MXPA01006862A - Therapy for human cancers using cisplatin and other drugs or genes encapsulated into liposomes - Google Patents

Abstract

A method for encapsulating cisplatin and other positively-charged drugs into liposomes having a different lipid composition between their inner and outer membrane bilayers is disclosed. The liposomes are able to reach primary tumors and their metastases after intravenous injection to animals and humans. The encapsulated cisplatin has a high therapeutic efficacy in eradicating a variety of solid human tumors including but not limited to breast carcinoma and prostate carcinoma. Combination of the encapsulated cisplatin with encapsulated doxorubicin or with other antineoplastic drugs are claimed to be of therapeutic value. Also of therapeutic value in cancer eradication are claimed to be combinations of encapsulated cisplatin with a number of anticancer genes including but not limited to p53, IL-2, IL-12, angiostatin, and oncostatin encapsulated into liposomes as well as combinations of encapsulated cisplatin with HSV-tk plus encapsulated ganciclovir.

Description

THERAPY FOR HUMAN CANCERES THAT USES CISPLATINE AND OTHER DRUGS OR GENES ENCAPSULATED IN LIPOSOMES FIELD OF THE INVENTION The present invention relates to drugs and delivery systems encapsulated in liposomes, specifically cisplatin encapsulated in liposomes. The drugs are useful for killing cancer cells in a variety of human malignancies after intravenous injection.

BACKGROUND OF THE INVENTION Throughout this application, reference is made to various publications, patents and patent specifications published by author and date, or by a patent identification number. Full bibliographic citations are provided for publications within this description or immediately preceding the claims. The descriptions of these publications, patents and published patent specifications are incorporated as well as reference in the present description, to describe in greater detail the state of the art to which this invention belongs. Cis-diaminodichloroplatinum (11), cis- [Pt (NH3) 2C12] 2 \ abbreviated as cisplatin or cis-DDP, is one of the antineoplastic drugs that is they use more widely for the treatment of testicular and ovarian carcinomas and against carcinomas of the head and neck. More than 90% of testicular cancers are cured by cisplatin. The most severe side effects are nephrotoxicity, bone marrow toxicity and gastrointestinal irritation (Oliver and Mead, 1993). Birateral optic neuropathy was observed in a patient affected by ovarian carcinoma treated with 160 mg / m2 of cisplatin and 640 mg / m2 of carboplatin (Caraceni et al., 1997). Oral treatment with hexamethylmelamine in a group of 61 patients with epithelial ovarian carcinoma with cis- or carboplatin resistance (relapse within 6 months after the end of that therapy), showed an objective response regimen of 14% (Vergote et al. , 1992). Cationic cholesterol derivatives have been used to deliver therapeutic agents. For example, they have been mixed with phosphatidylethanolamine and treated with sound to form small unilamellar vesicles which can be combined with DNA and measure their entry into the cytosol from the endosome compartment. One of the liposome formulations, DC-Chol liposomes, has been used in a clinical trial of gene therapy against melanoma. The transactivating protein gene of human immunodeficiency virus 1 (HIV-1) with a reporter gene under the control of the long terminal repeat of HIV-1 was cosuminized. Human tumor cells selected for cisplatin resistance or isolated from patients who had not shown response to cisplatin therapy were highly transfectable with cationic liposomes. These results suggested a serial therapy protocol with cisplatin and gene therapy for malignancy (Farhood et al., 1994). Several platinum complexes prepared from depots of 2-amino-methylpyrrolidine as vehicle ligands were tested for their antitumor activity against colon carcinoma 26 and P388 leukemia using subcutaneous and / or intraperitoneal injections in mice. 2-aminomethylpyrrolidine proved to be the most effective vehicle ligand in its amine derivatives (Morikawa et al., 1990). An optimal procedure was established by orthogonal test for the preparation of albumin and cisplatin microspheres (Cis-DDP-AMS) using the emulsion heating stabilization method (the average size was 148 microns). The distribution and elimination half-lives of platinum were prolonged 3.36 times and 1.23 times after hepatic arterial chemoembolization with Cis-DDP-AMS against Cis-DDP, respectively (Zhang et al., 1995). The search for platinum (II) -based compounds with improved therapeutic properties was accelerated to design and synthesize a new family of water-soluble third-generation cis-diaminodichloroplatinum (II) complexes bound to uracil and uridine. However, none of the synthesized compounds showed any significant cytotoxic activity against after cell lines that were treated (Kim et al., 1998). The newly developed bioreductor was found to be 4- [3- (2-nitroimidazolyl) -propylamino] -7-chloroquinoline hydrochloride (NLCQ-1), potentiates the antitumor effect of the chemotherapeutic agents melphalan (L-PAM), cisplatin (cisDDP) and cyclophosphamide (CPM), without concurrent intensification in bone marrow toxicity. The enhancement was strictly program dependent, and the optimal effect was observed (1.5 to 2 death records beyond the addition) when NLCQ1 was administered 45 minutes before cisDDP. These results support the classification of NLCQ-1, based on clinical studies, as a chemosensitizer (Papadopoulou treatment Second-line therapy in patients with non-small cell lung cancer (NSCLC), who had previously received first-line therapy with cisplatin, achieved a partial response (40%) in 14 patients (Stathopoulos et al., 1999). Abra et al., Patent of E.U.A. No. 5,945,122, issued August 31, 1999), describe a liposomal composition containing uncharged cisplatin trapped in primarily neutral lipids. However, the procedure of Abra et al. uses neutral lipids comparatively with the DPPG anionic lipid described in the present patent, for the capture of cisplatin. In this way, although previous reports indicate that the supply of cisplatin and other therapeutic drugs mediated by liposomes is possible, the therapeutic efficiency has been limited by the low aqueous solubility and the low stability of cisplatin. Therapeutic efficacy is also limited by the high toxicity of the drug. Thus, there is a need to reduce the difficulties involved in the processing of drugs containing cisplatin and the high toxicity of cisplatin when used therapeutically. This invention covers this need, and also provides related advantages.

BRIEF DESCRIPTION OF THE INVENTION In one aspect, this invention provides a method for encapsulating cisplatin and other positively charged drugs in liposomes that have a different lipid composition between their inner and outer membrane bilayers, and are capable of reaching primary tumors and their metastases after its intravenous injection to animals and humans. In one aspect, the method includes the formation of complexes between cisplatin with DPPG (dipalmitoyl phosphatidyl glycerol) or other lipid molecules to convert cisplatin to its aqueous form by hydrolysis, which has positive charge and is the active form of the cisplatin endowed of antineoplastic activity. In this step, fusion peptides of membranes and other molecules with fusogenic properties can be added to improve the entry of the complex through the cell membrane. The aqueous cisplatin micelles-DPPG are converted to liposomes by mixing with lipids forming vesicles, such as liposomes or lipids previously made, followed by dialysis and extrusion through membranes, trapping and encapsulating cisplatin at a very high yield. Doxorubicin or other positively charged compounds can replace cisplatin in these formulations. Encapsulated cisplatin has high therapeutic efficacy to eradicate a variety of human solid tumors including, but not limited to, breast carcinoma and prostate carcinoma. It is claimed that the combination of encapsulated cisplatin with doxorubicin encapsulated or with other antineoplastic drugs is of therapeutic value. It is also claimed that of therapeutic value in the eradication of cancer are combinations of cisplatin encapsulated with several anticancer genes including, but not limited to, p53, IL-2, IL-12, angiostatin and oncostatin encapsulated in liposomes, as well as also combinations of cisplatin encapsulated with HSV-tk plus encapsulated ganciclovir.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the encapsulation of cisplatin. Figure 2 shows the regression of MCF-7 tumors in SCID mice after 3 to 4 injections of encapsulated cisplatin. Figure 3 shows the histology of tumors in SCID mice with or without treatment with cisplatin. Figure 3A shows untreated MCF-7 tumors developed in SCID mice. 40X increase. Note the pattern of homogenous structures characteristic of the tumor tissue. Figure 3B shows mice treated with cisplatin (4 injections). The cells are apoptotic, there are groups of cells in the structures, and the nuclei are stained larger and darker, characteristic of apoptotic cells. Figure 3C shows tumors of untreated animals showing muscle invasion. Increase of 20X. Figure 3D shows mice treated with cisplatin. No invasion is observed. Increase of 20X. Figure 4 shows the macroscopic (visual) difference in tumor size between an animal treated with encapsulated cisplatin (A) and an untreated animal (B).

WAYS TO CARRY OUT THE INVENTION The practice of the present invention will use, unless otherwise indicated, conventional techniques of immunology, molecular biology, microbiology, cellular biology and recombinant DNA. These methods are described in the following publications. See, for example, Sambrook, et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al., Eds., (1987)); the METHODS IN ENZYMOLOGY series (Academic Press, Inc.); "PCR: A PRACTICAL APPROACH" (M. MacPherson, et al., IRL Press at Oxford University Press (1991)), PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)); ANTIBODIES, A LABORATORY MANUAL (Harlow and Lane, eds. (1988)); and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)). As used in the specification and in the claims, the singular form "a" "an" and "the" include plural references, unless the context clearly indicates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. The term "comprising" means that the compositions and methods include the elements indicated, but not excluding others. "Consisting essentially of" when used to define compositions and methods, must mean that it excludes other elements of some essential meaning for the combination. Thus, a composition consisting essentially of the elements as defined herein, would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as pH regulated saline with phosphate, preservatives, and the like. "Consisting of" should mean that it excludes more than trace elements from other ingredients and substantial method steps to administer the compositions of this invention. The modalities defined by each of these transition terms are within the scope of this invention. The terms "polynucleotide" and "nucleic acid molecule" are used reciprocally to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides and / or their analogues. The nucleotides they can have any three-dimensional structure, and can perform any function, known or unknown. The term "polynucleotide" includes, for example, single stranded and double stranded helical molecules, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNAs, recombinant polynucleotides, branched polynucleotides, plasmids , vectors, DNA isolated from any sequence, RNA isolated from any sequence, nucleic acid probes and primers. A nucleic acid molecule can also comprise modified nucleic acid molecules. A "gene" refers to a polynucleotide that contains at least one open reading frame that is capable of encoding a particular protein or polypeptide after being transcribed and translated. A "composition" means a combination of active agent and another compound or composition, inert (e.g., a detectable agent or label or a pharmaceutically acceptable carrier) or active, such as an adjuvant. A "pharmaceutical composition" includes the combination of an active agent such as a vehicle, inert or active, which forms the composition suitable for therapeutic or diagnostic use in vitro, in vivo or ex vivo. As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a saline solution regulated in its pH with phosphate, water and emulsions such as an oil in water or water emulsion. water in oil, and various types of wetting agents. The compositions may also include stabilizers and preservatives. For examples of vehicles, stabilizers and adjuvants, see Martin, REMINGTON'S PHARM. SCI., 15th. edition (Mack Publ Co., Easton (1975)). An "effective amount" is an amount sufficient to produce beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. A "subject", "individual" or "patient" is used reciprocally herein, and refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, apes, humans, farm animals, animals for sporting activities and pets. A "control" is an alternative subject or sample that is used in an experiment for comparative purposes. A control can be "positive" or "negative". For example, when the purpose of the experiment is to determine a correlation of an altered level of expression of a gene with a particular cancer type, it is generally preferred to use a positive control (a subject or a sample of a subject having said alteration and exhibits characteristic syndromes of that disease) and a negative control (a subject or a sample of a subject lacking the altered expression and clinical syndrome of that disease). A "gene delivery vehicle" is defined as any molecule that can carry polynucleotides inserted into a cell host Examples of gene delivery vehicles are liposomes, cationic liposomes, viruses such as baculoviruses, adenoviruses, adeno-associated viruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other vehicles typically used in the art, which have been described for expression in a variety of prokaryotic and eukaryotic cells and can be used for gene therapy, as well as for the simple expression of proteins. A "viral vector" is defined as a virus or recombinantly produced viral particle comprising a polynucleotide to be delivered in a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and the like. In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and the inserted polynucleotide. As used herein, the terms "retroviral mediated gene transfer" or "retroviral transduction" have the same meaning, and refer to the procedure by which a gene or nucleic acid sequences are stably transferred into the host cell in virtue of the virus that enters the cell, and integrates its genome in the genome of the host cell. The virus can enter the host cell by its normal mechanism of infection, or is modified so that it binds to a different ligand or receptor on the surface of the host cell to enter the cell. As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral entry mechanism or viral type entry mechanism. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse transcribed in the form of DNA that is integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. In aspects where gene transfer is mediated by a viral DNA vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a polynucleotide that is going to be inserted. Adenoviruses (Ads) are a homogenous group of relatively well characterized viruses that include more than 50 serotypes (see, for example, WO 95/27071). The Ads are easy to develop, and do not require integration into the genome of the host cell. Vectors derived from recombinant adenoviruses have also been constructed, particularly those that reduce the potential for recombination and generation of wild type viruses (see WO 95/00655 and WO 95/11984). The wild type AAV has high infectious character and high specific character, and is integrated into the genome of the host cells (Hermonat and Muzyczka (1984) PNAS USA 81: 6466-6470; Lebkowski, et al. (1988) Mol. Cell. Biol. 8: 3988-3996).

Vectors that contain a promoter and a cloning site in which a polynucleotide can be operably linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, Wl). To optimize expression and / or in vitro transcription, it may be necessary to remove, add or alter 5 'and / or 3' untranslated portions of the clones to eliminate alternative, inappropriate and potential extra start translation codons or other sequences that may interfere with the expression or reduce it, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5 'from the start codon to inactivate the expression. The gene delivery vehicles also include several non-viral vectors, including DNA / liposome complexes, and targeted viral protein DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof, can be used in the methods of this invention. To enhance delivery to a cell, the nucleic acid or proteins of this invention can be conjugated with antibodies or binding fragments thereof, which bind to cell surface antigens, for example, TCR, CD3 or CD4. Polynucleotides are inserted into the vector genomes using methods well known in the art. For example, DNA from the vector and the insert can be contacted, under appropriate conditions, with a restriction enzyme to create complementary ends in each molecule that can pair with each other and bind with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of restricted polynucleotides. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector DNA. In addition, an oligonucleotide containing a stop codon and an appropriate restriction site can be ligated for insertion into a vector containing, for example, the following elements or some of them: a selectable marker gene, such as the neomycin for selection of stable or transient transfectants in mammalian cells; intensifier / promoter sequences of the early human CMV immediate gene for high levels of transcription; RNA processing signals and SV40 transcription termination for stability of messenger RNA; origins of SV40 and ColEl polyoma replication for appropriate replication of episomes; versatile multiple cloning sites; 3 'stabilizing elements for the inserted polynucleotide, and T7 and SP6 RNA promoters for the in vitro transcription of sense and antisense RNA. Other means are well known and are available in the art. The term "host cells" is used to include any individual cell or cell culture that may be or have been recipient of vectors or the incorporation of exogenous proteins, polypeptides and / or polynucleotides. It is also used to include the progeny of an individual cell, and the progeny can not necessarily be completely identical (in morphology or in the complement of total or genomic DNA) to the original progenitor cell due to natural, accidental or deliberate mutation. The cells can be prokaryotic or eukaryotic and include, but are not limited to, bacterial cells, yeast cells, plant cells, insect cells, animal cells and mammalian cells, eg, murine, rat, simian or human cells. As used herein, the terms "neoplastic cells", "neoplasia", "tumor", "tumor cells", "cancer" and "cancer cells" (used reciprocally), refer to cells that exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation (i.e., deregulated cell division). The neoplastic cells can be malignant or benign. The "suppression" of tumor cell growth indicates a growth state that is shortened when compared to control cells. The growth of tumor cells can be assessed by any means known in the art including, but not limited to, measurement of tumor size, determination of whether tumor cells are proliferating using a 3H-thymidine incorporation assay, or cell count. tumor "Suppression" of tumor cell growth means any or all of the following states: diminution, delay and arrest of tumor growth, as well as tumor shrinkage.

DETAILED DESCRIPTION OF THE MODALITIES OF THE INVENTION Micelles, liposomes and procedures for obtaining them A new method for trapping cisplatin in lipids is claimed, which intensifies the content of cisplatin per unit volume, reduces its toxicity, is capable of directing primary tumors and their metastases after intravenous injection. , and shows contraction of the tumors and complete therapy of SCID mice that possess human tumors. Cisplatin is a heavy metal complex that contains two chlorine atoms and two amino groups in the cis position attached to an atom of the transient heavy metal platinum in its divalent form. It is a bifunctional alkylating agent, as well as a DNA intercalator that inhibits DNA synthesis. In one form, cisplatin is a yellow powder of molecular weight 300.1 and of limited solubility of 1 mg / ml. It is widely used for the treatment of cancer patients, especially those of squamous cell carcinomas of head and neck, ovarian, bladder, endometrial, testicular and lymphomas, breast carcinomas, and many other malignancies, often in combination with adriamycin, vinblastine, bleomycin, prednisone, vincristine, taxol, and other antineoplastic drugs, as well as radiation therapy. The claim is reduction in the total volume of cisplatin that is required for the treatment of patients due to an increase in its solubility in its lipid capture form. The volume that is used for intravenous injection is usually large (approximately 180 ml per adult patient or approximately 20-120 mg / m2) administered as a 24-hour infusion. It is cleared from plasma in a rapid phase of 25-80 min, followed by a slower secondary phase of 58-73 h; it is bound by plasma proteins and excreted by the kidneys (which explains the severe toxicity to the kidney in treated patients). The dose-related nephrotoxicity can be partially overcome with vigorous hydration, mannitol, furosemide and other drugs. Other toxicities incurred by cisplatin include ototoxicity, nausea and vomiting, anemia and moderate myelosuppression (The Merck Manual of Diagnosis and Therapy). The present invention overcomes the limitations of the methods and compositions of the prior art. Thus, in one aspect, this invention provides methods for producing cisplatin micelles, by combining cisplatin and a lipid derivative of phosphatidyl glycerol (PGL derivative) in a scale of 1: 1 to 1: 2.1, to form a mixture of cisplatin. In alternative modalities, the scale of cisplatin: derived from PGL is in scales of 1: 1.2; or 1: 1.4; or 1: 1.5; or 1: 1.6; or 1: 1.8 or 1: 1.9 or 1: 2.0 or 1: 2.1. The mixture is then combined with an effective amount of at least one 20% organic solvent, such as an ethanol solution to form cisplatin micelles.

As used herein, the term "lipid derivative of phosphatidyl glycerol (derivative of PGL)", is any lipid derivative having the ability to form micelles and have a head group with a net negative charge. It includes, but is not limited to, dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl glycerol and dicapryl phosphatidyl glycerol. In one aspect, phosphatidyl derivatives with a carbon chain of 10 to 28 carbons and having an unsaturated aliphatic side chain are within the scope of this invention. The combination of cisplatin with negatively charged phosphatidyl glycerol lipids that have variations in the molar ratio that gives the particles a positive (1: 1), neutral (1: 2) or slightly negative (1: 2.1) net charge will allow the location of different tissues in the body after administration. However, it has been shown that the combination of cisplatin with negatively charged PGL enhances the solubility of cisplatin, thus reducing the volume of the drug that is required for effective antineoplastic therapy. In addition, the combination of cisplatin and PGL with negative charge proceeds to a very high encapsulation efficiency, minimizing the loss of drug during the manufacturing process. These complexes are stable, do not form precipitates and retain their therapeutic efficacy after being stored at 4 ° C for at least 4 months. As used herein, the term "cisplatin" includes analogs. Examples include carboplatin, ormaplatin, oxaplatin, 2-aminomethylpyrrolidine (1,1-cyclobutane dicarboxylate) platinum, lobaplatin, 1- Cyclobutane-dicarboxylate (2 -) - (2-methyl-1,4-butanediamine-N, N ') platinum, zipiplatine, enloplatin, 254-S nedaplatin and JM-216 (bis-acetate-amine-dichlorocyclohexylamine-platinum (IV )). It will be understood, although not always explicitly stated, that other positively charged drugs, including, but not limited to, the antineoplastic drug doxorubicin, can replace cisplatin. Alternatively, other types of drug that are neutral can be used after their conversion into positively charged drugs by derivatization with positively charged groups. Modification of a neutral or negatively charged anticancer agent or other drug to a positively charged molecule can be achieved by a number of well-established methods in the organic synthesis technique. This can be achieved, for example, by replacing a hydroxyl group in the drug with an amino group, or by a trimethylamino group, thereby introducing a positive charge to the compound. The replacement of a ring hydroxyl group with an amino group is described in the US patent. 5,837,868, Wang, et al., Issued November 17, 1998. The above method does not require that the steps be carried out in the order indicated above. For example, the method can be practiced by combining cisplatin with an effective amount of at least one 20% organic solvent solution to form a solution. The solution is combined with a lipid derivative of phosphatidyl glycerol (PGL) on a scale of 1: 1 to 1: 2.1 to form a cisplatin micelle. How I know indicated above, the scale of cisplatin: derived from PGL is in scales of 1: 1.2; or 1: 1.4; or 1: 1.5; or 1: 1.6; or 1: 1.8 or 1: 1.9 or 1: 2.0 or 1: 2.1. Any organic solvent or ethanol formulation, or any other alcohol that does not form a two-phase system, or another organic solvent (ie, chloroform), that is miscible in 20% alcohol, is useful in the methods described herein. . For example, the alcohol solution can be any of at least 30%, 35%, 40%, 45% and up to 90%, including any increase between these values. Preferably, the alcohol solution is 30% ethanol for DPPG, and for other lipids the optimum percentage may be different. In one embodiment, the partial replacement of DPPG molecules by peptides with a net negative charge gives rise to cisplatin complexes that have fusogenic properties capable of crossing the target cell membrane. Fusogenic peptides can also be covalently bound at the free end and PEG for better exposure. The addition of a small amount of cationic lipids that replace the positive charges of aqueous cisplatin in the final cisplatin / DPPG complex also endows the complex with fusogenic properties; the percentage of positive charges that are to be replaced by cationic lipids (eg, DDAB, dimethyldioctadecyl ammonium bromide, DMRIE, N- [1- (2,3-dimyristyloxy) propyl] -N, N-dimethyl-N bromide - (2-hydroxyethyl) ammonium, DMTAP, 1,2-dimyristoyl-3-trimethylammonium propane, DOGS, dioctadecylamidoglycylspermine, DOTAP, N- (1- (2,3- dioleoyloxy) propyl) -N, N, N-trimethylammonium; DPTAP, 1,2-dipalmitoyl-3-trimethylammonium propane; DSTAP, 1,2-diesteroyl-3-trimethylammonium propane) is small due to the toxicity of cationic lipids. Some formulations contain an amount of the amphophilic amphiphilic lipid DOPE in the micelles. In another aspect, cisplatin micelles are encapsulated in lipids that form vesicles, for example, for use in the delivery of drugs. Lipid-encapsulated cisplatin has a high therapeutic efficacy for eradicating a variety of solid tumors in humans including, but not limited to, breast carcinoma, prostate carcinoma, glioblastoma multiforme, non-small-cell lung carcinoma, pancreatic carcinoma, cell carcinoma scaly head and neck and T cell lymphomas., in one aspect, the invention provides a method for the treatment of a variety of human malignancies using encapsulated cisplatin or, alternatively, other positively charged antineoplastic drugs in liposomes having a different lipid composition between their inner and outer membrane bilayers. The drugs encapsulated in liposomes are able to reach the primary tumors and their metastases, after their intravenous injection to animals and humans. A combination of cisplatin encapsulated with doxorubicin or other antineoplastic drugs has a greater therapeutic efficacy than cisplatin alone. Also of greater therapeutic efficacy in the eradication of cancer, are combinations of cisplatin encapsulated with a number of anticancer genes including, but not limited to, p53, IL-2, IL-12, angiostatin and oncostatin encapsulated in a similar type of liposomes, as well as cisplatin combinations encapsulated with HSV-tk plus encapsulated ganciclovir. In another aspect, the present invention provides a combination of the encapsulated cisplatin with the following genes: (i) a cDNA expression vector of the wild type p53 gene under the control of the CMV promoter, beta-actin and other promoters, and origins of replication in humans capable of sustaining the long-term expression of the p53 gene; Viral replication origins that require viral replication-initiating proteins such as T antigen for their activation are not suitable for p53 gene transfer because the p53 protein interacts strongly with the T antigen. (ii) an expression vector of cDNA PAX5, the only suppressor of the known p53 gene (mutant and wild-type p53 genes) that interacts with a short regulatory region (10 nucleotides) within intron 1 of the p53 gene. A major drawback in therapy with the p53 gene is the inactivation of the wild type p53 protein by the endogenous mutated forms of p53, which are overexpressed in tumors and are capable of tetramerizing with the wild-type p53 protein; the endogenous p53 genes will be suppressed by the expression of Pax5, a potent repressor of p53 gene transcription. The cDNA vector of the wild type p53 gene lacks intron 1, and consequently the suppressor region of PAX5 binding. It is important to suppress the expression of the endogenous mutant p53 gene and eliminate the mutant p53 gene from cancer cells to enhance the induction of apoptosis and tumor suppression. (iii) The thymidine kinase gene of herpes simplex virus (HSV-tk). The herpes simplex virus thymidine kinase (HSV-tk) suicide gene will also be included in combinations of the p53 and Pax5 genes that cause disruption in DNA synthesis after ganciclovir (GCV) treatment of the model in animals and patients humans; this is expected to increase chain breaks in cancer cells, and potentiate tumor suppressor functions of p53 that is known to bind to chain breaks and damaged DNA sites. In another embodiment, ganciclovir is combined and encapsulated in liposomes. Gene therapy is a new era of biomedical research focused on introducing therapeutic genes into somatic cells of patients (reviewed by Boulikas, 1998a, Martin and Boulikas, 1998). Two major obstacles prohibit the successful application of somatic gene transfer: (1) the small percentage of transduced cells, and (2) the loss of transcription signal from the therapeutic gene after approximately 3 to 7 days of injection in vivo. The first problem arises (i) from the inability of the delivery vehicles that possess the gene to reach the surface of the target cell (the vast majority of liposome-DNA complexes are rapidly eliminated from the bloodstream); (ii) of the difficulty in penetrating the cell membrane, and (iii) to release the DNA from the endosomes after incorporation by the cells; (V) of the inefficient incorporation in nuclei. Others have used stealth liposomes (Martin and Boulikas, 1998a), which persist in circulation for days and concentrate on tumors. However, classic stealth liposomes are not captured by cancer cells. In the present strategies are described that are designed to intensify the incorporation of liposomes (fusogenic peptides). The second problem arises from the loss of the plasmids in the nucleus due to degradation by the nuclease and the impossibility of replicating independently, leading to its dilution during cell poliferation between the cells of the progeny, or by inactivation of the introduced DNA after of the integration into the chromosomes of the host cell. Nevertheless, the use of human sequences capable of supporting the extrachromosomal replication of plasmids for prolonged periods (see U.S. Patent entitled "Cloning method for trapping human origins of replication" by Teni Boulikas No. 5,894,060), will overcome this limitation. Also claimed herein are tumor regression and tumor mass volume reduction of breast, prostate and other cancers in animal and human models after delivering encapsulated cisplatin (termed Lipoplatin ™) or encapsulated doxorubicin, and combinations of these drugs with genes including, but not limited to, p53, PAX5 and HSVtk / encapsulated ganciclovir, IL-2, IL-12, GM-CSF, angiostatin, IL-4-IL-7, IFN-gamma, TNF-alpha, RB, BRCA1, E1A, cystosine deaminase in combination with encapsulated 5-fluorocytosine, bcl-2 genes, MDR-1 , p21, p16, bax, bcl-xs, E2F, IGF-I, VEGF, TGF-beta, and the like. Accordingly, this invention also provides a method for delivering cisplatin or other therapeutic agent to a cell, which comprises contacting the cell with encapsulated drugs or other intermediates, obtainable by the methods of this invention. Also provided by this invention is a method for inhibiting the growth of a tumor in a subject, which comprises administering to the subject an effective amount of the encapsulated drugs obtainable by the methods of this invention. The methods can be practiced in vitro, ex vivo or in vivo. Thus, this invention also provides combination therapy using encapsulated drugs and polynucleotides. As used herein, a polynucleotide includes, but is not limited to, genes encoding proteins and polypeptides, as well as sequences encoding ribozymes and antisense sequences. Combination therapy is more effective in eradicating cancer than any treatment alone, because the two mechanisms are different and can achieve synergism. For example, the property of the p53 protein to bind to damaged DNA regions and the free ends of DNA is known and also triggers the mechanism of apoptosis in cells. damaged (reviewed by Boulikas, 1998a). It is expected that free ends of DNA will be produced in cancer cells after damage by cisplatin, intensifying the induction of an apoptotic pathway in these cells by expressing the transferred wild-type p53 protein (many tumors have mutated p53, and could be unable to effectively induce this path). The therapeutic polynucleotide can also be inserted into a gene transfer vector prior to its incorporation into the micelle. The transfer of the wild-type p53 gene has been used successfully to retard the proliferation of tumor cells in vivo and in cell culture in numerous studies. It has been shown that intratumoral injection using adenoviral / p53 vectors is effective against lung tumors in recent clinical trials (Roth et al., 1996) and against prostate tumors in animal models (reviewed by Boulikas, 1998a). However, the intratumoral injection method may not be applicable to metastases frequently associated with late stages of cancer. The systemic delivery of the p53 gene with encapsulated cisplatin and the localization of tumors in any region of the body, constitute an effective treatment for the cure of cancer. Strategies are sought to improve or partially overcome the four main obstacles to the successful transfer of somatic genes using the liposomal supply of wild-type p53, as well as pax5, HSV-tk, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-gamma, FNT-alpha, RB, BRCAL, E1A and cytosine deaminase, in combination with encapsulated 5-fluorocytosine, bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I, VEGF, TGF-beta, angiostatin and other genes in combination with encapsulated cisplatin and other drugs, to a variety of human cancers in models in animals and in patients that are tested in clinical trials. These strategies include: (i) concentration and encapsulation of the drug and inclusion of genes in liposomes, reducing their toxicity; (ii) localization of solid tumors and metastases by coating the surface of the complexes with polyethylene glycol (PEG), hyaluronic acid or other polymers; (iii) improvement in the uptake of drugs and plasmids by cancer cells due to fusogenic peptides or a small percentage of cationic lipids; and (iv) sustained expression of genes using human replication origins (ORIs) capable of sustaining episomal replication or long-term expression of therapeutic genes and high levels of expression. In another embodiment, the polynucleotides or genes further comprise regulatory DNA sequences that sustain the expression of the genes for months, rather than days. This results in fewer treatments and less suffering for the cancer patient. It can also exert a strong therapeutic effect due to higher levels of expression of the anticancer gene; the same gene put under the control of weak regulatory DNA will be ineffective. Several experimental strategies have been designed for the treatment of cancer using the p53 gene supply; the novelty of the present invention is that the endogenous mutant p53 forms, the which are overexpressed in more than half of the prostate malignancies in humans, especially those of advanced prostate cancer, are suppressed using the PAX5 expression vector. The mutated forms of p53 have amino acid substitutions mainly in their DNA binding domain, but are still capable of tetramerizing with the wild-type p53 form; p53 acts as a tetramer, and the presence of high levels of endogenous mutant p53 in human cancer cells, interferes with the tumor suppressor functions of the wild-type p53 form to be delivered. PAX5 is a homeodomain protein that determines body structures during development; PAX5 is expressed in the early stages of development of mammals and in adults during differentiation into hematopoietic stem cells; the expression of the p53 gene is eliminated by the PAX5 suppressor protein in the early stages of development, which allows the cells to multiply rapidly in the developing embryo. PAX5 is inactivated in later stages throughout adulthood, allowing p53 to be expressed and exert its tumor suppressor functions and regulate poptosis especially in the hematopoietic cell lineage. Numerous delivery systems are being used in the transfer of somatic genes, each associated with advantages and disadvantages. Recombinant adenoviruses do not replicate efficiently; the recombinant murine retroviruses are randomly integrated and are inactivated by chromatin environments; Recombinant AAVs are randomly integrated and can not achieve high titers for clinical utility. All have a maximum capacity of 3.5-7.5 kb of introduced DNA due to packaging limitations. Naked DNA is rapidly degraded (half-life of 5 minutes) after its systemic delivery. Cationic liposomes are toxic, do not survive in the circulation beyond a heartbeat, and are mainly directed to the endothelium of the lung, liver and heart. Until now, only "stealth" liposomes have been shown to be able to concentrate on tumor sites (also in the liver and spleen) and to survive for prolonged periods in the bloodstream (for example, one day comparatively with minutes for liposomes neutral non-stealth, and a few seconds for cationic liposomes). However, stealth liposomes are not rapidly absorbed by the tumor cells remaining in the extracellular space, where they release their charge for days after lysis (reviewed by Martin and Boulikas, 1998); however, the aspect of the invention described below modifies stealth liposomes with fusogenic peptides, or provides a partially cationic lipid composition or DOPE in its inner bilayer, which would allow it to enter the membrane of the tumor cell, causing disturbance of the lipid bilayer. Having achieved the concentration and uptake of the drug and the inclusion of genes in solid tumors in animals with stealth liposomes, the second step is the efficacy of the drug and the procedure for locating the drug. gen. A human clinical trial conducted at the MD Anderson Cancer Center uses wild-type p53 gene transfer in patients suffering from non-small cell lung cancer, and shows that p53 mutations occur in their tumors using local injection of recombinant adenovirus Ad5 / CMV / p53 in the tumor site in combination with cisplatin. The first results of this clinical trial are encouraging after the intratumoral injection of p53 (Roth et al., 1996, reviewed by Boulikas, 1998a). However, local injection is not applicable to metastases frequently associated with advanced stages of malignancies; in particular, prostate cancer metastasizes to bone by a mechanism that involves stimulation of prostate tumor proliferation by insulin-like growth factor I (IGF-I), which is secreted especially by bone cells. Therefore, the delivery system proposed herein, capable of concentrating on the mass of tumor cells after systemic injection, is probably not only to treat the primary tumor, but also its metastases. The proposed cisplatin liposomes will primarily locate tumors due to the nature of the delivery system of the present invention. The genes in the combination therapy will mainly target dividing cells due to the use of HSV-tk and ganciclovir that are incorporated into the replicating DNA, and mainly vascularizing tumors due to the use of stealth liposomes. In this way, liver and spleen cells that are not reached by the stealth liposomes will not be destroyed.

In another embodiment, the drugs encapsulated in liposomes described herein further comprise an effective amount of a fusogenic peptide. Fusogenic peptides belong to a class of helical amphipathic peptides characterized by a gradient of hydrophobic character along the long helical axis. This gradient of hydrophobic character causes the inclined insertion of the peptides in the membranes, thus destabilizing the lipid nucleus and, in this way, intensifying the fusion of membranes (Decout et al., 1999). Hemagglutinin (HA) is a surface homotrimeric glycoprotein of the influenza virus. During infection, it induces the fusion of membranes between viral and endosomal membranes at low pH. Each monomer consists of the HA1 domain of receptor binding and the HA2 domain of membrane interaction. The NH2-terminal region of the HA2 domain (amino acids 1 to 127), the so-called "fusion peptide", is inserted into the target membrane, and plays a crucial role by triggering fusion between the viral and endosomal membranes. Based on the substitution of 8 amino acids in the 5-14 region with cysteines and paramagnetic electron spin resonance, it was concluded that the peptide forms an alpha helix inclined approximately 25 degrees from the horizontal plane of the membrane, with a maximum depth of 15 Angstroms (Á) from the phosphate group (Macosko et al., 1997). The use of haegoglutinin HA2 fusogenic peptides of influenza virus greatly enhanced the uptake efficiency of the transferrin-polylysine-DNA complex by the cells; in in this case, the peptide was bound to polylysine, and the complex was delivered by transferrin-mediated endocytosis (reviewed by Boulikas, 1998a). This peptide had the sequence: GLFEAIAGFIENGWEGMIDGGGYC (SEQ ID NO: 1), and was able to induce the release of the fluorescent dye calcein from liposomes prepared with egg yolk phosphatidylcholine, which was higher at acidic pH; this peptide was also able to increase up to 10 times the anti-HIV potency of antisense oligonucleotides, at a concentration of 0.1-1 mM, using CEM-SS lymphocytes in culture. This peptide changes conformation in the slightly more acidic environment of the endosome, destabilizing and breaking the endosomal membrane (reviewed by Boulikas, 1998a). The presence of negatively charged lipids in the membrane is important for the manifestation of the fusogenic properties of some peptides, but not others; The fusogenic action of a peptide, which represents a domain of putative fusion of fertilin, a protein from the surface of the sperm that intervenes in the fusion of the sperm and the ovum, depended on the presence of negatively charged lipids. However, this did not occur for the HIV2 peptide (Martin and Ruysschaert, 1997). For example, to analyze the two domains in the fusogenic peptides of HA haemagglutinin of influenza virus, HA chimeras were designed in which the cytoplasmic tail and / or the transmembrane domain of HA were replaced with the corresponding glycoprotein domains. F fuselage of the Sendai virus. HE obtained constructs of HA in which the cytoplasmic tail was replaced by peptides of human neurofibromin type 1 (NF1) (residues 1441 to 1518) or c-Raf-1 (residues 51 to 131). The constructs were expressed in CV-1 cells using the transient expression system of the T7 polymerase of vaccinia virus. Membrane fusion between CV-1 cells and bound human erythrocytes (RBCs) mediated by parental or chimeric HA proteins showed that, after the pH decreased, a flux of calcein aqueous fluorophore of pre-loaded RBCs in the cytoplasm of CV-cells occurred. 1 that express the protein. This indicated that the fusion of membranes involves both layers of the lipid bilayers, and leads to the formation of an aqueous fusion pore (Schroth-Diez et al., 1998). A remarkable discovery was that HIV TAT protein is able to cross cell membranes (Green and Loewenstein, 1988), and that a TAT domain of 36 amino a, when chemically interlaced with heterologous proteins, conferred the ability to transduce into cells . It is useful to mention that the 11-amino afusogenic peptide of TAT (YGRKKRRQRRR (SEQ ID NO: 2)) is a nucleolar localization signal (see Boulikas, 1998b). Another HIV protein, glycoprotein gp41, contains fusogenic peptides. Linear peptides derived from the proximal membrane region of the gp41 ectodomain have potential applications as anti-HIV agents, and inhibit the infectious character by adopting a helical conformation (Judice et al., 1997). The N-terminal peptide of 23 amino acid residues of gp41 of HIV-1, has the ability to destabilize large unilamellar vesicles with negative charge. In the absence of cations, the main structure was an alpha helix of pore formation, whereas in the presence of Ca2 +, the conformation changed to a predominantly extended fusogenic beta structure. The fusion activity of HIV (ala) (which possesses R22 (substitution A), was reduced by 70%, whereas the fusogenic character was completely suppressed when a second substitution was included (V2 (E), holding that it is not a helix alpha, but an extended structure adopted by the fusion peptide of HIV-1, which actively destabilizes electrically neutral membranes containing cholesterol (Pereira et al., 1997) .Prion protein (PrP) is a functional glycoprotein It occurs in diseases such as bovine spongiform encephalopathy and Creutzfeldt-Jakob disease in humans, where the PrP is converted to an altered form (called PrPSc). of computer modeling, domains 120 to 133 and 118 to 135 of PrP are peptides inclined to association with lipids that are inserted in an oblique form in a lipid bilayer, and that are able to interact with liposomes to induce leakage of encapsulated calcein (Pillot et al., 1997b). The C-terminal fragments of Alzheimer's amyloid peptide (amino acids 29-40 and 29-42), have properties related to those of fusion peptides of viral proteins that induce liposome fusion in vitro. These properties could mediate a direct interaction of the amyloid peptide with cell membranes, and explain part of the cytotoxicity of the amyloid peptide. In view of the epidemiological and biochemical links between the pathology of Alzheimer's disease and the polymorphism of apolipoprotein E (apo E), the examination of the potential interaction between the three common apoE isoforms and the C-terminal fragments of the Amyloid peptide, showed that only apoE2 and apoE3, not apoE4, are potent inhibitors of the fusogenic and aggregation properties of the amyloid peptide. It was thought that the protective effect of apoE against the formation of amyloid aggregates is mediated by the formation of stable complexes of apoE / amyloid peptide (Pillot et al., 1997a, Lins et al., 1999). The fusogenic properties of a net negatively charged amphipathic peptide (WAE 11), consisting of 11 amino acids, were strongly promoted when the peptide was anchored to a liposomal membrane; the fusion activity of the peptide appeared to be independent of pH and membrane fusion, and the target membranes required a positive charge that was provided incorporating phosphatidylethanolamine (PE-K) coupled to lysine. While the coupled peptide could cause aggregation of vesicles by non-specific electrostatic interaction with PE-K, the free peptide could not induce aggregation of PE-K vesicles (Pecheur et al., 1997). Numerous studies suggest that the stabilization of a secondary structure of the alpha helix of the peptide after its insertion in lipid bilayers in cell membranes or liposomes, determines membrane fusion properties of peptides; Zn2 + enhances fusogenic activity than peptides because it stabilizes the alpha helix structure. For example, the HEXXH domain of the salivary antimicrobial peptide, located in the functional C-terminal domain of histatin-5, a recognized zinc binding motif, is in a helical conformation (Martin et al., 1999, Melino et al., 1999; Curtain et al., 1999). Fusion peptides have been formulated with DNA plasmids to create peptide-based gene delivery systems. A combination of the peptide YKAKnWK was used to condense plasmids into nanoparticles of 40 to 200 nm with the antipathetic peptide GLFEALLELLESLWELLLEA (SEQ ID NO: 3), which is a lytic agent responsive to pH, designed to facilitate the release of the plasmid from expression enhanced in endosomes containing the beta-galactosidase reporter gene (Duguid et al., 1998). DOPE (dioleyl phosphatidyl ethanolamine) is a fusogenic lipid; the unfolding of N-methoxy-succinyl-Ala-Ala-Pro-Val-DOPE by elastase converted this derivative to DOPE (full positive charge), to deliver an encapsulated fluorescent probe, calcein, into the cell cytoplasm (Pak et al., 1999). An oligodeoxynucleic sequence of 30 bases complementary to a region of beta-endorphin mRNA, induced inhibition of beta-endorphin-dependent concentration production in cell culture, after it was encapsulated within small unilamellar vesicles (50 nm) containing dipalmitol-DL-alpha-phosphatidyl-L-serine endowed with fusogenic properties (Fresta et al., 1998). Other fusogenic peptides useful in the methods of this invention are described in Table 1 below: Fusogenic peptide Protein source Properties References (LKKL) 4 Fusogenic peptide Gupta and Kothekar, amphiphile capable of 1997 interacting with four molecules of DMPC Residues 53-70 Apolipoprotein Induces the fusion of Lambert et al, (C-terminal helix) (apo) All unilamellar 1998 lipid vesicles and displaces apo Al from HDL and r-HDL Residues 90-1 1 1 Alfa PH-30 (a fusogenic activity of Niidome et al, protein that the membrane for 1997 works in the bilayer phospholipid fusion of sperm-egg acids) Term N of Nef Protein Nef of fusogenic activities Macreadie et al, immunodeficiency and perturbation of 1997 human type 1 membrane in (HIV-1) artificial membranes; causes the death of cells in E. coli and yeast Signal peptides of alpha s2- and beta- Interacts with Creuzenet et al liposomes, casein of dimyristoyl phosphatidyl-1997 glycerol and -coline; shows fusogenic and lithic activities F1 Polypeptide of F1 Polypeptide Can be used as a Particles et al, Amino-Measle Sequence Carrier System for 1996 Terminal Epitopes of CTL Virus (MV) 23 amino acids S virus protein A high degree of Rodriguez-Crespo hydrophobic in hepatitis B similarity with the et al, 1994 region amino- (HBV) terminal fusogenic peptides known from other viruses After the micelles are formed, they are mixed with an effective amount of a vesicle-forming lipid to form liposomes containing drugs. Lipids useful for this invention include neutral liposomes prepared in advance, powdered lipids, PEG-DSPE or hydrogenated soy phosphatidylcholine (HSPC). Vesicle-forming lipids are selected to achieve a specific degree of fluidity or stiffness of the final complex that provides the lipid composition of the outer layer. These may be composed of 10 to 60% cholesterol and the remaining amounts include bipolar phospholipids, such as phosphatidylcholine (PC) or phosphatidylethanolamine (PE), with a hydrocarbon chain length in the range of 14 to 22, and saturated with one or more double bonds C = C. A preferred lipid for use in the present invention is cholesterol (10 to 60%), hydrogenated soy phosphatidylcholine (HSPC) in 40-90%, and the vesicle-forming lipid derivative PEG-DSPE in 1-7%. The liposomes provide the outer lipid bilayer surfaces that are coated with the hydrophilic polymer, PEG. The PEG chains have a molecular weight between 1,000-5,000 Daltons. Other hydrophilic polymers include hyaluronic acid, polyvinylpyrrolidone, DSPE, hydroxyethylcellulose and polyaspartamide. PEG-DSPC and PEG-HSPC are commercially available from Syngena. Prior to mixing with vesicle forming lipid, the ethanol and other organic solvents can be removed by any method known in the art, for example dialysis of the micelles through permeable membranes.

Diagnosis and therapeutic methods Subject therapy is claimed, for example, mammals such as mice, rats, apes and human patients, with human cancers including, but not limited to, breast cancer, prostate, colon, non-cell lung small, pancreatic, testicular, ovarian, cervical carcinomas, squamous cell carcinomas of the neck and head. In one aspect, intravenous injection of cisplatin encapsulated in liposomes as well as by combinations of encapsulated cisplatin with doxorubicin encapsulated, fluorodeoxyuridine, bleomycin, adriamycin, vinblastine, prednisone, vincristine, taxol or radiation therapy, encapsulated oligonucleotides, ribosomes endowed with anticancer properties and a number of anti-cancer genes that include, but are not limited to, genes p53 / Pax5 / HSV-tk, are claimed. Our approach consists of two main parts: (i) the ability to locate cancer cells (ii) effectiveness of our approach to kill cancer cells. Accordingly, this invention also provides a method for delivering cisplatin or other therapeutic agent to a cell comprising contacting the cell with the encapsulated drugs obtainable by the methods of this invention. Also provided by this invention is a method for inhibiting the growth of a tumor in a subject, which comprises administering to a subject an effective amount of encapsulated drugs obtainable by the methods of this invention. Depending on the composition of the micelle / lipid formulation, they are also claimed in the present methods for identifying solid tumors and metastases in a subject by intravenous administration of an effective amount of encapsulated drugs and methods for penetrating the cell membrane of a tumor in a subject by administering an effective amount of the encapsulated drug, wherein the micelle contains a free fusogenic peptide or a lipido-fusogenic peptide conjugate. The methods can be practiced in vitro, ex vivo or in vitro. The in vitro practice of the method involves the removal of a tumor or culture biopsy from a sample of cells containing tumor cells. The final liposome complex or any intermediate product arising during the encapsulation of cisplatin (eg the micelles are shown in Figure 1A) makes contact with the cell culture under the conditions suitable for intracellular incorporation of the drug. The in vitro method is useful as a screen to determine the best drug therapy for each patient separately. Inhibition of cell growth or proliferation indicates that the cell or tumor is adequately treated by this therapy. The effective amount of drug for each therapy varies with the tumor being treated and with the subject to be treated. Effective amounts can be determined empirically by those skilled in the art. When given to an animal, the method is useful to reconfirm the efficacy of the drug or therapy for each type of tumor. As an example of suitable animal models, groups of SCID mice or hairless mice (Balb / c NCR nu / females nu, Simonsen, Gilroy, CA), can be subcutaneously inoculated with approximately 105 to 109 target or carcinogenic cells as defined herein. When the tumor has already been identified, the liposome is administered. As used herein, "administration, supply or administration" is intended to include any method that ultimately provides the liposome / drug complex to the tumor mass. Examples include, but are not limited to, topical application, intravenous administration, parenteral administration or subcutaneous injection around the tumor. Tumor measurements to determine tumor size reduction are carried out in two dimensions using Vernier calipers twice a week. For in vivo administration, the pharmaceutical compositions are preferably administered parenterally, ie, by intravenous, intraperitoneal, subcutaneous and intrathecal injection into the spinal cord, intramuscularly and intraarterially, injection into the portal vein or intratumorally. More preferably, the pharmaceutical compositions are administered intravenously or intratumorally by bolus injection. In other methods, the pharmaceutical preparations can make contact with the target tissue by direct application of the preparation to the tissue. The application can be carried out by "open or closed" topical procedures. By "topic" is meant the direct application of the pharmaceutical preparation to the tissue exposed to the environment, such as the skin, nasopharynx, external auditory canal, eye, inhalation to the lung, genital mucosa and the like. The "open" procedures are those procedures that include incision of the skin of a patient and direct visualization of the underlying tissue to which the pharmaceutical preparations are to be applied. This is usually achieved by a surgical procedure, such as a thoracotomy to access the lungs, abdominal laparotomy to access the abdominal viscera, or other direct surgical approach to the target tissue. The "closed" procedures are invasive procedures in which the internal target tissues are not directly visualized, but are accessed through insertion instruments through small skin wounds. For example, the preparations can be administered to the peritoneum by needle washing. Likewise, pharmaceutical preparations can be administered to the meninges or spinal cord by infusion during a lumbar puncture followed by an appropriate position of the patient as is commonly practiced for spinal anesthesia or metrazamide image of the spinal cord. Alternatively, the preparations can be administered through endoscopic devices. In vivo administration can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods for determining the most effective means and dosage of administration are well known to those skilled in the art and will vary with the compositions used for the therapy, the purpose of the therapy, the target cell to be treated, and the subject treat. The administrations Individual or multiple can be carried out with dose levels and patterns that are selected by the doctor who applies the treatment. Suitable dosage formulations and methods for administering the agents can be determined empirically by those skilled in the art. The agents and compositions of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions. Ideally, the lipid / drug formulation should be administered to achieve maximum concentrations of the active compound in the part suffering from the disease. This can be achieved, for example, by intravenous injection of the lipid / drug formula. Desirable blood levels of the drug can be maintained by continuous infusion to provide a therapeutic amount of the active ingredient within the damaged tissue. The use of the operative combinations is contemplated to provide the therapeutic combinations that require a low total dosage of each drug of the component that may be required when each individual therapeutic compound or drug is used alone, by means of which the adverse effects are reduced. While it is possible for the lipid / drug formula to be administered alone, it is preferable to present it as a pharmaceutical formulation comprising at least one active ingredient, as defined above, together with one or more pharmaceutically acceptable carriers thereof and optionally other therapeutic agents. Each vehicle can be "acceptable" in the sense of being compatible with other ingredients of the formulation and not dangerous to the patient. Designing the third generation of vehicles for the administration of anticancer drugs and genes for solid tumors as described herein was the result of five major improvements over existing technologies: 1) The encapsulation of antineoplastic drugs in sterically stabilized liposomes has been reduced many times the toxicity This is anticipated at the end of the nightmare of cancer patients undergoing chemotherapy. Most commonly used antineoplastic drugs suffer severe side effects such as hair loss, vomiting, weight loss and infarct cause as well as damage to the kidneys, brain, liver, and other vital tissues. The antineoplastic drugs described herein are hidden within the lumen of the lipid bilayer, are not visible to most tissues and concentrate on their tumor targets, not for every tissue in the body. In the uptake by the solid tumor they exert a specific cytotoxic effect for the cancer cells without damaging the normal cells. 2) Identification of solid tumors and their metastases over the entire body. More than 95% of cancer patients succumb to complications related to metastases, and not from the main tumor.

Our drug and gene delivery system has been designed to evade the immune system after intravenous administration of the drug and gene inclusion achieved not only to the main tumor but to each metastasis in the animal or human body regardless of the size of the tumor. This is based on the long circulation time of our gene and the inclusion of the drug carrying the vehicles and their extravasation through the vascular endothelium of the tumors due to their imperfections and leaks in their initial stage of formation (neoangiogenesis in tumor growth) as well as due to the differences in hydrostatic pressure between the growth of the solid tumor and the normal body tissues. The liposomes of this invention have a different composition between their outer and inner lipid layers allowing efficient encapsulation and identification of the tumor. 3) Capture of the inclusion of liposome by means of the cancer cell. The liposome inclusions are capable of promoting fusion with the cell membrane. Similar "stealth" inclusions developed everywhere are not able to cross the membrane barrier of cancer cells. 4) Achieve almost 100% of the efficacy of liposome encapsulation for anticancer drugs, oligonucleotides and genes as a main breakthrough. This means a minimal loss and an effective use of the cost of the gene drug. It also moves to simple steps in making anti-cancer inclusion.

) The unique technology described herein can identify regulatory DNA sequences that sustain the expression of genes in anticancer inclusions for months instead of days. This translates into short treatments and less suffering from the cancerous patient. A strong therapeutic effect can also be exerted due to the high levels of expression of the anticancer gene; the same gene placed under the control of weak regulatory DNA will not be effective. The following examples are intended to illustrate, and not limit the invention.

EXAMPLES Preparation of encapsulated lipid micelles and cisplatin A formula for encapsulation includes the steps of: (A) mixing cisplatin (powder or other form) with DPPG (dipalmitoyl phosphatidyl glycerol) or other negatively charged lipid molecules with a molar ratio of 1: 1 to 1: 2 at minus 30% in ethanol, 0.1 M Tris HCl, pH 7.5 to achieve approximately 5 mg / ml of the final cisplatin concentration. Variations in the molar ratio between cisplatin and DPPG are also of therapeutic value by identifying different tissues. (B) Heating at 50 ° C. During steps A and B the initial powder suspensions, which have to give a precipitate of yellow cisplatin powder, are converted to gel form (colloidal); during steps A and B there is a conversion of cisplatin into your aqueous form (by hydrolysis of the chlorine atoms and their replacements by aqueous molecules attached to cisplatin) which is positively charged and is the active form of cisplatin with antineoplastic activity; Aqueous cisplatin is composed simultaneously with the negatively charged lipid in the micelles in 30% ethanol. This electrostatic complex of cisplatin DPPG has improved the properties of free cisplatin in tumor establishment. (C) The properties of the complex (and of the final formulation after step D, see below) that pass through the tumor cell membrane after reaching its target are enhanced by the addition of peptides and other molecules that give the complex this property. (D) The cisplatin-micellar complex DPPG is converted into liposomes that encapsulate the cisplatin-DPPG monolayer (figure 1 above) or to another type of complexes by the direct addition of previously elaborated liposomes followed by dialysis against the saline solution and extrusion through the membranes to decrease the size of these from 100b to 160 nm in diameter (figure 1 below). It is the lipid composition of the aggregated liposomes that determines the composition of the outer surface of the final cisplatin formulation. The variation in steps (A) allows the encapsulation of doxorubicin or other antineoplastic compounds with positive charge. The addition of positively charged groups to neutral or negatively charged compounds allows their encapsulation in a similar way in liposomes.

Therapeutic application Ninety (90) day release hydrogen lozenges were implanted subcutaneously in female SCID mice. Mice were injected subcutaneously into the mammary adipose ball with 7.5 million MCF-7 (a human breast carcinoma available from ATCC) of cells in 0.1 ml PBS. After identifying the tumors, the mouse was injected intravenously into the tail vein with 0.1 ml of cisplatin liposomes. The results are shown in Figures 2 to 4. Since the invention has been described in detail with rence to the specific embodiments thereof, it will be apparent to those skilled in the art that different changes and modifications can be made therein. without departing from the spirit and scope of the invention.

RENCES Ban M, Hettich D, Huguet N (1994) Nephrotoxicity mechanism of cis-platinum (II) diamine dichloride in mice. Toxicol Lett, 71 (2): 161-8. Bellon SF, Coleman JH and Lippard SJ (1991) DNA Unwinding Produced by Site-Specific Intrastrand Cross-Links of the Antitumor Drug cis-Diamminedichloroplatinum (II). Biochemistry 30, 8026-8035. Bongartz J-P, Aubertin A-M, Milhaud PG, and Lebleu B (1994) I mproved biological activity of antisense oligonucleotides conjugated to a fusogenic peptide. Nucleic Acids Res 22, 4681-4688. Boulikas T (1992) Evolutionary consequences of preferential damage and repair of chromatin domains. J. Mol. Evol. 35, 156-180. Boulikas, T (1996a) The nonuniform repair of active and inactive chromatin domains. Int J Oncol 8, 65-75. Boulikas T (1996b) A unified model explaining the preferential repair of active over inactive genes and of the transcribed over the nontranscribed strand: a leading role for transcription factors and matrix anchorage, Int J Oncol 8, 77-84. Boulikas T (1996c) DNA lesion-recognizing proteins and the p53 connection. Anticancer Res 16, 225-242. Boulikas T (1998a) Status of gene therapy in 1997: molecular mechanisms, disease targets, and clinical applications. Gene Ther Mol Biol 1, 1-172.

Boulikas T (1998b) Nucleocytoplasmic traffícking: implications for the nuclear mport of plasmid DNA during gene therapy. Gene Ther Mol 1, 713-740. Brown SJ, Kellett PJ and Lippard SJ (1993) Ixrl, to Yeast Protein That Binds to Platinated DNA and Confers Sensitivity to Cisplatin. Science 261, 603-605. Bruhn SL, Pil PM, Essigman JM, Housman DE, and Lippard SJ (1992) Isolation and characterization of human cDNA clones encoding a high mobility group box protein that recognizes structural distortions to DNA caused by binding of the anticancer agent cisplatin. Proc Nati Acad Sci USA 89, 2307-2311. Buchanan RL and Gralla JD (1990) Cisplatin Resistance and Mechanism in a Viral Test System: SV40 Isolates That Resist Inhibition by the Antitumor Drug Have Lost Regulatory DNA. Biochemistry 29, 3436-3442. Caraceni A, Martini C, Spatti G, Thomas A, Onofrj M (1997) Recovering optic neuritis during systemic cisplatin and carboplatin chemotherapy. Acta Neurol Scand, 96 (4): 260-1. Chao CC-K, Huang S-L and Lin-Chao S (1991) Ca2 + -mediated inhibition of a nuclear protein that recognizes UV-damaged DNA and is constitutively overexpressed in resistant human cells: DNA-binding assay. Nucleic Acids Res. 19, 6413-6418.

Chu G and Chang E (1988) Xeroderma Pigmentosum Group E Cells Lack a Nuclear Factor That Binds to Damaged DNA. Science 242, 564-567. Chu G and Chang E (1990) Cisplatin-resistant cells increased increased levéis of a factor that comprises damaged DNA. Proc Nati Acad Sci USA 87, 3324-3327. Clugston CK, McLaughIin K, Kenny MK and Brown R (1992) Binding of Human Single-Stranded DNA Binding Protein to DNA Damaged by the Anticancer Drug c / s-Diamminedichloroplatinum (II). Cancer Res 52, 6375-6379. Creuzenet C, Durand C, Haertle T (1997) Interaction of alpha s2-and beta-casein signal peptides with DMPC and DMPG lipsomes. Peptides, 18 (4): 463-72. Curtain C, Separovic F, Nielsen K, Craik D, Zhong Y, Kirkpatrick A (1999) The interactions of the N-terminal fusogenic peptide of HIV-1 gp41 with neutral phospholipids. Eur Biophys J, 28 (5): 427-36. Decout A, Labeur C, Vanloo B, Goethals M, Vandekerckhove J, Brasseur R, Rosseneu M (1999). Contribution of the hydrophobicity to the secondary structure and activity of fusogenic peptides. Mol Membr Biol, 16 (3): 237-46. Donahue BA, Augot M, Bellon SF, Treiber KD, Toney JH, Lippard SJ and Essigmann JM (1990) Characterization of a DNA Damage-Recognition Protein from Mammalian Cells That Binds Specifically to Intrastrand d (GpG) and d (ApG) DNA Adducts of the Anticancer Drug Cisplatin. Biochemistry 29, 5872-5880. Duguid JG, Li C, Shi M, Logan MJ, Alila H, Rolland A, Tomlinson E, Sparrow JT, Smith LC (1998) A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy Biophys J. 74 (6): 2802-14. Eapen S, Green M, Ismail IM (1985) Kinetic studies on the diaqua form of cis-platin and various nucleobases, J. Inorg Biochem, 24 (3): 232-7. Elipoulos AG, Kerr DJ, Herod J, Hodgkins K, Krajewski S, Reed JC, Young LS (1995a) The control of apoptosis and drug resistance in ovarian cancer: influence of p53 and Bcl-2. Oncogene, 11 (7): 1217-28. Eliopoulos AG, Kerr DJ, Maurer HR, Hilgard P, Spandidos DA (1995b) Induction of the c-myc but not the cH-ras promoter by platinum compounds. Biochem Pharmacol, 50 (1): 33-8. Farhood H, Gao X, Son K, Yang YY, Lazo JS, Huang L, Barsoum J, Bottega R, Epand RM (1994) Cationic liposomes for direct gene transfer in therapy of cancer and other diseases. Ann N and Acad Sci, 716: 23-34; discussion 34-5. Fresta M, Chillemi R, Spampinato S, Sciuto S, Puglisi G (1998) Liposomal delivery of a 30-mer antisense oligodeoxinucleotide to inhibit proopiomelanocort expression. J Pharm Sci, 87 (5): 616-25.

Ghosh JK, Shai Y (1999) Direct Evidence that the N-Terminal Heptad Repeat of Sendai Virus Fusion Protein Participates in Membrane Fusion. J Mol Biol, 292 (3): 531-546. Green M and Loewenstein PM (1988) Autonomous functional domains for chemically synthesized human immunodeficiene and tat tat protein transactivator. Cell 55, 1179-1188. Gupta D, Kothekar V (1997) 500 picosecond molecular dynamics simulation of amphiphilic polypeptide Ac (LKKL) 4 NHEt with 1, 2 di-mysristoyl-sn-glycero-3-phosphorylcholine (DMPC) molecules. Indian J Biochem Biophys, 34 (6): 501-11. Holler E. Bauer R, Bernges F (1992) Monofunctional DNA-platinum (ll) adduets block frequently DNA polymerases. Nucleic Acids Res 1992 May 11; 20 (9): 2307-12. Hughes EN, Engelsberg BN and Billings PC (1992) Purification of Nuclear Proteins That Bind to Cisplatin-damaged DNA. J. Biol. Chem. 267, 13520-13527. Judíce JK, Tom JY, Huang W, Wrin T, Vennari J, Petropoulos CJ, McDowell RS (1997) Inhibition of HIV type 1 infectivity by constrained alpha-helical peptides: implications for the viral fusion mechanism. Proc Nati Acad Sci USA, 94 (25): 13426-30. Kim JC, Lee MH, Choi SK (1998) Synthesis and antitumor evaluation of cis- (1, 2-diaminoethane) dichloroplatinum (II) complexes linked to - and 6-methileneuracil and -uridine analogues. Arch Pharm Res, 21 (4): 465-9. Kono K, Nishii H, Takagishi T (1993) Fusion activity of an amphiphilic polypeptide having acidic amino acid residues: generation of fusion activity by alpha-helix formation and charge neutralization. Biochim Biophys Acta, 1164 (1): 81 -90. Lambert G, Decout A, Vanloo B, Rouy D, Duverger N, Kalopissis A, Vandekerckhove J, Chambaz J, Brasseur R, Rosseneu M (1998) The C-terminal helix of human apolipoprotein All promotes the fusion of unilamellar liposomes and displaces apolipoprotein Al from high-density lipoproteins. Eur J Biochem, 253 (1): 328-38. Lee S, Aoki R, Oishi O, Aoyagi H, Yamasaki N (1992) Effect of amphipathic peptides with different alpha-helical contents on liposome-fusion, Biochim Biophys Acta, 1103 (1): 157-62. Lelkes Pl, Lazarovici P (1988) Pardaxin induces aggregation but not fusion of phosphatidylserine vesicles. FEBS Lett, 230 (1-2): 131-6. Lins L, Thomas-Soumarmon A, Pillot T, Vandekerchkhove J, Rosseneu M, Brasseur R (1999) Molecular determinants of the interaction between the C-terminal domain of Alzheimer's beta-amyloid peptide and apolipoprotein E alpha-helices. J Neurochem, 73 (2): 758-69. Macosko JC, Kim CH, Shin YK (1997) The membrane topology of the fusion peptide region of influenza hemagglutinin determined by spin-labeling EPR, J Mol Biol, 267 (5): 1139-48.

Macreadie IG, Lowe MG, Curtain CC, Hewish D, Azad AA (1997) Cytotoxicity resulting from addition of HIV-1 Nef N-terminal peptides to yeast and bacterial cells. Biochem Biophys Res Commun, 232 (3): 707-11. Martin F and Boulikas T (1998) The challenge of liposomes in gene therapy. Gene Ther Mol Biol 1, 173-214. Martin I, Pecheur El, Ruysschaert JM, Hoekstra D (1999) Membrane fusion induced by a short fusogenic peptide is assessed by its insertion and orientation into target bilayers. Biochemistry, 38 (29): 9337-47. Martin I, Ruysschaert JM (1997) Comparison of lipid vesicle fusion induced by the putative fusion peptide of fertilin (a protein active in sperm-egg fusion) and the NH2-terminal domain of HIV2 gp41. FEBS Lett, 405 (3): 351-5. Massari S, Colonna R (1986) Gramicidin induced aggregation and size increase of phosphatidylcholine vesicles. Chem Phys Lipids. 39 (3): 203-20. McLaughIin K, Coren G, Masters J, Brown R (1993) Binding activities of cis-platin-damage-recognition proteins in human tumor cell lines. Int J Cancer, 53 (4): 662-6. Melino S, Rufini S, Sette M, Moreno R, Grottesi A, Pací M, Petruzzelli R (1999) Zn (2+) ions selectively induces antimicrobial salivary peptide histain-5 to fuse negatively charged vesicles. Identification and characterization of a zinc-binding motif present in the functional domain.

Biochemistry, 38 (30): 9626-33.

Midoux P, Monsign and M (1999) Efficient gene transfer by histidylated polylysine / pDNA complexes. Bioconjug Chem. 10 (3): 406-11. Morikawa K, Honda M, Endoh K, Matsumoto T, Akamatsu K, Mitsui H, Koizumi M (1990) Synthesis of platinum complexes of 2-aminomethylpyrrolidine derivatives for use as carrier ligands and their antitumor activities. Chem Pharm Bull (Tokyo), 38 (4): 930-5. Morikawa K, Honda M, Endoh K, Matsumoto T, Akamatsu K, Mitsui H, Koizumi M (1990) Synthesis of platinum complexes of 2-aminomethylpyrrolidine derivatives for use as carrier ligands and their antitumor activities. Chem Pharm Bull (Tokyo), 38 (4): 930-5. Murata M, Kagiwada S, Hishida R, Ishiguro R, Ohnishi S, Takahashi S (1991) Modification of the N-terminus of membrane fusion-active peptides blocks the fusion activity. Biochem Biophys Res Commun, 179 (2): 1050-5. Mymryk JS, Zaniewski E and Archer TK (1995) Cisplatin inhibits chromatin remodeling, transcription factor binding, and transcription from the mouse mammary tumor virus promoter in vivo. Proc Nati Acad Sci USA 92, 2076-2082. Niidome T, Kimura M, Chiba T, Ohmori N, Mihara H, Aoyagi H (1997) Membrane interaction of synthetic peptides related to the putative fusogenic region of HP-30 alpha, a protein in sperm-egg fusion, J. Pept Res, 49 (6): 563-9.

Oliver T and Mead G (1993) Testicular cancer. Curr Opin Oncol 5, 559-567. Ormerod MG, O'Neill C, Robertson D, Kelland LR, Harrap KR (1996) cis-diamminedichioroplatinum (ll) -induced cell death through apoptosis in sensitive and resistant human ovarian carcinoma cell lines. Cancer Chemother Pharmacol, 37 (5): 463-71. Pak CC, Erukulla RK, Ahi PL, Janoff AS, Meers P (1999) Elastase activated liposomal delivery to nucleated cells. Biochim Biophys Acta, 1419 (2): 111-26. Papadopoulou MV, J¡M, Bloomer WD (1998) NLCQ-1, a novel hypoxic cytotoxin: potentiation of melphalan, cisDDP and cyclophosphamide in vivo. Int J Radiat Oncol Biol Phys, 42 (4): 775-9. Párente RA, Nir S, Szoka FC Jr (1988) pH-dependent fusion of phophatidylcholine small vesicles. Induction by a synthetic amphipathic peptide. J. Biol Chem, 263 (10): 4724-30. CD matches, Vohra P, Steward MW (1996) Priming of measles virus-specific CTL responses after immunization with a CTL epitope linked to a fusogenic peptide. Vírology, 215 (1): 107-10. Pecheur El, Hoekstra D, Sainte-Marie J, Maurin L, Bienvenue A, Philippot JR (1997) Membrane anchorage brings about fusogenic properties in a short synthetic peptide. Biochemistry, 36 (13): 3773-81. Peelman F, Vanloo B, Perez-Mendez O, Decout A, Verschelde JL, Labeur C, Vinaimont N, Verhee A, Duverger N, Brasseur R, Vandekerckhove J, Tavernier J, Rosseneu M (1999) Characterization of functional residues in the nterfacial recognition domain of lecithin cholesterol acytransferase (LCAT). Protein Eng, 12 (1): 71-8. Pereira FB, Goni FM, Muga A, Nieva JL (1997) Permeailization and fusion of uncharged lipid vesicles induced by the HIV-1 fusion peptide adopting an extended conformation: dose and sequence effects. Biophys, J, 73 (4): 1977-86. Pil PM and Lippard SJ (1992) Specific Binding of Chromosomal Protein HMG1 to DNA Damaged by the Anticancer Drug Cisplatin. Science 256, 234-237. Pillot T, Drouet B, Queille S, Labeur C, Vandekerchkhove J, Rosseneu M, Pincon-Raymond M, Chambaz J (1999) The nonfibrillar amyloid beta-peptide induces apoptotic neuronal cell death: involvement of its C-terminal fusogenic domain. J. Neurochem, 73 (4): 1626-34. Pillot T, Goethals M, Vanloo B, Lins L, Brasseur R, Vandekerckhove J. Rosseneu M (1997a) Specific modulation of the fusogenic properties of the Alzheimer beta-amyloid peptide by apolipoprotein E isoforms. Eur J Biochem, 243 (3): 650-9. Pillot T, Lins L, Goethals M, Vanloo B, Baert J, Vandekerckhove J, Rosseneu M, Brasseur R (1997b) The 118-135 peptide of the human prion protein forms amyloid fibrils and induces liposome fusion. J Mol Biol, 274 (3): 381-93.

Prasad KN, Hernández C, Edwards-Prasad J, Nelson J, Borus T, Robinson WA (1994) Modification of the effect of tamoxifen, cis-platin, DTIC, and interferon-alpha 2b on human melanoma cells in culture by a mixture of vitamins Nutr Cancer, 22 (3): 233-45. Rodríguez-Crespo I, Gomez-Gutierrez J, Nieto M, Peterson DL, Gavilanes F (1994) Prediction of a putative fusion peptide in the S protein of hepatitis B virus. J. Gen Virol, 75 (Pt 3): 637-9. Rodríguez-Crespo I, Nuñez E, Yelamos B, Gomez-Gutierrez J, Albar JP, Peterson DL, Gavilanes F (1999) Fusogenic activity of hepadnavirus peptides corresponding to downstream of the putative cleavage site. Virology, 261 (1): 133-42. Roth, J.A. et al (1996), "Retrovius-mediated wild-type p53 gene transfer to cancer tumors with cancer" Nature Med. 2: 985-991. Schroth-Ten B, Ponimaskin E, Reverey H, Schmidt MF, Herrmann A (1998) Fusion activity of transmembrane and cytoplasmic domain chimeras of the influenza virus glycoprotein hemagglutinin. J Virol, 72 (1): 133-41. Stathopoulos GP, Rigatos S, Malamos NA (1999) Paclitaxel combined with cis-platin as second-line treatment in patients with advanced non-small cell lung cancers refractory to cis-platin. Oncol Rep, 6 (4): 797-800. Suenaga M, Lee S, Park NG, Aoyagi H, Kato T, Umeda A, Amako K (1989) Basic amphipathic helical peptides induces destabilization and fusion of acidic and neutral liposomes. Biochim Biophys Acta, 981 (1): 143-50.

Toney JH, Donahue BA, Kellett PJ, Bruhn SL, Essigmann JM and Lippard SJ (1989) Isolation of cDNAs encoding human protein that binds selectively to DNA modified by the anticancer drug c / 's-diammine-dichloroplatinum (II). Proc Nati Acad Sci USA 86, 8328-8332. Tournois H, Fabrie CH, Burger KN, Mandersloot J, Hilgers, P, van Dalen H, de Gier J, de Kruijff B (1990) Gramicidin A induced fusion of large unilamellar dioleoylphophatidylcholine vesicles and its relation to the induction of type II nonbilayer structure . Biochemistry, 29 (36): 8297-307. Ulrich AS, Tichelaar W, Forster G, Zschorning O, Weinkauf S, Meyer HW (1999) Ultrastructural characterization of peptide-induced membrane fusion and peptide self-assembly in the lipid bilayer. Biophys J, 77 (2): 829-41. Vergote I, Himmelmann A, Frankendal B, Scheistroen M, Vlachos K, Trope C (1992) Hexamethylmelamine as second-line therapy in platin-resistant ovarian cancer. Gynecol Oncol, 47 (3): 282-286. Vilpo JA, Vilpo LM Szymkowshi DE, O'Donovan A and Wood RD (1995) An XPG DNA repair defect causing mutagen hypersensitivity in mouse leukemia L1210 cells. Mol Cell Biol 15, 290-297. Voneche V, Callebaut I, Kettmann R, Brasseur R, Burny A, Portetelle D (1992) The 19-27 amino acid segment of gp 51 adopts an amphiphilic structure and plays a key role in the fusion events induced by bovine leukemia virus. J Biol Chem, 267 (21): 15193-7.

Zhang YQ, Jiang XT, Sun QR, Zhang GQ, Wang Y (1995) Studies on CDDP-albumin microspheres for hepatic arterial chemoembolization. Yao Hsueh Hsueh Pao, 30 (7): 543-8 [Article in Chínese]

Claims (28)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for producing cisplatin micelles, characterized in that it comprises: a) combining cisplatin with a lipid derivative of phosphatidyl glycerol in a range of 1: 1 to 1: 2 to form a mixture of cisplatin; and b) combining the mixture of step a) with an effective amount of at least 30% of an ethanol solution to form the cisplatin micelles.

2. A method for producing cisplatin micelles characterized in that it comprises: a) combining cisplatin with an effective amount of at least one solution of 30% ethanol to form a solution of cisplatin / ethanol; and b) combining the solution with a lipid derivative of phosphatidyl glycerol derivatized in a range of 1: 1 to 1: 2 to form the cisplatin micelles.

3. The method according to claim 1 or 2, further characterized in that the lipid derivatives of phosphatidyl glycerol are selected from the group consisting of dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl glycerol (DMPG), dicaproyl phosphatidyl glycerol (DCPG) ), distearoyl phosphatidyl glycerol (DSPG) and dioleyl phosphatidyl glycerol (DOPG).

4. The method according to claim 1 or 2, further characterized in that the molar ratio is 1: 1.

5. The method according to claim 1 or 2, further characterized by additionally comprising the combination of an effective amount of a free fusogenic peptide, a lipido-fusogenic peptide conjugate or a fusogenic peptide-PEG-HSPC conjugate with the step a) wherein the fusogenic peptide is derived with a stretch of 1 to 6 amino acids with negative charge at the N or C terminal and thus, be able to electrostatically bind to aquaplatine.

6. The method according to claim 5, further characterized in that the free fusogenic peptide or lipid-fusogenic peptide conjugate comprises DOPE or DOPE / cationic lipid.

7 '.- A cisplatin micelle characterized in that it is obtained by the method as claimed in claims 1 or 2.

8. A cisplatin micelle characterized in that it is obtained by the method as claimed in claim 5.

9.- A method for encapsulating cisplatin micelles, characterized in that it comprises mixing an effective amount of the vesicle-forming lipid with the cisplatin micelles as claimed in claim 1 or 2.

10. Encapsulated cisplatin which is obtainable by the method as claimed in claim 9.

11. The method according to claim 9, further characterized in that the lipid is selected from previously prepared neutral liposomes, composed of 10 to 60% cholesterol, Hydrogenated soy phosphatidylcholine (HSPC) from 40 to 90% and polyethylene glycol (PEG) -HSPC from 1 to 7% or lipids in solution, powdered lipids and PEG-DSPE.

12. The method according to claim 9, further characterized in that the lipid comprises 10 to 60% cholesterol.

13. A method for obtaining a cisplatin / lipid complex capable of evading macrophages and cells of the immune system when administered to a subject, characterized in that it comprises mixing an effective amount of cisplatin micelles according to claim 9 with an effective amount of PEG-DSPE, PEG-DSPC or hyaluronic acid-DSPE.

14. The method according to claim 1 or 2, further characterized in that it further comprises removing the ethanol from the cisplatin micelles.

15. The method according to claim 14, further characterized in that the removal of the ethanol is done by means of dialysis of the micelles through the permeable membranes to remove the ethanol.

16. Encapsulated cisplatin obtainable by the method as claimed in claim 11.

17. Encapsulated cisplatin obtainable by the method as claimed in claim 13.

18. A method for supplying cisplatin to a cell characterized in that it comprises contacting the cell with the encapsulated cisplatin as claimed in claim 15.

19. - A method for supplying cisplatin to a cell characterized in that it comprises contacting the cell with the encapsulated cisplatin as claimed in claim 17.

20. The use of the encapsulated cisplatin as claimed in claim 16 to prepare a pharmaceutical composition for inhibiting the growth of a tumor in a subject.

21. The use of encapsulated cisplatin as claimed in claim 17 for preparing a pharmaceutical composition for inhibiting the growth of a tumor in a subject.

22. The use of encapsulated cisplatin as claimed in claim 16 or 17 for preparing a pharmaceutical composition for identifying solid tumors and metastases in a subject, wherein said pharmaceutical composition is for intravenous administration.

23. The use of the cisplatin micelle obtainable by the method as claimed in claim 7 for preparing a pharmaceutical composition for penetrating the cell membrane of a tumor in a subject.

24. The use of the encapsulated cisplatin as claimed in claim 10 in combination with a gene selected from the group consisting of p53, pax5 and HSV-tk genes to prepare a pharmaceutical composition for inhibiting tumor growth in a subject.

25. The use as claimed in claim 24, wherein said pharmaceutical composition is provided together with encapsulated ganciclovir. 26 -.

26 - The use as claimed in claim 24, wherein the genes to be combined with cisplatin are any of, or combinations of IL-2, IL-4, IL-7, IL-12, GM- CSF, IFN-gamma, TNF-alpha, RB, BRCA1, E1A, cystosine deaminase encapsulated in combination with 5-fluorocytosine bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I, VEGF , Encapsulated TGF-beta, and the like.

27. A composition characterized in that it comprises the encapsulated cisplatin according to claim 10 and encapsulated oligonucleotides, ribozomes, triplex or PNA.

28. A composition characterized in that it comprises the encapsulated cisplatin according to claim 10 and a drug selected from the group consisting of doxorubicin, fluorodeoxyuridine, bleomycin, adriamycin, vinblastine, prednisone, vincristine, and taxol.