MXPA97005572A - Stable complexes for the supply of pharmacosque contain lipids and methods for your production - Google Patents

Abstract

New stable complexes are described for the delivery of drugs, concentrates, biologically active, ready to be used and containing lipids, methods for their use. The biological activity of the complexes produced is comparable to that of the formulations prepared according to the prior art method based on the joint mixing, and, upon being purified, the complexes produced by the method of the present invention have a concentration 50 to 500 times that of the components of the complexes formed by the joint mixing. The method described herein, allows the large-scale production of drug delivery systems and containing lipids, useful for gene-based therapy, and for other applications.

Description

"STABLE COMPLEXES FOR THE SUPPLY OF DRUGS CONTAINING LIPIDS AND METHODS FOR THEIR PRODUCTION" Field of the Invention The present invention relates to cationic lipids and their use as vehicles for the transfer of nucleic acids or other macromolecules such as proteins, inside the cells. More specifically, the present invention relates to complexes for the delivery of drugs and containing lipids, which complexes are stable, biologically active, capable of being concentrated; and also refers to the methods for the production of said complexes. BACKGROUND OF THE INVENTION The development of new therapeutic forms that resort to macromolecules such as proteins or nucleic acids as therapeutic agents, has created the need to develop new and effective means to deliver or deliver said macromolecules to their appropriate objectives. cell phones. Therapeutics based on the use of specific polypeptide growth factors, whether specific genes to replace missing or defective genes, are examples of the therapy that these new delivery systems may require. The clinical application of these therapies depends not only on the efficacy of the new delivery systems but also on their safety and the ease with which the technologies on which these systems are based can be adapted for large-scale pharmaceutical production. , in its capacity to be stored, and in the commercial distribution of therapeutic formulations. Gene-based therapy has been transformed into a modality of REF: 25321 increasing importance to treat several pathological conditions of genetic type. The potential capacity to provide effective treatments, and even cures, has served as a stimulus to make an intensive effort to apply this technology to some diseases for which there was no effective treatment. Recent advances in this field have indicated that gene-based therapy can have a significant impact not only in the treatment of pathological conditions related to a single gene, but also in more complex diseases, such as cancer. However, one of the significant obstacles in the achievement of an efficient therapy based on genes has been the difficulty of designing new and effective means to deliver (deliver) the therapeutic nucleic acids to the target cells. Therefore, an ideal vehicle for the delivery of exogenous genes to cells and tissues, should be highly efficient in nucleic acid delivery, safe to use, easy to produce in large quantities, and have sufficient stability to be practical as a chemical.

The non-virosic vehicles, which are mainly represented by cationic lipids, are a type of vehicle that for the following reasons have been taken into account to be used in gene-based therapy. First, the plasmid DNA required for gene-based and lipid-mediated therapy can be prepared extensively and routinely on a large scale, and its use is simpler and less risky than the use of viral vectors such as retrovirus.

Second, the delivery of genes, mediated by retroviruses, can deliver either RNA or DNA. Therefore, DNA, RNA or an oligonucleotide can be introduced directly into a cell. On the other hand, cationic liposomes are not toxic, they are not immunogenic, so they can be used repeatedly in vivo, as evidenced by the successful in vivo delivery of genes to catheterized blood vessels (Nabel, EG and Other (s) ) (1990) Science 249: 1285-1288), epithelial lung cells (Brigham, L., and Others), (1989) Am. J. Respir. Cell Mol. Biol .. 195-200, Stribling, R. and Other (s) (1992) Proc. Nati Acad. Sci. U.S. A .. 89: 1 1277 - 1 1281), and other systemic uses (Zhu, N., and Other (s) (1993) Science. 261: 209-21 1, Philip, R., and Other (s), (1993), Science. 261: 209-211) of cationic liposomes. While a variety of cationic liposome formulations, including the commercially available cationic liposome reagent, DOTMA / DOPE (Nl- (2,3-dioleoyloxy) propyl-N, N, N-trimethyl ammonium / dioleoyl phosphatidylethanolamine) are known in the art (Felgner, PL and Other (s) (1997) Proc. Nati, Acad. Scie. USA 84: 7413-7417), a formulation of cationic liposome, designated as DC-Chol / DOPE (3BN- (N'.N'-dimethylaminoethane) carbamoyl cholesterol) / (dioleoyl phosphatidy-ethanolamine) has been shown to be relatively non-toxic and more efficient than DOTMA / DOPE, in in vitro tests (Gao, X. and Huang, L. (1991) Biochem Biophvs, Res. Commun 179: 280-285). On the other hand, after extensive in vivo studies (Plautz, GE and Other (s) (1993), Proc. Natl.Acad. Scie. USA. 90: 4645-4649, Stewart, MJ and Other (s) (1992) ) Hum. Gene Ther .. 3: 267-275) in which it was demonstrated that the DC-Chol / DOPE was both safe and effective as a system for the delivery of a nucleic acid, said formulation has been approved by the US Food and Drug Administration (FDA) and by the U.K. Medicines Control Agency (MCA), and has been used in two clinical trials of gene therapy, separately (Nabel, G.J. and Other (s), (1993), Proc. Nati Acad. Scie. U.S.A .. 90: 11307-13311, Caplen, N.J. USA, and Other (s) (1995), Nature Medici ne. 1: 39-46. However, the use of DC-Chol / DOPE and other currently existing cationic liposomes, as vehicles for delivering nucleic acids to cellular targets, are inconvenient for large-scale therapeutic applications, for a variety of reasons. First, the relationships: liposome / nucleic acid, used to form the nucleic acid / liposome complexes in the previous method, based on the joint mixing, results in the formation of complexes that have a large diameter, so their stability is relatively low .

Therefore, none of the currently used cationic liposome formulations, including DC-Chol / DOPE, is designed as a ready-to-use stable pharmaceutical formulation of a nucleic acid / liposome complex. This limitation of the method of joint mixing, requires the user to prepare the complex before each use, which is a drawback that requires specially trained personnel. In addition, the preparation of the complex by mixing together before each use, creates the possibility of a possible source of variations in the doses, which prevents the evaluation of the treatments made with these complexes, due to a possible sub- or super-dosage given to the receiver. Second, the method of joint mixing, of the prior art, for preparing the nucleic acid / cationic liposome complexes before each use, requires using a diluted solution of nucleic acid (less than 4 μg / ml) and a diluted dispersion of liposome (lower at 50 μM), to prepare the nucleic acid / liposome complex, in order to reduce the possibility of formation of large and less active aggregates. This limitation makes it difficult to prepare small biologically active complexes, unless practically optimal conditions are used, such as reducing the amount of liposomes (which causes a lower activity in nucleic acid transfer) or increasing the amount of liposome (which increases the toxicity). On the other hand, the fact that the complex must be prepared in diluted concentrations, is a significant drawback for its clinical applications, particularly in the case of intratumoral injection of the complex, since only a small volume of the complex can be injected into each one of the sites (Nabel, GJ, and Other (s), (1993), Proc.Natl.Acad.Scie. USA. 90: 11307 - 11311). Therefore, the object of the present invention is to provide stable, biologically active complexes, comprising lipids, for the delivery of drugs, which complexes must be capable of being concentrated; and it is also that of providing methods to produce sayings. Brief Description of the Invention The present invention provides methods for producing lipid-containing complexes for the delivery of drugs, which complexes have a net positive charge and / or a positively charged surface. By the term "drug" as used in the present specification and claims, reference is made to any molecular entity, which is monomeric or oligomeric, and which, when bound in complex form with a lipid or a lipid and a polycation, is administered to an individual in order to provide a therapeutic effect on the recipient. Therefore, it would be expected that macromolecules having a net negative overall charge, or regions of negativity, would be capable of forming the delivery complexes of the present invention. Macromolecules that are particularly suitable for use in conjunction with the complexes of the present invention are, for example, DNA, RNA, oligo-nucleotides or negatively charged proteins. However, it would also be expected that macromolecules having a positive charge ((eg, a large cationic protein) would be capable of forming the complexes of the present invention by binding the cationic macromolecule with the lipid or anionic polymer, and then the cationic lipid, so as to form a complex The complexes of the present invention encompass a drug / lipid complex formed by mixing the drug to be delivered, with cationic liposomes, in a relationship: drug / lipid, such that the drug complex / lipid formed has a net positive charge and a complex: drug / lipid / polycation formed by mixing the drug with cationic liposomes and polycation at a lipid / polycation ratio, such that the drug / lipid / polycation complex formed has a net positive charge. expression "positive net charge", applied to the drug / lipid complex, refers to an excess of positive charge, of the lipid with respect to the beg The expression "net positive charge", applied to the drug / lipid / polycation complex, indicates that the positive charges of cationic lipid and polycation exceed the negative charge of the drug. However, it should be understood that the present invention also encompasses the drug lipid and drug / lipid / polycation complexes having a positively charged surface, regardless of whether the net charge of the complex is positive, neutral or even negative. It is possible to measure a positively charged surface of a complex, by migrating the complex in an electric field, known to those with expertise in the art, such as "Zeta Potential Measurement". (Martion, A., Swarick, J., and Cammarata, A., Physical Pharmacy &Physical Chemical Principles in the Pharmaceutical Sciences, 3rd edition, Lea and Febinger, Philadelphia, USA 1 ° 83), or through the binding affinity of the complex with all surfaces. Complexes that have a positively charged cell surface have a greater affinity to cell surfaces than complexes that have a neutral or negatively charged surface. Therefore, the present invention relates to methods for producing those drug / lipid and drug / lipid / polycation complexes, which comprise mixing the drug to be delivered, with cationic liposomes, and optionally polycation, in a ratio such that the Complex formed has a positive net charge and / or a positively charged surface.

In another embodiment of the present invention, the methods for producing drug / lipid or drug / lipid / polycation complexes can also comprise the oasis of purification of said complexes from free excess components (drug, lipid, polycation). ), after its production. The drug / lipid and drug / lipid / polycation complexes of the present invention are generally stable, capable of being produced at a relatively high concentration, and retain their biological activity over time, under storage conditions. Said complexes are used in the delivery (delivery) of nucleic acids, proteins and other macromolecules, to cells and tissues. DESCRIPTION OF THE FIGURES Figure 1 shows a typical distribution of the sizes (average diameter) of nucleic acid / liposome complexes prepared in the form of a joint mixture from DC-Chol / DOPE (3: 2) liposomes and DNA of plasmid pRSVL (2 μg), with the lipid / DNA ratios, indicated. Figures 2A and 2B show the distribution of the liposome marker, 3H-cholesteryl hexadecyl ether (o) and the 2P-DNA marker (•), selected from the fractions of the sucrose gradients. The locations of each of the fractions in the sucrose gradients, of both Figures 2A and 2B, is indicated in the upper part of Figure 2 A. The distribution of the H markers is shown in Figure 2A and after the centrifugation of the free liposomes (10 μmoles of DC-Chol DOPE (2: 3) in 2 ml volume) or free DNA (50 μg of pRSVL DNA in a volume of 2 ml) through a density gradient of Sucrose Figure 2B shows the distribution of the H markers and P after the ultra-centrifugation of the DNA-liposome complex (formed through the mixing of 20 μmoles of DC-Chol / DOPE lipids (2: 3) and 0.4 mg of pRSVL DNA in a volume of 2 ml) through of a gradient of sucrose densities. Figure 3 shows the transfection activities in the CHO cells, of the complex DNA / liposome mixed complex (o), of the mixed DNA / liposome poly-L-lysine complex (PLL), of the complex (D) of the DNA / lipid complex (•), and of the DNA / lipid PLL (B) complex. The DC-Chol / DOPE liposomes used to form the above-mentioned complexes contained a variable amount of mol% of DC-Chol, as indicated in the bottom of Figure 3. The DNA / lipid complexes (•) and DNA lipid / PLL (B), were purified in a gradient of sucrose densities before being tested to establish transfection activity. The activity of transfection is indicated on the vertical axis, in the form of luminous units of luciferase activity. Figure 4 shows the transfection activities of the mixed complex of liposome DNA (o) and the mixed DNA / liposome / PLL (D) complex compared to the transfection activities of the DNA / lipid complexes (•) and DNA / lipid / PLL stored at a temperature of 4 ° C for 130 days after purification in a gradient of sucrose densities. The DC-Chol / DOPE liposomes used to form the above-mentioned complexes contained variable mol% amounts of DC-Chol, as indicated in the lower part of Figure 4. The transfection activity is indicated on the vertical axis , in the form of luminous units of luciferase activity. Figure 5 shows the concentration of the protein that can be extracted from the CHO cells, 36 hours after the cells were treated with a mixed DNA / liposome complex (o); mixed liposome DNA / PLL complex (D); DNA / lipid complex (•); or DNA / lipid / PLL complex (B). The DNA lipid and DNA / lipid / PLL complexes were purified in a gradient of sucrose densities, before being tested to evaluate the activity of the transfection. The DC-Chol / DOPE liposomes used to form the aforementioned complexes contained varying amounts, expressed in mol%, of DC-Chol, as indicated in the bottom of Figure 5. The results are shown in Figure 6. of the CAT assays of tumor extracts prepared from mice having ovarian tumors. An amount (2 x 10E6) of human ovarian carcinoma cells was injected subcutaneously in SCID mice on day 0.

On day 14, 100 μl solutions containing DNA of pUCCMVCAT (containing the chloramphenicol acetyl transferase gene of the _ coii) (30 μg), which formed a complex DC-Chol liposomes (30 nmol) in the form of a mixed mixture (avenues (lanes) 1 and 2, samples in duplicate), or the same amount of DNA in the form of purified complex (prepared from liposome DNA: DC-Chol in a ratio 1 μg / 25 nmol, avenues 3 and 4, samples in duplicate), were directly injected into some tumors. Forty-eight hours after transfection, the mice were sacrificed, and the tumor extracts containing 100 μg of protein were assayed to establish CAT activity.

Avenue 5 shows a control CAT activity, positive, for the standard CAT of E. coli. Figures 7A-7C show the transfection activities of the DNA / lipid complex mixing pool and purified and unpurified complexes of DNA / lipid / PLL in "293" cells (Figure 7A), C3 cells (FIG. Figure 7B) and BL6 cells (Figure 7C). The activity of transfection is indicated on the vertical axis of the Figures 7A-7C, in the form of light units of luciferase activity. Description of the Invention The present invention relates to complexes for delivering drugs and containing lipids, which complexes have a positive net charge and / or a positively charged surface a pH of 6.0 - 8.0. These complexes comprise cationic liposomes, drugs, and optionally also comprise polycations. The present invention further relates to a method for producing these complexes, the method further optionally including the step of purifying these formulations by eliminating the individual components present in excess. For the production of the drug / lipid complexes of the present invention, the inclusion of the purification step is a preferred embodiment. It should be understood that when the step of purification is applied to the drug / lipid / polycation complexes, the recovery of said complexes in a pure state free of excess components, after purification, is lower than the recovery of the drug / complexes. lipid after its purification, since the peak containing the drug / lipid / polycation complex after the purification of sucrose after density centrifugation, is broader than the peak containing the drug / lipid complexes, so that overlaps the peaks of the free components. The drug delivery and lipid-containing complexes of the present invention are stable, capable of being produced at relatively high concentrations, and retain the biological activity of the drug component over time under storage conditions. The method to produce these complexes, is based on a model of linkage between two polymers of opposite charges (ie, negatively charged nucleic acid and positively charged lipids) in which the formation of large unstable aggregates is prevented by neutralizing the charge Negative of the drug by using an excess amount of positive charge in the form of cationic liposomes and polycation.

It has been observed that the complexes of the present invention retain their initial diameter and bioactivity for 4 months under storage conditions in a 10% sucrose buffer. The "drugs" that are contained in the drug delivery and lipid-containing complexes of the present invention can be nucleic acids, polyanionic proteins, polysaccharides, and other macromolecules that can complex directly cationic lipids. However, cationic drugs (eg, large canonical proteins), can directly form complexes an anionic lipid or form sequentially complexes first an anionic lipid or polymer followed by a cationic lipid. The use of this process, allows delivery of the drug, positively or neutrally charged, to the cells, by means of the complexes of the present invention. To produce the drug lipid and drug / lipid / polycation complexes with a net positive charge, the excess of positive charge of the lipid with respect to the drug, or of the lipid and polycation with respect to the drug, can be an excess of approximately 30 times of the positive charge in the complex of total lipids / drug, or of lipid and polycation and drug, preferably an excess of charge of 2 to 10 times, and more preferably, an excess of the charge, of 2 to 6 times the charge. Complexes that have a positive charge on their surfaces can have similar preferred ranges of excess surface charge with respect to the drug. To produce a nucleic acid / lipid complex having an excess of positive charge of lipid with respect to the nucleic acid, the molar amounts of cationic liposomal lipid to be mixed with 1 μg of nucleic acid to produce a nucleic acid / lipid complex having a positive charge in excess of lipid to nucleic acid at pH 6.0-8.0, can vary from about 0.1 nmol to about 200 nmol of lipid, preferably from about 5 nmol to about 100 nmol of lipid, depending on the content of the positive charge of the cationic liposome. Of course, if the drug were a protein, the amount of lipid to be mixed with 1 μg of negatively charged protein would be at least ten times less than the amount of lipid to be mixed with 1 μg of DNA, as shows in what precedes, inasmuch as proteins have a charge of lower density than nucleic acids. Those of ordinary skill in the art will readily understand that, depending on the positive content of the charge of the cationic liposomes, it would be necessary to mix different molar amounts of cationic liposomes with an equivalent amount of drug, in order to produce an excess of cationic liposomes. positive charge of the lipid with respect to the drug. When a complex drug / lipid / polycation having a net positive charge and / or a positively charged surface is produced, the inclusion of the polycation reduces the amount of lipid to be mixed with the drug, as long as the positive charge due to the lipid, it may be lower than the negative charge due to the drug. This reduction in the amount of lipid reduces the toxicity of the formulations containing polycation. The molar amounts of cationic liposomes to be used in the formulation of nucleic acid / lipid / polycation complexes can vary in the range from about 0.1 nmol to about 200 nmol of lipid per 1 μg of nucleic acid, more preferably, of about 1 to about 25 nmoles of lipid per 1 μg of nucleic acid, depending on the positive charge content of the cationic liposomes. In general terms, it should be understood that in the production of the nucleic acid / lipid and nucleic acid / lipid / polycation complexes of the present invention, the molar amount of lipids required to produce these complexes will increase as the concentration of the nucleic acid mixed with the liposomes. Those of ordinary skill in the art will readily understand that when the complexes of the present invention are purified, the excess positive charge of the cationic liposomes with respect to the drug or the cationic liposomes and the polycation with respect to the drug immediately before mixing, it will be greater than the excess of positive charge in the purified complexes of the lipids with respect to the drug, or of the lipid and polycation, as the step of the purification, results in the removal of excess free lipids and / or free polycation. In order to illustrate how the charges attributed to the cationic liposome, drug and polycation, can be determined at pH 6, 0 - 8.0 the following example is provided. Assuming that the drug to be delivered is DNA, we proceed to determine the negative charge of the DNA to be delivered, for which the amount of DNA to be delivered, or the amount of DNA in the complex, is divided by 330, which is the molecular weight of an isolated nucleotide; a nucleotide is equal to a negative charge. Therefore, the negative charge for 1 μg of DNA is 3.3 nmols. For 10 nmol of DC-Chol / DOPE lipids, one calculates the effective lipid load, for which the amount of liposomal lipid (10 nmol) is multiplied by 0.4 (40% of the liposomal lipid is the cationic liposome DC-Chol). ), obtaining 4 nmol of DC-Chol lipid in the liposomes. Since at pH 6-8, one molecule of DC-Chol has a positive charge, the effective positive charge of liposomal lipid at the time of mixing, or in the complex, is 4.0 nmol. Of course, those of ordinary skill in the art would readily understand that there are other cationic liposomes that could have a higher or lower positive charge per molecule of cationic liposome at pH 6.0 -8.0 than the DC- Chol. Assuming that the polycation to be mixed to form the complex, is a bromide salt of poly-L-lysine (PLL), the positive charge of PLL at the time of mixing, is obtained by dividing the amount of PLL to be mixed, by 2QZ, which is the molecular weight of a lysyl residue; A lysyl residue is equivalent to a positive charge. Therefore, the positive charge for 1 μg of PLL is approximately 5.0 nmols. To calculate the positive charge contributed by the lysyl residues in a complex formed, the amount of lysine present in the complex is divided by the molecular weight of a lysyl residue, taking into account the weight of a counter-ion, if present. . The application of the calculations just explained, to the data presented in Table 1 included here (see Example 3), illustrates how a positive / negative charge ratio can be calculated, both at the time of mixing the DNA and the liposome and , after the purification of the complex produced by the mixing of the DNA with the liposome. In Table 1 of Example 3, 0.4 mg of DNA are mixed with 20 μmols of cationic DC-Chol / DOPE liposomes, obtaining a DNA / lipid complex. For cationic liposomes having a DC-Chol / DOPE ratio of 4: 6, the positively charged content of the liposomal lipid, taken equal to 8000 nmol and the negative charge content of the 0.4 mg of DNA, to be mixed with the liposomes, it is taken as 1320 nmols, based on the calculations with the samples, presented in the previous paragraphs.

Therefore, the ratio between positive and negative charge at the time of mixing is 6.06 (8000 divided by 1320). However, once the complex has been purified, the ratio between the liposome and the DNA of this purified complex was 12.7 nmol of liposome μg of DNA, as shown in Table 1 (see "row"). 4: 6"). This ratio of 12.7 translates to a positive to negative charge ratio of 1.5, which shows that the purification removed the excess positive charge from the free liposomes. Also in Table 1, in which the DNA / lipid / PLL complex was prepared by mixing 4 μmol of liposomes (DC-Chol / DOPE 4: 6) and 1 mg of PLL with 0.4 mg of DNA, it is possible to calculate the relationship between positive and negative charge at the time of mixing, as follows.

Based on the calculations in the samples, presented in the preceding paragraphs, the liposomal lipid (4 μmol) provides 1600 nmol of positive charge, PLL (1 mg) provides 5000 nmol of positive charge, and DNA (0.4 mg) ) provide 1,320 nmol of negative charge. Therefore, the relationship between positive and negative charge, at the time of mixing of the liposomes, PLL and FDNA, is 5 (1600 + 5000) / 1320 In addition, those with expertise in the art should understand that the net charge of the complex can be determined by measuring the amount of DNA, lipid and, if present, polycation, in the complex, by using an adequate analytical technique such as the use of radio-isotopic labeling of each of the components or through elemental analysis. Once the amounts of each of the components (DNA, lipid, and, if present, polycation) in a complex with a given pH are known, one could then calculate the approximate net charge of said complex with the pH given, taking into account the pK's of the components that can be known or determined analytically. In a preferred embodiment, the drug is a nucleic acid sequence, preferably a nucleic acid sequence that encodes a gene product that has a therapeutic utility. In one embodiment of the invention, a method for producing nucleic acid / lipid complexes having a net positive charge and / or a positively charged surface at pH 6.0-8.0 comprises combining nucleic acids with cationic liposomes. in a relationship: acid 10 nucleic / lipid, such that the nucleic acid / lipid complex formed, has an excess of positive charge of lipid to nucleic acid. In an alternative embodiment, the nucleic acid and the cationic liposome can be mixed with the polycation in a ratio: nucleic acid / polycation, such that the nucleic acid / polycation complexes formed have a positive charge in excess of the lipid and polycation with with respect to the nucleic acid, with a pH = 6-8. In a preferred embodiment, the nucleic acid / lipid and nucleic acid / lipid / polycation complexes are produced by the slow addition of nucleic acid to the liposome or liposome plus polycation, and mixing with a stir bar when the mixing is allowed to take place in the second term. Alternatively, the liposome, or the liposome / polycation mixture, can be added in a single chamber, from a first entry, at the same time that the nucleic acid is added to the chamber through a second entry.

The components are then mixed simultaneously by mechanical means, in a common chamber. Cationic liposomes mixed with the drug, or with drug and polycation, to form the complexes of the present invention, may contain a cationic liposome alone or a cationic liposome and combined with a neutral phospholipid. Suitable cationic liposome species include, but are not limited to, the following: 1,2-bis (oleoyloxy) -3- (trimethyl ammonium) propane (DOTAP); N-1, (2,3-dioleoyloxy) propyl-N, N, N-trimethyl ammonium chloride (DOTMA), or other N- (N, N-dialkoxy) alkyl-N, N, N-substituted ammonium surfactants; l, 2-dioleoyl-3- (4'-trimethylammonium) butanoyl-sn-glycerol (DOBT) or the cholesteryl (4'-rimethylammonium) butanoate (ChOTB) in which the trimethylammonium group is connected by a separating arm of butanoyl, whether the double chain (for DOTB) is the cholesteryl group (for ChOTB); DORI (DL-1, 2-dioleoyl-3-dimethylamino? ropil-B-hydroxyethylammonium) O DORIE (DL-1, 2-0-dioleoyl-3-dimethylaminopro? il-β-hydroxyethylammonium) (DORIE), or analogs of the same, such as is disclosed in WO 93/03709; 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate ester (ChOSC); the Upo polyamines such as the doctadecylamide glycyl spermine (DOGS) and the dipalmitoyl phosphatidi ethanolamido spermine (DPPES) or the cationic lipids disclosed in U.S. Patent No. 5,283,185, the cholesteryl-3β-carboxylamido-ethylenetrimethylamino iodide, the iodide of l-dimethylamino-3-trimethylammonium-DL-2-propyl-cholesteryl carboxylate, cholesteryl- 36 -carboxyamido ethylene amine, cholesteryl-3β-oxysuccinamido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-tri-methyl ammonium-DL-2-propyl-cholesteryl-3β-oxysuccinate, 2- (2-trimethylammonium) ethylmethylamino ethylcol steryl-3β-oxysuccinate iodide, 3ßN- (N ', N'-dimethyl amino ethane) carbamoyl cholesterol (DC -Chol), and 3ß-N- (polyethyleneimine) -carbamoylcholesterol. Examples of preferred cationic lipids include: cholesteryl-3β-carboxymethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylamino-DL-2-propyl-cholesterylcarboxylate iodide, iodide of teril-3β-carboxyamido ethylene ketones amine, the iodide of choles teril-3ß-oxisuccina-midoethylene methylammonium, the iodide of l-dimethylamino-3-trimc.tamino-DL-2-propyl-cholesteryl-3β-oxysuccinate, the iodide of 2- (2-tri-methylammonium) ethylmethyl amino ethyl-cholesteryl-3β-oxysuccinate, 3ßN- (N ', N'-di methyl aminoethane) -carbamoyl-cholesterol (DC-Chol), and 3ßN- (polyethylene imine) -carbamoyl cholesterol. Since one of the attributes of the complexes of the invention, is its stability during storage (ie, its ability to retain a small diameter and to retain a biological activity over time after its formation), those that have the usual skill in the art will understand that the preferred cationic lipids are those lipids in which the bonds between the lipophilic group and the amino groups are stable in the aqueous solution. While such bonds found in cationic lipids include amide bonds, ester linkages, ether linkages and carbamoyl bonds, the preferred cationic lipids are those that have a carbamoyl bond. An example of a preferred cationic lipid having a carbamoyl bond is DC-Chol. Those of ordinary skill in the art will readily understand that liposomes containing more than one cationic lipid species can be used to produce the complexes of the present invention. For example, liposomes have been discovered comprising two species of cationic lipids, lysyl-phosphatidyethanolamine and β-alanyl cholesterol ester (Brunette, E. and Other (s), (1992), Nucí. Acids. Res .. 20: 1151).

Furthermore, it should be understood that in the selection of suitable cationic liposomes to be used in the joint mixing with the drug and optionally with the polycation, to form the complexes of the present invention, the methods of the present invention are not restricted to the use of the lipids already mentioned herein; on the contrary, any lipid composition can be used, as long as a cationic liposome is produced. Therefore, in addition to the cationic lipids, the cationic liposomes used to form the complexes of the invention may contain other lipids in addition to the cationic lipids. Said lipids include, but are not limited to, the following: the smooth lipids of which lysophosphatidylcholine (1-oleoylidephosphatidylcholine) is an example, cholesterol, or neutral phospholipids that include diolel phosphatidyl ethanolamine (DOPE) or diolel phosphatidylcholine (DOPC). The lipid complexes of the present invention may also contain negatively charged lipids as well as cationic lipids, as long as the net charge of the complexes formed is positive and / or the surface of the complex is positively charged. The negatively charged lipids of the present invention are those which comprise at least one lipid species having a net negative charge at or near physiological pH, or combinations thereof. Suitable negatively charged lipid species comprise phosphatidyl glycerol and phosphatidic acid or a similar phospholipid analogue. On the other hand, it is considered that in the cationic liposomes used to form the complexes of the invention, the ratio of the lipids can be varied so as to include a majority of cationic lipids in combination with cholesterol or with mixtures of smooth lipids or lipids. neutral When the chosen cationic lipid is to be combined with lipid, a preferred lipid is a neutral phospholipid, more preferably, DOPE.

The methods for producing the liposomes to be used in the production of the lipid-comprising complexes that serve to deliver the drugs of the present invention are known to those skilled in the art. A review of the methodologies for the preparation of liposomes can be found in: Liposome Technology (CFC Press NY 1984); Liposomes by Ortro (Marcel Schher, 1987); Methods Biochem Anol. 33: 337-462 (1988) and U.S. Patent No. 5,283,185. Such methods include freeze-thaw extrusion and sonication. Both unilamellar liposomes (average diameter, less than about 200 nm) and multilamellar liposomes (average diameter, greater than about 200 nm), can be used as starting components to produce the complexes of this invention. In the cationic liposomes used to produce the drug: lipid complexes of the present invention, the cationic lipid is present in the liposome in from about 10 to about 100 mol% of complete liposomal lipid, preferably from about 20 to about 80 mol%. , and preferably still, from about 20 to about 60 mol%. The neutral lipid, when included in the liposome, may be present in a concentration of about 0 to about 90 mol% of the complete liposomal lipid, preferably from about 20 to about 80 mol%, more preferably still, of about 40 to 80 % mol. The negatively charged lipid, when included in the liposome, may be present in a concentration ranging from about 0 mol% to about 49 mol% of the complete liposomal lipid, preferably from about 0 mol% to about 40 mol%. . In a preferred embodiment, the liposomes contain a cationic lipid and a neutral lipid, more preferably, DC-Chol and DOPE, in ratios of between about 2: 8 to about 6: 4. It should further be understood that the complexes of the present invention may contain modified lipids, proteins, polycation or receptor binders that function as a targeting factor that directs the complex towards a particular type of cells or tissues. Examples of aiming factors include, but are not limited to, asiallycoprotein. insulin, low-density lipoprotein (LDL), folate, and monoclonal and polyclonal antibodies directed against molecules on the surface of cells. Potential targets include, but are not limited to: liver, blood cells, endothelial cells and tumor cells. Furthermore, it should be understood that the positive charge of the complexes of the present invention can be affected not only by the lipid composition of the complex but also by the pH of the solution in which the drug / lipid complexes are formed. For example, if the pH is increased (it becomes more basic), the positive charge of the tertiary amine of the cationic lipid DC-Chol is gradually neutralized. In a preferred embodiment, the complexes of the present invention are produced, and stored, with a pH such that the complexes have a net positive charge and / or a positively charged surface. A preferred range for pH is pH 6.0-8.0, more preferably 7.0-7.8. When a polycation is to be mixed with nucleic acid and cationic liposomes, the polycation can be selected from among the organic polycations having a molecular weight of between about 300 and about 200,000. Said polycations preferably also have a valence of between about 3 and about 1000, at pH = 7.0. The polycations can be amino acids, peptides, proteins, polyamines, carbohydrates, natural and synthetic, and any synthetic cationic polymers. Non-limiting examples of polycations include: polyarginine, polyomitin, protamines and polylysine, polybrene (hexadimethrine bromide), histone, cationic dendrimers, spermine, spermine and polypeptides derived from SV40 antigen or large T having an excess of positive charges and represents a nuclear localization signal. A preferred polycation is poly-L-lysine (PLL). In the production of the complexes: nucleic acid / lipid / polycation, of the present invention, the ratio between the polycation and the nucleic acid is maintained. fixed, while the amount of liposome is varied.

However, those of skill in the art will recognize that the relationship between the polycation and the nucleic acid will be affected by the density of the lipoin charge to be mixed together with the nucleic acid and the polycation. For example, if the density of the liposome charge decreases as a result of changes in the lipid composition of the liposome (for example, if the ratio between the cationic lipid and the neutral lipid in the liposome is decreased), the amount of polycation to be mixed together with the nucleic acid and the liposome will be increased to compensate for the decrease in positive charge contributed by the liposomes. However, if polycation is used, it is preferred to use sub-saturating amounts of polycation (ie, amounts that do not saturate the entire negative charge of the nucleic acid) in order to allow the cationic liposomes to complex with the nucleic acid. Therefore, in a preferred embodiment of the present invention, an excess of positive charge of lipid with respect to the nucleic acid is used, even when there is a polycation mixed with the lipid and the nucleic acid. The amounts of polycation that can be mixed with 1 μg of nucleic acid and varying amounts of cationic liposomes in the present invention, vary between about 0.01 μg and about 100 μg polycation per μg nucleic acid, preferably about 0.1 μg to approximately 10 μg of polycation per μg of nucleic acid. When the purification of the nucleic acid / lipid and nucleic acid / lipid / polycation complexes is desired, by removing the excess free DNA, the excess free liposomes and the excess free polycation, the purification can be carried out by centrifugation through a gradient of sucrose densities, or other means that are suitable to form a density gradient. However, it is understood that other purification methods such as chromatography, filtration, phase partitioning, precipitation or absorption could also be used. In a preferred embodiment, the purification is used through centrifugation by a gradient of sucrose densities. The sucrose gradient may vary from about 0% sucrose to about 60% sucrose, preferably from about 5% sucrose to about 30% sucrose. The buffer in which the sucrose gradient is carried out, can be any aqueous buffer that is suitable for the storage of the fraction containing the complexes and it is preferable that it is a suitable buffer for the administration of the complex to the cells and tissues . A preferred buffer is the pH 7.0-8.0 Hepes. It is understood that in the present invention, the preferred nucleic acid sequences are those that are capable of directing the expression of the proteins. Said sequences can be inserted by routine techniques, in plasmid expression vectors, known to those having skill in art, prior to mixing with cationic liposomes or cationic lipids and polycation, so as to form the complexes comprising lipids. and that deliver the drugs of the present invention. The amount of nucleic acid mixed together with the cationic liposomes or with the cationic liposomes and the polycation can vary from about 0.01 μg to about 1 mg, preferably from about 0.1 μg to about 1.0 mg. It is understood that when the nucleic acid of interest is contained in the plasmid expression vectors, the amount of nucleic acid referred to above, refers to the plasmid containing the nucleic acid of interest. The purification of the nucleic acid / lipid and nucleic acid / lipid / polycation complexes of the present invention serves to concentrate the nucleic acids and lipids contained in the resulting complexes, from about 50 to about 500 times, such that the content of lipid contained in the complexes can reach up to about 40 μmol / ml and the nucleic acid content can reach up to about 2 mg / ml . The diameter of the complexes produced by the methods of the present invention is less than about 400 nm, preferably less than about 200 nm, and more preferably even less than 150 nm. The complexes formed by the methods of the present invention are stable for up to about a year when stored at a temperature of 4 ° C. Complexes can be stored in a 10% sucrose solution when recovered from the sucrose gradient, or can be lyophilized and then reconstituted 2Q in a 10% sucrose solution, prior to use. In a preferred embodiment, the complexes are stored in solution. The stability of the complexes of the present invention is measured by specific tests in order to determine physical stability and biological activity over time, under storage conditions. The physical stability of the complexes is measured by determining the diameter of the complexes, for which recourse is made to methods well known to those of ordinary skill in the art, which includes, for example, electron microscopy, filtration chromatography of gel, or by the almost-elastic light scattering, for which a Coulter N4SD particle sizer is used, as described in the examples. The physical stability of the complex is "substantially unchanged" during storage when the diameter of the stored complexes is not increased by more than 100%, preferably by no more than 50%, and more preferably by no more than 30%, with with respect to the diameter of the complexes as they are determined at the moment in which the complexes have been purified. The tests used in the determination of the biological activity of the complexes vary depending on which is the drug contained in the complexes. For example, if the drug is a nucleic acid encoding a gene product, it is possible to determine the biological activity by treating the cells in vitro under transfection conditions used by those having the usual skill in the art for the transfection of mixing complexes. set, of DNA and cationic liposomes. Cells that can be transfected by the complexes include those cells that can be transfected by complex DNA / liposome pooling complexes. The activity of the stored complexes is then compared with the transfection activity of the complexes prepared by joint mixing. If the drug is a protein, then it is possible to determine the activity by an appropriate bioassay for that protein. It is also understood by those skilled in the art that the complexes of the present invention can be used in vivo as vectors in gene-based therapy. Therapeutic formulations in which the complexes of the present invention are used, preferably comprise the complexes in a physiologically compatible buffer such as for example physiological phosphate-buffered saline, isotonic saline or low ionic strength buffer such as sucrose 10% in water (pH 7.4-7.6) or in Hepes (pH 7-8, with a more preferred pH, 7.4-7.6). The complexes can be administered in the form of aerosols or with liquid solutions for intratumoral, intravenous, intratracheal, intraperitoneal and intramuscular administration. All articles or patents referred to herein are incorporated by reference. The following examples illustrate various aspects of the present invention, but are in no way intended to limit the cationic alipid thereof. EXAMPLES Materials The DOPE was purchased from Avanti Polar Lipid, Inc. (Alabaster, AL, USA). PRSVL, which is a plasmid encoding the luciferase gene under the control of the long terminal repeat of the Rous sarcoma virus (De Wet, JR and Other (s), Mol.Cell.Bio 7: 725- 737), was expanded in E. coli and purified by using the standard ultracentrifugation method CsCl-ErtBr (Sambrook, J. Fritsch, EF and Maniatis, T., Molecular Cloning: A Laborato ry Manual (2nd edition), Cold Spring Harbor Laboratory Press: New York, USA. (1989). All media for tissue culture were provided by Gibco BRL (Gaithersburg, MD). Lipids 293 of human embryonic kidney, CHO cells (Chinese Hamster ovarian cells), BL6 and BHK cells (hamster calf kidney cells), were provided by the American Type Culture Collection (Rockville, MD). Mouse lung cells (MLC), are primary culture cells originally derived from the lung of a Balb / c mouse by Dr.

S. Kenned (Oak Ridge National Laboratory, TN). 293, BL6, BHK and MLC cells were cultured with DMEM medium, CHO cells were cultured with F12 medium, and C3 Hela cells were cultured in RPMI-1640 medium. All media were supplemented with their 10% fetal bovine rooster (Hyclone Laboratories, Inc., UT), and 100 units of penicillin / ml and 100 μg of streptomycin per ml. Poly-L-lysine hydrobromide (MW 3000 and MW 25,600), and other chemicals, were provided by Sigma (St. Louis, MI). The DC-Chol was synthesized according to Gao's method and Huang (1991) (Gao, X., and Huang, L. (1991) Biochem. Biophvs, Res. Commun. 179, 280-285), with modifications in the steps of purification, as follows: after the reaction they cooled 10 ml of hexane were added, and the mixture was extracted three times with 10 ml of water. The organic phase was combined and dried under vacuum at a temperature of 4 ° C. The resulting solid was dissolved in a minimum amount of absolute ethanol under heat, and "crystallized from acetonitrile at a temperature of 0 ° C. The purity of the DC-Chol was at least over 95%, as analyzed by the TLC and H-NMR method, and the yield was about 70%, which is a significant improvement over the yield. of the aforementioned method of Gao, X., and Huang, L. (1991), Biochem. Biophvs. Commun .. 179: 280-285). Methods Preparation v Purification of the Complexes Cationic liposomes were prepared with a total lipid complex, of 20 nM, from DC-Chol and DOPE, with various ratios, by a sonication method, according to a published procedure ( Gao, X., and Huang., L. (1991) Biochem. Biophvs. Res. Commun. 179: 280-285.

A vestigial amount of 3 H cholesteryl hexadecyl ether (Amersham, Arlington Heights, IL) was included for quantification purposes. The size of the liposomes was between 100 and 150 nm in diameter, as determined by the quasi-elastic scattering of the light, for which a Coulter N4SD particle sizer (Coulter Electronics, Inc., Hialeah, FL). Unless otherwise indicated in the following examples, the lipid DNA complexes were prepared on a typical laboratory scale by the addition of amounts of DC-Chol / DOPE free liposomes, as indicated in each of the examples in a volume of 1 ml of 2 nM Hepes buffer (pH 7.6) to a 15 x 7.5 polystyrene culture system (Baxtere, McGraw Pare, IL); A micro-magnetic stirrer was placed in the tube, and the solution was well mixed. Quantities of pRSVL DNA, as indicated in each of the examples, were then placed dropwise from a stock solution (0.2 mg / ml, in 2 mM Hepes buffer, pH 7.6) to the liposome solution for a period of 3 min. A trace amounts of pRSVL labeled with p by a nick transport device (Promega, Madison, Wl) and p dCTP (Amersham, Arlington Heights, IL) were included for quantification purposes. To prepare the purified lipid / PLL / DNA complexes, an amount of the above 0.2 mg / ml DNA solution, as indicated in each of the examples, was added to 1 ml of PLL / liposome mixture which contained amounts of liposomes and PLL, as indicated in each of the examples. The DNA lipid complexes were placed on the top of a step gradient of sucrose, composed of 0.5 ml each of the following: sucrose at 5%, 7.5%, 10% and 15% (weight / weight), the DNA lipid / PLL complexes were placed on the top of a step gradient of sucrose compound of 0.5 ml of each of the following: 5% sucrose, 10%, 15%, 20% and 30% strength. % ([weight / weight). The DNA lipid and DNA / lipid / PLL complexes were then purified by removing the free lipid and the free PLL, by ultra-centrifugation at 100,000 g for 30 minutes at a temperature of 4 ° C. After centrifugation, from the upper part of the tube to its bottom, fractions of 200 μl were extracted. Aliquots of each of the fractions were evaluated to establish the radioactivity H and p, for which a scintillation counter was used. The fractions containing peak values of p, frieron recovered and reunited. Said assembled fractions were then tested to verify the size of their particles and the activity of the transfection. In Vitro Evaluation of Transfection The biological activity of the aforementioned complexes was evaluated by in vitro transfection of lipids in the Examples, as follows. Briefly, cells cultured in 48-well plates were incubated with a DNA / lipid complex diluted in 0.5 ml of CHO-S-SFM (Gibco BRL) or with a mixed DNA / liposome complex prepared according to Gao and Huang (1991) (Gao, X., and Huang, L. (1991), Biochem. Biophvs, Res. Commun. 179: 280-285). For the transfection of pRSVL DNA using liposomes from DC-Chol in the presence of PLL, the liposomes were first mixed with PLL, and then joined with DNA, forming complexes with it. All transfections were carried out for 4 hours at a temperature of 37 ° C. After transfection, the lipids were subjected to further culture for 36 hours in the appropriate medium containing bovine serum. fetal to 10%. The cells were then washed with PBS and used with 100 μl of 1X lysis buffer provided by a portable evaluation kit (Promega, Madison, Wl). A sample of 4 μl of the lysate was tested to verify the transferase activity, for which 100 μl of 15 substrate solution taken from the reconstituted luciferase evaluation equipment and an AutoLumat LB953 luminometer (Berthold) were used. , Germany). The concentration of the proteins of each of the lysates was evaluated by a method of Coomassie blue staining, according to the manufacturer's protocol (Pierce, Rockford, IL). 2nd EXAMPLE 1 Determination of DNA / Lipid Complex Size. Through the Relationship: DNA / Lipid. This experiment was carried out to demonstrate that the size of the DNA / lipid complex formed by the joint mixture changed _ as the ratio of the DNA mixed with the liposome varied. Briefly, the plasmid DNA pRSVL (2 μg) was mixed with various amounts of DC-CHOL / DOPE (3: 2) liposomes in 2 nM Hepes buffer at pH 7.6 in a final volume of 500 μL and at After 7 minutes, the size of the complex was determined by a Coulter N4SD scattered laser light particle sizer, which operated in the single-mode mode.

As shown in Figure 1, no large aggregates were formed when the DNA was in excess (ratios: liposome / DNA, less than 7); but with lipid / DNA relationships that presented neutral charges (approximately 10), the size of the complex reached a maximum. In addition, when the liposome / DNA ratio was kept constant at 10 nmol / μg, the size of the complex increased as the concentration of the DNA and liposome increased, and finally precipitates formed. However, when the liposome / DNA ratio was increased, the size of the complex was progressively reduced until the size of the complex became constant (250 0 300 nm) when the liposome / DNA ratio exceeded 25 nmol of lipid / μg of DNA. This result may be due to the fact that the DNA was perhaps coated by excess liposomes and therefore the aggregation between the complexes did not occur. Based on the data presented in Figure 1, the lipid / DNA complexes were prepared using a liposome / DNA ratio of 50 nmol / g by slow addition of a 200 μg / ml DNA solution to an excess amount. (10 μmols) of liposome. The size of the complex formed was approximately 250 nm.

When the ratio of the liposome DNA was changed to 25 nmoles / μg, the size of the complex increased to approximately 350 nm. The complexes formed using either the 25 nmol / μg ratio or the 50 nmol / g ratio appeared to be physically stable since no precipitates formed during storage for 4 weeks at a temperature of 4 ° C. EXAMPLE 2 Purification of the DNA / Lipid Complexes When the DNA was mixed with liposomes in a ratio of 1 μg / 50 nmol, it was observed that there was an excess of free liposomal lipids together with the DNA / lipid complex. Since the excess free lipids are toxic to the cells, an experiment was carried out to determine if the liposomal lipids could be separated from the DNA / lipid complex by a density gradient ultra centrifugation method. Briefly, the free liposomes (10 μmoles of DC-Chol / DOPE (2: 3) in a volume of 2 m); Free DNA (50 μg of pRSVL in a volume of 2 ml) and DNA lipid complex formed by mixing 20 μmol of DEC-Chol / DOPE ((2: 3) and 0, 4 mg plasmid DNA of pRSVL (50 nmol / μg), were each subjected to centrifugation for 30 minutes at a temperature of 4 ° C in a gradient consisting of 0.5 ml of sucrose with each of the following concentrations: 5%, 7.5%, 10% and 15% (weight / weight). Fractions of 200 μl were then collected from the upper portion of the tube at the bottom of the tube and assayed to establish the distribution -1 DNA marker (p, B) and lipid marker (3H o). Figures 2A and 2B show the results of typical separations of free liposomal lipid, free DNA (Figure 2A) and DNA / lipid complex (Figure 2B), in the gradient of sucrose. The results presented in Figure 2B show that after centrifugation, the complex formed a larger band in the 10% sucrose layer. For comparison, Figure 2A shows that most of the radioactivity of free DNA or free liposomal lipids was distributed in the upper half of the tube, and that they did not enter the gradient of sucrose. Furthermore, although the peak of H and the peak of p in Figure 2B coexisted in fraction No. 16, there was a significant amount of H distributed in fractions 1 to 10, which indicates that the excess liposomal lipids were well separated from the DNA / lipid complex. AX PLO 3 Physical Stability of the Complexes Lipid / DNA v Lipid / PLL / DNA. Purified DNA / lipid complexes were formed by mixing 20 μmoles of liposomes of various DC-Chol / DOPE compositions (see Table 1) with 0.4 mg of pRSVL plasmid DNA, in a ratio of 1 μg of DNA per 50 nmoles of lipid . The lipid / PLL / DNA complexes were formed by mixing 4 μmoles of liposomes of various DC-Chol / DOPE compositions, with 1 mg of PLL (MW = 3000) and 0.4 mg of pK VL plasmid DNA.

Both complexes were then purified by removing free lipids, free DNA and free PLL, by centrifugation of sucrose gradient, as described above in the Chapter: Methods. The peak fractions were recovered and collected. The collected samples were then evaluated for their diameters, immediately after recovery (day O) or after storage in 10% sucrose at a temperature of 4 ° C for 120 days. In the following Table 1 the results of these evaluations are shown.

TABLE 1.- PHYSICAL STABILITY OF COMPLEXES LIPID DNA AND LIPID PLL / DNA. PURIFIED The data shown in Table 1 show that the purified lipid / DNA and lipid / PLL / DNA complexes had a small size (less than 200 nm) at day 0 and that their size did not increase dramatically with storage. On the other hand, the DNA / lipid ratios in the purified complexes were between 10 and 23 nmoles of lipid / μg of DNA, depending on the composition of the liposomes used, and this relationship did not change after a storage of 120 days . An inverse relationship was also observed between the DC-Chol concentration in the liposomes and the amount of lipid present in the complex, which is an indication that the liposomes enriched with DC-Chol show a stronger DNA binding or a neutralizing activity of the charge, more energetic than liposomes less enriched with DC-Chol. The columns on the far right show DNA recovery labeled with 32p in the DNA / lipid and DNA / lipid / PLL complexes after purification in the sucrose density gradient. The results show that DNA recovery in complexes that do not contain PLL was higher than that observed for complexes containing PLL. EXAMPLE 4 Biological Activity of Purified Complexes, in Various Cells Since the DNA / lipid complexes formed by a mixture of lipids to DNA having a high lipid / DNA ratio were both small and stable over time, experiments were carried out to carry out the transfection activity of said complexes with the activity of the DNA / lipid complex prepared by the method of joint mixing. In one of the experiments, the CHO cells grown on plates of 48 cavities were treated for 4 hours with a mixed pool of either 1 μg pRSVL and 10 nmol of DC-Chol / DOPE liposomes of different lipid compositions only (o) or together with 1 μg of PLL (MW = 3,000) ( D), or, the cells were treated with a purified DNA / lipid complex (•) or with a DNA / lipid / PLL (fl) complex formed by mixing 1 μg of DNA with 50 nmoles of DC-Chol / DOPE liposomes (DNA complex / lipid) or with 19 nmol of DC-Chol / DOPE liposomes and 1 μg of PLL (DNA / lipid / PLL complex) followed by centrifugation through a gradient of sucrose densities, as described in the Chapter: Methods. 36 hours after the treatment, the cells were used in 100 μl of lysis buffer, and 4 μl of the lysate was evaluated to establish the activity of the luciferase, for which 100 μl of a luciferase substrate solution was used. The luciferase activity was then subjected to counting for a period of 20 seconds. The results presented in Figure 3 show that the most preferred liposome composition for transfecting the CHO cells was 40% DC-Chol and 60% DOPE. Furthermore, in the presence of an additional 1 μg of poly-L-lysine (PLL, MW = 3,000), a 2 to 6-fold reinforcement of the transfection activity was observed in most cases. It is particularly interesting that the activity of the complex Purified DNA / lipid was similar to that of the complex DNA / complex pool when the same amount of DNA was added to the cells.

However, the transfection activity of the purified complex DNA / lipid / LPL, was approximately 30% to 50% lower than the DNA / liposome / PLL complex prepared by the whole mixing procedure. In order to establish that the results obtained in the cells of CHO were not specific for certain cells, the transfection activities of the DNA / lipid and DNA / lipid / PLL complexes, purified, in two other cells, BHK and mouse lung cells (MLC), were compared with those of the DNA / liposome complexes formed by joint mixing. Briefly, the cells (either BHK or MLC) grown in 48 cavity plates with a 60% confluence, were fried transfected with 1 μg of pRSVL which formed a complex with 10 nmol of DC-Chol 10 liposomes (whole mixing complex) , with the same amount of DNA mixed with liposomes with a DNA / liposome ratio of 1 μg / 50 nmol in order to produce purified DNA / lipid complex or with purified DNA / lipid / PLL complex prepared with a DNA / liposome / PLL ratio of 1 μg / 10 nmoles / 2 μg. The cells were then harvested at 36 hours after transfection, and the luciferase activity of the lysates from the transfected cells was determined as described in the Chapter: Methods. The results of these experiments are shown in Tables 2 and 3. - 'TABLE I EXPRESSION PEL GENE PE LVCIFERASE IN PH CELLS TRANSFECTAPAS WITH pRSVL Luciferase activity (relative units of lus? O'3) Mixed Composition Complex Complex Purifide Purifide Complex Liposome Complex DNA / DNA lipid complex / (DC-Chol / DOPE) DNA / PLL liposome 2: 8 91.8 ± 9.5 110.1 ± 5.2 214.6 ± 41.1 3: 7 61.2 ± 19.9 1886.8 ± 266.7 151.7 ± 62.9 4: 6 438.2 ± 14 , 4 1638.8 ± 63.9 446.3 ± 16.9 5: 5 837.8 ± 8 1015.0 ± 41.2 234.2 ± 46.4 TABLE 3.- EXPRESSION OF THE LUCIFERASE GENE IN PULMONARY MOUSE CELLS TRANSFECTED WITH pRSVL Luciferase activity (relative units of light x 10'3> Composition Mixed Complex Purified Complex Puride Liposome together with DNA / DNA fused DNA / (DC-Chol / DOPE) lipid lipid lipid / PLL DNA / liposome 2: 8 1.1 ± 0.7 0.4 ± 0.2 0.3 ± 0.1 3: 7 1.5 ± 1.0 0.3 ± 0.0 4.1 ± 1.3 4: 6 3.1 ± 0.2 2.0 ± 0.3 14.6 ± 3.1 : 5 0.1 ± 0.0 1.5 ± 1.2 10.1 ± 2.3 It is interesting to note that for the strain of BHK cells, the transfection activity of the purified DNA / lipid complex was substantially higher than that of the DNA / liposome complex formed by joint mixing. For cells such as MLCs, which are difficult to transfect, the purified complexes made from DNA / liposome / PLL mixtures, were apparently superior to the complex mixing complexes and the purified lipid DNA complexes made without PLL. In order to determine whether the lipid / PLL / DNA complexes could be made by different ratios: lipid / nucleic acid and with a PLL of different molecular weight than that used in the previous examples, the following experiment was carried out. A lipid / poly-L-lysine / DNA complex was prepared from 20 μg of pRSVL plasmid DNA, 10 μg of poly-L-lysine (MW 25,600), and DC-Chol / DOPE liposomes (molar ratio: 4.5 / 5.5), with the lipid DNAS ratios shown in Table 4. The resulting complexes were then purified by sucrose gradient ultra centrifugation, as described in the Chapter: Methods. An aliquot of the purified complex containing 0.5 μg of DNA was used to transfect the CHO cells, and then the activity of the luciferase was measured. The results of this experiment are shown in the following Table 4. TABLE 4- EFFECT OF THE RELATIONSHIP; LIPID / DNA IN THE PURIFIED OUE COMPLEX CONTAINS POLY-L-LYSINE (M . 600? Relation Composition of Activity Size (nol Complex Purifexplex of trans-lipid / cado (nmoles of purified feccidn μg DNA) lipid / μg (nm) (counts \ SO) xl? ° 3.3 1.1 89 108 (5) 6,6 2,5 98 6,065 (604) 12,5 4,3 101 5,846 (668) 20, 0 9,6 35 7,633 (977) The results show that in the presence of greater amounts of polycation, it is possible to use ratios: lipid / DNA, lower to produce DNA / lipid / polycation complexes that have an appreciable transfection activity. EXAMPLE 5 Transfection Activity of Stored Complexes A few CHO cells cultured in 48-well plates were treated for 4 hours with a pooled mixture of 1 μg pRSVL and 10 nmol of DC-Chol / DOPE liposomes of lipid compositions only (o) or together with 1 μg of PLL (MW = 3,000) (D), or, the cells were treated with a purified DNA / lipid complex (•) or with a DNA / lipid / PLL (I) complex stored at a temperature of 4. ° C for 130 days in 10% sucrose. The purified complexes had been formed by mixing 1 μg of pRSVL and 50 nmol of DC-Chol / DOPE liposomes of different compositions of DC-Chol / DOPE alone (DNA / lipid) or with 1 nmol of DC-Chol DOPE liposomes and 1 μg of PLL (DNA / lipid / PLL complex) followed by centrifugation through a gradient of sucrose densities, as described in the Chapter: Methods. The results show that the activity of luciferase in the lysates of cells prepared from cells transfected with the complexes Lipid DNA and DNA / lipid / PLL, stored, was comparable with the luciferase activity observed in the lysates of transfected cells with the corresponding complexes prepared by pooling. EXAMPLE 6 1 ° Comparison of the Cytotoxicity of the DNA / Lipo soma Complexes Prepared by Mixing together, with that of the purified DNA / Lipid Complexes The toxicity of the different complexes on the cells was studied in CHO cells, as follows. CHO cells were treated with whole DNA / liposome (o) pooling complex, liposome / PLL pooling complex (3); DNA / purified lipid complex (•); or DNA / lipid / purified PLL complex (B). The whole pool complexes were formed by mixing 1 μg of pRSVL DNA with 10 nmoles of DC-Chol / DOPE liposomes or different DC-Chol / DOPE compositions, alone or together with 1 g of PLL (mw = 3,000). The purified complexes were formed by mixing 1 μg of pRSVL DNA with 50 nmol of DC-Chol / DOPE liposomes only (DNA / lipid complex) or with 10 nmol of DC-Chol / DOPE liposomes and 1 μg of (PLL DNA lipid complex) followed by centrifugation through a sucrose density gradient, as described in the Methods Chapter. 36 hours after the treatment, the cells were used, the protein was extracted and then quantified by a Coomassie blue dyeing method. Figure 5 shows the results of this experiment in which the amount of extractable protein recovered at the end of the experiment serves as an indicator of the portion of the cells that have survived after the indicated treatment. The data presented show that while the purified complex appeared to be slightly more toxic to the cells than the mixed liposome DNA complex, morphologically, the transfection did not cause any serious toxicological effects on the cells treated with either the joint-mixing complexes purified complexes, with the proviso that cells treated with purified complexes containing a high mole% of DC-Chol, were less confluent at the end of the experiment. EXAMPLE 7 In Vivo Transfection of Tumors Using Purified DNA / Lipid Complexes An amount (3 x 10) human ovarian carcinoma cells were injected simultaneously into SCID mice on day 0. Fourteen days later, 100 μl of solutions containing pUCCMVCAT DNA (30 μg) that formed a complex with DC-Chol liposomes (DC-Chol / DOPE = 3: 2) in the form of a mixed pool (avenues 1 and 2), or the same amount of DNA in the form of purified DNA lipid complex 40 (prepared from DNA and DC-Chol liposomes with ratios of 1 μg of DNA / 25 nmol of lipid, fried directly injected into the tumors. Animals were sacrificed two days later, and extracts of tumors containing 100 μg of protein were challenged to establish CAT activity at a temperature of 37 ° C according to Ausubel and Other (s) (Wiley, Boston , USA), Vol. 1, pp. 9.6.2-9.6.5). The results show that the purified complex, although it had been prepared under non-optimal conditions, exhibited a transfection activity in vivo. EXAMPLE 9 Comparison of the Transfection Activity of DNA / Lipid / PLL Complexes. Purified and Unpurified, with that of the DNA / Mixing Lipid Complex as a whole The transfection activities of the DNA / lipid / PLL complex, purified and unpurified, and of the mixed mixing complex FDNA / lipid, in three strains of cells (293, BL6 and C3), were measured as follows: the DNA / lipid / PLL complex, purified, was formed by mixing 1 μg of pRSVL DNA with 10 nmol of DC-Chol / liposomes. DOPE (2: 3 mol / mol) and 1 μg of PLL (MW = 25,600) followed by purification by centrifugation through a density gradient of sucrose, as described in the Chapter: Methods. Unpurified DNA / lipid / PLL complexes were formed by mixing 100 μg of pRSVL DNA with 80 μg of PLL (MW = 25,600) and 1702 nmol of DC-Chol / DOPE liposomes (2: 3 mol / mol) (i.e. ratio of lipid DNA PLK, of 1 μg of DNA 17 nmol of lipid / 0.8 μg of PLL) in a final volume of 500 μl of water. 20 μl of the unpurified DNA / lipid / PLL complex (ie, 4 μg of DNA, 3.2 μg of a serum-free medium, suitable for the cell strain to be transfected.) The DNA / lipid complex mixed complex, was formed by mixing 1 μg of pRSDVL DNA with 10 nmols of DC-Chol / DOPE liposomes (3: 2 mol / mol), 293, C3 and BL6 cells, cultured at 80% confluence in plates of 24 cavities, were transfected for 4 hours at a temperature of 37 ° C with 1 μg of DNA in the form of purified DNA / lipid / PLL complex, mezo DNA lipid complex or FDNA / lipid / PLL complex without purification . After transfection, the cells were subjected to a subsequent culture for 36 to 48 hours in the appropriate medium containing 10% fetal calf serum. The activity of the transferase was then measured, as described in the Chapter: Methods. The results presented in Figures 7A (cells 293), 7B (C3 cells) and 7c (BL6 cells) demonstrate that the unpurified DNA / lipid PLL complex exhibited the highest transfection activity in all of the three strains of cells tested. While in the foregoing we have described a number of embodiments of the present invention, it is evident that their basic constructions may be altered in order to provide other embodiments ****. which resort to the methods and devices of this invention.

Therefore, it should be appreciated that the scope of the invention is defined by the claims appended hereto, rather than by the specific embodiments that have been presented in the present by way of example.

Claims (30)

1. A method for producing drug / lipid complexes having lipids with an excess of positive charge with respect to the drug, characterized in that it comprises mixing said drug with cationic liposomes with a relationship between the drug and the lipid, such that said drug / lipid complexes are formed.
2. 2. The method according to claim 1, characterized in that said ratio between the drug and the lipid for the purpose of forming said complex, is from about 1 μg / 0.1 nmol to about 1 μg / 200 nmol.
3. 3. The method according to claim 2, characterized in that said method further comprises the step of purifying said complexes.
4. 4. The method according to claim 3, characterized in that said step of the purification, is the centrifugation through a density gradient of sucrose.
5. 5. The method according to claim 1, characterized in that the drug is a nucleic acid and the liposomes are DC-Chol / DOPE liposomes.
6. 6. A method for producing drug / lipid / polycationic complexes that have an excess of positive charge of the lipid and polycation with respect to the drug, characterized in that it comprises: mixing said drug with cationic liposomes and at least one polycation, in a relation: drug / lipid / polycation, such that said complexes are formed according to claim 1.
7. 7. - The method according to claim 6, characterized in that said ratio: drug / lipids, is from about 1 μg / 0.1 nmol to about 1 μg / 200 nmol.
8. 8. The method according to claim 6, characterized in that said polycation is present in an amount of about 0.01 μg to about 100 μg.
9. 9. The method according to claim 8, characterized in that the polycation is poly-L-lysine having a molecular weight of about 300 to about 200,000 Daltons.
10. 10. The method according to claim 6, characterized in that the cationic liposome comprises a cationic lipid and a neutral phospholipid. 1.
11. The method according to claim 10, characterized in that the cationic lipid is DC-Chol.
12. 12. The method according to claim 1, characterized in that the neutral phospholipid is the dioloeil phosphatidyl-ethanolamine.
13. 13. The method according to claim 12, characterized in that the drug is nucleic acid.
14. 14. The method according to claims 1 or 6, characterized in that said complex has an average diameter of less than 300 nm.
15. 15. The method according to claim 14, characterized in that the average diameter of said formulation is maintained without substantial changes for up to one year under storage conditions.
16. 16. A drug / lipid complex according to claim 1, characterized in that it comprises: at least one species of lipid and a drug; the relationship between said lipid species and said drug being such that said complex has an excess of positive charge, of the lipid with respect to the drug.
17. 17. The complex according to claim 16, characterized in that said drug is nucleic acid.
18. 18. The complex according to claim 16, characterized in that said ratio between the drug and the lipid is from about 1 μg / 0.1 nmol to about 1 μg / 200 mmol.
19. 19. The complex according to claim 16,; characterized in that said lipid species is a cationic lipid.
20. 20. The complex according to claim 19, characterized in that said complex also comprises a kind of neutral phospholipid.
21. 21. - The complex according to claim 16, characterized in that said complex is purified of the excess free drug and the free excess lipid species.
22. 22. A drug / lipid / polycation complex according to claim 16, characterized in that it comprises at least one lipid species, at least one polycation and one drug; the relationship between said drug, said lipid species and said polycation being such that said complex has an excess of positive charge of the lipid and polycation, with respect to the drug.
23. 23. The complex according to claim 22, characterized in that said ratio between the lipid and the DNA, is from about 1 μg / nmol to about 1 μg / 200 nmol.
24. 24. - The complex according to claim 22, characterized in that said polycation is present in approximately .01 μg to approximately 100 μg.
25. 25. The complex according to claim 24, characterized in that said polycation is poly-L-lysine having a molecular weight of about 300 to 200,000.
26. 26. The complexes according to claims 16 and 22, characterized in that the positive charge of said complexes exists with a pH of 6.0 to 8.0.
27. 27. The complexes according to claim 26, characterized because the lipid species are DC-Chol and DOPE.
28. 28. A method for delivering drugs to an individual, characterized in that it comprises administering the complex according to any one of claims 16 or 22, in a therapeutically active amount, to an individual in need of treatment.
29. 29. The method according to claim 28, characterized in that the complex is a drug / lipid complex.
30. 30. The method according to claim 28, characterized in that the complex is a drug / lipid / polycation complex.