Patent Publication Number: US-7223887-B2

Title: Multivalent cationic lipids and methods of using same in the production of lipid particles

Description:
This application claims the benefit of U.S. Provisional Application Ser. No. 60/341,661, filed Dec. 18, 2001, which is incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     This application relates to a new class of cationic lipids which can be used in lipid particles and in the formation of lipid-nucleic acid complexes for cell transfection. 
     Monovalent cationic lipids are known in the art, for example as described in U.S. Pat. Nos. 6,320,017; 5,976,567; 5,965,542 and 5,785,992, which are incorporated herein by reference. Plasmid DNA-cationic lipid complexes are the leading non-viral gene therapy vectors used in the clinical setting. (1–4). However, the levels of gene expression that can be achieved using these complexes are substantially lower than can be achieved using viral vectors. Considerable effort has therefore been devoted to synthesizing new cationic lipids that have improved transfection properties over those currently available. (5–10). 
     SUMMARY OF THE INVENTION 
     The present invention provides a new class of cationic lipids for use forming nucleic acid complexes for transfection of the nucleic acid. The cationic lipids of the invention are multivalent lipids (generally divalent) of the general formula: 
                         
wherein n is an integer 1 or greater, the R groups, which may be the same or different from one another, are optionally substituted alkyl or alkenyl (1 to 4 double bonds) groups of from 1 to 24 carbon atoms, X is CH 2 , CH 2 CH 2 , NH (in which case the cationic lipid is trivalent), O or S and Y is an alkyl group of from 1 to 6 carbon atoms, a hydroxyalkyl of from 1 to 6 carbon atoms or a hydroxy group. In preferred cationic lipids of the invention, n is from 1 to 2, X is CH 2  or CH 2 CH 2 , Y is methyl and the R groups are all the same and are partially unsaturated alkenyl groups.
 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the structures of two multivalent cationic lipids in accordance with the invention. 
         FIG. 2  shows a reaction scheme for synthesis of multivalent cationic lipids in accordance with the invention. 
         FIG. 3  illustrates the transfection properties of complexes in accordance with the invention. 
         FIG. 4  illustrates the transfection properties of complexes in accordance with the invention. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides a new class of cationic lipids for use in forming nucleic acid complexes for transfection of the nucleic acid. This class of compounds, which are referred to herein at times as “catiolipins,” contain four hydrocarbon chains and a bridging group linking two ammonium moieties. The bridging group may be all carbon atoms, or it may include hetero atoms. When the bridging group includes a amine nitrogen, the total charge on the cationic lipid is increased. 
     The catiolipins are multivalent aminolipids, that is they have a net charge of +2 or greater. In specific embodiments, the catiolipins are divalent. The positive charges are the result of two quaternary amine groups linked by the bridging moiety. The positive charge on these quaternary amine groups is independent of pH. If additional nitrogens are incorporated in the bridging moiety, they may be titratable, such that the additional charge is pH dependent, or they may be quaternary amines with pH-independent positive charges. 
     Catiolipins in accordance with the invention are suitably incorporated into lipid particles containing the catiolipin to promote DNA transfection. The ability of cationic lipids to facilitate intracellular delivery of DNA is related to their ability to induce the non-bilayer hexagonal H ∥  phase in combination with anionic lipids. (11). The catiolipins of the invention have been designed structurally to maximize their ability to induce H ∥  phase organization. 
     The ability of lipids to adopt the H ∥  phase has been related to their dynamic molecular shape properties. Within this framework, lipids with small cross-sectional area in the headgroup region and a larger acyl chain cross-sectional area exhibit a “cone” shape compatible with H ∥  phase organization. (12). Increasing the cone-shape character of a given lipid can be achieved by increasing the number of alkyl chains associated with the headgroup. Thus, broadly stated, the multivalent cationic lipid of the invention has a positively-charged head group comprising at least two quaternary amine groups and a hydrophobic portion comprising four hydrocarbon chains, which may be the same or different and which are optionally substituted alkyl or alkenyl groups. The cross-sectional size of the headgroup is varied in the catiolipins of the invention by varying the length of the bridge separating the quaternary amines. 
     The physical properties of catiolipins having a three carbon bridge (TODMAC3) and having a six-carbon bridge (TODMAC6) are quite different. TODMAC6 readily forms bilayer structures, both in the presence and absence of DOPE, on dispersion in an aqueous medium, whereas TODMAC3 does not. Further, when mixed with equimolar (with respect to charge) amounts of DPPS, TODMAC6 forms a homogeneous dispersion that collectively adopts non-bilayer structure in the region of 30° C. TODMAC3 on the other hand is able to interact with only a limited subset of the DPPS, but is able to induce the H ∥  phase for that subset at temperatures as low as 15° C. At higher TODMAC3/DPPS ratios (2:1 on a charge basis)) TODMAC3 is able to induce apparently complete H ∥  organization at temperatures of 25° C. or higher. These results are consistent with steric effects that limit the ability of TODMAC3 to interact with more than one DPPS molecule due to the small size of the TODMAC3 headgroup. 
     In a specific embodiment, the multivalent cationic lipids of the invention have the formula: 
                         
wherein n is an integer 1 or greater for example 1–5, the R groups, which may be the same or different from one another, are optionally substituted alkyl or alkenyl (1 to 4 double bonds) groups of from 1 to 24 carbon atoms, X is CH 2 , CH 2 CH 2 , NH, O or S and Y is an alkyl group of from 1 to 6 carbon atoms, a hydroxyalkyl of from 1 to 6 carbon atoms or a hydroxy group. In specific preferred embodiments, n is 1 or 2, X is CH 2  or CH 2 CH 2 , and/or Y is methyl and the R groups are all the same and are partially unsaturated alkenyl groups. Two specific catiolipins in accordance with the invention are N,N,N′,N′-tetraoleyl-N-N′-dimethyl-1,3-propanediammonium chloride and N,N,N′,N′-tetraoleyl-N-N′-dimethyl-1,6-hexanedammonium chloride, the structures of which are shown in  FIG. 1 .
 
     The cationic lipids of the invention can be synthesized by nucleophilic substitution of secondary amines using an alkyl bromide under phase-transfer catalytic condition as outlined in  FIG. 2 . The strongly basic amines generally require high alkaline reaction conditions so that they remained deprotonated to react with alkyl bromide. Thus, one aspect of the invention is a method for making the catiolipins of the invention comprising the step of reacting a diamine of the general formula: 
                         
with an alkyl bromide or a mixture of alkyl bromides in a basic phase transfer catalysis reaction medium (toluene, 1 M NaOH), optionally containing t-butyl ammonium hydrogen sulfate. When a mixture of alkyl bromides is used, the product has a random mixture of different alkyl (R) groups dependent on the relative amounts of the alkyl bromides included in the reaction mixture. The reaction is continued under reflux conditions for periods of 1 to 9 days to alkylate the diamine with the alkyl bromide.
 
     The cationic lipids of the invention may be used alone, or they may be formulated with other lipid components to form lipid particles, including liposomes. In addition to the cationic lipids of the invention, the lipid particles may contain neutral lipids or sterols, modified lipids, such as polyamide oligomers or PEG-modified lipids. Such additional components are described in the US patents mentioned above for use in combination with monovalent cationic lipids. For example, as shown in the examples below, the cationic lipids of the invention can be formulated with dioleylphosphatidyl ethanolamine (DOPE) into lipid particles useful for nucleic acid transfection. 
     The multivalent cationic of the invention may be used in place of or in combination with monovalent cationic lipids for applications known in the art, for example as described in U.S. Pat. Nos. 6,320,017; 5,976,567; 5,965,542 and 5,785,992 mentioned above. Such uses include, without limitation, the introduction of polyanionic materials into cells, both in vivo and in vitro; and as drug delivery compositions for the systemic or local delivery of therapeutic agents or bioactive agents and for use in diagnostic assays. 
     In a preferred use, the multivalent cationic lipids of the invention are used in the formulation of lipid particles which complex with nucleic acid polymers, which are anionic at physiological pH, and which ate useful for transfection of the nucleic acid polymer into cells. As used in the specification and claims of this application, the term “nucleic acid polymer” refers to any polymeric nucleic acid structure, be it single or double stranded, for which intracellular delivery is desired and encompasses DNA, cDNA, RNA, and mRNA. The term specifically encompasses ribozymes; nucleic acid cloning and/or expression vectors such as plasmids; genetically engineered viral genomes, expression cassettes, and chromosomes from mammalian (especially human) sources. For example, the nucleic acid polymer may be in the form of a plasmid, which includes a genetic sequence to be delivered, as well as promoter and optionally marker elements to confirm transfection and inducer or suicide elements to provide control of the transfected cells after plasmid introduction. Alternatively, the nucleic acid polymer may be a relatively short oligomer, for example having a length of at least 10 nucleotides, which may be useful for genetic immunization, antisense therapy, RNAi therapy or the like. 
     Transfection is accomplished using standard methods known in the art for transfection with other types of cationic lipid DNA complexes. Since transfection with the multivalent cationic lipids of the invention is generally more efficient, lower levels of lipid-DNA complexes, and shorter times may still provide suitable results. 
     The invention will now be described with reference to the following, non-limiting examples. 
     EXAMPLE 1 
     Synthesis of N,N,N1,N1-Tetraoleyl.N,N′-dimethyl-1,3-propanediammonium Chloride (TODMAC3) 
     A solution of N,N′-dimethylpropanediamine (0.253 g, 2.5 mmol) and oleylbromide (5 g, 15.1 mmol) in toluene (20 ml) and a solution of tetrabutylammonium hydrogen sulfate (0.4 g, 1.2 mmol) in 1 M sodium hydroxide (20 ml) were stirred under reflux. After 4 days, 2.5 g oleylbromide (7.5 mmol) was added and the reaction continued for another 5 days. The water phase was extracted 2 times with toluene. The combined organic phases were evaporated. The residue was dissolved in CH 2 CI 2  and washed six times with 3% hydrochloric acid and three times with saturated aqueous NaCl. The organic phase was evaporated and the remaining material purified by silica gel chromatography (CH 2 Cl 2 /MeOH from 10/0 to 9/1). TODMAC3 was obtained as a pale yellow oil at a yield of 1.15 g (0.98 mmol, 39%). The product was washed six times with 3% hydrochloric acid and three times with saturated aqueous NaCI again. 
     The cationic lipids were purified by column chromatography using silica gel 60 (Merck, 70–230 mesh) and dichloromethane (CH 2 Cl 2 )/methanol (MeOH) 9/1 mixtures (v/v). The reaction progress was followed by thin layer chromatography (TLC) on silica plates FZ54 (Merck). The following developing systems were used: UV light, H 2 SO 4 /EtOH (1/1 v/v), Dragendorff reagent (Sigma), ninhydrin reagent (Sigma). The observed Rf was 0.43.  1 H,  13 C,  31 P NMR spectra were obtained employing a Bruker MSL 200 spectrometer operating at 200.13, 50.3 and 81.02 MHz, respectively. Deuterated chloroform (CDCl 3 ) was used as the solvent in the NMR experiments. Chemical shifts were measured relative to CHCl 3  (87.24 ppm) for 1H, and relative to CDCl 3  (877.16 ppm) for  13 C. The spectra confirmed to that anticipated for TODMAC3. 
     EXAMPLE 2 
     N,N,N′,N′-Tetraoleyl-N,N′-dimethyl-1,6-hexanediammonium chloride (TODMAC6) was synthesized using the same procedure as described above in Example 1, substituting N,N′-dimethylhexanediamine for N,N′-dimethylpropanediamine. The product had an observed Rf of 0.61 and an NMR consistent with that expected for the structure. 
     EXAMPLE 3 
     As noted above, the ability of cationic lipids to induce the hexagonal H ∥  phase in combination with anionic lipids correlates well with their transfection properties in plasmid DNA-cationic lipid complexes. The relative ability of cationic lipids to induce the H ∥  phase can be ascertained by measuring the bilayer-to-non-bilayer transition temperature (Tc) of the cationic lipid in equimolar mixtures (with respect to charge) with an anionic phospholipid such as 1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine] (DPPS). The bilayer-to-non-bilayer transition can be conveniently monitored employing  31 P NMR techniques (12). 
     To evaluate the polymorphic phase properties of mixtures of TODMAC3 and TODMAC6 with DPPS, lipid mixtures containing 40–50 mg DPPS and appropriate molar ratios of cationic lipids N,N-dioleyl-N-N-dimethylammonium chloride (DODAC), TODMAC3, and TODMAC6 were prepared by drying from chloroform solution, placed in 10 mm NMR tubes and hydrated with 1.5 mL distilled water. Free induction decays (FIDs) corresponding to 1000 scans were obtained by using a 3.0 J.1S 60° pulse with a 1 μs interpulse delay and a spectral width of 25,000 Hz. A gated two-level proton decoupling was used to ensure sufficient decoupling with minimum sample heating. An exponential multiplication corresponding to 50 Hz of line broadening was applied to the FIDs prior to Fourier transformation. The sample temperature (±1° C.) was regulated using a Bruker B-VT1000 variable temperature unit. Chemical shifts were referenced to 85% phosphoric acid as an external standard. 
     In the first series of experiments the temperature dependent polymorphism of TODMAC6/DPPS mixtures was compared to the behaviour of DODAC/DPPS mixtures. At 25° C. both mixtures exhibited  31 P NMR lineshapes indicative of bilayer structure. However at 30° C. the mixture containing TODMAC6 has largely adopted the H ∥  phase or a structure giving rise to a narrow isotropic  31 P NMR peak, whereas the dispersion containing DODAC remained in a bilayer organization at 35° C. On the basis of this behaviour it would be expected that TODMAC6 should exhibit transfection properties that are comparable to or slightly better than DODAC when used in plasmid-DNA-cationic lipid complexes. 
     Comparable experiments on dispersions of TODMAC3/DPPS (0.5:1 mol:mol) showed that at temperatures as low as 15° C. an H ∥  component is clearly visible, however the large bulk of the phospholipid remains in the bilayer organization and the proportion in the H ∥  phase does not increase as the temperature is raised. This behaviour is consistent with a limited solubility of the TODMAC3 in the DPPS component in the lipid mixture. This maybe due to steric constraints associated with the need to form ion pairs between the cationic lipid and two DPPS molecules in order to form the H ∥  phase. For example, if only one DPPS molecule was able to form an ion pair with each TODMAC3, 50% of the DPPS would be unpaired and thus remain in the bilayer organization. If this is the case it would be expected that TODMAC3/DPPS systems containing higher proportions of TODMAC3 should exhibit increased preference for non-bilayer structure. The phase properties of a mixture containing equimolar proportions of TODMAC3 and DPPS were therefore investigated. This system exhibited an increased preference for H ∥  phase structure. On the basis of this behaviour TODMAC3 would not be expected to be as effective a transfection agent as DODAC on a charge for charge basis, but nevertheless should be useful as a transfection agent. 
     EXAMPLE 4 
     To prepare large unilamellar vesicles (LUV), mixtures of cationic lipids and DOPE in chloroform were dried under a stream of nitrogen gas and the residual solvent removed under high vacuum for 2 hours. The resulting lipid films were hydrated with distilled water. LUVs were obtained by extruding the lipid dispersions 10 times through two stacked 100-nm pore size polycarbonate filters (Costar Nuclepore polycarbonate membrane, Lipex Biomembranes extruder) (15). Phospholipid concentrations were determined by the inorganic phosphorus assays according to Fiske and Subbarow (16). 
     EXAMPLE 5 
     Lipoplexes were prepared in distilled water at pDNA concentrations ranging from 12.5–25 μg pDNA/ml at positive-to-negative charge ratios between 0.5–5. Plasmid DNA was added under vortexing to cationic LUVs (cationic lipid/DOPE 1:1). In the absence of the helper lipid DOPE, lipoplexes were prepared by addition of the cationic lipids from ethanol solutions to an aqueous solution containing the pDNA. The amount of ethanol in these formulations was between 0.4–2.3%. The lipoplexes were incubated at room temperature for 30 min, diluted to a final concentration of 2.5 μg pDNA/ml with serum-free media (Dulbecco&#39;s Modified Eaglels Medium, DMEM) and subsequently applied to BHK cells. 
     EXAMPLE 6 
     In vitro Transfection of BHK Cells 
     BHK cells, grown in DMEM supplemented with 10% fetal calf serum (FCS), 100 U/ml of penicillin and 100 μg/ml of streptomycin, were seeded in 96-well plates at a density of 1×10 4  cells per well 24 h before transfection. The next day media was removed and replaced with 100 μl of lipoplexes dispersed in serum-free DMEM (0.25 μg pDNA/100 μl), After 4 hr, lipoplexes were replaced with serum-containing DMEM and cells further incubated for 20 h. After that, the culture medium was aspirated, the cells washed twice with 100 μl of PSS and then lysed through addition of 50 μl of lysis buffer (1% Triton X-100 in PBS). Luciferase activity was measured with a Dynex Luminometer (Dynex model ML3000) using the Luciferase Assay Kit from Promega. The total protein concentration per well was determined using the BCA assay from Pierce. Means and standard deviations were calculated from three independent experiments. 
     DNA plasmid complexes containing TODMAC3 and TODMAC6 are usually formed by mixing plasmid DNA with preformed vesicles containing the cationic lipid mixed with equimolar amounts of the- “helper” lipid- dioleoyfphosphatidylethanolamine (DOPE) (17, 1–8). However it was found that vesicles could not be prepared consisting of TODMAC3/DOPE mixtures, as lipid films prepared of this lipid mixture were extremely difficult to hydrate and formed clumps that could not be extruded. Vesicles of TODMAC6/DOPE could be readily prepared and the transfection properties of complexes formed employing TODMAC6 and DODAC and containing the LO18 plasmid coding for the luciferase gene were tested in vitro using baby hamster kidney (BHK) cells.  FIG. 3  illustrates the transfection properties of complexes prepared at cationic lipid-to-DNA charge ratios (positive to negative) ranging from 0.5 to 5. It may be observed that the transfection potency of complexes containing TODMAC6 are comparable to or slightly lower than complexes containing DODAC at charge ratios or two or lower, but are greater at higher charge ratios. 
     In order to test the relative transfection potencies of DODAC, TODMAC6 and TODMAC3 a different procedure was used to associate the cationic lipid with the plasmid to form the complexes. In this method the cationic lipid dissolved in ethanol was introduced into an aqueous solution of the plasmid and DOPE was not present. The transfection properties of complexes formed in this manner are shown in  FIG. 4 . It may be noted that at charge ratios of two and lower DODAC exhibits superior tranfection properties, however at charge ratios above two TODMAC6 exhibits transfection properties that are superior to both DODAC and TODMAC3. Specifically, for charge ratios˜2.5, TODMAC6 complexes gave rise to luciferase expression levels 5–20 fold higher than could be achieved with DODAC or TODMAC3. 
     The following reference are cited herein and are incorporated herein by reference. 
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