abelcet ™, Ambisome "*, Amphotec ™) by the Food and Drug Administration (FDA), have recently been approved, or are subject of applications to the NDA and are under review by the FDA In this regard, considerable effort has been made for the development of lipid-mediated methods for the efficient and efficient transport of genetic material directly to a biological cell. For example, gene therapy techniques alleviate morbid states by transfecting target cells from a patient with nucleic acid constructs that are capable of affecting the processes of gene replication, transcription and translation in a therapeutically desirable manner. of nucleic acid consist of polyanionic molecules of high molecular weight for which it is normally required Transport mediated by transporters for the successful transfection of cells, either in vivo, ex vivo or in vi tro. See, for example, U.S. Patent No. 5,264,618, in which various techniques for the use of lipid-based transporters are described, as well as these same pharmaceutical compositions in clinical application. These transfection methods are also used in the development of new cell lines and of animals that produce proteins with commercial interest. Recently, several lipid transporters have been discovered for the transport of plasmids. See U.S. Patent Nos. 4,897,355; 4,946,787; 5,049,386; 5,366,737; 5,545,412 to Felgner et al., U.S. Patent Nos. 5,264,618; 5,283,185 (for Epand et al., Describing DC-chol); 5,334,761; PCT Publications WO 95/14381; WO 96/01840; WO 96/1841; and WO 96/18372, and Felgner et al., Methods in Enzymology, 5, 67-75 (1993). Although the compounds described in the above references facilitate the entry of biologically active substances into cells, the development of additional lipid transporters that provide higher uptake efficiencies, greater specificity and less toxicity is still desirable. This invention satisfies these needs and other related ones.

One aspect of the invention relates to a cationic guanidine lipid derivative of Formula I: R! R2N-C (0) -A-X Formula I wherein: Ri and R2, which may be the same or different, are C? 0-C26 hydrocarbyl groups; A is a hydrocarbylene group in which one or more methylene groups are optionally replaced by a group Y (as long as none of the groups Y are adjacent to each other), wherein each Y is independently in the direction shown -O- , -OC (O) -, -C (0) 0-, -NR5-, -NR5C (0) -, -C (0) NR5-, NR5C (0) NR5-, -NR5C (0) 0-, -0C (0) NR5-, -S (0) "- (where n is 0, 1 or 2), or -NZ-C (= NZ) NZ-, where each Z is independently H or - where m is an integer from 1 to 10, and each R 5 is independently H or lower alkyl; X is: (1) a trihydrocarbylammonium group, wherein each hydrocarbyl group is the same or different from the others, or (2) -NH-C (= NR3) NHR, wherein R3 and R4 are independently hydrocarbyl, haloalkyl , hydroxyalkyl, O-protected hydroxyalkyl, alkoxyalkyl, haloalkoxyalkyl, aryloxyalkyl, aminoalkyl, mono- or disubstituted aminoalkyl, N-protected aminoalkyl, acyl, alkoxycarbonyl, aryloxycarbonyl, -C (0) NR6R7 (where R6 and R7 are independently H or hydrocarbyl), a nitrogen protecting group, or R3 and R4 together with the atoms to which they are attached form an optionally substituted monocyclic or bicyclic ring, provided that when Ri and R2 are both identical Ce alkyl groups and X is -NH-C (= NH) NH2, A is not a butylene chain; and salts, solvates, resolved or unresolved enantiomers, diastereomers and mixtures thereof. Preferably X is -NH-C (= NR3) NHR4. In other preferred aspects Ri and R2 are identical and are monounsaturated alkenyls. In other preferred aspects, R3 and R are identical and are H, a protected nitrogen, aminoalkyl or aminoalkyl protected in N, hydroxyalkyl or hydroxyalkyl protected in O. Frequently R3 and R4 are aminoalkyl groups represented by - (CH2) P- NH2, where p is an integer from 2 to 10. The amino group can also be protected, preferably by a tert-butyloxycarbonyl group. It will also be recognized that the alkylamino groups may be quaternized or present as the corresponding N oxides. In related aspects, the invention provides lipid-polyanion complexes comprising the cationic lipids of Formula I, methods for their preparation and their use in the transport of biologically active substances to cells. In another aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid, a cationic lipid of Formula I and an optional pharmaceutically acceptable excipient.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows representative cationic lipid compounds of the invention, represented as lipoamido tail groups possessing the structures shown attached to head groups X. Figure 2 shows head X groups representative of the cationic lipid compounds of the invention. Figures 3A and 3B demonstrate the comparative efficiency of DNA transfection of human endothelial cells between commercially available compounds, Lipofectin ™ (Lp) and LipofectAmine ™ (Lf), and compounds of the present invention in serum-free medium (Fig. 3A) and in medium containing serum (Fig. 3B). Figures 4A and 4B demonstrate the comparative efficiency of DNA transfection of human bronchial epithelial cells between commercially available compounds, Lipofectin ™ (Lp) and LipofectAmine ™ (Lf), and compounds of the present invention in serum-free medium (FIG. Fig. 4A) and in medium containing serum (Fig. 4B). Figures 5A and 5B demonstrate the comparative efficiency of DNA transfection of murine 3T3 fibroblast cells between commercially available compounds, Lipofectin ™ (Lp) and LipofectAmine ™ (Lf), and compounds of the present invention in serum-free medium (FIG. Fig. 5A) and in medium containing serum (Fig. 5B).

Figure 6 demonstrates the effect of co-lipids cholesterol and DOPE on the transfection efficiency of the new compound 2-guandino-N, N-di-octadec-9-enyl-propionamide (2B) in human endothelial cells in medium free of serum (Fig. 6B). The proportions in parentheses are the proportions of 2B: cholesterol: DOPE in each experiment. Figure 7A demonstrates the transfection efficiency of compounds of the present invention in relation to other lipid compounds, Lipofectin ™ (Lp), LipofectAmine ™ (Lf) and GS-2888 (lln, used as the free base, lln / bo as the chloride salt, lln / Cl) in epithelial cells. Fig. 7B demonstrates the activity of various of the compounds tested in vascular endothelial cells in culture. Figure 8A shows the in vivo transfection efficiency of compounds of this invention in relation to DOTMA ™ when administered by instillation through the airways in rats.

DEFINITIONS AND GENERAL PARAMETERS A. DEFINITIONS The following definitions are enunciated to illustrate and define the meaning and scope of the various terms used to describe this invention. The term "hydrocarbyl" refers to a monovalent hydrocarbon radical formed by carbon chains or rings of up to 26 carbon atoms, to which hydrogen atoms are attached. The term includes alkyl, cycloalkyl, alkenyl, alkynyl and aryl groups, groups possessing a mixture of saturated and unsaturated bonds, carbocyclic rings, and includes combinations of said groups. It can refer to a linear chain, a branched chain, cyclic structures, or combinations thereof. The term "hydrocarbylene" refers to a divalent hydrocarbyl radical. Representative examples include alkylene, phenylene, cyclohexylene, dimethylenecyclohexyl, 2-butene-1,4-diyl, and the like. Preferably, the hydrocarbylene chain is completely saturated, and / or has a chain of 1 to 10 carbon atoms. The term "trihydrocarbylammonium" refers to the group (R) 3N + -, wherein each R is independently a hydrocarbyl radical, preferably lower alkyl. An aliphatic chain includes the alkyl, alkenyl and alkynyl classes defined below. A linear aliphatic chain is limited to unbranched carbon chain radicals. Alkyl refers to a fully saturated branched or unbranched carbon chain radical, which has the specified number of carbon atoms, or up to 26 carbon atoms if not specifically specified. For example, an alkyl of 1 to 8 carbon atoms refers to radicals such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl and octyl, and to those radicals that are positional isomers of these radicals. "Lower alkyl" refers to an alkyl of 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. An alkyl of 6 to 26 carbon atoms includes hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, trichosyl and tetracosyl. Alkenyl refers to any branched or unbranched unsaturated carbon chain radical having the specified number of carbon atoms, or up to 26 carbon atoms if limitation in the number of carbon atoms is not specified; and that has one or more double bonds in the radical. An alkenyl of 6 to 26 carbon atoms is exemplified by hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosenyl, docosenyl, tricosenyl and tetracosenyl, in their various isomeric forms, in which the unsaturated bond or bonds can be located anywhere in the radical and can have either the (Z) or (E) configuration in the double bonds. Alkynyl refers to hydrocarbon radicals with the same scope as for alkenyl, but which possess one or more triple bonds in the radical. The term "lower alkoxy" refers to the group -O-R ', where R' is lower alkyl. The term "polymethylene" refers to the group - (CH2) n- where n is an integer from 2 to 10. The term "methylene" refers to the group -CH2-.

The term "butylene" refers to the group - (CH2) 4-. The term "alkylene" refers to a divalent saturated aliphatic radical. The term "carbonyl" refers to the group -C (0) -. The term "hydroxycarbonyl" or "carboxy" refers to the group -C (0) OH. The term "lower alkoxycarbonyl" refers to the group -C (0) OR 'where R' is lower alkyl. The term "acyl" refers to the group -C (0) R ', where R' is hydrogen or hydrocarbyl, for example methylcarbonyl, ethylcarbonyl, benzoyl, naphthoyl, and the like. The term "carbamoyl" refers to the group -C (0) NR'R where R and R 'are independently hydrogen or lower alkyl, for example when R is hydrogen and R' is lower alkyl, the group is lower alkyl-carbamoyl; when R and R 'are lower alkyl, the group is di-lower alkyl-carbamoyl. The term "monosubstituted amino" refers to the group -NHR, where R is hydrocarbyl or acyl. The term "disubstituted amino" refers to the group NR'R ", where R 'and R" are independently hydrocarbyl or acyl. The term "halo" refers to fluoro, bromo, chloro and iodo. The term "aryl" refers to an aromatic monovalent mono- or polycarboxylic radical. The term "(lower alkyl) -hydroxymethyl" refers to the group -CH (OH) - (lower-alkyl).

The term "arylmethyl" refers to the group aryl-CH2-. Aralkyl refers to an organic radical derived from an alkyl radical in which a hydrogen atom is replaced by an aryl group. Representative examples are benzyl, phenethyl, 3-phenylpropyl, and the like. Monocyclic rings generally have 3 to 8 atoms per ring. Bicyclic rings generally have 7 to 14 atoms per ring. The carbocyclic rings are those ring systems in which all the ring atoms are carbon. Heterocyclic rings (heterocycles, heterocycle, etc.) are those ring systems in which at least one ring atom is a heteroatom, typically O, N or S (0) n (where n is 0, 1 or 2). The term "protecting group" refers to a grouping of atoms such that, when bound to a reactive group in a molecule, it masks, reduces or prevents the reactivity of this group. Examples of protecting groups can be found in T. W. Greene et al., Protective Groups in Organic Chemistry (Wiley, 2nd ed., 1991) and Harrison and Harrison et al., Compendium of Synthetic Organic Methods, vols. 1-8 (John Wiley and Sons, 1971-1996). Representative amine protecting groups include the formyl group, or the lower alkanoyl groups having from 2 to 6 carbon atoms, in particular the acetyl or propionyl group, the trityl or trityl substituted groups, such as the monomethoxytrityl group, dimethoxytrityl groups such such as 4,4'-dimethoxytrityl or 4,4'-dimethoxytriphenylmethyl group, trifluoroacetyl, allyloxycarbonyl, t-butyl carbamate (t-BOC), 1-adamantylcarbamate, benzyl carbamate (Cbz), 9-fluorenylmethyl carbamate (FMOC), nitro-veratriloxycarbamate (NVOC), the phthalyl group and the like. Representative hydroxyl protecting groups are those in which the hydroxyl is either acylated or alkylated, and include the benzyl and trityl esters, as well as the alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers. Biologically active substance refers to any molecule or mixture or complex of molecules that exerts a biological effect in vi tro and / or in vivo, including pharmaceuticals, drugs, proteins, vitamins, steroids, polyanions, nucleosides, nucleotides, polynucleotides, nucleic acids, etc. The taps described in this discovery include "Tris", "HEPES" and "PBS". "Tris" is tris (hydroxymethyl) -aminomethane, and for the purposes of the preferred aspects of this invention, it is used at a pH of about 7. "HEPES" is N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, also used here at a pH of about 7. Phosphate buffered saline, or "PBS", is 10 mM sodium phosphate and 0.9% by weight NaCl, used as an isotonic physiological buffer at a pH of 7.4. A polyanion is a biologically active polymeric structure such as a polypeptide, polynucleotide, nucleic acid, or other macromolecule, wherein more than one unit of the polymer has a negative charge and the net charge of the polymer is negative. A complex (or a liposome complex) is defined as the product obtained after mixing pre-formed liposomes including a lipid of Formula I with a polya-nion. Such a complex is characterized by an interaction between the polyanion and lipid components which results in the elution of the polyanion and the liposome together as substantially the same entity through a gel filtration column which separates based on the Stoke radius or by other process from separation. A charge ratio refers to a quantitative relationship between the net positive charges contributed by the lipid and the negative net charges contributed by the polya-nion in a complex. The charge ratio in this invention is expressed as positive with respect to negative, that is, 5: 1 means five net positive charges in the lipid for a net negative charge in the polyanion. A liposome-polyanion complex is a composition of matter produced by contacting a polyanion solution with a preparation of liposomes produced from a lipid of Formula I (with optional co-lipids if appropriate). Optional or optionally means that the event or circumstance described below may or may not take place, and that the description includes cases in which said event or circumstance takes place and cases in which it does not.

Optional substituents include alkyl, cycloalkyl, alkenyl, alkynyl, aryl, haloalkyl, hydroxy, amino, halo, nitro, cyano, carboxy, carbamoyl, alkoxy, haloalkoxy, mono- and di-substituted amino, acyl, alkoxycyl, aryloxyacyl and the like. An optional co-lipid should be understood as a structure capable of producing a stable liposome, either alone or in combination with other lipid components including the cationic guanidino lipids of this invention. It can be neutral, or it can be positively or negatively charged. Double layer complexes are prepared from liposome complexes that have a net positive charge. Liposome complexes possessing a net positive charge are prepared using a higher molar amount of positively charged lipid than the molar amount of negative charge contributed by the polyanion. These positively charged complexes are mixed with negatively charged lipids to produce double layer complexes. If enough negatively charged lipid is added, the final complex will have a net negative charge. This definition includes lipo-somas that possess additional modifications in the surface, such as the incorporation of antibodies or antigens in it. DNA means deoxyribonucleic acid, and may optionally comprise non-natural nucleotides. The DNA can be single-stranded, double-stranded or present in the form of a triple helix.

RNA means ribonucleic acid, and may additionally include non-natural nucleotides. The RNA can be single chain or double chain. A polynucleotide is DNA or RNA that contains more than one nucleotide. The polynucleotides can be obtained by synthetic chemical methodology, available to people normally knowledgeable in the field, or by the use of recombinant DNA technology, or by a combination of the two, and include those that incorporate non-natural nucleotides. "Antisense" refers to a nucleotide sequence that is complementary to a specific sequence of nucleotides in DNA or RNA. The term "nucleic acid" refers to DNA (e.g., genomic DNA, cDNA), RNA (e.g. mRNA, ribose-mal RNA, tRNA, antisense RNA), ribozymes, oligonucleotides, polynucleotides, mixed duplexes and triple helices of DNA and RNA, plasmids, expression vectors, etc., including those sequences containing non-natural nucleotides. Drug refers to any therapeutic or prophylactic agent other than a food, which is used in the prevention, diagnosis, relief, treatment or cure of a disease in an animal or in man. (Therapeutically useful polynucleotides, nucleic acids and polypeptides are within the scope of this drug definition). A pharmaceutical formulation is a composition of matter that includes a drug, for therapeutic administration to a human or animal. 1 A pharmaceutically acceptable anion is an anion which by itself is non-toxic and is otherwise pharmaceutically acceptable, and whose presence does not result in the compound being pharmaceutically unacceptable. Examples of such anions are chloride, bromide and iodide halides. Inorganic anions such as sulfate, phosphate and nitrate can also be used. Organic anions can be derived from simple organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid and the like. A stable transfectant is a living cell into which DNA that has been integrated into the genomic DNA of said cell has been introduced. Topical administration includes application on any surface of the body, including ocular administration and administration on the surface of any body cavity. Transdermal administration is administration through the skin with a systemic effect. Transfection is concerned, for the purposes of this invention, to the introduction of DNA or RNA into a living cell. Unnatural nucleotides include those commercially available or that can be manufactured directly by known means from those of ordinary skill in the art. The term "pharmaceutically acceptable salt" refers to any salt derived from an inorganic or organic acid or base. As used herein, the terms "treatment" or "treating" a condition and / or a disease in a mammal means: (i) avoiding the condition or disease, i.e. avoiding any clinical symptom of the disease; (ii) inhibit the condition or disease, that is, stop or reduce the development or progression of clinical symptoms; and / or (iii) relieving the condition or disease, that is, causing regression of clinical symptoms. As used herein, the term "therapeutically effective amount" refers to that amount of a biologically active substance that, when administered to a mammal in need, is sufficient for the treatment to take place. The amount that constitutes a "therapeutically effective amount" will vary depending on the substance, state or disease and its severity, as well as the mammal to be treated, although it can be determined routinely by a person normally understood in the field. taking into account current knowledge and this invention. All temperatures are given in degrees Celsius (ie, ° C).

Unless otherwise specified, the reactions described herein take place at atmospheric pressure in a temperature range from about -78 ° C to about 150 ° C, more preferably from about 10 ° C to about 50 ° C. , and more preferably at about room temperature, for example at about 20 ° C. Unless otherwise specified, the time and temperature ranges described herein are approximate, for example, "from 8 to 24 hours at 10 ° C to 100 ° C", meaning approximately 8 to 24 hours at about 10 ° C to 100 ° C. The isolation and purification of the compounds and intermediates described herein can be carried out, if desired, by any suitable separation or purification process, such as, for example, filtration, extraction, crystallization, column chromatography, high pressure liquid chromatography. preparative (preparative HPLC), thin layer chromatography or thick layer chromatography, or by a combination of these procedures. Specific illustrations of suitable separation and isolation procedures can be obtained by reference to the examples described below. However, other equivalent separation or isolation procedures may also be used. In the following examples some representative compounds are named: [CH3 (CH2) -CH = CH. { CH2) 8] 2N-C (O) CH2NHC (= NH) NH2 2-guanidino-N, N-dioactadec-9-enyl-acetamide [CH3 (CH2) 7CH = CH (CH2) g] 2N-C (O) CH2CH2NHC (= NH) NH2 3-guanidino-N, N-dioctadec-9-enyl-propionamide [CH3 (CH2) 7CH = CH (CH2) "] 2N-C (O) CH2CH2NHC (= NBOC) NHBOC 3- [N ', N' '-bis (tert-butyloxycarbonyl) guanidino] -N, N-dioctadec-9-enyl-propionamide [CH3 (CH2) 7CH = CH (CH2) 8] 2N-C (O) CH2CH2NHC (= NC2H4NHBOC) -NHC2H4NHBOC 3- [N ', N "-bis (2-tert-butyloxycarbonylaminoethyl) -guanidino] -N, N-dioctadec-9-enyl-propionamide [CH3 (CH2) 7CH = CH (CH2) 8] 2N-C ( O) CH2CH2NHC (= NC3H6NHBOC) -NHC3H6NHBOC 3- [N ', N "-bis (2-tert-butyloxycarbonylaminopropyl) -guanidino] -N, N-dioctadec-9-enyl-propionamide [CH3 (CH2) 7CH = CH (CH2) 8] 2N-C ( O) CH2CH2NHC (= NCH2CH2NH2) -NHCH2CH2NH2 3- [N ', N "-bis (2-aminoethyl) -guanidino] -N, N-dioctadec-9-enyl-propione ida [CH3 (CH2) 7CH = CH ( CH2) a] [C ??H35] NC (O) CH2CH2NHC (= NH) NH2 3-guanidino-N-octadec-9-enyl-N-octadecyl-propionamide The compounds are also represented and identified in reference to the structures shown in Figures 1 and 2. In this scheme, the compounds are represented in the form of a lipoamide tail group attached to a cationic head X group. Figure 1 shows the representative lipoamide tail groups 1-14, and Figure 2 shows the structures of representative cationic head groups A-U. Accordingly, a compound defined as IB refers to the compound in which the lipoamido tail group 1 is attached to the cationic head group B, ie, 2-guanidino-N, N-dioctadec-9-enyl-acetamide.

SYNTHESIS OF FORMULA COMPOUNDS I As used in the Reaction Schemes, Rir R? R3? R4 / Rs, Re and R are as described in the Summary of the Invention. Reaction Scheme A illustrates a representative scheme for the preparation of new cationic guanidine lipid derivatives, ie, compounds of Formula I. Reaction Scheme B illustrates a representative scheme for the preparation of asymmetric amines R? R2NH used as materials starting in Reaction Scheme A.

REACTION SCHEME A NH HOOC NHBOC R ') PEPTIDE COUPLING BOP / DI EA / DMF STAGE 1 NHBOC DISPROTECTOR AGENT EXfiPA 2 (Ha > t 1 \ NH2.HQ *. { STAGE 3 REACTION SCHEME B l ^ NH, RCOOH IDICO OR R, HN AR AGENT. REDUCER LiAIH4 / THF R * NH < STARTING MATERIALS With reference to Reaction Schemes A and B, the starting materials are available from Aldrich Chemicals Co., Inc., Fluka Chemical Corporation, K &; K Chemi-cals, Eastman Kodak Chemicals, Lancaster Synthesis Ltd., Karl Industries, Maybridge Chemical Co. Ltd., or Tokyo Kasai International. The long chain acids are preferably obtained from Nu Chek Prep Inc., (Elysian, MN). Those compounds that are not commercially available can be prepared by persons normally skilled in the art following the established procedures in references such as "Fieser and Fieser's Reagents for Organic Synthesis", Volumes 1-15, John Wiley and Sons, 1991; "Rodd 's Chemistry of Carbon Compounds", Volumes 1-5 and Supplements, Elsevier Science Publishers, 1989; and "Organic Reactions", Volumes 1-40, John Wiley and Sons, 1991.

PREPARATION OF AMINO R? R2NH Referring to Reaction Scheme B, intermediates of structure R? R2NH can be synthesized by first coupling an amine of formula RNH2 with a carboxylic acid of formula RiCOOH in the presence of an activating group such as N -hydroxysuccinimide, p-nitrophenol, pentachlorophenol, pentafluorophenol and the like, and a coupling agent such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), N-hydroxybenzotriazole (HBOT), hydrochloride N-hydroxybenzotriazolephosphoryl, isobutyl chloroformate, N, N'-carbonyldiimidazole or the like. The coupling is generally carried out in an anhydrous non-hydroxylic organic solvent. The resulting amide is then reduced with a reducing agent such as a metal hydride (for example, lithium aluminum hydride, diborane or the like) to yield the desired amine RXR2NH. The RNH2 amines and the RCOOH acids are generally commercially available. The amines RNH2 can also be prepared by reduction of a carboxamide precursor (available in turn from the corresponding acid via the acid chloride). RiCOOH acids can be purchased, or are available by oxidation of the precursor alcohols, which are frequently available.

PREPARATION OF FORMULA COMPOUNDS I The bifunctional linkers HOC (0) -A-NH2 (where A as defined above) carry an amine at one end and a carboxylic acid at the other are commercially available as well. from suppliers such as Sigma Chemical Company (St. Louis, MO), or can be prepared using standard methods known to those skilled in the art. Referring to Reaction Scheme A, the amine is protected and the carboxyl group of the resulting N-protected linker is then coupled to an amine of formula RLR2NH in step 1 under conditions similar to those described above. In step 2, the nitrogen protecting group is removed under suitable conditions (acid treatment, hydrogenolysis, photolysis, etc.) and the resulting free amine is condensed in step 3 with a thiourea or an isothiouronium salt to yield a compound of Formula I. Thioureas and isothiouronium salts are commercially available, or can be prepared using the synthetic procedures described in Org. Syn. Coil., Vol. II (S-methyl thiourea sulfate), Org. Syn. Coil., Vol. III (S-ethylthiourea) and Chem. Reviews, 55, 181 (1955). Compounds of Formula I in which X is trihydrocarbyl onium can be obtained by alkylation of the free amine obtained in step 2 with the necessary alkylating agents, for example a trihydrocarbyl iodide, p-toluenesulfonate, mesylate and the like, sequential if necessary. The compounds of this invention can conveniently be represented in the form of a lipoamide tail group attached to a cationic head group X. Representative lipid group tail groups are shown in Figure 1, where X represents the cationic head group. The representative cationic head groups X are shown in Figure 2. Frequently, the cationic head group is a guanidino moiety. Employing the representation of Figures 1 and 2, Table 1 presents a representative sample of the compounds of this invention prepared using the methods described above and in the Examples.

PREFERRED COMPOUNDS Presently preferred are compounds of Formula I in which Ri and R2 are CH3 (CH2) 7CH = CH (CH2) 8-. Especially preferred is the compound of Formula I wherein R3 and R4 are alkylamino (optionally N-protected) or tert-butyloxycarbonyl, and Ri and R2 are CH3 (CH2) 7CH = CH (CH2) 9-.

UTILITY The cationic lipids of this invention are commonly used as carriers of various biologically active substances, such as drugs or nucleic acids. In particular, the cationic lipids can be used alone or in combination with other lipids in formulations for the preparation of lipid complexes for the intracellular transport of said biologically active substances. The contemplated uses for the cationic guanidino lipids of this invention include transfection procedures corresponding to those currently practiced with commercial cationic lipid preparations., such as Lipofectin ™, and other published techniques employing conventional cationic lipid transport technology. The cationic lipids of this invention can be used in pharmaceutical formulations to transport therapeutic agents by various routes, in vivo or ex vivo, and to various locations in a mammal to achieve a desired therapeutic effect. In vi tro can also be used to transfect and prepare cell lines expressing proteins of commercial interest.

PREPARATION OF LIPIDIC COMPLEXES The liposomes containing the cationic lipids of this invention are prepared by methods known to those skilled in the art. In general, a solution of the cationic lipid (with one or more additional co-lipids) in an organic solvent is dried to provide a lipid film that is rehydrated to obtain a suspension of liposomes. It will be understood that a suitable solvent for the preparation of a dry lipid film from the desired lipid components is any solvent which can dissolve all the components and which can then be conveniently removed by evaporation or lyophilization. Examples of solvents are chloroform, dichloromethane, diethyl ether, cyclohexane, cyclopentane, benzene, toluene, methanol or other aliphatic alcohols such as propanol, isopropanol, butanol, tert-butanol, isobutanol, pentanol and hexanol. Mixtures of two or more solvents can be used for the practice of the invention. An aqueous medium suitable for the formation of liposomes from the dried lipid film will be understood to be, for example, water, an aqueous buffer solution, or a tissue culture medium. For example, a suitable buffer is phosphate buffered saline, i.e., 10 mM potassium phosphate at pH 7.4 in 0.9% NaCl solution. The pH of the medium should be in the range of from about 2 to about 12, but preferably from about 5 to about 9, and more preferably at a pH of about 7. In some situations, the biologically active substances will be included in the rehydrating medium. tion, while in other cases, as for nucleic acids, they will subsequently be added to the rehydration / formation of the liposomes. Examples of co-lipids and materials related to optional phospholipids are lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, dio-leoylphosphatidylcholine (DOPC). , dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphos-fatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate ( DOPE-bad). Additional lipids that do not contain phosphorus are, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, iso-propyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkyl aryl sulfate, acid amides. polyethoxylated fatty acid, dioctadecyldimethyl ammonium bromide and steroids such as cholesterol, ergosterol, ergosterol Bl, B2 and B3, androsterone, cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid and the like. The preferred co-lipids are cholesterol and / or DOPE. Optionally, the suspension can be subjected to sonication, reverse phase evaporation, freeze-thaw or extrusion processes to produce liposomes of a specified size range. Preferably, unilamellar liposomes of about 50 to about 200 μm in diameter will be prepared. The biologically active substance to be transported is then mixed with the liposome suspension to produce a lipid complex of the biological substance. The net charge in the complex is determined by the charge on the liposomes, the charge on the biological substance and the relative amounts used of both. Accordingly, either cationic or anionic lipid complexes can be prepared. It is generally preferable to avoid neutrality when complexes are prepared, especially when preparing for the in vivo transport of a nucleic acid. Typically, this is achieved by adding the less represented component (based on the charge) to the most represented component with vigorous stirring to avoid local concentration gradients. In this way, when an anionic complex is prepared, the suspension of cationic liposomes is added to the nucleic acid, whereas when a cationic complex is prepared, the order of addition is the inverse. It has been generally observed that anionic complexes are more suitable for the transport by air of nucleic acids, while intravenous transport is carried out more effectively with cationic complexes.

PHARMACEUTICAL FORMULATIONS The present invention provides pharmaceutical compositions which include a cationic lipid as described above and one or more biologically active substances. Such pharmaceutical compositions facilitate the intracellular entry of biologically active molecules into tissues and organs, such as the epithelium of the airways, the lung, the heart, the gastric mucosa and solid tumors. Additionally, these compositions facilitate the entry of biologically active substances into cells maintained in vi tro.

Biologically active substances The biologically active substances included in the pharmaceutical formulations of this invention include drugs and nucleic acids. As described herein, nucleic acids include genomic DNA, cDNA, RNA, mRNA, ribosomal RNA, antisense RNA, ribozymes, mixed duplexes and triple helices of RNA and DNA and plasmids. The nucleic acids also include those species in which the bases, carbohydrate residues and / or phosphodiester bonds that are found naturally have been modified, as well as peptide nucleic acids. Such modifications include phosphorothioates, substitutions by non-natural bases, and the like, including, but not limited to, those species discovered in PCT Publications WO 96/1840 and WO 96/1841. Frequently, nucleic acids will encode significant proteins from a diagnostic or therapeutic point of view. Such proteins include histocompatibility antigens, cell adhesion molecules, growth factors (e.g., vascular endothelial growth factor for peripheral arterial disease), recombinant human Factor VIII, hormones (insulin, growth hormone, growth hormone), cytokines (e.g., IL-12), chemokines, antibodies, antibody fragments, cell receptors, intracellular enzymes, transcription factors (e.g. NF-B, I? B), toxic peptides ( such as the ricin A chain, diphtheria toxin, etc., which are capable of eliminating diseased or malignant cells) or any fragment or modification of any of them. It will be understood that such proteins include truncated forms and muteins of wild proteins and that they can act as agonists or antagonists of the wild-type variant depending on the therapeutic need. The nucleic acids can also include expression control sequences and will generally possess a transcriptional unit comprising a transcription promoter, a stimulator, a transcription terminator, an operator or other control sequence. It will often be desirable to possess a tissue-specific promoter that will ensure that the protein is specifically expressed in the target tissue. Nucleic acids encoding diagnostically significant proteins will often carry additional sequences encoding selectable or diagnostic markers (eg, lacZ, β-galactosidase, chloramphenicol transferase, etc.). The cationic lipid-nucleic acid formulations of this invention may also contain ligands and / or receptors capable of binding to a component of the cell. A ligand is any compound of interest that can specifically bind to another molecule, called a receptor, with the ligand and the receptor forming a complementary pair. For example, a ligand may be an antibody against a cell surface receptor, such as the antigens of the major histocompatibility complex, HLA-A. Such formulations allow specifically targeting nucleic acids against a particular subgroup of cells that express the receptor on their surface. Alternatively, a ligand may be a small molecule, such as an inhibitor of an intracellular enzyme, such as the angiotensin converting enzyme. Ligands can be covalently bound to the cationic lipid, or be inserted non-covalently in the liposomal membrane. Transfection The cationic lipid-nucleic acid complexes of the present invention were used to transfect cells both in vi tro and in vivo. The in vivo experiments were performed both in the presence and in the absence of serum. Transfection efficiencies were determined in relation to commercial cationic lipid formulations, such as Lipofectin ™, DOTMA ™, as well as recently discovered cationic lipids Cytofectin GS-2888 (JG Lewis et al., Proc. Nati. Acad. Sci. USA , vol 93, 3176-3181 (1996) Transfection efficiencies were determined using the pCMVβ plasmid carrying a detectable lacZ / β-galactosidase marker As shown in more detail in the Examples section, the efficiency of The transfection of the cationic lipids of this invention in the presence of serum was consistently superior to those of both Lipofectin ™ and GS-2888. Although the invention is not intended to be restricted by any particular theory, it is believed that this surprising superiority provided by cationic lipids of guanidino discovered here is due to the firmer union of guanidino head groups to DNA, thus being less accessible bind to the serum proteins. Cell types that can be transfected using lipid mediated transport using the cationic lipids of this invention include, but are not limited to, endothelial cells, epithelial cells (in particular, lung epithelial cells), alveoli, bronchial cells, keratinocytes, and synovial cells. It was also surprisingly observed that the length of the linker connecting the guanidino head group with the lipoamide portion played a role in transfection efficiency, with the chains having spacers of two or three carbon atoms between the amido function of the lipoamide -na and the guanidino head group had the highest transfection efficiencies with respect to the three or four carbon atom chains. Table 1 shows the efficiency of transfection of cationic lipids with an oleyl glue and a guanidino head group having chain link lengths of 1 to 4 atoms in murine fibroblast cells (3T3), epithelial cells (HBE) and vascular endothelial cells (IVEC). The transfection efficiencies are expressed in relation to that observed for DOTMA ™.

TABLE 1 It was also surprisingly observed that substituents on the amino group of the guanidino head group also provided unexpectedly high transfection efficiencies. Table 2 shows the efficiency of transfection of cationic lipids with an olefin tail, an ethylene linker chain and different substituents in the guanidino head group in murine fibroblast cells (3T3), epithelial cells (HBE) and vascular endothelial cells (IVEC). The transfection efficiencies are expressed in relation to that observed for DOTMA ™.

TABLE 2 Transfection procedures can be carried out by direct injection into the cells of an animal in vi vo. Alternatively, the transfection can be carried out in vi tro with explanted cells of the animal, proceeding next to the reintroduction of the cells in the animal (ex vivo methods). Protocols for in vivo and ex vivo transfection can be found in the clinical setting in Human Gene Therapy, 7, 1621-1642 (1996). Tissues that can be transfected in vi ve include the epithelium of the airways and the vascular endothelium. Administration can be carried out via any systemic or local route, for example, parenterally, orally (in particular for infant formulations), intravenous, nasal, bronchial inhalation (ie, aerosol formulation), transdermal or topical, in dosage forms solid, semi-solid or liquid, such as for example, tablets, suppositories, pills, capsules, powders, solutions, suspensions, aerosols, emulsions or the like, preferably in unit dosage forms suitable for the simple administration of precise doses. The compositions will include a conventional pharmaceutical carrier or excipient, a biologically active substance, a cationic lipid of Formula I and, additionally, may contain other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc. The carriers can be selected from various oils, including those of animal, vegetable, petroleum derivative or synthetic origin, for example, peanut oil, soy bean oil, mineral oil, sesame oil, and the like. Preferred liquid transporters are water, saline, aqueous glucose and glycols, in particular for solutions in injectable. Suitable pharmaceutical carriers include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt or rice flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, skimmed milk powder, glyce -rol, propylene glycol, water, ethanol, and the like. Other suitable pharmaceutical carriers and their formulations are described in "Remington 's Pharmaceutical Sciences", by E.W. Martin. If desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffering agents and the like, such as for example sodium acetate, sorbitan monolaurate, triethanolamine oleate. , etc. The lipid-polyanion complexes of this invention are generally administered as a pharmaceutical composition which includes a pharmaceutical excipient in combination with a polyanion and a cationic lipid of Formula I. As described above, it will be particularly useful to carry nucleic acids. that encode meaningful prns from a therapeutic point of view. The concentration of the nucleic acid and the cationic lipid in a formulation can vary within the entire range employed by those skilled in the art. For in vi tro administration, the nucleic acid may be in the range of about 0.5 to 100 μM, preferably about 1.5 to 30 μM, and the cationic lipid may be in the range of about 1 to 200 μM, preferably from about 5 to 120 M. For in vivo administration, the nucleic acid may be in the range of about 0.1 to 10 mM, preferably about 0.2 to 2 mM, and the cationic lipid may be in the range from about 0.1 to 20 mM, preferably from about 0.2 to 10 M.

INTRAVENOUS ADMINISTRATION Intravenous injection has proven to be an important route of administration of therapeutic agents. Pharmaceutical formulations containing cationic lipids of the present invention can be administered by this route, for example, by preparing lipid complexes as described above and dispersing them in an acceptable infusion liquid. A typical daily dose of a compound of the invention can be administered by an infusion, or by a series of infusions spaced at periodic intervals. Liquid pharmaceutical formulations of controlled release liposomes are described for injection or oral administration in U.S. Pat. 4,016,100. Liposome applications have been suggested for the oral administration of drugs from a lyophilized liposome / peptide drug mixture encapsulated in intestine capsules, see U.S. Pat. 4,348,384. The above is incorporated herein by reference.

AEROSOL ADMINISTRATION Aerosol administration is an effective means for the administration of a therapeutic agent directly in the respiratory tract. Some of the advantages of this method are: 1) it avoids the effects of enzymatic degradation, poor absorption from the gastrointestinal tract, or loss of the therapeutic agent due to the first hepatic effect; 2) therapeutic agents that otherwise would not reach their sites of action in the airways are administered due to their molecular size, charge or affinity for extrapulmonary sites; 3) provides rapid absorption into the body via the alveoli of the lungs; and 4) it avoids the exposure of other organic systems to the therapeutic agent, which is important in cases in which exposure could cause undesirable effects. For these reasons, aerosol administration is particularly advantageous for the treatment of asthma, local infections of the lung and other diseases or morbid conditions of the lung and respiratory tract. There are three types of pharmaceutical inhalation systems: nebulizer inhalers, metered-dose inhalers (MDI) and dry powder inhalers (DPI). The nebulization devices produce a jet of air at high velocity which causes the therapeutic agent (which has been formulated in liquid form) to be sprayed as a mist which is introduced into the patient's airway. The MDIs normally have the formulation packaged with a compressed gas. When they act, the device discharges a fixed amount of the therapeutic agent by the compressed gas, thereby providing a reliable method of administering a fixed amount of agent. Historically, MDIs had used chlorofluorocarbons (CFCs) as compressed gas to propel the therapeutic agent. In recent years, CFCs have been linked to the disappearance of the earth's ozone layer. As a result, alternative propellants are being investigated that do not endanger the ozone layer as substitute substances for CFCs. DPIs administer therapeutic agents in the form of a free-flowing powder that can be dispersed in the airflow of the patient's inspiration while being aspirated through the device. In order to obtain a free flowing powder, the therapeutic agent is formulated with an excipient, such as for example lactose. A measured amount of the therapeutic agent is deposited in a capsule and is dispensed with each actuation. Examples of DPIs currently in use are Spin aler® (for the administration of disodium cromoglycate), Rotahaler® (for albuterol) and Turbuhaler® (for terbutaline sulfate). All of the above methods can be used for the administration of the present invention, in particular for the treatment of asthma or other similar or related airway disorders. • SUPPOSITORIES For systemic administration by suppositories, traditional binders and transporters are included, for example, polyalkylene glycols or triglycerides [for example PEG 1000 (96%) and PEG 4000 (4%)]. These suppositories can be obtained from mixtures containing active ingredients in the range from about 0.5% w / w to about 10% w / w, preferably from about 1% w / w to about 2% w / w.

LIQUIDS Pharmaceutical compositions administrable in liquid form can be prepared, for example, by dissolving, dispersing, etc., an active compound (from about 0.5% to about 20%), as described above, and optional pharmaceutical adjuvants, in a transporter, such as, for example, water, saline, aqueous glucose, glycerol, ethanol and the like, to thereby form a solution or suspension. Current methods for the preparation of these dosage forms are known, or will be apparent, to those skilled in the art; for example, see "Remington's Pharmaceutical Sciences," Mack Publishing Company, Easton, Pennsylvania, 16th ed., 1980. The composition to be administered will, in any case, contain an amount of the active compound or compounds in a pharmaceutically effective amount. for the relief of the particular state to be treated, according to what is described in this invention.

EXAMPLES The following examples are presented in order to allow those experts in the field a clearer understanding, as well as the implementation of the present invention. They should not be considered as limiting the scope of the invention, but simply as illustrative and representative of it. Abbreviations: BOC - terbutyloxycarbonyl CDI - carbonyldiimidazole thio - CDI - thiocarbonyldiimidazole PYBOP - (benzotriazol - 1 - yloxy) tripyrrolidinophosphonium hexafluorophosphate DIEA - diisopropylethylamine EXAMPLE 1 - PREPARATION OF FORMULA COMPOUNDS I A. Preparation of 3- [N ', N "-bis (2-tert-butyloxy-carbonyl-aminoethyl) guanidino] -N, N-dioctadec-9-enyl-propionamide (Compound 2-Q with reference to Figures 1 and 2) The specific reaction sequence for the synthesis of 2B is shown in Reaction Schemes C and D. The intermediates described in the experimental procedure presented below are numbered as shown in these Reaction Schemes.

REACTION SCHEME C CDI CH2C12 II LiAIH * RCO, H RCHJNHJ R "^^ NH, NH4OH THF Bu4NBr CDI O LiAIH, RCH, RC02H CHJCIJ RCHjN.?, ** NH H '^ R ^ RCi¿ RCH, PyBOP BOCNHtCH ^ COjH - »- N -" - CH2CH2NHBOC DIEA / DMF RCHj R = CH3 (CH2) 7CH = CH (CH2) 7 REACTION D SCHEME Di oxano HjNCH ^ H ^ + rY H ^ CH ^ CHjNHBOC Tduert > II a. + i. - BOCNHCH2CH2NH-- --HNCH2CH2NHBOC eleven Compound 1 To a solution at room temperature of oleic acid (99 +%, 12.48 g, 44.2 mmol) in methylene chloride (200 mL) was added 1.1 '-carbonyldiimidazole (8.24 g, 50.2 mmol). After stirring for 30 minutes, con centrated ammonium hydroxide (50 ml) and tetrabutylammonium bromide (1.42 g, 4.42 mmol) were added, and the resulting mixture was stirred rapidly for 2 hours. After stirring with water (100 ml), the organic phase was separated, washed twice with water, dried (magnesium sulfate), and dried in vacuo. The crude product (12.88 g) was recrystallized from hexane to yield 12.35 g of compound 1 as a white solid. LH NMR (CDC13) d 0.86 (t, J = 6.6 Hz, 3H), 1.27-1.80 (m, 22H), 2.0 (m, 4H), 2.22 (t, J = 8.0, 2H), 5.30 (m, 2H); MS m / z 281 (M +). Compound 2 To a solution at room temperature of compound 1 (10 g, 35.5 mmol) in dry tetrahydrofuran (100 ml) under an argon atmosphere was added dropwise for one minute by syringe lithium aluminum hydride (39 ml, 39 mmol one molar in THF). A slight exotherm was observed. The milky mixture was stirred one hour at room temperature and then at 50 ° C for 6 hours. The mixture was cooled in ice under rapid stirring and water was carefully added dropwise (1.5 ml), followed by aqueous sodium hydroxide (15%, 1.5 ml), and then water (3.5 ml). The resulting white granular solid was filtered and washed with methylene chloride (30 ml). After drying the methylene chloride solution, the resulting colorless liquid (9.7 g) was flash chromatographed on silica gel (eluting with 5% to 7% to 10% methanol in methylene chloride) to yield anhydrous. , 4 g of a colorless liquid. 4: H NMR (CDCl 3) d 0.88 (t, J = 6, 6 Hz, 3H), 1.27-1.64 (m, 36H), 2.00 (m, 4H), 2.68 ( t, J = 7.0 Hz, 2H), 5.36 (m, 2H); MS m / z 267 (M +). Compound 3 To a room temperature solution of oleic acid (99%, 28.25 g, 100 mmol) in methylene chloride (400 ml), 1,1 '-carbonyldiimidazole (17.84 g, 110 mmol) and stirred under argon atmosphere for 30 minutes. Compound 2 (26.75 g, 100 mmol) was added and stirred under argon atmosphere for two additional hours. Water (200 ml) was added and the mixture was stirred for a few more minutes. The methylene chloride layer was separated, dried (magnesium sulfate), and concentrated in vacuo to yield a white fat (57.5 g). X H NMR (CDCl 3) d 0.88 (t, J = 6.5 Hz, 6 H), 1.27 r-l, 73 (m, 54 H), 2.01 (m, 8 H), 2.15 (t, J = 6.0 Hz, 2H), 3.26 (q, J = 7.0 Hz, 2H), 5.35 (m, 2H); MS m / z 531 (M +). Compound 4 To a solution at room temperature of compound 3 (9.67 g, 18.25 mmol) in dry tetrahydrofuran (100 ml) under an argon atmosphere, lithium aluminum hydride was added dropwise over a minute with syringe. 20 ml, 20 mmol one molar in THF), and then stirred under an argon atmosphere at 60 ° C overnight. The mixture was cooled with ice and under rapid stirring water (0.7 ml) was carefully added dropwise, followed by aqueous sodium hydroxide (15%, 0.7 ml), and then water (2.1 ml). The resulting granular white solid was filtered and washed with ether (50 ml), dried (anhydrous magnesium sulfate), and concentrated in vacuo. The crude yellow oil (9.45 g) was flash chromatographed on silica gel (25% ethyl acetate / hexanes plus 1% triethylamine), yielding 4.2 g of a pale yellow oil and 0.89 g. g of a slightly impure product.

XH NMR (CDC13) d 0.88 (t, J = 2.6 Hz, 6H), 1.25-1.98 (m, 46H), 2.02 (m, 8H), 2.58 (t, J = 2.5 Hz, 4H), 5.34 (m, 4H); MS m / z 518 (M +). Compound 5 To a mixture at room temperature of compound 4 (10.36 g, 20 mmol), Nt-BOC-β-alanine (3.78 g, 20 mmol) and PyBOP (12.49 g, 24 mmol) under atmosphere of argon, dimethylformamide (75 ml) was added and stirred for five minutes. To the solution was added diisopropylethylamine (10.45 ml, 60 mmol), followed by stirring for an additional 45 minutes. To the brown solution under stirring was added water (500 ml) and ethyl acetate (250 ml). The organic phase was separated and the aqueous phase was extracted twice with ethyl acetate (100 ml). The combined organic portions were washed with water (100 ml), dried (magnesium sulfate) and concentrated in vacuo. The crude brown oily solid was flash chromatographed on silica gel (5% to 10% ethyl acetate / hexanes) to yield 13.45 g of a colorless oil. : H NMR (CDC13) d 0.88 (t, J-6.7, 6H), 1.26-1.42 (m, 45H), 1.42 (s, 9H), 1.43-1, 75 (m, 6H), 2.00 (m, 8H), 2.50 4 (t, J = 4.8, 2H), 3.16 (br t, 2H), 3.30 (br t, 2H) ), 3.41 (br t, 2H), 5.35 (m, 2H); MS m / z 688 (M +). Compound 6 To a solution of compound 5 (7.75 g, 11.2 mmol) in dry dioxane (20 ml) under argon atmosphere was added 4N HCl in dioxane (25 ml) and stirred at room temperature overnight . The solution was dried, acetonitrile (50 ml) was added, dried, and the process repeated twice more. The residue was stirred with ethyl acetate (200 ml) and 10% aqueous sodium hydroxide solution (100 ml) for two hours. The ethyl acetate layer was separated, the aqueous layer was extracted with ethyl acetate (50 ml), the combined portions of ethyl acetate were dried (magnesium sulfate) and concentrated in vacuo. The unrefined oil (6.6 g) was flash chromatographed on silica gel (3% and then 10% methanol / methylene chloride) to yield 6.29 g of a very pale yellow oil. lH NMR (CDC13) d 0.88 (t, J = 7.0 Hz, 6H), 1.27-1.60 (m, 54H), 2.01 (m, 8H), 2.55 (t, J = 5.9 Hz, 2H), 2.95 (m, 2H), 3.10 (br t, 2H), 3.15 (br t, 2H), 3.25 (br t, 2H), 5 , 34 (m, 4H); MS m / z 589 (M +). Compound 7 To a solution at 65 ° C of compound 6 (0, 589 g, 1.0 mmol), thiourea (95 mg, 1.25 mmol), triethylamine (0.42 mL, 3.0 mmol), and tetrahydrofuran (25 mL) was added mercuric chloride (0.34 g, 1.25 mmol) and stirred at 65 ° C under argon atmosphere for seven days. The white suspension gradually turned black. The suspension was cooled to room temperature, filtered through celite and dried. The unrefined dense oil was flash chromatographed on silica gel (from 5% to 8% to 10% to 15% methanol / methylene chloride plus 1% concentrated ammonium hydroxide). The starting material (425 mg) was recovered, and the pure product (170 mg) was obtained as a pale yellow oil. The oil was dissolved in 10% methanol / methylene chloride and IN HCl in ether (1 ml) was added, and was dried to obtain a cloudy fat. XH NMR (CDC13) d 0.88 (t, J = 6.5 Hz, 6H), 1.27-1.78 (m, 60H), 2.00 (m, 8H), 2.56 (m, 2H), 3.18 (m, 2H), 3.28 (br t, 2H), 3.54 (br t, 2H), 5.34 (m, 4H), 8.29 (br t, 1H); MS m / z 630 (M +). Compound 8 To a room temperature solution of ethylene diamine (21 g, 0.349 mol) in dry dioxane (120 ml) was added dropwise for 3 hours di-t-butyl-dicarbonate (9.8 g, 26.6 mmol). ) in dioxane (120 ml). The cloudy mixture gradually clarified. The mixture was stirred overnight at room temperature. After drying, the residue was stirred with water (200 ml) and the white precipitate was filtered. The filtrate was extracted with methylene chloride (200 ml) three times, dried (magnesium chloride) and dried giving a colorless oil (4.8 g). X H NMR (CDCl 3) d 1.44 (s, 9 H), 2.83 (t, J = 6 Hz, 2 H), 3.17 (q, J = 6 Hz).

Compound 9 To a mixture at room temperature of compound 8 (0.8 g, 5 mmol) and ethyl acetate (35 ml) was added 1,1'-thiocarbonyldiimidazole (1.07 g, 6 mmol) and stirred throughout the night. After drying, the residue was flash chromatographed on silica gel with 30% to 50% ethyl acetate / hexane to yield 0.66 g of a solid. H NMR (CDC13) d 1.53 (s, 9H), 3.59 (t, J = 8.4 hz, 2H), 4.10 (t, J = 7 Hz, 2H). Compound 1 A solution of compound 8 (0.275 g, 1.72 mmol), compound 9 (0.347 g, 1.72 mmol) and toluene (4 ml) was stirred at 75 ° C under an argon atmosphere overnight. After drying, the residue was flash chromatographed on silica gel with 2% to 3% methanol / methylene chloride to yield an oil (0.37 g). X H NMR (CDCl 3) d 1.45 (s, 9 H), 3.34 (t, J = 5, 8 Hz, 2 H), 3.54 (m, 2 H). Compound _11 To a solution at 65 ° C of compound 6 (0.29 g, 0.494 mmol), compound 10 (0.21 g, 0.596 mmol), triethylamine (0.27 mL, 1.98 mmol), and tetrahydrofuran ( 25 ml) was added mercuric chloride (0.16 g, 0.596 mmol) and stirred at 65 ° C under an argon atmosphere for one day. The white suspension gradually turned black. The suspension was filtered through celite, and the solution was dried. The crude unrefined oil was flash chromatographed on silica gel (5% to 10% methanol / methylene chloride plus 1% concentrated ammonium hydroxide) to give a dense oil (0.21 g). lK NMR (CDC13) d 0.88 (t, J = 6, 8 Hz, 6H), 1.2-1.6 (m, 72H), 2.1 (m, 10H), 2.78 (m, 2H), 3.2-3.55 (m, 14H), 3.68 (m, 2H), 5.35 (m, 4H); MS m / z 916 (M +). B. Preparation of other compounds of Formula I Using the procedures described above and replacing oleic acid with the appropriate acid and with the appropriate thiourea or isothiouronium salt, the compounds listed below are also prepared. The compounds are defined in reference to the lipoamido tail groups and the cationic head groups as shown in Figures 1 and 2. For example, the compound IA refers to the compound 1 shown in Figure 1 wherein X is the head group A of Figure 2.

New lipids for gene therapy EXAMPLE 2 - PREPARATION OF LIPOSOMES In the following examples, cationic liposomal vesicles containing a 1: 1 molar ratio of the indicated cationic lipid and the neutral lipid dioleoylphosphatidylethanolamine (DOPE) were used. For example, 9.19 mg of 2-guanidino-N, N-dioctadec-9-enyl-propionamide (compound 2B) dissolved in methylene chloride were mixed with 10.81 mg of DOPE in chloroform. The solvents were removed by rotary evaporation and the lipid film was dried under vacuum. The films were rehydrated with 20 ml of sterile water to a concentration of 1 mg / ml, heated to 45 ° C and sonicated in a high potency sonication bath or extruded to form multilamellar liposomal vesicles. The particles were measured by laser light scattering using a Coulter Submicron Par-ticle Sizer, N4M (Coulter, Hialeah, Florida). The average particle size of the new liposome preparations was 296 ± 40 nm.

EXAMPLE 3 - PREPARATION OF COMPLICATIONS OF LIPIDS AND NUCLEIC ACIDS In the following examples, the plasmid pCMVß (Clontech, Palo Alto, CA), which codes for the gene for lacZ / β-galactosidase, was stored at -20 ° C in water at a concentration of 1 mg / ml. The complexes for in vivo transport were carried out with the plasmid pCT0129 coding for the CAT gene linked to the CMV promoter. The plasmid DNA was diluted in serum-free Optimem medium (Life Technologies, Ga-thersburg, Md) at a concentration of 8 μg / ml, and added to an equal volume of cationic liposome solution. The complexes were formed for 30 to 45 minutes before their use in the transfection experiments. The complexes were prepared with lipid / DNA ratios of 1: 1, 1: 5 and 1:25 by weight. The lipid / DNA complexes were also prepared using two commercially available lipid transfection agents, Lipofectamine ™ and Lipofectin ™ (Life Technologies, Gathersburg, Md). These agents were used to evaluate the transfection efficiency in relation to commercially available lipids routinely used for transfection.

EXAMPLE 4 - DETERMINATION OF TRANSFER EFFICIENCIES The transfection efficiencies obtained using the guanidino-containing liposomes of the present invention, in relation to that of the liposomes containing quaternary amines, were compared in serum-containing or serum-free medium (Figures 3 to 5). The effect of the co-lipid variation (DOPE or cholesterol), as well as the molar ratio of co-lipid to cationic guanidino lipid on the transfection efficiency was also determined (Figure 6). Comparison data of the lipid transfection efficiency of the present invention with respect to that of DOTMA ™ and GS-2888 were also determined both in vi tro (Figure 7) and in vivo (Figure 8). The new lipid / DNA complexes were routinely tested in a 96-well microtiter plate format to determine the efficiency of gene transfer using three different cell lines, murine fibroblasts NIH3T3 (ATCC # CTRL 1658), human endothelial cells IVEC (provided by Dr. Denise Paulin, Institute Pasteur, Paris, France, J. Cellular Physiol., 457, 41-51 (1993), and human epithelial cells 16-HBE14o, Am. J. Physiol., 268, L347- L360 (1995) Cells were grown in Costar microtitre plates (Cambridge, MA) coated with 0.5% collagen (Collaborative Biomedical, Bedford, MA) Cells were seeded at 20,000 cells / well in complete culture medium 48 hours before transfection: On the day of transfection, the medium was aspirated, the cells were washed three times with Optimem medium, 50 μl of Optimem medium with or without 10% FBS (BioWhittaker, Walker) was added to each plate d microtiter, and 50 μl of the lipid / DNA complex was added to the appropriate wells, to obtain the proper final DNA concentration. The cells were incubated with the DNA and lipid complex for 5 hours at 37 ° C. The medium was then aspirated, replaced with 100 μl of culture medium containing whole serum, and the cells were cultured for an additional 48 hours. To evaluate the efficiency of transfection, we used Cells used, and β-galactosidase activity was determined using the fluorogenic substrate 4-methyl-umbelliferyl-β-D-galactosidase (MUG) (Sigma, St. Louis, MO), in accordance with the manufacturer's instructions. The amount of hydrolyzed substrate was measured fluoro-electrically using a CytoFluorlI fluorometer (Millipore, Bedford, MA). The total cellular protein was determined in those used using a BCA assay (Pierce, Rockford, IL). The data are presented as fluorescence units per μg of protein, and each point represents the average of three sample measurements. A. Transfection of human endothelial cells (IVEC): IVEC cells (2x10 *) were transfected with 2 μg / ml of Plasmid DNA pCMV-ß complexed with 0, 2, 10 or 50 μg / ml of cationic liposomes in serum-free medium (Figure 3A) or in medium containing serum (Figure 3B). The β-galactosidase activity was measured 48 hours after transfection using the fluorescent substrate (MUG) and the activity was normalized by μg of protein in the cell lysate. Each point of the data represents the average of tripled samples. In the presence of serum, as illustrated in Figure 3B, several of the new compounds transfect with an efficiency of 2 to 10 times better than the commercially available compound. B. Transfection of human bronchial epithelial cells (16HBE): 16HBE cells (2xl04) were transfected with 2 μg / ml pCMV-β plasmid DNA complexed with 0, 2, 10 or 50 μg / ml of cationic liposomes in serum-free medium (Figure 4A) or in medium containing serum (Figure 4B). The β-galactosidase activity was measured 48 hours after transfection using the fluorescent substrate (MUG) and the activity was normalized by μg of protein in the cell lysate. Each point of the data represents the average of tripled samples. The data of Figure 4A show that in human bronchial epithelial cells, in the absence of serum, several of the new compounds transfect with an efficiency rate comparable to that of the commercially available compound Lipofectin ™, and one of the new compounds transfectates with approximately 30% more efficiency. Additionally, in the presence of serum, as illustrated in Figure 4B, several of the new compounds transfect with efficiencies approximately several times better than the commercially available compound.

C. Transfection of 3T3 murine fibroblast cells: 3T3 fibroblasts (2xl04) were transfected with 2 μg / ml pCMV-β plasmid DNA complexed with 0, 2, 10 or 50 μg / ml of cationic liposomes in serum-free medium (Fig. 5A) or in medium containing serum (Figure 5B). The β-galactosidase activity was measured 48 hours after transfection using the fluorescent substrate (MUG) and the activity was normalized by μg of protein in the cell lysate. Each point of the data represents the average of tripled samples. The data of Figure 5A show that, in murine 3T3 fibroblast cells, in the absence of serum, several of the new compounds transfect with an efficiency of about 30 to 50% higher than that of the commercially available compound, Lipofectin ™ , shown on the right side of the graph. Additionally, in the presence of serum, as illustrated in Figure 5B, several of the new compounds transfect with efficiencies of 4 to 10 times better than the commercially available compound. D. Effect of co-lipids on the transfection efficiency of the new compound 2-guanidino-N, N-dioctadec-9-enyl-propionamide (2B) in human endothelial cells: Cationic liposomes containing 2B: cholesterol: DOPE were prepared various molar proportions. IVEC cells (2xl04) were transfected with 2 μg / ml pCMV-β plasmid DNA complexed with 0, 2, 10 or 50 μg / ml of cationic liposomes in serum-free medium or in medium containing serum (Figure 6). The β-galactosidase activity was measured 48 hours after transfection using the fluorescent substrate (MUG) and the activity was normalized by μg of protein in the cell lysate. Each point of the data represents the average of tripled samples. The data in Figure 6 show that in human endothelial cells, in the presence of serum, the new compound 2B combined with cholesterol and DOPE in the proportions of 1: 0: 1 and 0.5: 0.25: 0.25 transfected with approximately 10 to 20 times more effective than the commercially available compounds Lipofectin ™ and Lipofectamine ™, shown on the left side of the graph. In the absence of serum, as shown in Figure 6, the new compound 2B combined with DOPE at a ratio of 1: 1 transforms at an efficiency approximately 40% better than the commercially available compound Lipofectamine ™. E. Transfection activity of the new RS compounds in relation to Cytofectin GS2888 BOC-protected GS-2888 was prepared in two ways, as the chloride salt and as the free base. The lipid ratio of cationic lipid to neutral lipid was 1: 1. Figure 7B serves as a control, showing that GS-2888 was active. The compounds employed in both Figures 7A and 7B were prepared from the same batch, on the same day, using the same DNA in different cells. 2 × 10 4 cells were transfected with 2 μg / ml pCMV-β plasmid DNA complexed with 0, 2, 10 or 50 μg / ml of cationic liposomes in serum-containing medium (Fissure 7B).

GS-2888 was formulated with neutral lipid at molar ratios of 1: 1 and 2: 1. The β-galactosidase activity was measured 48 hours after transfection using the fluorescent substrate (MUG) and the activity was normalized by μg of protein in the cell lysate. Each point of the data represents the average of tripled samples. Figure 7A illustrates the transfection efficiency in IVEC endothelial cells; Figure 7B illustrates the transfection efficiency in 16-HBE epithelial cells. Figure 7A shows that in HBE cells several of the new guanidino lipids are 10 times more efficient than GS-2888. F. Efficiency of in vivo transfection of the new compounds of this invention A reproducible process was established to prepare DNA / lipid complexes at concentrations and volumes suitable for transport in vi ve. This process was developed with liposomes containing the cationic lipid DOTMA ™ or cationic lipids of the present invention formulated with the neutral lipid DOPE. The DNA was in the form of circular polyanionic plasmids. Complex formation was achieved by slow infusion of the minor component in a rapidly stirred solution of the predominant component. This process was carried out in a sterile environment and proved to be reproducible with respect to the size of the complex in a wide range of concentrations (DNA 0.15-1.5 mM; lipids 0.2-8.5 mM) • and loading ratios.

In vitro testing was performed by intratracheal instillation of lipid / DNA complexes in anesthetized rats. A plasmid (pCT0129) encoding the chloramphenicol acetyl transferase (CAT) gene linked to a CMV promoter was used for all in vivo evaluations of transfection efficiency. The transfection complexes typically contained 200 μg of DNA complexed with 0.8 to 3.5 mM liposomes and 5% glucose. 250 μl of the complex was administered to the lungs of rats by instillation through a PÍO tube connected to a 30 gauge needle. The animals were sacrificed 40 hours later, the lungs were extracted and analyzed for the enzymatic activity using a CAT assay in ELISA format (Boehringer Mannheim). The results are shown in Figures 8A for different sets of cationic lipids, compared to DOTMA ™. The data are expressed as the average pg of CAT protein / mg lung protein (n = 8) and show that CAT protein levels detected in animals receiving DNA complexed with lipids of the invention were 50 times higher than those detected in animals. who received DNA complexed with DOTMA ™. This invention has been described in detail by way of illustration and example, for purposes of clarification and better understanding. It will be apparent to persons skilled in the art that changes and modifications may be made within the scope of the appended claims. Accordingly, it should be understood that the foregoing description is intended to be illustrative and not restrictive.

Accordingly, the scope of the invention will be determined not with reference to the foregoing description, but will instead be determined by reference to the following appended claims, together with the full scope of equivalents contemplated by said claims. All patents, patent applications and publications cited in this application are hereby incorporated by reference in their entirety for all purposes with the same effect as if each patent, patent application and individual publication had been individually cited. 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 (16)

1. Rei indications 1. A compound of Formula I R! R2N-C (0) -A-X Formula I characterized in that R: and R2, which may be the same or different, are C10-C26 hydrocarbyl groups; A is a hydrocarbylene group in which one or more methylene groups are optionally replaced by a group Y (as long as none of the groups Y are adjacent to each other), wherein each Y is independently in the direction shown - O-, -OC (O) -, -C (0) 0-, -NR5-, -NR5C (0) -, -C (0) NR5-, NR5C (0) NR5-, -NR5C (0) 0 -, -0C (0) NR5-, -S (0) n- (where n is 0, 1 or 2), or -NZ-C (= NZ) NZ-, where each Z is independently H or - being m an integer from 1 to 10, and each R5 is independently H or lower alkyl; X is: (1) a trihydrocarbylammonium group, wherein each hydrocarbyl group is the same or different from the others, or (2) -NH-C (= NR3) NHR4, wherein R3 and R4 are independently hydrocarbyl, haloalkyl , hydroxyalkyl, O-protected hydroxyalkyl, alkoxyalkyl, haloalkoxyalkyl, aryloxyalkyl, aminoalkyl, mono- or disubstituted aminoalkyl, N-protected aminoalkyl, acyl, alkoxycarbonyl, aryloxycarbonyl, -C (0) NR6R7 (where R6 and R? are independently H or hydrocarbyl ), a nitrogen protecting group, or R3 and R together with the atoms to which they are attached form an optionally substituted monocyclic or bicyclic ring, provided that when Ri and R2 are both identical C1 alkyl groups and X is -NH-C (= NH) NH2, A is not a butylene chain; and salts, solvates, resolved or unresolved enantiomers, diastereomers and mixtures thereof.
2. 2. An ascompound according to claim 1, characterized in that X is -NH-C (= NR3) NHR4.
3. 3. A compound according to claim 1 or 2, characterized in that Rx and R2 are alkyl groups or monounsaturated alkenyl groups.
4. 4. A compound according to any of the racial reivi-t of 1 to 3, character i zato by ue Ri and R2 are identical.
5. 5. A compound according to any of claims 1 to 4, characterized in that RL and R2 are CH3 (CH2) 7CH = CH (CH2) 8-.
6. 6. A compound according to any of claims 1 to 5, characterized in that A is an alkylene group.
7. 7. A compound according to any of claims 1 to 6, characterized in that A is a methyl or ethylene group, and R3 and R are both H or aminoalkyl protected in N.
8. 8. A compound according to any of the claims of 7, characterized in that R3 and R4 are N-protected aminoalkyl groups selected from the group consisting of 2- (t-butyloxycarbonylamino) ethyl and 3- (t-butyloxycarbonyl) propyl.
9. 9. A polyanion and lipid complex, characterized in that it includes a compound according to any of Claims 1 to 8 and a polyanion.
10. 10 A polyanion and lipid complex of Claim 9, characterized in that the polyanion is a nucleic acid. eleven .
11. A polyanion and lipid complex according to claims 9 or 10, characterized by additionally includes a targeting ligand.
12. 12 A polyanion and lipid complex according to any of claims 9 to 11, characterized in that the nucleic acid is an expression plasmid.
13. 13. A polyanion and lipid complex of Claim 12, characterized in that said expression plasmid codes for IL- 12 or I? B.
14. 14. A method for obtaining a polyanion and lipid complex, characterized in that it includes the steps of: forming a liposome that includes a compound according to any of Claims 1 to 8 and optional colipids; and contacting the liposome with a polyanion to form a polyanion and lipid complex according to any one of Claims 9 to 13.
15. 15. A method of transporting a polyanion to a cell, characterize it because it includes: the formation of a complex according to the Claim 14 and contacting the cell with the complex.
16. 16. A pharmaceutical formulation, "characterized in that it includes a polyanion and lipid complex according to any of Claims 9 to 13, and pharmaceutically acceptable excipients.