MXPA00001855A - Novel compositions for the delivery of negatively charged molecules - Google Patents

Abstract

This invention features permeability enhancer molecules, and methods, to increase membrane permeability of negatively charged polymers thereby facilitating cellular uptake of such polymers.

Description

NOVEDOUS COMPOSITIONS FOR THE SUPPLY OF NEGATIVELY LOADED MOLECULES Background of the Invention The following is a brief description of the supply of biopolymers. This summary is not intended to be complete, but is provided only for the understanding of the invention that follows. This summary is not an admission that all work described below is the prior art of the claimed invention. The traffic of large charged molecules within living cells is highly restricted by the complex membrane systems of the cell. Specific transporters allow selective entry of regulatory molecules, while excluding most exogenous molecules such as nucleic acids and proteins. The two main strategies for improving the transport of foreign nucleic acids within cells are the use of viral or cationic lipid vectors and the related cytofectins. Viral vectors can be used to transfer genes efficiently within some cell types, but they can not be used to introduce chemically synthesized molecules into cells. An alternative approach is to use release formulas that incorporate cationic lipids, which interact with nucleic acids through one end and with lipids or membrane systems through the other (for a review see Felgner, 1990 Advanced Drug Delivery Reviews, 5, 162-187; Felgner, 1993, J. Liposome Res., 3, 3-16). Synthetic nucleic acids, as well as plasmids, can be sent using cytofectins, although their utility is often limited by the specificity of the cell type, requirement for low serum during transfection, and toxicity. Since the first description of liposomes in 1965, by Bangham (J. Mol. Biol. 13, 238-252), there has been a sustained interest and effort in the area of the development of lipid-based carrier systems for the supply of compounds pharmaceutically active Liposomes are attractive drug carriers since they protect biological molecules from degradation, while improving their cellular uptake. One of the classes of liposome formulations that are most commonly used for the supply of polyanions (eg, DNA), is one that contains cationic lipids. Lipid aggregates can be formed with macromolecules using cationic lipids alone or including other lipids and amphiphiles such as phosphatidylethanolamine. It is well known in the art that both the composition of the lipid formulation, as well as its method of preparation, have an effect on the structure and size of the resulting anionic macromolecule-cationic lipid aggregate. These factors can be modulated to optimize the supply of the polyanions to the specific cell types in vi tro and in vivo. The use of cationic lipids for the cellular supply of biopolymers has many advantages. The encapsulation of the anionic compounds using the cationic lipids is essentially quantitative due to the electrostatic interaction. Furthermore, it is believed that cationic lipids interact with the membranes of negatively charged cells that initiate cell membrane transport (Akhtar et al., 1992, Trends Cell Bio 2, 139; Xu et al., 1996, Biochemistry, 35, 5616 ). The transmembrane movement of negatively charged molecules such as nucleic acids can therefore be markedly improved by co-administration with cationic lipids or other permeability enhancers (Bennet et al., 1992, Pharmacol., 41, 1023- 33; Capaccioli et al., 1993, BBRC, 197, 818-25; Ramila et al., 1993, J ". Biol. Chem., 268, 16087-16090; Stewart et al., Human Gene Therapy, 3, 267-275) Since the introduction of the DOTMA cationic lipid and its liposomal formulation Lipofectin (Felgner et al. 1987, PNAS 84, 7413-7417; Eppstein et al., U.S. Patent No. 4,897,355), a number of other lipid-based release agents have been described, primarily for transfecting mammalian cells with plasmids or antisense molecules (Rose, U.S. Patent Number 5,279,833; Eppand et al., U.S. Patent No. 5,283,185; Gebeyehu et al., U.S. Patent Number 5,334,761; Nantz et al., U.S. Patent Number 5,527,928; Bailey et al. of the United States of America Number 5,552,155; Jesse, Patent of the United States of America Number 5,578,475). However, each formulation is of limited utility because it can release plasmids within some, but not all cell types, usually in the absence of serum (Bailey et al., 1997, Biochemistry, 36, 1628). Generally, concentrations (charge and / or mass ratios) that are suitable for plasmid delivery (-5,000 to 10,000 bases in size) are not effective for oligonucleotides such as synthetic riboenzymes or antisense molecules (-10 to 50). bases) . Also, recent studies indicate that the optimal release conditions for antisense oligonucleotides and riboenzymes are different, even in the same cell type. However, the number of available release vehicles that can be used in the screening procedure is highly limited, and there continues to be a need to develop carriers that can improve the introduction of nucleic acid into many cell types. Eppstein et al., US Pat. No. 5,208,036, discloses a liposome, LIPOFECTIN ™, which contains an amphipathic molecule having a positively charged (water-soluble) choline main group attached to a diacyl glycerol group (no. soluble to water). Promega (Wisconsin) trades with another cationic lipid, TRANSFECTAMMR, which can help introduce the nucleic acid into a cell. Wagner et al., 1991, Proc. Nat. Acad. Sci. , USA 88, 4255; Cotten et al., 1990, Proc. Na t. Acad. Sci. , USA 87, 4033; Zenke et al., 1990, Proc. Nat. Acad. Sci. , USA 87, 3655; and Wagner et al., Proc. Nat. Acad. Sci. , USA 87, 3410, describe the transferrin-polymer conjugates which can improve the uptake of DNA within cells. They also describe the characteristic of a receptor-mediated endocytosis of the transferrin-polycarboxylated conjugates to introduce the DNA into the hematopoietic cells. Wu et al., J. Biol. Chem., 266, 14338, describes the receptor-mediated gene delivery, in which a asialoglycoprotein-polycarboxylic conjugate consisting of asialoorosomucoid, is coupled to the poly-L lysine. A complex of soluble DNA was formed capable of specifically targeting the hepatocytes by means of the asialoglycoprotein receptors present in the cells. The PCT Publication of the Biospan Corporation International Number WO 91/18012, describes the covalently internalizable covalent conjugates of the cell having an "intracellularly dissociable bond" such as a "disulfide disulfide bond", or an ester linkage labile enzyme Brigham, U.S. Patent No. 5,676,954 discloses a method for expressing the nucleic acid after transfection within an objective organ consisting of mammalian cells. The references cited above are different from the present claimed invention, since they do not describe or contemplate the release vehicles of the current invention.

SUMMARY OF THE INVENTION This invention features lipid-based compositions to facilitate the delivery of negatively charged molecules within a biological system, such as animal cells. The present invention describes the design, synthesis, and cellular testing of novel agents for the delivery of negatively charged molecules in vi tro and in vivo. Tracing procedures are also described to identify the optimal release vehicles for any given nucleic acid and cell type. In general, the conveyors described here were designed to be used either individually or as part of a multi-component system. It is expected that the compounds of the invention that are generally shown in Figure 1, improve the delivery of negatively charged molecules within a number of cell types that originate from different tissues, in the presence or absence of serum. It is intended that "negatively charged molecules" include molecules such as nucleic acid molecules (eg, RNA, DNA, oligonucleotides, mixed polymers, peptide nucleic acid, and the like), peptides. (e.g., polyamino acids, polypeptides, proteins, and the like), nucleotides, pharmaceutical and biological compositions having negatively charged groups that can be matched by ions with the positively charged major group of the cationic lipids of the invention. In a first aspect, the invention features a cationic lipid having the formula I: wherein n is 1, 2, or 3 carbon atoms; n1 is 2, 3, 4 or 5 carbon atoms; R and R1 independently represent an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by the double bonds 1-4; and R2 and R3 are independently H, acyl, alkyl, carboxamidine, aryl, acyl, substituted carboxamidine, polyethylene glycol (PEG) or a combination thereof. In a second aspect, the invention features a cationic lipid having the formula II: Main Group wherein n is 1, 2, or 3 carbon atoms; n1 is 2, 3, 4 or 5 carbon atoms; R and R? independently represent an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by the double bonds 1-4; and Alk represents methyl, hydroxyalkyl (e.g., hydroxymethyl and hydroxyalkyl) or a combination thereof. In a third aspect, the invention features a cationic lipid having the formula III: R NHR2 I NH wherein R and Rx independently represent an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by the double bonds 1-4; and R2 is H, PEG, acyl or alkyl. In a fourth aspect, the invention features a cationic lipid having the formula IV: Main Group wherein n is 1-6 carbon atoms; R and R? independently represent an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by the double bonds 1-4; and R2 is H, carboxamidine, alkyl, acyl, aryl, PEG, and substituted carboxamidine wherein R3 is H or P03H3 and R1 is OH, NH2 or = 0, In a fifth aspect, the invention features a cationic lipid having the formula V: wherein n is 1-6 carbon atoms; X and Xx independently represent an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by the double bonds 1-4; B is a nucleic acid base or H; and R5 is H, PEG, or carboxamidine. In a sixth aspect, the invention features a cationic lipid having the formula VI: wherein n is 1, 2 or 3 carbon atoms, wherein n is 1-6 carbon atoms; R and R1 independently represent an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by the double bonds 1-4; R and R1 independently represent an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by the double bonds 1-4; and R2 and R3 are independently H, polyethylene glycol (PEG) or For the formulas referred to above, N, 0 and H are nitrogen, oxygen and hydrogen, in accordance with the abbreviations well known in the art. In a seventh aspect, the invention features a cationic lipid having the formula VII: R6-LL-Cholesterol wherein R6 is selected from the group consisting of arginylmethyl ester, arginylamide, homoarginylmethyl ester, homoarginylamide, ornithinmethyl ester, ornithinamide, lysylmethyl ester, lysilamide, triethylenetetramine (TREN, for its acronym in English), N, N '-di -carboxamidine TREN, N-benzylhistidylmethyl ester, pyridoxyl and aminopropylimidazole, L? is a linker represented by R7P02, wherein R7 is H, CH3, or CH2CH3. Examples of this group of compounds are: PH55933, PH55938, PH55939, PH55941, PH55942, PH55943 and PH55945. In an eighth aspect, the invention features a cationic lipid having the formula VIII: R8-L2-Cholesterol wherein R8 is selected from the group consisting of arginyl ester, N-Boc arginyl, homoarginyl, N-Boc homoarginyl, ornithine, N-Boc ornithine, N-benzylhistidyl, lysyl, N-Boclisyl, N-methylarginyl, N-methylguanidine, guanidine and pyridoxyl, L2 is a linker represented by NH, glycine, N-butyldiamine or guanidine. Examples of this compound are Boc-argininelesteryl amide (DS46596), N-guanylcholesteryl amide (DS57511). In a ninth aspect, the invention features a cationic lipid having the formula IX: -NHCOR -NHCOR -COR, wherein R is independently an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by the double bonds 1-4; and R is represented by TREN, N, N'-di-carboxamidine TREN, lysyl, arginyl, ornithyl, homoarginyl, histidyl, aminopropylimidazole, spermine carboxylic acid. By "Main Group" is meant an amino-containing moiety which is positively charged and which can form ion pairs with the negatively charged regions of the biopolymers, such as the nucleic acid molecules. By "lipophilic group" is meant a group that contains a hydrophobic lipid that facilitates transmembrane transport of the cationic lipid. By "linker" is meant a chain of 1-6 carbon atoms that links the main group with the lipophilic group. By "ion pairs" is meant a non-covalent interaction between the oppositely charged groups. Specifically, an "alkyl" group refers to a saturated aliphatic hydrocarbon, which includes straight chain, branched chain and cyclic alkyl groups. Preferably, the alkyl group has from 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably from 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted, the substituted group (s) is (are) preferably hydroxy, cyano, alkoxy, N02 or N (CH3) 2, amino, or SH. By "alkoxy", it is meant an OR group, wherein R is an alkyl. An "aryl" group refers to an aromatic group, which has at least one ring having a? T conjugated electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent (s) on the aryl groups is (are) halogen, trihalomethyl, hydroxyl, SH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. The term "alkenyl" group refers to unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight chain, branched chain and cyclic groups. Preferably, the alkenyl group has from 1 to 12 carbons. More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably from 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted, the substituted group (s) is (are) preferably hydroxyl, cyano, alkoxy, N02, halogen, N (CH3) 2, amino, or SH. The term "alkynyl" refers to an unsaturated hydrocarbon group containing at least one triple carbon-carbon bond, which includes straight chain, branched chain and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7 carbons, more preferably from 1 to 4 carbons. The alkynyl group can be substituted or unsubstituted. When substituted, the substituted group (s) is (are) preferably hydroxyl, cyano, alkoxy, = 0, = S, N02 or N (CH3) 2, amino or SH. An "alkylaryl" group refers to an alkyl group (as described above) covalently linked to an aryl group (as described above). The "carbocyclic aryl" groups are groups wherein the ring atoms in the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. The "heterocyclic aryl" groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the rest of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, pyrrolo, pyrimidyl, pyrazinyl, imidazolyl, and the like, all optionally substituted. By "acyl", it is meant groups -C (0) R, wherein R is an alkyl or aryl. In a preferred embodiment, the invention presents the processes for the synthesis of the compounds of the formulas I-IX. In another preferred embodiment, a series of multi-domain cell transport vehicles (MCTVs) are described, which include one or more lipids of the formulas I-IX, which improve cellular uptake and transmembrane permeability. negatively charged molecules in a variety of cell types. The lipids of the invention are used either alone or in combination with other compounds with a neutral or negative charge including, but not limited to, the neutral lipid and / or the target components, to improve the effectiveness of the lipid formulation in the supply and target setting of the negatively charged polymers to the cells. Another objective is to describe the utility of these delivery vehicles to increase the transport of other impermeable and / or lipophilic compounds within the cells. By "compounds with neutral charge", it is meant compositions that are neutral or uncharged at a neutral or physiological pH. Examples of these compounds are cholesterol (ie, a steroidal alcohol, as defined by Lehninger, Biochemistry, 1982 ed., Worth Pub., Page 315) and other steroids, cholesteryl hemisuccinate (CHEMS, for its acronym in English ), dioleoylphosphatidylcholine, distearoylphosphotidylcholine (DSPC), fatty acids such as oleic acid, phosphatidic acid and its derivatives, phosphatidyl serine, polyethylene glycol conjugated phosphatidylamine, phosphatidylcholine, phosphatidylethanolamine and related variants, pre-phenyl compounds they include farnesol, polyprenols, tocopherol, and their modified forms, diacyl succinyl glycerols, fusogenic or pore forming peptides, dioleoylphosphotidylethanolamine (DOPE), ceramide and the like. Target compounds include ligands for cell surface receptors, including peptides and proteins, glycolipids, lipids, carbohydrates, and their synthetic variants. In yet another preferred embodiment, the cationic lipid molecules of the invention are provided as a lipid aggregate, such as a liposome, and co-encapsulated with the negatively charged polymer to be delivered. Liposomes, which can be unilamellar or multilamellar, can introduce the encapsulated materwithin a cell by different mechanisms. See Ostro, Scientific American, 102, January 1987. For example, the liposome can directly introduce its encapsulated materinto the cytoplasm of the cell by fusing with the cell membrane. Alternatively, compartments may be made in the liposome within an acidic vacuole (i.e., an endosome) having a pH below 7.0. This low pH makes it possible to make ion pairs with the encapsulated increments and the negatively charged polymer, which facilitates diffusion of the enhancer complex: polymer outside the liposome, the acidic vacuole, and within the cell cytoplasm. By "lipid aggregate" is meant a composition containing a lipid (i.e., a composition comprising a lipid according to the invention), wherein the lipid is in the form of a liposome, mycelium (non-lamellar phase) or other aggregates with one or more lipids. In yet another preferred embodiment, the invention features a lipid aggregate formulation that includes phosphatidylcholine (variable chain length); for example, egg yolk phosphatidylcholine), cholesterol, a cationic lipid, and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-poly-tile-glycol-2000 (DSPE-PEG2000), The cationic lipid component of this lipid aggregate can be any cationic lipid known in the art, such as 1,2-diacyl-3-trimethyl-ammonium-dioleoyl propane (DOTAP, for its acronym in English). In a preferred embodiment, this cationic lipid aggregate comprises a cationic lipid which is described in any of the formulas I -IX. In yet another preferred embodiment, polyethylene glycol (PEG) is covalently bound to the cationic lipids of the present invention. The bound PEG can have any molecular weight, but preferably has between 2000-5000 daltons.

The molecules and methods of the present invention are particularly convenient for introducing nucleic acid molecules into a cell. For example, the invention can be used for the delivery of riboenzymes wherein its target site of action exists intracellularly. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Description of Preferred Modes First, the drawings will be briefly described.

Drawings; Figure 1 describes the different classes of cationic lipids of the current invention. Figure 2 depicts a scheme for the synthesis of cationic diaminobutyric and guanidinium-based lipids. Figure 3 describes a scheme for the synthesis of Boc-argininelesteryl amide (12; DS46596). Figure 4 depicts a scheme for the synthesis of cholesterol-lysin-methyl-ester-methylphosphonamidate (PH55933; 15) and cholesterol-homoarginine-methyl-ester-methylphosphonamidate (PH55938; 16).

Figure 5 depicts a scheme for the synthesis of cholesterol-lysin-amide-methyl-phosphonoamidate (PH55939; 17). Figure 6 depicts a scheme for the synthesis of cholesterol-TREN-methylphosphonamidate (PH55941; 18) and cholesterol-TREN-bis-guanidinium-methylphosphonamidate (PH55942; 19). Figure 7 depicts a scheme for the synthesis of cholesterol-histidine-methylphosphonamidate (PH55943; 20). Figure 8 depicts a scheme for the synthesis of cholesterol-aminopropyl-imidazole-methylphosphono-amidate (PH55945; 21). Figure 9 describes a scheme for the synthesis of vitamin B6 and cationic lipids based on beta-alanine. Figure 10 depicts a scheme for the synthesis of cationic lipids based on 2'-aminouridine. Figure 11 describes a scheme for the synthesis of the vitamin B6-cholesterol conjugate. Figure 12 shows the model of the secondary structure for seven different classes of enzymatic nucleic acid molecules. The arrows indicate the dissociation site. indicates the target sequence. It is intended that the lines interspersed with points indicate the tertiary interactions, it is intended that - indicate the base paired interaction. Group I Intron: P1-P9.0 represents different stem-cycle structures (Cech et al., 1994, Na ture Struc. Bio., 1, 273). Rnasa P (M1ARN): EGS represents the external leader sequence (Forster et al., 1990, Science, 249, 783, Pace et al., 1990, J. Biol. Chem., 265, 3587). Group II Intron: 5'SS means 5 'splice site; 3 'SS means splice site 3'; IBS stands for intron fixation site; EBS means exon fixation site (Pyle et al., 1994, Biochemis try, 33, 2716). RNA VS: It is intended that I-VI indicate six stem-cycle structures; shaded regions indicate tertiary interaction (Collins, PCT International Publication Number WO 96/19577). Riboenzyme HDV: I-IV is intended to indicate four stem-cycle structures (Been et al., U.S. Patent Number 5,625,047). Riboenzyme Hammerhead: It is intended that I-III indicate three stem-cycle structures; the I-III stems can be any length and can be symmetrical or asymmetric (Usman et al., 1996, Curr. Op. Struct. Bio., 1, 527) Passer Riboenzyme: It is intended that H1-H4 indicate the helices 1- 4; the helices 1 and 4 can be of any length; helix 2 is between 3 and 8 base pairs in length; And it's a pyrimidine; B is guanosine, ditidine or uridine; V is adenosine, guanosine, or cytidine (Burke et al., 1996, Nucleic Acids &Mol. Biol., 10, 129; Chowrira et al., United States Patent Number 5,631,359). Figure 13 describes the structure of the fluorescein conjugated riboenzyme and its substrate mRNA sequence. The fraction of fluorescein (fluorite), bound through an amino linker, does not reduce the enzymatic activity of the riboenzyme. Cationic lipids are bifunctional reactants (cationic main group conjugated to a lipid tail) that include a positively charged group that can be ion-matched with an anionic group present in a negatively charged polymer, such as a phosphate group present in a linkage. nucleophilic acid phosphodiester or a group of phosphorothioate present in the nucleic acid having a modified phosphodiester bond. Preferably, the cationic group is ion-paired with a negatively charged polymer, to form a lipid: polymer complex, such as a complex with a polynucleotide or a polyamino acid (eg, RNA, DNA, and protein). More preferably, the cationic group is paired by ions with an RNA having enzymatic activity, such as a riboenzyme. Ion pair formation increases the intrinsic hydrophobicity of the negatively charged polymer and facilitates diffusion of the polymer through a cell membrane within the cell. The lipid: polymer complex may contain more than one lipid molecule. Preferably, a lipid: polymer complex contains the lipid in an amount to be ion-matched with at least 50 percent of the anionic groups of a negatively charged polymer. More preferably, a lipid: polymer complex contains the lipid in an amount to be ion-matched with at least 90 percent of the anionic groups of a negatively charged polymer. The lipid of a lipid: polymer complex can be the same or different. For example, the complex may contain lipids that differ in the cationic groups. The amount of the cationic lipid and the negatively charged polymer that must be combined to achieve the desired amount of ion pairings depends on the environment in which the lipid and polymer are mixed, the type of lipid, and the type of polymer. The degree of pairing by ions can be measured by techniques known in the art (see, for example, U.S. Patent No. 5,583,020, the contents of which are incorporated by reference herein). As a guideline, the lipid molecule should be provided in an amount of at least two to ten times by negative charge in the polymer molecule. Cationic lipids represent a subset of compounds in the broader class of multiple domain cell transport vehicles (MCTVs). The MCTV family of the invention includes single-component, as well as multi-component delivery systems, which incorporate the structural domains designated to improve the permeability of the membrane, the cell bank, while reducing nonspecific interactions and toxicity of the incoming compound. In addition to cationic lipids, examples of MCTVs include carriers such as facial amphiphiles and other amphiphilic compounds, carriers with targeting elements such as glycolated fractions, peptides and vitamins, and liposomes with fusogenic elements, pegylated lipids, and / or sensitive components at pH. By "nucleic acid molecule" as used herein, is meant a molecule having nucleotides covalently linked to one another and in the general size range of 2 nucleotide bases up to 100 kilobases or more, preferably at the size range of 10 bases up to 50 kb., and preferably in the size range of 100 bases up to 10 kb and more preferably, in the size range of 1 kb to 5 kb. The nucleic acid can be single-stranded, double-stranded or multi-stranded and can comprise modified or unmodified nucleotides or non-nucleotides of different mixtures and combinations thereof. Non-limiting examples of the nucleic acid molecules according to the invention, are riboenzymes, plasmid DNA, antisense oligonucleotides, 2-5A antisense chimera, triple-forming oligonucleotides, peptide nucleic acid molecules, nucleotides and others. By "oligonucleotide" or "polynucleotide" as used herein, is meant a molecule comprising two or more nucleotides covalently linked. By "riboenzyme" is meant a nucleic acid molecule that can catalyze (alter the rate and / or proportion of) a variety of reactions including the ability to repeatedly dissociate other separate nucleic acid molecules (endonuclease activity) , in a specific manner of nucleotide base sequence. This molecule with endonuclease activity can have complementarity in a region of substrate binding with a specific gene target, and also has an enzymatic activity that specifically dissociates the RNA or DNA in the target. That is, the nucleic acid molecule with endonuclease activity can dissociate the RNA or DNA intramolecularly or intermolecularly, and thereby deactivate a target RNA or DNA molecule. These complementary functions allow for sufficient hybridization of the enzyme RNA molecule with the target RNA or DNA to allow dissociation to occur. A 100 percent complementarity is preferred, but a complementarity as low as 50-75 percent may also be useful in this invention. The nucleic acids in the base, sugar, and / or phosphate groups can be modified. The term "enzymatic nucleic acid" is used interchangeably with phases such as riboenzymes, catalytic RNA, enzymatic RNA, catalytic DNA, catalytic oligonucleotides, nucleoenzyme, DNAsezyme, enzymatic DNA, RNA enzyme, endoribonuclease, endonuclease, minienzyme, guiding enzyme, oligoenzyme, or DNA enzyme. All these terminologies describe nucleic acid molecules with enzymatic activity. The specific enzymatic nucleic acid molecules that are described in the current application are not limiting in the invention and those skilled in the art will recognize that all that matters is that in a nucleic acid molecule of this invention, it is that it has a specific site. of specific substrate binding, which in the case of an endonuclease, has sequences complementary to one or more of the target nucleic acid regions, and having nucleotide sequences within or around the substrate binding site, which imparts a catalytic activity to the molecule. By "enzymatic portion" or "catalytic domain" is meant the portion / region of the essential riboenzyme to catalyze a reaction such as the dissociation of a nucleic acid substrate. By "substrate binding arm" or "substrate binding domain", it is meant that the portion / region of a riboenzyme that interacts with the substrate can be paired base with a portion of a nucleic acid substrate that is complementary. In general, this complementarity is 100 percent, but it can be less if desired. For example, they can be paired by bases as few as 10 bases of 14. These arms are generally shown in Figure 12 and Table 1. That is, these arms contain sequences within a riboenzyme that are supposed to bind to the riboenzyme and the objective through the complementary interactions of the base pairing. The riboenzyme of the invention may have attachment arms that are contiguous or non-contiguous and may be of varying lengths. The length of the fixation arm (s) is preferably greater than, or equal to, four nucleotides; specifically from 12-100 nucleotides: more specifically from 14-24 nucleotides in length. If a riboenzyme with two fixation arms is selected, then the length of the fixation arms is symmetrical (ie, each of the fixation arms is of the same length - for example, six and six nucleotides or seven and seven nucleotides in length), or asymmetric (ie, the fixation arms are of different lengths, for example, six and three nucleotides or three and six nucleotides in length). By "complementary" as used herein, is meant a nucleic acid that can form hydrogen bonds with another nucleic acid sequence by either the traditional Watson-Crick or other non-traditional types (for example, the Hoogsteen type) of pairing interactions by bases. By "antisense", is meant a non-enzymatic nucleic acid molecule that binds to a target RNA by means of RNA-RNA or RNA-DNA or RNA-NPC interactions (protein nucleic acid; Egholm et al., 1993, Nature 365, 566) and alters the activity of the target RNA (for a review see Stein and Cheng, 1993, Science, 261, 1004). By "2-5A antisense chimera" is meant an antisense oligonucleotide containing adenylated, linked, 5 'phosphorylated 2'-5' residues. These chimeras bind to the target RNA in a sequence-specific manner and activate a 2-5A-dependent ribonuclease which, in turn, dissociates the target RNA (Torrence et al., 1993, Proc. Nati. Acad. Sci. USA). 90, 1300). By "triple-forming oligonucleotides (TFO)" is meant an oligonucleotide that can be fixed with a double-stranded DNA in a sequence-specific manner to form a triple-stranded helix. It has been shown that the formation of this triple helix structure inhibits the transcription of the target gene (Duval-Valentin et al, 1992, Proc Nati Acad Sci USA 89, 504). The "biological system" as used herein can be a eukaryotic system or a prokaryotic system, it can be a bacterial cell, plant cell, or a mammalian cell, or it can be of plant origin, origin of mammal, origin of yeast, origin of Drosophila, or archaebacterial origin. By "cation", we mean a positively charged molecule. By "vitamin", is meant a small molecule, such as riboflavin, nicotinamide, biotin, thiamin, lipoic acid, retinal, pyridoxal, folate, pantothenic acid, cyanocobalamin, aminopterin, and their respective analogs, which bind to the specific protein and participate directly in the catalysis of the enzyme.Method of Use The cationic lipid molecules of the current invention can be used to administer negatively charged polymers, which act as pharmaceutical agents. Pharmaceutical agents avoid, inhibit the occurrence, or treat (alleviate a symptom to some degree, preferably, all symptoms) of a disease state in a patient. By "patient", it is meant an organism that is a donor or recipient of explanted cells or of the cells themselves. "Patient" also refers to an organism to which the components of the invention can be administered. Preferably, a patient is a mammal, for example, a human being, primate or a rodent. Generally, these molecules are used in solution with the negatively charged polymer to be administered (e.g., RNA, DNA or protein) and introduced by any standard means, with or without stabilizers, pH regulators, and the like, to form a pharmaceutical composition, when it is desired to use a liposome delivery mechanism, standard protocols for the formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for the injectable administration; and similar. The present invention also includes pharmaceutically acceptable formulations of the compounds described above, preferably in combination with the negatively charged polymer to be delivered. These formulations include salts of the above compounds, for example, acid addition salts, for example, salts of hydrochloric acid, hydrobromic acid, acetic acid, and benzenesulfonic acid. A pharmaceutical composition of the formulation, refers to a composition or formulation in a form suitable for administration, for example, systemic administration, within a cell or patient, preferably a human being. The appropriate forms, in part, depend on the use or the route of entry, for example oral, transdermal, or by injection. These forms should not prevent the composition or formulation from reaching the target cell (i.e., a cell to which it is desired to deliver the negatively charged polymer). For example, pharmacological compositions that are injected into the bloodstream should be soluble. Other factors are known in the art, and include considerations such as toxicity and the ways that prevent the composition or formulation from exerting its effect. By "systemic administration" is meant the systemic absorption in vivo or the accumulation of drugs in the bloodstream followed by distribution throughout the body. Administration routes that lead to systemic absorption include, without limitation, intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these routes of administration exposes the negatively charged polymers that are desired, eg, nucleic acids, to an accessible diseased tissue. It has been shown that the rate of entry of a drug into the circulation is a function of weight or molecular size. The use of a liposome or other drug carrier comprising the compounds of the present invention, can potentially locate the drug, for example, in certain types of tissue, such as the tissues of the reticular endothelial system (RES, for its acronym in English). ). A liposome formulation that can facilitate the association of the drug with the surface of the cells, such as lymphocytes and macrophages, is also useful. This approach can provide an improved delivery of the drug to the target cells by taking advantage of the specificity of the immune recognition of the macrophage and the lymphocyte of the abnormal cells, such as cancer cells. In the preferred embodiment, the invention features the use of the cationic lipids of the invention in a composition comprising surface modified liposomes containing poly (ethylene glycol) lipids.

(Liposomes modified by PEG, or long circulation or liposomes of subreption). These formulations offer a method to increase the accumulation of drugs in objective tissues. This class of drug carriers resists opsonization and elimination by means of the mononuclear phagocytic system (MPS or RES), enabling, by the same time, longer blood circulation and improved tissue exposure to the encapsulated drug (Lasic et al., C e_T! Rev., 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull., 1995, 43, 1005-1011). It has been shown that these liposomes accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science, 1995, 1275-1276; Oku et al., 1995, Biochim. Biophys. , 1238, 86-90). Prolonged circulation liposomes improve the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly in comparison to conventional cationic liposomes, which are known to accumulate in MPS tissues (Liu et al., J. Biol. Chem. , 1995, 42, 24848-24870, Choi et al, PCT International Publication Number WO 96/10391, Ansell et al, PCT International Publication Number WO 96/10390, Holland et al., PCT International Publication Number WO 96/10392; are incorporated as reference to the present). It is also likely that prolonged circulation liposomes protect drugs from nuclease degradation to a greater degree compared to cationic liposomes, which are based on their ability to prevent the accumulation in metabolically aggressive MPS tissues, such like the liver and the spleen. All of these references are incorporated herein by reference. The present invention also includes compositions that are prepared for storage or administration, which includes a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R.

Gennaro edit. , 1985) which is incorporated herein by reference. For example, condoms, stabilizers, inks and flavoring agents may be provided. Id. in 1449. These include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used. Id. A pharmaceutically effective dose is that dose which is required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some degree, preferably all symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition that is used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, the concurrent medication, and other factors that those skilled in the art. medical techniques will recognize. Generally, an amount of between 0.1 milligrams / kilogram and 100 milligrams / kilogram of body weight / day of the active ingredients is administered, depending on the potency of the negatively charged polymer. The examples provided herein illustrate the different aspects and embodiments of the present invention. Although the examples presented here relate primarily to the supply of riboenzymes and plasmid DNA, one skilled in the art will recognize that any nucleic acid, protein, lipid, or other molecule can be delivered, either alone or in combinations to a objective biological system, using the teachings of the present invention. These examples are not intended to limit the described invention in any way.

Example 1: Synthesis of diaminobutyric acid and cationic lipids based on quanidinium (Figure 2) Synthesis of palmi tiloleilamina (1) -. 1-Bromohexadecane (15.27 grams, 50 mmol) was added rapidly to oleylamine (26.75 grams, 100 mmol) at 100 ° C. The reaction mixture was heated at 120 ° C for 30 minutes and then cooled to room temperature. Chloroform (200 milliliters) was added followed by 1 N NaOH (50 milliliters). The mixture was then extracted with H20 (200 milliliters), the organic layer was dried (Na2SO4) and concentrated to a syrup. Silica gel column chromatography using a gradient of 15-20 percent methanol in dichloromethane, produced 20.5 grams of palmitylolethylamine as a syrup (yield, 83 percent).

The identity of the product was confirmed using spectroscopy NMR. XK NMR (CDC13) d 5.34 (m, 2H, CH = CH), 2.58 (m, 4H), 2.00 (m, 4H), 1.47 (m, 4H), 1.25 (m, 48H), 0.86 (m, 6H). FAB-MS: 493 [M + H] "(Other reagents could include oleylbromide and hexadecanamine).

Synthesis of N-palmiyl-N-oleyl-N-CBZ-glycinamide (2): (1) (2.46 grams, 5 mmol) was added to a solution of N-CBZ-glycine-N-hydroxysuccinimide ether (3.06 grams) , 10 mmol) suspended in dichloromethane (1.39 milliliters) containing triethylamine (TEA) (10 mmol). The reaction mixture was stirred at room temperature overnight and then concentrated to an oil under vacuum. Silica gel column chromatography using a gradient of 1-5 percent methanol in dichloromethane yielded 1.54 grams of N-palmityl-N-oleyl-N-CBZ-glycinamide (yield, 45 percent). - "? NMR (CDC13) d 7.35 (m, phenyl), 5.83 (br s, NH), 5.35 (m, CH = CH), 5.12 (s, 2H, CH2Ph), 4.00 (m, 2H, glycyl), 3.31 (m, 2H), 3.13 (m, 2H), 2.00 (m, 4H), 1.53 (m, 4H), 1.25 (m, 48H), 0.88 (m, 6H).

Synthesis of N-palmiyl-N-oleyl-glycinamide (3): 10 percent palladium on carbon (Pd / C) was added to the N-palmityl-N-oleyl-N-CBZ-glycinamide (0.5 grams, 0.73 mmol) dissolved in absolute ethanol (3 milliliters) under argon gas. The flask was immersed in a water bath at 20 ° C before the addition of 1,4-cyclohexadiene (0.66 milliliters). The reaction mixture was stirred at room temperature overnight, the catalyst was filtered and the filtrate was evaporated to dryness, yielding 0.3 grams of the product (yield, 75 percent). 1 H NMR (acetone-d 6) d 5.41 (m, 2 H, CH = CH), 4.07 (br s, 2 H, glycyl), 3.36 (m, 2 H), 3.29 (m, 2 H), 2.80 (br s, NH 2) , 2.05 (m, 2H), 1.98 (m, 2H), 1.63 (m, 2H), 1.25 (m, 48H), 0.87 (m, 6H). FAB-MS: 549 [M + H] ~.

Synthesis of N-palmityl-N'-oleyl-alpha, gamma-bis-Boc-diaminobutyryl-glycinamide (4): The mixture of (3) (1.12 grams, 2.04 mmol), N-alpha-N-gamma acid was stirred -di-Boc-diaminobutyric (631 milligrams, 2.24 mmol), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (553 milligrams, 2.24 mmol) in CH 2 Cl 2 for 1 hour at room temperature. The mixture was then concentrated to a syrup and 1.2 grams of the product was isolated by column chromatography, using 20-50 percent gradients of hexanes in ethyl acetate (yield, 69 percent). XH NMR (CDC13) d 7.14 (br s, NH), 5.38 (m, 2 H, CH = CH), 5.28 (br s, 1 H, NH), 5.12 (br s, ÍH NH), 4.02 (m, 2H, glycyl), 3. 42 (m, ÍH), 3.31 (m, 2H), 3.15 (m, 2H), 3.02 (m, 2H), 1.95 (m, 4H), 1.77 (m, 2H), 1.53 (m, 4H), 1.25 (m, 48H). FAB-MS: 850 [M + H] ~.

Synthesis of N-palmityl-N'-oleyl-alpha, gamma-diamino-butyryl-glycinamide (JA59311) (5): Compound (4) was dissolved in dioxane (6 milliliters) followed by the addition of 4M HCl in dioxane ( 6 milliliters). The reaction mixture was stirred at room temperature for 2 hours, then concentrated in vacuo and azeotroped twice with toluene. The residue was partitioned between CH2Cl2 and 0.2 N NaOH. The organic layer was washed with saturated NaHCO 3 solution, and then dried (Na 2 SO 4) and evaporated to dryness. Flash silica gel chromatography using CH2Cl2 / methanol / conc. NH40H 40: 10: 2 produced 200 milligrams of compound 5 (yield, 75 percent). 1H NMR (CDC13) d 8.06 (br s, ÍH, NH), 5.38 (m, 2H, CH = CH), 4.04 (m, 2H, glycyl), 3.54 (m, ÍH), 3.31 (m, 2H), 3.17 ( m, 2H), 2.86 (m, 2H), 1.93 (m, 4H), 1.67 (m, 2H), 1.54 (m, 4H), 1.41 (br s, 4H, NH2), 1.25 (m, 48H), 0.87 (m, 6H). FAB-MS: 650 [M + H] ".

Synthesis of N'-palmiyl-N '-oleyl-N-gamma-carboxamidin-alpha -gamma-diaminobutyryl-glycinamide (JA59312) (6): 1H-pyrazole-1-carboximidine hydrochloride (70 milligrams, 0.48 mmol) was added ) to the solution of (5) (0.16 grams, 0.25 mmol) and diisopropylethylamine (DIPEA) (83 milliliters) in THF / methanol 1: 1 (0.8 milliliters), under argon gas. The reaction mixture was stirred at room temperature overnight and then concentrated to a syrup. Column chromatography on silica gel using CH2C12, followed by CH2Cl2 / methanol / conc. NH 4 OH 40: 10: 2 yielded 50 milligrams of compound 6 (yield, 29 percent). XH NMR (CDC13) d 5.37 (m, 2H, CH = CH), 4.07 (m, 2H, glycyl), 3.94 (m, ÍH), 3.42 (m, 2H), 3.21 (m, 4H), 2.01 (m, 6H), 1.58 (m, 4H), 1.46 (m, 2H, NH2), 1.25 (m, 48H), 0.87 (m, 6H). FAB-MS: 692 [M + H] ".

Synthesis of N '-palmyl tyl-N' -oleyl-alpha, gamma-bis-trimethylammonium butyryl-glycinamide (JA59316) (7): A (5) dihydrogen chloride salt (130 milligrams, 0.2 mmol) was dissolved in methanol (4). milliliters) and combined with KHC03 (0.2 grams) and CH3I (0.2 milliliters). The mixture was then stirred at room temperature for 3 days. The reaction mixture was then filtered through the Celite bed, followed by filtration through a 0.45m PTFE filter. The filtrate was then evaporated to dryness, yielding 160 milligrams of the desired product (yield, 94 percent). "" "H NMR (CDCl 3) d 3.58 [s, 9 H, (CH 3) 3], 3.44 [s, 9 H, (CH 3) 3].

Synthesis of Nf -palmy-N'-oleyl-N-carboxamidin-glycinamide-9HCl (JA59314) (8): Using the same procedure as described above for the preparation of (6), except that crystallization was used from methanol, instead of column chromatography for purification, (8) was prepared in 5 percent yield. "" "H NMR (CDC13) d 7.70-7.25 (m, 5H, NH), 5.38 (m, 2H, CH = CH), 4.25 (m, 2H, glycyl), 3.27 (m, 4H), 1.96 (m , 4H), 1.53 (m, 4H), 1.25 (m, 48H), 0.87 (m, 6H), FAB-MS: 592 [M + H] ".

Synthesis of N'-palmiyl-N '-oleyl-guanidine (JA59317) (9): The mixture of a hydrochloride salt of (1) (285 milligrams, 0.54 mmol), cyanamide (50 milligrams, 1.19 mmol) and 1-butanol (2 milliliters) was stirred at 120 ° C for 2 hours. The cooled mixture was diluted with CH2C12 (50 milliliters) and washed with saturated aqueous NaCl (saline) / methanol 1: 1 (50 milliliters).

The organic layer was then dried (Na2SO4), evaporated to an oil and subjected to column chromatography on silica gel using CH2Cl2 / methanol / conc. NH4OH 40: 10: 2 which gave 80 milligrams of the desired material (yield, 28 percent).

XH NMR (CDC13) d 7.08 (br s, ÍH, NH), 5.34 (m, 2H, CH = CH), 3.29 (m, 4H), 2.00 (m, 4H), 1.62 (m, 4H), 1.25 (m, 48H), 0.88 (m, 6H). FAB-MS: 535 [M + H] ".

Example 2; Synthesis of DS 46596 (12) Synthesis of Cholesterylamine (10): With reference to Figure 3, cholesterylchloride (10 grams, 25 mmol) was partially dissolved in dry methanol (50 milliliters) and the solution was heated with stirring at 155 ° C for 18 hours at 500 psig using a 300 milliliter Parr pump apparatus loaded with dry ammonia gas. The pump was cooled to room temperature and the methanol was removed by steam distillation on a rotary evaporator. Compound (10) was purified using Merck silica chromatography by levigation with dichloromethane / methanol (4: 1 v / v) to yield 4 grams of the ninhydrin positive product (yield, 60 percent). The identity was confirmed by ES-MS.

Synthesis of Boc 3argininNHcoles ter i lamida (11): A 200 milliliter pear-shaped flask was charged with a stir bar, with a mixture of (10) (1 gram, 2.6 mmol), Boc3arginine (1.2 grams, 2.6 mmol), diisopropylcarbodiimide (450 milliliters, 2.9 mmol) and dichloromethane (70 milliliters). The reagent mixture was stirred at room temperature for two hours. After the reaction, the solution was washed with aqueous sodium bicarbonate (5 percent w / v) and the organic layer was separated and dried to a solid using a rotary evaporator. Compound (11) was dissolved in 5 milliliters of dichloromethane, before purification using silica gel chromatography (yield, 90 percent). The identity was confirmed by ES-MS. 1H NMR (dmso-d6): 9.32 (bd), 6.63 (d), 5.30 (m), 4.00 (m), 3.80 (m), 3.40 (m), 1.539 (s, tBoc), 1.517 (s, tBoc) ), 1,497 (s, tBoc).

Synthesis of Bocarginin NHcholesterilamide (DS4659) (12): Compound (11) (50 milligrams, 60 mmol) was dissolved in anhydrous 1,4-dioxane (300 milliliters) and combined with 4M HCl in dioxane (400 milliliters). The mixture was left at room temperature for 2 hours and the reaction was stopped by removing all the solvent and HCl, using a stream of dry nitrogen gas. Compound (12) was isolated using a wide pore C18 silica column and a water-levigating isocratic methanol (88:12) with detection at 210 nm. The fractionation allowed a recovery of 20 milligrams of the compound (12) (yield, 44 percent). The identity was confirmed by ES-MS. ^? NMR (dmso-d6): 7.82 (d), 6.36 (bs), 3.51 (m), 3.43 (m), 3.33 (m), 3.15 (m), 1.472 (s, tBoc).

Example 3; Synthesis of PH55933 (15) and PH55938 (16) Synthesis of (13): With reference to Figure 14, 4-dimethylaminopyridine (DMAP) (0.31 grams, 2.5 mmol) was added to a solution of methylphosphonic dichloride (0.332 grams, 2.5 mmol, 31 P NMR s, 43.93 ppm), stirring at room temperature under positive pressure argon. The resulting clear, colorless solution was cooled to -70 ° C and a solution of cholesterol (0.97 grams, 2.5 mmol) suspended in anhydrous dichloromethane (20 milliliters) was added via a syringe with vigorous stirring for a period of one hour. The mixture was allowed to warm to room temperature and was maintained at room temperature for 18 hours, at which time the 31P NMR analysis of a small aliquot of the reaction mixture indicated the complete reaction (d, 39.08 ppm) . The (13) crude was treated with additional DMAP (0.31 grams, 2.5 mmol) and the reaction mixture was cooled to -70 ° C while stirring under positive pressure argon. H-Lys (Z) -OCH3 (0.66 grams, 2.25 mmol) in anhydrous dichloromethane (20 milliliters) was added dropwise by syringe over a period of one hour. The reaction mixture was warmed to room temperature and stirred for an additional 18 hours (complete reaction by 31 P NMR). Direct charging on flash silica followed by a gradient of 0 to 10 percent EtOAc / hexanes, then 5 percent EtOH / dichloromethane, gave 1.12 grams of (14) (yield, 60 percent during two steps). 31 P NMR (s, 30.98 ppm).

Synthesis of cholesterol-lyin-methyl phosphonoamidate (PH55933) (15): Compound (14) (1.0 gram, 1.35 mmol) was dissolved in anhydrous EtOH and cooled to 0 ° C with an ice / water bath while stirring under argon. 10 Pd / c (1.0 gram, 1 eq. Mass) was added to the reaction mixture followed by a dropwise addition of 1,4-cyclohexadiene (1.27 milliliters, 13.5 mmol). After warming to room temperature, the reaction was complete after 4 hours, as determined by TLC (15 percent MeOH / dichloromethane). The reaction mixture was filtered over celite and dried in vacuo. Flash chromatography using a gradient of 5 to 15 percent MeOH / dichloromethane, 1 percent TEA, yielded 0.64 grams of (15): (yield, 78 percent) 31P NMR (s, 30.88 ppm), specific mass calculated = 606.87, found = 607.47.

Synthesis of cholesterol -homoarginine-methyl phosph Onoamidate (PH55938) (16): 1-H-pyrazole-l-carboxamidine «HCl (32 milligrams, 0.216 mmol) was added to a solution of (15) (0.131 grams, 0.216 mmol) , stirring at room temperature under argon in anhydrous DMF (2.0 milliliters), followed by diisopropylethylamine (28 milliliters, 0.216 mmol). The reaction mixture was lightly stripped on a rotary evaporator and then rotated overnight without vacuum at room temperature. After removing the DMF in vacuo, the reaction residue was dissolved in dichloromethane and applied to a column of silica gel. An isocratic system of 20 percent MeOH / dichloromethane, 2 percent NH40H, followed by treatment with Dowex (300 milligrams), gave 80 milligrams of the desired product (yield, 57 percent). 31P NMR (s, 31.99 ppm), calculated specific mass = 648.91, found = 649.48.

Example 4: Synthesis of PH55939 (17) Synthesis of cholesterol amide-lyin-metil f Osf onoamidate (PH55939) (17): With reference to Figure 5, compound (15) (76 milligrams, 0.125 mmol) was treated with a solution of methanolic ammonia saturated at 0 ° C at room temperature for 18 hours (some ventilation required). The reaction mixture was evaporated in vacuo and then purified by flash silica gel chromatography to give 45 milligrams of the product (yield, 61 percent). 31P NMR (s, 32.17 ppm), calculated specific mass = 591.86, found = 592.23.

Example 5; Synthesis of PH55941 (18) and PH55942 (19) Synthesis of cholesterol-TREN-methyl phosphonoamidate (PH55941) (18): With reference to Figure 6, 4-dimethylaminopyridine (DMAP) (0.31 grams, 2.5 mmol) was added to a solution of methylphosphonic dichloride (0.332 grams, 2.5 mmol, 31P NMR s, 43.93 ppm) was stirred at room temperature under positive pressure argon gas. The resulting clear, colorless solution was cooled to -70 ° C and a solution of cholesterol (0.97 grams, 2.5 mmol) in anhydrous dichloromethane (20 milliliters) was added via syringe with vigorous stirring for a period of one hour. The mixture was allowed to warm to room temperature and was maintained for 18 hours, at which time the 31P NMR analysis of a small aliquot of the reaction mixture indicated the complete reaction (d, 39.08 ppm). The (13) crude was treated with additional DMAP (0.31 grams, 2.5 mmol) and the reaction mixture was cooled to -70 ° C while stirring under positive pressure argon. Tris (2-aminoethyl) amine (TREN) (0.37 milliliters, 2.5 mmol) in anhydrous dichloromethane (20 milliliters) was added dropwise by syringe over a period of two hours. The reaction mixture was warmed to room temperature and stirred for an additional 18 hours (complete reaction by 31 P NMR). Direct loading on flash silica followed by a gradient of 0 to 20 percent MeOH / dichloromethane with 1 to 4 percent NH40H gave 0.442 grams of (18) as a white foam: (yield, 28 percent during two steps ), 31P NMR (d, 32.57 ppm), calculated specific mass = 592.89, found = 593.49.

Synthesis of cholesterol-TREN-guanidium-methylf osf onoamidate (PH55942) (19): Compound (18) was dissolved in anhydrous DMF (1.0 milliliters) and anhydrous dichloromethane (5.0 milliliters). L-H-pyrazole-l-carboxamidine »HCl (73 milligrams, 0.50 mmol) was added to the reaction mixture, followed by diisopropylethylamine (87 milliliters, 0.50 mmol). The dichloromethane was stripped from the reaction mixture on a rotary evaporator and then the rotation was continued overnight without vacuum at room temperature. After removing the DMF in vacuo, the reaction residue was dissolved in dichloromethane and applied to a column of flash silica gel. A gradient of 5 to 20 percent MeOH / dichloromethane with 0.5 to 20 percent NH4OH followed by treatment with Dowex OH "(300 milligrams) in MeOH gave the (19) pure: 0.11 grams, 65 percent, 31P NMR (d, 33.83 ppm), calculated specific mass = 676.97, found = 677.54.

Example 5: Synthesis of PH55943 Synthesis of cholesterol-lisin-methylf Osf onoamidate (PH55943) (20): With reference to Figure 7, the (13) crude was treated with additional DMAP (0.31 grams, 2.5 mmol) and the reaction mixture was cooled to -70 ° C while stirring under argon. positive pressure H-His (Bzl) -0CH3 (0.65 grams, 2.5 mmol) in anhydrous dichloromethane (20 milliliters) was added dropwise by syringe over a period of one hour. The reaction mixture was then warmed to room temperature and stirred for 18 hours (complete reaction by 31 P NMR). Direct charging on flash silica followed by a gradient of 2 to 10 percent EtOAc / hexanes, then 5 percent EtOH / dichloromethane, gave 0.53 grams of (20) (3 percent during two steps). 31P NMR (d, 31.39 ppm), calculated specific mass = 705.96, found = 706.47.

Example 6: Synthesis of PH55945 (21) Synthesis of cholesterol-histidine-methyl phenoxyamide (PH55945) (21): With reference to Figure 8, the (13) crude was treated with additional DMAP (0.31 grams, 2.5 mmol) and the reaction mixture was cooled to -70. ° C while stirring under positive pressure argon. 1- (3-Aminopropyl) -imidazole (0.30 milliliters, 2.5 mmol) in anhydrous dichloromethane (20 milliliters) was added dropwise by syringe over a period of one hour. The reaction mixture was warmed to room temperature and stirred at room temperature for an additional 18 hours (complete reaction by 31 P NMR). The direct charge on flash silica after the saturated bicarbonate bath, followed by a gradient of 0 to 10 percent EtOAc / hexanes, then 0 to 10 percent MeOH / dichloromethane that gave 0.77 grams of (21) (yield, 54 percent during two steps). 31P NMR (d, 32.47 ppm), calculated specific mass = 571.82, found = 572.33.

Example 7: Synthesis of vitamin B6 and cationic lipids based on beta-alanine Synthesis of N-CBZ-beta-alanine-N-hydroxy-succinimide ester (22): With reference to Figure 9, the compound was prepared in accordance with Lewis et al. PNAS 1996, 93, 3176-3181 (which is incorporated herein by reference). Performance, 80 percent. 1 H NMR (DMSO-d 6) d 7.42, (t, ÍH, NH), 7.33 (m, 5H, benzyl), 5.009 (s, CH2), 1.47 (m, 4H), 3.32 (m, 2H, CH2NH), 2.86 (t, 2H, CH2CO), 2.79 (s, 4H, CH2CH2).

Synthesis of N'-palmiyl-N'-oleyl-N-CBZ-beta-alanine-amide (23): Compound (22) (1.0 gram, 2.03 mmol) was added to a solution of N-CBZ-ester beta-alanine-N-hydroxy-succinimide (0.16 grams, 0.5 mmol) in CH2Cl2 containing Et3N (0.42 milliliters, 3 mmol). The reaction mixture was stirred at room temperature overnight with dichloromethane and washed with saturated NaHCO 3 and saline. The organic layer was dried over sodium sulfate and evaporated to dryness. The residue was purified by flash chromatography on silica, using the mixture of EtOAc-hexanes (1: 3) as a levigant to give 1.32 grams of (23) as a yellow oil (yield, 93 percent). 1 H NMR (CDCl 3) d 7.34 (m, phenyl), 5.62 (t, NH), 5.35 (m, CH = CH), 5.082 (s, 2H, CH2Ph), 3.49 (m, 2H, CH2NH), 3.27 (m , 2H), 3.15 (m, 2H), 2.5 (m, 2H, CH2CO), 2.00 (m, 4H), 1.49 (m, 4H), 1.25 (m, 48H), 0.87 (m, 6H).

Synthesis of N'-palmiyl-N'-oleyl-beta ta-alaninamide (AK 524-68) (POABA) (24): Compound (23) (1.2 grams, 1.72 mmol) was dissolved in absolute ethanol (10 milliliters) ) and 10 percent Pd-C was added under argon. The flask was then immersed in a water bath at 20 ° C before the addition of 1,4-cyclohexadiene (1.6 milliliters). The reaction mixture was stirred at room temperature for 40 hours, the catalyst was filtered and the filtrate was evaporated to dryness. The residue was purified by flash chromatography on silica, levigating with the linear gradient of MeOH (5 percent to 10 percent) in dichloromethane, to give 0.68 grams of (24) (yield, 70 percent). XH NMR (CDC13) d 5.37 (m, 2H, CH = CH), 3.26 (m, 4H), 3.16 (m, 2H), 2.75 (t, NH2), 1.95 (m, 2H), 1.51 (m, 4H), 1.25 (m, 48H), 0.87 (m , 6H). FAB-MS: 563.6 [M + H] ".

Synthesis of N'-palmiyl-N'-oleyl-N-carboximidin-beta-alaninamide (AK 524-73) (GPPOA) (25): The mixture of (24) (60 milligrams, 0.11 mmol), hydrochloride was stirred of pyrazole carboxamidine (16 milligrams, 0.11 mmol) and diisopropylethylamine (20 milliliters, 0.12 mmol) in 0.5 milliliters of THF-MeOH (1: 1) overnight at room temperature. It was then evaporated to dryness, dissolved in dichloromethane and washed with aqueous ammonia. The organic layer was dried over sodium sulfate to give (25) as a yellowish oil. Near quantitative performance. ^? NMR (CDC13) d 7.70-7.25 (m, 5H, NH), 5.38 (m, 2H, CH = CH), 3.43 (m, 2H, CH2NH), 3.23 (m, 2H), 3.17 (m, 2H), 2.54 (m, 2H, CH2CO), 1.96 (m, 4H), 1.49 (m, 4H), 1.25 (m, 48H), 0.87 (m, 6H). FAB-MS: 605.6 [M + H] +.

Synthesis of N (N "-palmyl-N" -oleyl-amidopropyl) -pyridoxamine (AK 524-74) (POCAEP) (26): Compound (26) was prepared analogously to compound (27) ( Yield, 78 percent). FAB-MS: 714.6 [M + H] +.

Example 8; Synthesis of AK524-76 (27) N-cholesteryl-pyridoxamine (AK524-76) (CCAEP) (27): With reference to Figure 11, the suspension of pyridoxal hydrochloride (0.1 grams, 0.5 mmol) in ethanol was brought to a pH of 7 with INN NaOH, followed by the addition of aminocholesterol (0.19 grams, 0.5 mmol). The pH of the resulting bright yellow solution was adjusted to 8 (INN NaOH) and set aside for 10 minutes. Then sodium borohydride (20 milligrams, 0.5 mmol) was added to the reaction mixture, resulting in the immediate disappearance of the color. After 15 minutes, the reaction mixture (pH 6) was acidified with IN HCl, diluted with dichloromethane and washed with aqueous ammonia and water. The organic layer was dried over sodium sulfate and evaporated to dryness. The residue was purified by flash chromatography on silica, using 10 percent MeOH in dichloromethane as a levigant to give 0.25 grams (93 percent) of (27). XH NMR (CDC13) d 7.78 (s, ÍH, H-6 Pyr), 4.58 (s, 2H, CH20), 4.06 (AB-quartet, 2H, CH2N), 2.4 (s, 3H, 2-CH3), 2.2 -0.24 (m, cholesteryl fraction). FAB-MS: 537.4 [M + H] +.

Example 9: Synthesis of cationic lipids based on 2'-aminouridine 2'-deoxy-2 '- (n-Fmoc-beta-l aninamido) uridine (28): With reference to Figure 10, EEDQ (4.2 grams, 17 mmol) was added to the solution of 2'-amino-deoxyuridine (4 grams, 16.45 mmol) and N-FMOC-beta-alanine (5.1 grams, 16.45 mmol) in methanol and the reaction mixture was boiled for two hours. Subsequent flash chromatography on silica using a linear gradient of methanol (5 percent to 10 percent), in dichloromethane, provided 6 grams of 2'-deoxy-2 '- (n-Fmoc-beta-alaninamide) uridine (77 percent) . ^ NMR (CDCl 3 -DMSO-d 6) d 10,398 (s, ÍH, N 3 -H), 6.98-7.63 (m, H 6, Fmoc), 6.16 (t, ÍH, NHFmoc), 5.73 (d, ÍH, Hl ', Jlí / 2, 8.4), 5.34 (d, ÍH, H5), 4.22 (m, ÍH, 2'-H), 3.98 (dd, 2H, CH2), 3.88 (m, ÍH, 3'-H), 3.77 (br s, ÍH, 4'-H), 3.43 (m, 2H, 5'-CH2), 3.1 (m, 2H, CH2NHFmoc), 2.07 (t, 2H, CH2C0).

Synthesis of 3 ', 5' -di-palmyl-2'-deoxy-2 '- (N-Fmoc-beta-alaninamide) uridine (29): Palmitoyl chloride was added (1.55 milliliters, 1.8 mmol) to a solution of the nucleoside (28) in abspiridine and the reaction mixture was stirred overnight at room temperature. The solution was then cooled with MeOH, evaporated to dryness, dissolved in dichloromethane and washed with saturated aqueous sodium bicarbonate and saline. The organic phase was dried over sodium sulfate and evaporated to dryness. The residue was purified by flash chromatography on silica (EtOAc: hexanes, 1: 1), yielding 0.5 grams of (29) (yield, 65 percent).

Synthesis of 3 ', 5'-di-palmi-tyl-2'-deoxy-2' - (beta-alaninamido) uridine (AK 524-71) (30): Morpholine (1 milliliter) was added to the solution of (29) ) (0.5 grams, 0.49 mmol) in dichloromethane (5 milliliters) and the reaction mixture was stirred at room temperature for 36 hours. Flash chromatography on subsequent silica using the linear gradient of methanol (5 percent to 10 percent) in dichloromethane gave 0.22 grams of the desired product (yield, 56 percent) of (30). FAB-MS: 791.6 [M + H] +.

Synthesis of 3 ', 5'-di-palmi-tyl -2'-deoxy-2' - (N-carboxamidin-beta-alaninamido) uridine (AK 524-75) (31): The compound (31) was prepared in a manner analogous to compound (25) (Yield, 80 percent). FAB-MS: 833.6 [M + H] +.

Example 10: Preparation of lipid-based formulations including cationic lipids and DOPE For each cationic lipid, four aqueous suspensions were prepared, three containing the fusogenic neutral lipid DOPE (dioleoylphosphatidylethanolamine), and one containing only the cationic lipid (Table II). For this, the cationic lipids were dissolved in chloroform and the aliquots were transferred to 5 milliliter glass tubes with Teflon-lined lids. DOPE was also dissolved in chloroform, then added to the individual tubes at 1: 1, 2: 1 or 3: 1 molar proportions (ratio of cationic lipid to DOPE). The lipid mixture was deposited as a film in the glass tube by evaporating the solvent with a stream of argon. The lipid film was hydrated with water (1 milliliter per milligram of total lipid), and then resuspended by sonication, using a bath sonifier (three or four 15 s treatments with intermittent vortex mixing). The formulations were stored at 4 ° C until they were used (usually in 8 weeks). The residue was purified by flash chromatography on silica (EtOAc-Hexanes 1: 1), giving 0.5 grams of 29 (65 percent yield).

Synthesis of 3 '-5' -Di-palmi toyl -2 '-deoxi -2' - (beta-alaninamido) uridine (AK 524-71) (30): To the solution of 29 (0.5 grams, 0.49 mmol) in dichloromethane (5 milliliters), morpholine (1 milliliter) was added and the reaction mixture was stirred at room temperature for 36 hours. Subsequent flash chromatography on silica, using the linear gradient of methanol (5 percent to 10 percent), in dichloromethane, gave 0.22 grams of the desired product (56 percent yield) of 30. FAB-MS: 791.6 [M + H] +.

Synthesis of 3 ', 5' -Di-palmi toyl -2 '-deoxy -2' - (N-carboxamidine-beta-alaninamide) uridine (AK 524-75) (31): Compound 31 was prepared analogously to compound 25 Yield of 80 percent. FAB-MS: 833.6 [M + H] +.

Example 10: Preparation of lipid-based formulations including cationic lipids and DOPE For each cationic lipid, four aqueous suspensions were prepared, three containing the fusogenic neutral lipid DOPE (dioleoylphosphatidylethanolamine), and one containing only the cationic lipid (Table II) . For this, the solid cationic lipids were dissolved in chloroform, and the aclícuotas were transferred to 5 milliliter glass tubes, with Teflon-coated lids. The DOPE, also dissolved in chloroform, was then added to the individual tubes at molar ratios of 1: 1, 2: 1, or 3: 1 (ratio of the cationic lipid to DOPE). The lipid mixture was deposited as a film in the glass tube, by evaporating the solvent with a stream of argon. The lipid film was hydrated with water (1 milliliter per milligram of total lipid), and then resuspended by sonication, using a bath sonicator (three or four 15-second treatments with intermittent vortex mixing). The formulations were stored at 4 ° C until they were used (usually within the next 8 weeks).

Example 11: Cell culture and synthesis of anionic polymers Cell delivery and efficacy assays were performed on single-layer cultures of cells originating from normal tissues or tumors (Table II, III and V). The cells were maintained in humidified incubators, using growth medium recommended by the supplier. Hammerhead ribozymes were synthesized, and purified using standard protocols (Wincon et al., 1995, 23, 2677; Beigelman et al., 1995, J. Biol. Chem. 270, 25702, both incorporated herein by reference). . Nuclease-resistant bonds were incorporated at specific sites in the ribozymes, modifications that markedly increased the serum half-life from a few minutes to many hours. For cell delivery studies, 32 -mer labeled ribozymes were prepared with fluorophore, by attaching a fluorescein or rhodamine fraction to the curve portion through a base modified by amino-linker (Figure 13). An expression plasmid encoding the humanized Fluorescent Green Protein (plasmid pEGFP-Cl) was obtained with Clontech.

Example 12: Cell Ribozyme Delivery For the supply studies, subconfluent cultures of mammalian cells were seeded in 24-well plates (~ 20,000 cells / well), one day before the start of the assay. In a typical delivery assay, 100 μl of a 1 μM fluorescein or rhodamine conjugated with rhodamine (ie, 10 X ribozyme diluted in water) was placed in a polystyrene tube, and an aliquot of the lipid formulation was added. cationic at room temperature, to allow complex formation. The appropriate growth medium was added to each tube (0.9 milliliters), and then the contents were mixed and added to each well. The final concentration of the ribozyme was 100 nM, and the concentration of the transport vehicle was varied from 2.5 to 20 μg / milliliter. After an incubation of 3-4 hours, the medium was replaced with normal growth medium, and the location of the ribozyme associated with the cells was evaluated, by fluorescence microscopy, using a Nikon stage microscope, equipped with an objective of 40 X and a ccd camera. In some studies, the ribozyme associated with the cells was quantified by FACS analysis.

Example 13: Cellular Plasmid Delivery Cells were subconfluent cultures seeded in 24-well plates (~ 10,000 cells / well). In typical transfection studies, 100 ng of the plasmid was previously mixed in 0.1 milliliter of water, with individual lipids in a polyethylene tube, and incubated at room temperature for ~ 10 minutes, to allow complex formation.

Then 0.9 milliliters of growth medium was added (serum free) to each tube, the content was mixed, and administered to individual cell wells for 3-4 hours. The medium was then replaced with normal growth medium, and the cells were left for ~ 24 hours. The expression of GFP was monitored by fluorescence microscopy. The transport vehicles that led to the expression of GFP in the highest percentage of cells were identified for each cell line (Table IV).

Example 14: Cytotoxicity analysis The toxic effects of the lipid formulated compositions on the cells were determined in three ways. First, cell morphology was evaluated in relation to normal, untreated cells and significant abnormalities or reduction in cell numbers were noted. Secondly, to evaluate the general toxicity, propidium iodide was added to the medium, and the cells were examined for the presence of pink-red fluorescence in the nucleus, indicating the presence of perforated or damaged membranes. Finally, the long-term effect was quantified, using a sensitive MTS proliferation assay (Promega).

Example 15: Proliferation assay of c-m? B The proto-oncogene c-myb is a transcription factor that participates in the regulation of the proliferation of soft muscle cells. It has been shown that cells in which c-myb levels have been reduced by ribozyme-Lipofectamine treatment do not proliferate well in response to subsequent serum stimulation. Two ribozymes directed against c-myb are called "active" and "inactive" (Jarvis et al., 1995, RNA, 2, 419). Both ribozymes can recognize the target mRNA, but only the "active" can dissociate it. The "inactive" ribozyme serves as a negative control. In principle, the active ribozyme can reduce the expression of c-myb by catalyzing the specific dissociation to the mRNA sequence, leading to a reduction in cell proliferation. This assay was used to validate the utility of the delivery formulations, 3H-Thymidine Incorporation Assay. In typical cell proliferation, rat aortic soft muscle cells (RASMC) were plated in 48-well plates in DMEM supplemented with amino acids, Hepes, and 10 percent fetal bovine serum (5000 cells / well / 0.5 milliliters of medium). The next day the cells were stripped of the serum, to inhibit proliferation, by replacing the medium with low serum medium (0.5 percent serum) for ~ 2 days.

The stripped cells were then treated with ribozyme-bearing formulations, usually 100 nM of ribozymes previously mixed with 2.5-10 μg / milliliter of carrier lipid, in serum-free medium for ~ 2 hours, followed by "trafficking" in serum medium. low (0.25 percent serum) for ~ 20 hours. The set in triplicate of wells was exposed to each treatment. The cells were then stimulated to proliferate for 12 hours in medium containing 10 percent serum. This was followed by another 8 hour incubation in medium + 10 percent serum + 3H-thymidine (~ 1 μCi / milliliter). The cells were then fixed with ice-cold 10% triroacetic acid, washed twice with water, and the 3 H-thymidine incorporated into a new DNA was measured by scintillation counting. In Table V inhibition of proliferation is shown using different formulations of ribozymes.

Example 16: Cellular transport of lipophilic compounds The dioleoylphisphatidylethanolamine (DOPE) conjugated with rhodamine was mixed, with different cationic lipids, and was administered to cells seeded in 24-well plates. After an incubation of ~ 3 hours, the cellular distribution of the fluorescent rhodamine-DOPE was examined by fluorescence microscopy. All cells contained rhodamine (red fluorescence), indicating that the lipids were efficiently delivered to the cells. Then, ribozymes conjugated with fluorescein were packaged, and co-administered to the cells using the same vehicles, using the procedures described above. Again, every cell was labeled with rhodamine, while a subset contained incorporated ribozymes (green fluorescence). These observations suggested that lipid transporters can be used to deliver lipophilic as well as hydrophilic compounds to cells.

Example 17: Provision of anti-sense molecules A long phosphorothioate oligodeoxynucleotide of 21 nucleotides with a bound fluorescein fraction was synthesized by standard procedures. The oligonucleotide (100 nM) was formulated with different concentrations (2.5 to 10 μg / milliliter) of each transport vehicle, and was administered to the cells. The subcellular distribution of the incorporated material was evaluated by fluorescence microscopy. The results indicated that the optimal transporter concentrations are different for the anti-sense oligonucleotides compared to the ribozymes.

Example 18: Synthesis of N2, N3-di-oleyl- (N, N'-diguanidinoethyl-aminoethane) -2, 3-diaminopropionic acid (36) Referring to Figure 14, the Applicant describes that an acid reaction 2, 3 Diaminopropionic 32 with oleoyl chloride, in the presence of dimethylaminopyridine (DMAP) and triethylamine (TEA), can give the peracylated derivative 33, oleyl. Reaction of 33 with triethylenetetramine (TREN) 34, followed by reaction with 1H-Pyrazole-1-carboxamidine hydrochloride, can give the title compound 36.

Example 19: Preparation of PC liposome formulation: CHOL: DOTAP: DSPE2000 EPC formation: CHOL: DOTAP: DSPE-PEG2000. Egg yolk phosphatidylcholine (EPC), cholesterol, and DOTAP were purchased with Avanti Polar Lipids. DSPE-PEG2000 (1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol -200) was purchased with Shearwater polymers. The extruder was purchased with Lipex Biomembranes. FPLC was purchased with Pharmacia. The radioactive compounds were purchased with NEN and the ether with Sigma. The following lipids suspended in chloroform were mixed together in a 50 milliliter round bottom flask: phosphatidylcholine (egg yolk) (85.5 milligrams), cholesterol (21.8 milligrams), DOTAP (23.7 milligrams), ceramide-PEG C20 (61.8 milligrams) ), resulting in a molar ratio of 50: 25: 15: 10. A radioactive indicator of cholesterylhexadecyl ether (CHE) (26 μCi) was added to monitor the lipid concentration. and the lipids were dried by rotary evaporation, and then resuspended in ether (9 milliliters). Ribozyme (25 milligrams) suspended in IX of saline solution (3 milliliters) was added pH-regulated with phosphate to the ether / lipid mixture, and mixed in an emulsion. The ribozyme was quantified using a radiolabelled internally labeled ribozyme indicator 32 P (160 μCi). Liposome vesicles were formed by removing the ether under vacuum. The residual ether was removed by bubbling argon gas through the lipid-ribozyme mixture for 10 minutes. The vesicles were then passed through polycarbonate filters with pores of 200 nm and 100 nm consecutively 6-10 times, using an Extruder (Lipex Biomembranes, Vancouver, B.C.). The liposomes were purified from the unencapsulated material, using an FPLC column packed with DEAE Sepharose CL-6B. The concentration of ribozymes and lipids was determined by reading an aliquot of purified liposomes in a scintillation counter for both tritium and 32P. The counts indicated that 5.75 milligrams of ribozyme were present inside the liposome vesicles (23 percent encapsulation).

Example 20: Blood evacuation study using the EPC liposome: CHOL: DOTAP: DSPE2000 In this study C57B1 / 6J females weighing 20-25 grams were used, with 3 mmol of lipid (36 milligrams of ribozyme) by vein injection from the tail. The time points observed were 15 minutes, 1 hour, 4 hours, and 24 hours, with 3 mice per group. Euthanasia was performed on animals by asphyxia with C02. After cessation of breathing, the chest cavity was opened and the blood (200-500 μL) of the heart was sampled. The sampled blood was added to a heparinized microfuge tube, and centrifuged for 10 minutes to separate the plasma and blood cells. Plasma samples were treated with proteinase K containing pH buffer (100 mM NaCl, 10 mM tris (pH of 8), 25 mM EDTA, 10% SDS). A portion of the sample was flashing and counted. The remaining sample was resolved on a polyacrylamide gel, and intact ribozyme bands were quantified, using a phosphoroformer (molecular devices). The results are shown in Figure 15. The formulation of ribozyme with EPC: CHOL: DOTAP: DSPE: PEG C18 greatly increases the circulation time of the intact ribozyme in the plasma. Twenty-four hours after an intravenous bolus injection of 2 milligrams / kilogram of ribozyme formulated with EPC: CHOL: DSPE: PEG2000, more than 6 percent of the dose remained in the plasma. The average concentrations fell from an average of 6631 ng / milliliter at 15 minutes, to 2305 ng / milliliter at 24 hours. Since the plasma concentrations were relatively high 24 hours after an injection, it can be assumed that the elimination half-life is in the order of hours, if not days. In comparison, an intravenous bolus injection of 30 milligrams / kilogram is no longer detected after approximately 3 hours. The elimination half-life of the unformulated ribozyme is approximately 30 minutes in the mouse.

Example 21: Delivery of Plasmid DNA into Culturing Cells One day prior to transfection, the target cells were plated to a final confluence of 50 to 60 percent in a 48-well plate. The cell types tested under serum-free conditions are RT-4 (human bladder carcinoma), EJ (human bladder carcinoma), PC-3 (prostate cancer cell line), and MCF-7 (cancer cell line) of chest). The following cell types were tested in the presence of 10 percent serum in the medium: RT-4, PC-3 and MCF-7 cells. DNA (1 μg of the C-terminal protein fusion vector pEGFP-Cl (Clontech)) was added to a polystyrene tube, followed by the addition of 1 milliliter of the desired medium. After stirring, the cationic lipid was added to the tube (1.25, 2.5, 5 or 10 μg of lipid / μg of DNA), incubated at room temperature for 15 minutes, and then mixed by vortexing. The medium was aspirated from the cells plated, and then washed either with serum-free medium or with normal growth medium. 200 μL of the DNA / cationic lipid mixture was added to each well of a 48-well plate. The cells were incubated at 37 ° C for 3 to 5 hours, for free uptake of serum, and 18 to 24 hours for uptake in the presence of serum. The fluorescent cells were then counted using fluorescence microscopy. The transfection rate was determined by comparing the number of fluorescence positive cells with the total number of cells in the microscope field. Toxicity was determined by the addition of 5 μL of 0.5 milligrams / milliliter of a solution of propidium iodide (Pl) origin (Boehringer Mannheim) before examination by microscopy. The migration of the red dye into the nucleus of a cell indicated toxicity and loss of cell viability. The results of the plasmid delivery in the serum-free medium are shown in Table VI. The results of the plasmid delivery in the presence of serum are shown in Table VII. FACS analysis (fluorescence activated cell sorting) was performed on PC-3 cells, using many formulations, and the results are shown in Table VIII. Transfection was achieved using the protocol described above with cells that were incubated with DNA for 4 hours under serum-free conditions. The cells were trypsinized off the plate, pooled in growth medium containing serum, and rotated down for 5 minutes at 800 revolutions per minute. The supernatant was removed and taken up in 500 μL of pH regulator FAC (4 percent FBS in Hank's balanced salt solution (HBSS)). 10 μL of 0.5 milligrams / milliliter of Pl were added, before classification by FACS. The results indicated that the applicant's formulations improve the supply of macromolecules compared to other compounds that are commercially available.

Example 22: Preparation of Cationic Lipids conjugated with polyethylene glycol via amide bond and dissolved cationic lipid (100 milligrams), methoxypolyoxyethylenecarboxylic acid (725 milligrams), and 1,3-dicyclohexylcarbodiimide (DCC) (30 milligrams) ), in chloroform (30 milliliters) and the solution was allowed to react at 50 ° C overnight. The reaction mixture was filtered and hexane was added to the filtrate for purification by precipitation. The product was re-precipitated (N'-palmityl-N'-oleyl-ce-amino,? -PEGamino-glycinamide), using the same procedure, and then dried in vacuo to obtain a lipid conjugated with PEG. In addition to the carboxy-terminated PEG, the activated N-hydroxysuccinimide ester of PEG can be used in the above conjugation methods. The conjugation can also be performed by the formation of a carbamate link. The reaction would be initiated by reacting glycol of methoxypolyethylene terminated in hydroxy, activated with imidazolylcarbonate, with the amino groups of the cationic lipids described above. The methods described herein are not limiting. Those skilled in the art will recognize that PEG can be rapidly conjugated to cationic lipids using techniques known in the art, and are within the scope of this invention. Other modalities are among the following claims.

TABLE I Characteristics of naturally occurring ribozymes Group I Introns • Size: ~ 150 a > 1000 nucleotides. • require a U in the target sequence immediately 5 'from the dissociation site. • Fix 4-6 nucleotides on the 5 'side of the dissociation site. • Reaction mechanism: they attack by the 3 '-OH of the guanosine to generate dissociation products with 3' -0H and 5'-guanosine. • In some cases, additional protein cofactors are required to help folding and maintaining the active structure. • More than 300 known members of this class. It was found as an intermediate sequence in Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, T4 phage, blue-green algae, and others. • Major structural features widely established through phylogenetic comparisons, mutagenesis, and biochemical studies t, 1]. • Complete kinetic structure established for a ribozyme [, 3,4,5]. 1Michel, Francois; Westhof, Eric. Slippery substrates Nat. Struct. Biol. (1994), 1 (1), 5-7. Lisacek, Frederique; Diaz, Yolande; Michel, Francois. Automatic identification of group I introns nuclei in genomic DNA sequences. J. Mol. Biol. (1994), 235 (4), 1206-17. 2 Herschlag, Daniel; Cech, Thomas R .. Catalysis of dissociation of RNA by means of the thermophilic ribozyme Tetrahymena. 1. Kinetic description of an RNA substrate complementary to the active site. Biochemistry (1990), 29 (44), 10159-71. 3Herschlag, Daniel; Cech, Thomas R .. Catalysis of dissociation of RNA by means of the thermophilic ribozyme Tetrahymena .. 2. Kinetic description of the reaction of a • Studies of folding of the ribozyme and of the coupling of the substrate on the way [6,7,8] . • Investigation of chemical modification of important residues well established [9,10]. • The small (4-6 nt) binding site can make this ribozyme too non-specific for the dissociation of RNA substrate that forms a bad coupling in an active site. Biochemistry (1990), 29 (44), 10172-80. 4Krutt, Deborah S; Herschlag, Daniel. Dependencies of the pH of Ribozima Tetrahymena Reveal an Unconventional Origin of an Apparent pKa. Biochemistry (1996), 35 (5), 1560-70. 5 Bevilacqua, Philip C; Sugimoto, Naoki; Turner, Douglas H .. A mechanistic structure for the second step for the splicing catalyzed by the ribozyme Tetrahymena. Biochemistry (1996), 35 (2), 648-58. 6Li, Yi; Bevilacqua, Philip C; Mathews, David; Turner Douglas H .. Parameters of thermodynamics and activation for the fixation of a substrate labeled with pyrene, by means of the ribozyme Tetrahymena: the coupling is not controlled by diffusion and is triggered by a favorable entropy change. Biochemistry (1995), 34 (44), 14394-9. 7Banerjee, Aloke Raj; Turner, Douglas H .. The time dependence of the chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry (1995), 34 (19), 6504-12. 8Zarrinkar, Patrick P .; Williamson, James R .. The peripheral extension P9.1-P9.2 helps guide the folding of the Tetrahymena ribozyme. Nucleic Acids Res. (1996), 24 (5), 854-8. 10Strobel, Scott A .; Cech, Thomas R .. The exocyclic amine of the Preserved Pair G.cntdot.U in the Dissociation Site of the Ribozima Tetrahymena Contributes to the Selection of the 5 'Splice Site and to the Transition State Stabilization. Biochemistry (1996), 35 (4), 1201-11.

Target RNA, however, the Tetrahimena group I intron has been used to repair a "defective" galactosidase message by ligating new sequences of β-galactosidase on the defective message t11].

RNA P seRNA (Ml RNA) • Size: ~ 290 to 400 nucleotides. • Portion of RNA from a ubiquitous ribonucleoprotein enzyme. • Dissociates tRNA to tRNA precursors in mature form t12] • Reaction mechanism: possible attack by M2 + -OH to generate dissociation products with 3 '-0H and 5'-phosphate. • SeRNA P was found throughout prokaryotes and eukaryotes. The RNA subunit has been sequenced from bacteria, yeast, rodents, and primates. • The recruitment of seRNA P for therapeutic applications is possible, through the hybridization of a 1: lSullenger, Bruce A.; Cech, Thomas R .. Repair mediated by ribozymes of defective mRNA, by means of the objective transempalme. Nature (London) (1994), 371 (6498), 619-22. 12Robertson, H. D. Altman, S; Smith, J. D. . J. Biol. Chem. 247, 5243-5251 (1972).

External Guide Sequence (EGS) to target RNA [13,14]. • Contacts of important phosphate and 2 'OH recently identified [15,16].

Introns of Group II • Size: > 1000 nucleotides. • Transempalme of recently demonstrated target RNAs [17,18]. • The sequence requirements are not completely determined. 13Forster, Anthony C; Altman, Sidney. External guide sequences for an RNA enzyme. Science (Washington, D.C., 1883-) (1990), 249 (4970), 783-6. 14Yuan, Y .; Hwang, E.S .; Altman, S .. Directed dissociation of mRNA by human seRNA P. Proc. Nati Acad. Sci. USA (1992) 89, 8006-10. 15Harris, Michael E .; Pace, Norman R .. Identification of phosphates involved in the catalysis by the seRNA RNA of the ribozyme. RNA (1995), 1 (2), 210-18. 16Pan, Tao; Loria, Andrew; Zhong, Kun. Probing of tertiary interactions in RNA: contacts with 2'-hydroxyl base between the RNA of seRNA P and pre-tRNA. Proc. Nati Acad. Sci. U.S.A. (1995), 92 (26), 12510-14. 17Pyle, Anna Marie; GReen, Justin, B .. Building a Kinetic Structure for the Ribozyme Activity of Introns of Group II: Quantification of the Fixation of Interdomain and Reaction Speed. Biochemistry (1994), 33 (9), 2716-25. 18Michels, William J. Jr.; Pyle, Anna Marie. Conversion of an Intron from Group II to a New Ribozyme of Multiple Changes that selectively dissociate Oligonucleotides: Clarification of the Reaction Mechanism and the Relations of Structure / Function. Biochemistry (1995), 34 (9), 2965-77.

• Reaction mechanism: the 2 '-0H of an internal adenosine generates dissociation products with 3' -0H and a "loop" RNA that contains a branch point in 3'-5 'and 2'-5'.

• Only natural ribozyme with demonstrated participation in DNA dissociation [19,20] in addition to the dissociation and ligation of RNA. • Major structural features widely established through phylogenetic comparisons [21].

• Important contacts of 2'OH begin to be identified [22]. • Kinetic structure under development [3]. 19 Zimmerly, Steven; Guo, Huatao; Eskes, Robert; Yang, Jian; Perlman, Philip S .; Lambowitz, Alan M .. A group of group II introns RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell (Cambridge, Mass.) (1995), 83 (4), 529-38. 20 Griffin, Edmund A., Jr .; Qin, Zhifeng; Michels, Williams J., Jr .; Pyle, Anna Marie. Ribozymes from group II introns that dissociate the DNA and RNA bonds with similar efficiency, and lack contacts with the 2'-hydroxyl groups of the substrate. Chem. Biol. (1995), 2 (11), 761-70. 21Michel, Francois; Ferat, Jean Luc. Structure and activities of group II introns. Annu. Rev. Biochem. (1995), 64, 435-61. 22Abramovitz Dana L .; Friedman, Richard A .; Pyle, Anna Marie. Catalytic role of the 2'-hydroxyl groups within an active site of group II introns. Science (Washington, D.C.) (1996), 271 (5254), 1410-13. 23Daniels, Danette L .; Michels, William J., Jr .; Pyle, Anna Marie. Two competing pathways for the autoempalme by means of group II introns: a quantitative analysis of in vitro reaction rates and products. J. Nol.

Biol. (1996), 256 (1), 31-49.

Neuroespora VS RNA • Size: ~ 144 nucleotides. • Transdisociation of recently demonstrated hairpin RNAs [24]. • Sequence requirements not completely determined. • Reaction mechanism: attack by 2 '-0H 3' to the cleavable link, to generate dissociation products with 2'-3'-cyclic phosphate and 5'-OH ends. • Fixing sites and structural requirements not completely determined. • Only 1 known member of this class. It was found in Neuroespora VS RNA.

Ribozima of Hammerhead (see text for references) • Size: ~ 13 to 40 nuleotids. • Requires the target sequence UH immediately 5 'from the dissociation site. • Fixes a variable number of nucleotides on both sides of the dissociation site. • Reaction Mechanism: attack by 2 '-OH 5' at 24Guo, Hans C.T .; Collins, Richard A .. Efficient transdisociation of strain cycle RNA substrate, by means of a ribozyme derived from the neurospore VS RNA. EMBO J. (1995), 14 (2), 368-76. cleavable link, to generate dissociation products with 2'-3'-cyclic phosphate and 5'-OH ends. • 14 known members of this class. Found in a member of plant pathogens (virusoids) that use RNA as the infectious agent. • Broadly defined essential structural features, including 2 crystal structures [5,26]. • Minimum demonstrated ligation activity (for design through in vi tro selection) [27]. • Complete kinetic structure, established for two or more ribozymes [28]. • Investigation of the chemical modification of important residues well established [29]. 25Scott, W.G., Finch, J.T., Aaron, K. The crystal structure of an all-RNA hammerhead ribozyme: A proposed mechanism for the catalytic dissociation of RNA. Cell, (1995), 81, 991-1002. 26McKay. Structure and function of the hammerhead ribozyme: an unfinished story. RNA, (1996), 2, 395-403. 27Long, D., Uhlenbeck, O., Hertel, K .. Ligation with hammerhead ribozymes. Patent of the United States of North America Number 5,633,133. 28Hertel, K.J., Herschlag, G., Uhlenbeck, O .. A kinetic and thermodynamic structure for the reaction of the hammerhead ribozyme. Biochemistry, (1994) 33, 3374-3385. Beigelman, L., and collaborators, Chemical modifications of the hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-25708. 29 Beigelman, L., et al. Chemical modifications of the hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-25708.

Hairpin Ribozyme • Size: ~ 50 nucleotides. • Requires the GUC target sequence immediately 3 'from the dissociation site. • Fixed 4-6 nucleotides on the 5 'side of the dissociation site, and a variable number on the 3' side of the dissociation site. • Reaction Mechanism: attack by 2 '-0H 5' to the cleavable link, to generate dissociation products with 2'-3'-cyclic phosphate and 5'-OH ends. • 3 known members of this class. Found in three plant pathogens (satellite RNA from tobacco ring spot virus, arabis mosaic virus and yellow spotted chicory virus) that use RNA as the infectious agent. • Broadly defined essential structural characteristics [30, 31, 32, 33]. 30Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz, Phillip. 'Hairpin' catalytic RNA model: evidence for helices and sequence requirements for the RNA substrate. Nucleic Acids Res. (1990), 18 (2), 299-304. 31Chowrira, Bharat M.; Berzal-Herranz, Alfredo; Burke, John M .. Novel guanosine requirement for catalysis by hairpin ribozyme. Nature (London) (1991), 354 (6351), 320-2. 32Berzal-Herranz, Alfredo; Joseph, Simpson; Chowrira, Bharat M.; Butcher, Samuel E.; Burke, John M .. Sequences of essential nucleotides and secondary structure elements of the hairpin ribozyme. EMBO J. (1993), 12 (6), 2567-73.

• The ligation activity (in addition to the dissociation activity) makes the ribozyme willing to accept design through in vi tro selection [34]. • Complete kinetic structure, established for a ribozyme [35]. • The investigation of the chemical modification of important residues has begun [36,37].

Ribozyme of the Hepatitis Delta Virus (HDV) • Size: ~ 60 nucleotides. 33Joseph, Simpson; Berzal-Herranz, Alfredo; Chowrira, Bharat M.; Butcher, Samuel E .. Substrate selection rules for hairpin ribozyme, determined by in vitro selection, mutation, and analysis of mismatched substrates. Genes Dev. (1993), 7 (1), 130-8. 34Berzal-Herranz, Alfredo; Joseph, Simpson; Burke, John M. In vitro selection of active hairpin ribozymes by means of sequential reactions of dissociation and ligation of catalyzed RNA. Genes Dev. (1992), 6 (1), 129-34. 35 Hegg, Lisa A.; Fedor, Martha J .. Kinetics and Thermodynamics of the Intermolecular Catalysis of Fork Ribozymes. Biochemistry (1995), 34 (48), 15813-28. 36Grasby, Jane A .; Mersmann, Karin; Singh, Mohinder; Gait, Michael J .. Functional Purine Clusters in Essential Residues of Hairpin Ribozyme Required for RNA Catalytic Dissociation. Biochemistry (1995), 34 (12), 4068-76. 37Schmidt, Sabine; Beigelman, Leonid; Karpeisky, Alexander; Usman, Nassim; Sorensen, Ulrik S.; Gait, Michael J .. Base and sugar requirements for the dissociation of RNA from essential nucleoside residues in the internal cycle B of the hairpin ribozyme: implications for the secondary structure. Nucleic Acids Res. (1996), 24 (4), 573-81.

• Transdisociation of target RNAs demonstrated [38].

• Fixing sites and structural requirements not completely determined, although no 5 'sequence of the dissociation site is required. The folded ribozyme contains a pseudonymous structure [39]. • Reaction Mechanism: attack by 2 '-0H 5' to the cleavable link, to generate dissociation products with 2'-3'-cyclic phosphate and 5'-OH ends. • Only 2 known members of this class. Found in human HDV. • The circular shape of HDV is active and shows increased nuclease stability [40]. 38 'P, errotta, Anne T .; Been, Michael D .. Dissociation of oligoribonucleotides by a ribozyme derived from the RNA sequence of the hepatitis delta virus. Biochemistry (1992), 31 (1), 16-21. 39 Perrotta Anne T .; Been, Michael D .. A pseudonudo-like structure required for efficient autodisociation of the delta hepatitis virus RNA. Nature (London) (1991), 350 (6317), 434-6. 40Puttaraju, M.; Perrotta, Anne T .; Been, Michael D .. A ribozyme of the circular transactuating delta hepatitis virus. Nucleic Acids Res. (1993), 21 (18), 4253-8.

Table II: Formulations of Cationic Lipids and Cell Uptake Table III Table III: Lipid-mediated supply of ribozymes to different cell lines. The cells were treated with ribozymes conjugated with 100 nM fluorescein, formulated with a panel of cationic lipids (selected from nc21, nc25, nc26, nc49, nc51, ncl02, ncllO, see Methods). The subcellular distribution of the ribozyme was determined by means of fluorescence microscopy. The presence of fluorescence in the nucleus indicated that the ribozyme had been transported through the cell membrane (unconjugated fluorescein does not remain in the nucleus). The lipid formulations that led to high nuclear delivery without significant toxicity are shown.

Table IV Table IV: Lipid-mediated supply of plasmids to different cell lines. The cells were treated with 0.1-1 μg / milliliter of a green fluorescent protein (GFP) expression plasmid, formulated with 2.5-15 μg / milliliter of selected lipid formulations (Table II), as described (Methods). The expression of GFP was monitored by fluorescence microscopy ~ 20 hours after transfection. The formulations that resulted in the expression of GFP in ~ 5 percent or more of the cells are indicated.

Table V Lipid (formulation) Nuclear Capture? % Inhibition JA59312 (ncl02) Y > 50% 40% JA59312 (nc49) Y > 40% 38% JA59317 (nc98) Y > 10% 23% JA59311 (nclOl) Y > 5% 21% JA59311 (nc20) Y > 10% 0% PH55942 (nc48) N (dotted) 18% AK52468 (nc33) N (dotted) 16% JA59316 (nc97) N (dotted) 0% PHF55933 (ncl3) N 0% PZH55938 (nc22) N 0% Table V. Inhibition of cell proliferation by different formulations of ribozymes, and correlation with cellular and nuclear supply. An anti-myb ribozyme and its active version (control), with different lipids (Table II), were formulated and administered to rat soft muscle cells as described (Methods). The relative activity of the active ribozyme against the inactive one (percentage of inhibition of proliferation) is shown. In parallel experiments, the cells were treated with identical formulations of a ribozyme conjugated to fluorescein, and their subcellular localization was observed by fluorescence microscopy (Y, nuclear delivery (% of positive cells); N, no visible nuclear supply; dotted, fluorescence dotted cytoplasmic). In general, the formulations that led to the improved supply of the ribozyme to the cell and the nucleus, also led to improved efficacy.

Table VI. Supply of Green Fluorescent Protein Containing Plasmid within the Cells in the Crop Type of Compound Name Rate of Dose Toxicity Cell Formulation Optimal transfection of lipid PC-3 nc 19 JA 59311 DOPE at 1: 1 40-64% 2.5μg / ml < 10% PC-3 nc 20 JA 59311 DOPE at 2: 1 40-50% 2.5μg / ml < 5% PC-3 nc 101B JA 59311 70-75% 5μg / ml < 10% PC-3 nc 102 JA 59312 30-45% 2.5μg / ml 10% PC-3 nc 110D AK 52468 60% 2.5μg / ml 5% PC-3 nc 122 JA 59317 and JA 59311-2: 1 55-65% 2.5μg / ml 5-13% PC-3 nc 123 JA 59317 and JA 59311-3: 1 30-50% 2.5μg / ml 5% PC-3 nc 128 JA 59317 and PH 55942-2: 1 35-70% 2.5μg / ml < 3-15% PC-3 nc 144 JA 59311 and JA 59312-2: 1 40-60% 2.5μg / ml < 3-10% PC-3 nc 145 JA 59311 and JA 59312-3: 1 50-70% 2.5μg / ml < 3-20% PC-3 nc 146B JA 59311 and AK 52468-1: 1 40-69% 2.5μg / ml < 3-8% PC-3 nc 148B JA 59311 and AK 52468-3: 1 25-70% 2.5μg / ml 3-20% PC-3 nc 156B JA 59312 and JA 59311 at 3: 1 40-65% 1.5μg / ml < 5% PC-3 nc 168 AK 52468 and JA 59312 at 2: 1 33-75% 2.5μg / ml 5-10% PC-3 nc 169 AK 52468 and JA 59312 at 3: 1 50-79% 2.5μg / ml 5-10% fifteen twenty fifteen Table VII. Supply of Green Fluorescent Protein Containing Plasmid within the Cells in the Culture, in the Presence of 10% Serum fifteen See e

Claims (110)

1. A cationic lipid having the formula I: wherein n is 1, 2, or 3 carbon atoms; n ± is 2, 3, 4, or 5 carbon atoms; R and Rx independently represent an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by 1-4 double bonds; and R2 and R3 are independently H, acyl, alkyl, carboxamide, aryl, acyl, carboxamidine, polyethylene glycol (PEG), or a combination thereof.

2. A cationic lipid having the formula II: > xn; HxNAl: 3+ wherein n is 1, 2, or 3 carbon atoms; nx is 2, 3, 4, or 5 carbon atoms; R and R- represent independently an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by 1-4 double bonds; and Alk represents methyl, hydroxyalkyl or a combination thereof.

3. A cationic lipid having the formula III , NHR2 R. ^ NH wherein R and R-L independently represent an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by 1-4 double bonds; and R2 is H, PEG, acyl, or alkyl.

4. A cationic lipid having the formula IV: wherein n is 1-6 carbon atoms; R and R1 independently represent an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by 1-4 double bonds; and R2 is H, PEG, carboxamidine, alkyl, acyl, aryl, substituted carboxamidine, wherein R3 is H, or P03H2 and R4 is OH, NH2 u = 0.

5. A cationic lipid having the formula V: wherein n is 1-6 carbon atoms; X and Xx independently represent an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by 1-4 double bonds; B is a nucleic acid base or H; and R5 is H, PEG, or carboxamidine.

6. A cationic lipid having the formula VI: wherein n is 1, 2, or 3 carbon atoms; R and R1 independently represent an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by 1-4 double bonds; and R2 and R3 are independently H, acyl, alkyl, carboxamide, aryl, acyl, carboxamidine, polyethylene glycol (PEG), or

7. A cationic lipid having the formula VII: R 6 -LL-Cholesterol wherein R 6 is selected from the group consisting of arginylmethyl ester, arginyl amide, homoarginylmethyl ester, homoarginyl amide, ornithinmethyl ester, ornithylamide, lysylmethyl ester, lysyl amide, triethylenetetramine (TREN), N, N'-dicarboxamidine TREN, N-benzylhistidylmethyl ester, pyridoxyl and aminopropylimidazole; and L-L is a linker represented by R7P02, wherein R7 is H, CH3, or CH2CH3.

8. A cationic lipid having the formula VIII: R8-L2-Cholesterol wherein R8 is selected from the group consisting of arginyl, N-Boc arginyl, homoarginyl, N-Boc homoarginyl, ornithine, N-Boc ornithine, N -benzylhistidyl, lysyl, N-Boc lysyl, N-methylarginyl, N-methylguanidine, guanidine and pyridoxyl; and L2 is a linker represented by NH, glycine, N-butyldiamine or guanidine.

9. The cationic lipid of claim 1, wherein said cationic lipid is N '-palmityl-N' -oleyl-alpha, gamma-diaminobutyri-glycinamide.

10. The cationic lipid of claim 1, wherein the cationic lipid is N '-palmityl-N' -oleyl-N-gamma-carboxamidine-alpha, gamma-diaminobutyri-glycinamide.

11. The cationic lipid of claim 2, wherein the cationic lipid is N '-palmityl-N' -oleyl-alpha, gamma-bis-trimethylammoniobutyryl-glycinamide.

12. The cationic lipid of claim 3, wherein the cationic lipid is N '-palmityl-N' -oleyl-N-carboxamidine-glycinamide.

13. The cationic lipid of claim 3, wherein said cationic lipid is N '-palmityl-N' -oleyl-guanididine.

14. The cationic lipid of claim 8, wherein said cationic lipid is Boc arginine-cholesteryl amide.

15. The cationic lipid of claim 7, wherein the cationic lipid is cholesterol ester-methyl-methyl ester phosphonamidate.

16. The cationic lipid of claim 7, wherein the cationic lipid is homoarginine-methylphosphono-amidate of cholesterol-methyl ester.

17. The cationic lipid of claim 7, wherein the cationic lipid is cholesterol-lysine-amide-methylphosphonamidate amidate.

18. The cationic lipid of claim 7, wherein the cationic lipid is cholesterol-TREN-methylphosphonoamidate.

19. The cationic lipid of claim 7, wherein the cationic lipid is cholesterol methyl phosphonamidate-TREN-bis-guanidinium.

20. The cationic lipid of claim 7, wherein the cationic lipid is cholesterol-histidine-methyl-phosphonoamidate.

21. The cationic lipid of claim 7, wherein the cationic lipid is cholesterol-aminopropylimidazole-methyl-phosphonoamidate.

22. The cationic lipid of claim 4, wherein said cationic lipid is N '-palmityl-N' -oleyl-beta-alaninamide.

23. The cationic lipid of claim 4, wherein the cationic lipid is N '-palmityl-N' -oleyl-N-carboxamidine-beta-alaninamide.

24. The cationic lipid of claim 4, wherein the cationic lipid is N (N "-palmityl-N" -oleyl-amidopropyl) beta-pyridoxane.

25. The cationic lipid of claim 8, wherein said cationic lipid is N-cholesteryl-pyridoxamine.

26. The cationic lipid of claim 8, wherein the cationic lipid is 3 ', 5'-Di-palmitoyl-2'-deoxy-2' - (N-carboxamidine-beta-alaninamide) uridine.

27. The cationic lipid of any of claims 1-8, wherein the cationic lipid pairs the ions to a negatively charged polymer, selected from the group consisting of RNA, DNA and protein.

28. The cationic lipid of claim 27, wherein the RNA and DNA comprise one or more modifications.

29. The cationic lipid of claim 27, wherein the RNA is an enzymatic RNA.

30. The cationic lipid of claim 27, wherein said DNA is an enzymatic DNA.

31. A lipid aggregate comprising the cationic lipid of any of claims 1-6.

32. The lipid aggregate of claim 31, wherein the lipid aggregate is a liposome.

33. The lipid aggregate of claim 32, wherein the liposome is a furtive liposome.

34. The lipid aggregate of claim 31, wherein the lipid aggregate further comprises a negatively charged polymer.

35. The lipid aggregate of claim 34, wherein the negatively charged polymer is selected from the group consisting of RNA, DNA and protein.

36. The lipid aggregate of claim 35, wherein the RNA is an enzymatic RNA.

37. The lipid aggregate of claim 35, wherein the DNA is an enzymatic DNA.

38. The lipid aggregate of claim 35, wherein said RNA and said DNA comprise one or more modifications.

39. A pharmaceutical composition comprising the lipid of any of claims 1-8.

40. A pharmaceutical composition comprising the lipid aggregate of claim 31.

41. A pharmaceutical composition comprising the lipid aggregate of claim 34.

42. A cell comprising the cationic lipid of any of claims 1-8.

43. A cell comprising the lipid aggregate of claim 31.

44. A cell comprising the lipid aggregate of claim 34.

45. A cell comprising the pharmaceutical composition of claim 39.

46. A cell comprising the pharmaceutical composition of claim 40.

47. A cell comprising the pharmaceutical composition of claim 41.

48. The cell of claim 42, wherein said cell is a mammalian cell.

49. The cell of claim 48, wherein the cell is a human cell.

50. The cell of claim 44, wherein the cell is a mammalian cell.

51. The cell of claim 50, wherein the cell is a human cell.

52. The cell of claim 45, wherein said cell is a mammalian cell.

53. The cell of claim 52, wherein the cell is a human cell.

54. The cell of claim 46, wherein the cell is a mammalian cell.

55. The cell of claim 54, where the cell is a human cell.

56. A method for facilitating the transfer of a negatively charged polymer within a cell, comprising the step of contacting the cell with a mixture of the negatively charged polymer, with the cationic lipid of any of claims 1-8, under conditions suitable for the transfer of said negatively charged polymer within the cell.

57. The method of claim 56, wherein the negatively charged polymer is a nucleic acid molecule.

58. The method of claim 57, wherein the nucleic acid molecule is an enzyme nucleic acid molecule.

59. A method for facilitating the transfer of a negatively charged polymer within a cell, comprising the step of contacting said cell with the cationic lipid aggregate of claim 34, under conditions suitable for the transfer of the negatively charged polymer. inside the cell.

60. The method of claim 59, wherein the negatively charged polymer is a nucleic acid molecule.

61. The method of claim 60, wherein the nucleic acid molecule is an enzyme nucleic acid molecule.

62. The cationic lipid aggregate of claim 31, characterized in that it also comprises a neutral lipid.

63. The cationic lipid aggregate of claim 62, wherein the neutral lipid is dioleoylphosphotidylethanolamine.

64. The cationic lipid aggregate of claim 34, characterized in that it also comprises a neutral lipid.

65. The cationic lipid aggregate of claim 64, wherein the neutral lipid is dioleoyl-phosphotidylethanolamine.

66. A cationic lipid having the formula IX: -NHCOR -NHCOR ^ -COR, wherein R is independently an alkyl chain of 12 to 22 carbon atoms, which are saturated or unsaturated, wherein the unsaturation is represented by 1-4 double bonds; and R-L is selected from the group consisting of TREN, N.N '-di -carboxamidine TREN, lysyl, arginyl, ornithil, homoarginyl, histidyl, aminopropylimidazole, and sperminicarboxylic acid.

67. The cationic lipid of claim 66, wherein said cationic lipid is N2, N3-di-oleyl- (N, N'-diguanidinoethyl-aminoethane) -2,3-diaminopropionic acid.

68. A lipid aggregate comprising the cationic lipid of claim 66.

69. The lipid aggregate of claim 69, wherein the lipid aggregate is a liposome.

70. The lipid aggregate of claim 69, wherein the liposome is a furtive liposome.

71. The lipid aggregate of claim 68, wherein said lipid aggregate further comprises a negatively charged polymer.

72. The lipid aggregate of claim 71, wherein the negatively charged polymer is selected from the group consisting of RNA, DNA and protein.

73. The lipid aggregate of claim 72, wherein the RNA is an enzymatic RNA.

74. The lipid aggregate of claim 72, wherein the DNA is an enzymatic DNA.

75. The lipid aggregate of claim 72, wherein the RNA and DNA comprise one or more modifications.

76. A pharmaceutical composition comprising the lipid of claim 66.

77. A pharmaceutical composition comprising the lipid aggregate of claim 68.

78. A pharmaceutical composition comprising the lipid aggregate of claim 71.

79. A cell comprising the cationic lipid of claim 66.

80. A cell comprising the lipid aggregate of claim 68.

81. A cell comprising the lipid aggregate of claim 71.

82. A cell comprising the pharmaceutical composition of claim 76.

83. A cell comprising the pharmaceutical composition of claim 77.

84. A cell comprising the pharmaceutical composition of claim 78.

85. The cationic lipid of claim 5, wherein the cationic lipid is 3'-5'-Di-. palmitoyl-2'-deoxy-2 '- (beta-alaninamido) uridine.

86. The cationic lipid of claim 5, wherein the cationic lipid is 3'-5'-Di-palmitoyl-2'-deoxy-2 '- (N-carboxamidine-beta-alaninamido) uridine.

87. A lipid aggregate formulation comprising phosphatidylcholine, cholesterol, a cationic lipid and DSPE-PEG2000.

88. The lipid aggregate of claim 87, wherein the phosphatidylcholine is derived from the egg yolk.

89. The lipid aggregate of claim 87, where the cationic lipid is DOTAP.

90. The lipid aggregate of claim 87, wherein said cationic lipid is the cationic lipid according to any of claims 1-8 and 66.

91. The cationic lipid of any of claims 1-8, wherein the Cationic lipid is linked to polyethylene glycol (PEG).

92. The cationic lipid of claim 91, wherein the PEG is between about 2000-5000 daltons inclusive.

93. A lipid aggregate comprising the cationic lipid of claim 91.

94. A lipid aggregate comprising JA 59317 and JA 59311 in a ratio of 2: 1.

95. A lipid aggregate comprising JA 59317 and JA 59311 in a ratio of 3: 1.

96. A lipid aggregate comprising JA 59311 and PH 55942 in a ratio of 2: 1.

97. A lipid aggregate comprising JA 59311 and JA 59312 in a ratio of 2: 1.

98. A lipid aggregate comprising JA 59311 and JA 59312 in a ratio of 3: 1.

99. A lipid aggregate comprising JA 59311 and AK 52468 in a ratio of 1: 1.

100. A lipid aggregate comprising AK 52468 and JA 59312 in a ratio of 3: 1.

101. A lipid aggregate comprising JA 59311 and DOPE in a ratio of 1: 1.

102. A lipid aggregate comprising JA 59311 and TweendO in a ratio of 4: 1.

103. A lipid aggregate comprising JA 59311 and Tween80 in a ratio of 3: 1.

104. A lipid aggregate comprising JA 59349 and TweendO in a ratio of 4: 1.

105. The lipid aggregate of claim 92, wherein the lipid aggregate is a liposome.

106. The lipid aggregate of claim 92, wherein the lipid aggregate further comprises a negatively charged polymer.

107. The lipid aggregate of claim 106, wherein said negatively charged polymer is a nucleic acid molecule.

108. The lipid aggregate of claim 107, wherein the nucleic acid molecule is an enzyme nucleic acid molecule.

109. The lipid aggregate of claim 107, wherein the nucleic acid molecule is a DNA molecule.

110. The lipid aggregate of claim 109, wherein the DNA molecule is a plasmid DNA molecule.