MXPA99008343A - Cationic lipid compositions targeting angiogenic endothelial cells - Google Patents

Abstract

Angiogenic endothelial cells are selectively targeted with lipid/DNA complexes or cationic liposomes containing a substance which affects the targeted cells by inhibiting or promoting their growth. A site of angiogenesis can be precisely located by administering cationic liposomes containing a detectable label. The complexes may comprise nucleotide constructs which are comprised of promoters which are selectively and exclusively activated in the environment of an angiogenic endothelial cell.

Description

CATIONIC LIPIDIC COMPOSITIONS FOCUSING ANGIOGENIC ENDOTHELIAL CELLS BACKGROUND OF THE INVENTION The present invention can be applied to the treatment and diagnosis of a variety of different diseases and abnormalities. Although the present invention is not limited to such, it can be used in the treatment of cancer, wound healing and a variety of chronic inflammatory diseases. In general, each is currently treated directly by physical means, such as surgical removal of cancerous tissue, suturing of wounds and surgical removal of inflamed joints. Additionally, each can be treated by chemical means. Chemotherapy is applied to cancer, growth hormones are applied to wound healing and anti-inflammatory medications are applied to treat chronic inflammatory conditions. These, and related treatments, are directed, in general, to treat cancerous, injured or inflamed tissue directly. In order to provide an understanding of how the present invention departs from conventional treatment modalities, a brief and general description of current treatment technologies in these areas is provided.

TREATMENT OF CANCER The term "cancer" covers a spectrum of diseases that vary in treatment, prognosis and curability. The approach to diagnosis and treatment depends on the site of origin of the tumor, the degree of relaxation, sites of involvement, the physiological state of the patient, and prognosis. Once diagnosed, the tumor is usually "staggered," a process which involves using the techniques of surgery, physical examination, histopathology, imaging, and laboratory evaluation to define the degree of disease and to divide the patient population from cancer in groups in order of decreasing probability of cure. Such systems are used both to plan the treatment and to determine the prognosis for the patient (Stockdale, F., 1996, "Principies of Cancer Patient Management", In: Scientific American Medicine, vol. 3, Dale, DC, and Federman, DD (eds.), Scientific American Press, New York). The type or stage of the cancer can determine which of the three general types of treatment will be used: surgery, radiation therapy, and chemotherapy. A combination, aggressive mode treatment plan can also be chosen. To this end, surgery can be used to remove the primary tumor, and the remaining cells are treated with radiation therapy or chemotherapy (Rosenberg, SA, 1985, "Combined-modality therapy of cancer: what is it and when does it work? "(Combined cancer modality therapy: what is it and when does it work ?, New Engl. J. Med. 312: 1512-14.) Surgery plays a central role in the diagnosis and treatment of cancer. requires a surgical approach for biopsy, and surgery can be the definitive treatment for most cancer patients.The surgery is also used to reduce tumor mass, to resect metastases, to resolve emergencies, to relieve and rehabilitate. The primary surgical technique for cancer treatment has involved the development of an operative field where tumors are resected under direct visualization, current techniques allow some resections to be performed under Endoscopic studies A primary concern in the treatment of cancer is the consideration of operative risk (Stockdale, F., supra). Radiation therapy plays an important role both in the primary and palliative treatment of cancer. Both teletherapy (megavoltage radiation therapy) and brachytherapy (interstitial radiation and intracavity) are in common use. Electromagnetic radiation in the form of x-rays is very commonly used in teletherapy to treat common malignancies, while gamma rays are also used, a form of electromagnetic radiation similar to x-rays but emitted by radioactive radioactive isotopes, cobalt and other elements. Radiation therapy transfers energy to tissues as discrete packets of energy, called photons, which damage both malignant and normal tissues by producing ionization within cells. The target for ions is very commonly DNA; radiation therapy exploits the fact that radiation damage is not uniform between malignant and non-malignant tissues - rapidly dividing cells are more sensitive to DNA damage than quiescent cells (Pass, H. I., 1993, "Photodynamic therapy in oncology: mechanisms and clinical use" (Photodynamic therapy in oncology: mechanisms and clinical use), J. Nati, Cancer Institute 85: 443-56). Radiation therapy is associated with unique benefits as well as important toxicities. Radiation is preferred in certain anatomical areas (eg, the mediastinum), where radiation may be the only feasible local means of treatment, and radiation may also be used when the patient finds the surgery unacceptable, or when the medical condition of the patient forbids a surgical procedure. Radiation treatment involves tissue damage, which can lead to early and late radiation effects. Early effects (acute toxicity of radiation therapy) include erythema of the skin, desquamation, esophagitis, nausea, alopecia, and milosuppression, while late effects include tissue necrosis and fibrosis, and usually determine the limiting toxicity of the therapy. radiation (Stockdale, F., supra). Almost all currently used chemotherapeutic agents interfere with DNA synthesis, with the condition of precursors for DNA and RNA synthesis, or with mitosis, and in this way focus on proliferating cells (Stockdale, F., "Cancer growth and chemotherapy ", (Growth of cancer and chemotherapy), supra). Research and human clinical trials have shown that combinations of drugs produce greater proportions of objective response and greater survival than simple agents (Frei, E. III, 1972, "Combination cancer therapy: presidential address" (Combination therapy for cancer: presidential address), Cancer Res. 32: 2593-2607). Drug combination therapy uses the different mechanisms of action and cytotoxic potentials of multiple medications, including alkylating agents, antimetabolites, and antibiotics (Devita, VT, et al., 1975, "Combination versus single agent chemotherapy: a review of the cancer treatment basis for selection "(Combination chemotherapy versus simple agent: a review of the basis for cancer treatment treatment selection), Cancer 35: 98-1 10). The physiological condition of the patient, the characteristics of tumor growth, the heterogeneity of the tumor cell population, and the multidrug resistance stage of the tumor influence the efficacy of the chemotherapy. In general, chemotherapy is not focused (although these techniques are being developed, for example, Pastan, I. et al., 1986, "Immunotoxins", Cell 47: 641-648), and side effects may result. , such as bone marrow depression, gastroenteritis, nausea, alopecia, liver or lung damage, or sterility.

HERI DAS CU RATION Wound healing is a complex and slow process of tissue repair and remodeling involving many different types of cells, which requires finely tuned control of several cascades of biochemical reactions to balance regenerative processes. Wound healing is generally divided into three phases: inflammation, proliferation and maturation (Waldorf, H., and Fewkes, J., 1995, "Wound Healing", Adv. Dermatol., 10: 77-96 ). The process involves the migration of different types of cells in the wound region, stimulation of growth of epithelial cells and fibroblasts, formation of new blood vessels, and the generation of extracellular matrix. The correct functioning of these processes depends on the biological activation of several cytokines (Bennett, NT, and Schultz, GS, 1993, "Growth factors and wound healing: biochemical properties of growth factors and their receptors" (Growth factors and wound healing : biochemical properties of growth factors and their receptors), Am. J. Surg. 1 65: 728-37). Nutrition, immune system, oxygen, blood volume, infection, immunosuppression, and a decrease in red blood cells are all factors influencing wound healing (Witney, JD, 1989, "Physiological effects of tissue oxygenation on wound healing"). Physiological effects of tissue oxygenation in wound healing, "Heart Lung 18: 466-474.) The quality as well as the rate of wound healing are generally dependent on the type and degree of the original lesion. process to treat wounds, each of which is aimed at curing damaged tissue Wound closure is most commonly accompanied by suture, although tapes, adhesive fabrics or electrocautery can also be used (Wheeless, CR, 1996, Wheeless) Textbook of Orthaedics) (Garrett, WE et al., 1984, J. Hand, Surg. 9 (5): 683-92). Appointments for skin and various sutures each exhibit certain benefits and disadvantages in the brain. primary wound repair. Skin tapes cause less inflammatory reaction, but fail to close the spaces of subepithelial wounds, while the inflammatory reaction and subsequent scarring caused by several sutures depends on the size of the suture needle, the diameter of the suture material, and if it is a monofilament or woven suture (Simpson, WR, 1977, "Physiological principles of therapy in head and neck cutaneous wounds", Laryngoscope 87: 792-816). In a wound, the size of an inoculum of microorganisms, the virulence of the organisms, and host antimicrobial defense mechanisms determine whether an infection will develop. In this way, antibiotics can also be of therapeutic value in the treatment of wounds (Edlich, RF, et al., 1986, "Antimicrobial treatment of minor soft tissue lacerations: a critical review", (Antimicrobial treatment of soft tissue lacerations. minor: a critical review), Emergency Medical Clinics of North America 4 (3): 561-80). The pharmacological action of each antibiotic must be understood in order to choose the appropriate antibiotic, its route of administration, and to avoid side effects (Simpson, W. R., supra). Recent results suggest that antibiotic therapy allows cell proliferation and differentiation to proceed more rapidly and thus may be useful in increasing wound repair (Barrow, RE et al., 1994, "Efficacy of cefazolin in promoting ovine tracheal epithelial repair, "(Efficacy of cefazolin to promote epithelial repair of ovine trachea), Respiration 61: 231-5; Maeder, K., et al., 1 993," Methicillin-resistant Staphylococcus aureus (MRSA) colonization in patients with spinal cord injury "(Colonization of methicillin-resistant Staphylococcus aureus (MRSA) in patients with spinal cord injury), Paraplegia 31: 639-44). Proteolytic enzymes have also been used as adjuncts to antibiotic treatment of contaminated wounds (Rodeheaver, GT, et al., 1978, "Mechanisms by which proteolytic enzymes prolong the golden period of antibiotic action" (Mechanisms by which proteolytic enzymes prolong the golden period of antibiotic action), Am. J. Surg. 1 36 (3): 379-82). Topical administration of several cytokines, including Bfgf, EGF, PDGF and TGF-beta, either alone or in combination, can greatly accelerate wound healing (Moulin, V., 1995, "Growth factors in skin wound healing"). of growth in wound healing in skin), Eur. J. Cell. Biol. 68: 1-7). The growth factors attract cells to the wound, stimulate their proliferation, and have a profound influence on the deposition of extracellular matrix. Since the development of the ability to mass produce these cytokines by recombinant techniques, many studies have shown that growth factors can increase all aspects of tissue repair in normal and uneven healing models (eg, Schultz, GS et al. ., 1987"Epithelial wound healing enhanced by transforming growth factor-alpha and vaccinia growth factor" (Enhanced epithelial wound healing by transforming growth factor alpha and vaccinia growth factor), Science 235: 350-2; Deuel, T. F., et al. , 1991, "Growth factor and wound healing: platelet derived growth factor as a model cytokine" (Growth factor and wound healing: platelet-derived growth factor as a model cytokine), Annu. Rev. Med. 42: 567-84). Although preliminary clinical trials have shown that growth factor treatment has occasionally led to statistically significant improvements in tissue repair, it is not clear that these results are clinically meaningful, and it has been suggested that new clinical trials should focus on of target growth for specific types of uneven healing (Greenhalgh, DG, 1 996, "The role of growth factors in wound healing", J. Trauma 41: 1 59-67) .

CHRONIC INFLAMMATION Cellular, humoral, and natural immune mechanisms have all been implicated in the pathogenesis of chronic inflammatory diseases (Seymour, GJ et al., 1979, "The Immunopathogenesis of Progressive Chronic Periodontal Disease," The Immunopathogenesis of Periodontal Disease. progressive chronic inflammatory), 1979, J. Oral Pathol., 8: 249-65). Autoimmune diseases result from abnormalities in lymphocyte function. Abnormalities in T cell function may be responsible for the disease through cell-mediated immunity, and the activity of helper T cells in the production of antibodies may contribute to the formation of autoantibodies. The central role of helper T cells in autoimmune disease is supported by the association of many of these diseases with certain HLA molecules. Failure of one or more steps in maintaining tolerance could result in autoimmunity (Robinson, DR, 1996, "Immunologic Tolerance and Autoimmunity", in: Scientific American Medicine, Vol. 2, Section VI, Scientific American Press, New York, p.1 -1 1).

Several types of treatment are used in autoimmune disease, all of which are aimed at decreasing the immune response in the affected tissue. For example, treatment for rheumatoid arthritis, an autoimmune disease, may use anti-inflammatory agents such as nonsteroidal anti-inflammatory agents (NSAIDs) or glucocorticosteroids, remission-inducing agents, such as, gold salts, and / or immunosuppressive drugs, such as, cyclophosphamide. Orthopedic surgery can also be used to replace damaged joints during the inflammatory process (see Gilliland, BC, and Mannik, M., 1 983, "Rheumatoid Arthritis" (Rheumatoid Arthritis), in: Harrison's Principles of Internal Medicine, McGraw Hill, New York , P. 1 977-1 984). Recent work has suggested the possibilities of new treatments, also directed at the affected tissue, such as the use of TNF-alpha in the treatment of rheumatoid arthritis (Brennan, FM, et al., 1 995, "Cytokine expression in chronic inflammatory disease" (Expression of cytokines in chronic inflammatory disease), Br. Med Bull. 51: 368-384). Allergy refers to a condition in which the immune response to environmental antigens causes tissue inflammation and organ dysfunction. As in autoimmune diseases, the data suggest an interaction of several components of the immune system in allergic diseases. The expression diversity of allergic diseases arises from different immunological effector mechanisms, which cause specific patterns of tissue damage (Beer, DJ et al., 1996, "Allergy", in: Scientific American Medicine, Vol. 2, Section VII, Scientific American Press, New York, P. 1-29). The clinical characteristics of each allergic disease reflect the inflammatory response immunologically mediated in the affected organs or tissues (for example, asthma reflects an inflammatory response in the lungs). Several treatment strategies are used to treat immuno-mediated allergic diseases, all of which are aimed at decreasing the immune response in the inflamed tissue. For example, in the treatment of asthma, therapy may involve environmental control, pharmacotherapy, and allergen immunotherapy (Beer, DJ, et al., 1996, "Allergy" (Allergy), in: Scientific American Medicine, Vol. 2, Section VII, Scientific American Press, New York, P. 1-29). In the treatment of asthma, the elimination of the causative agent is the most successful means of preventing inflammation. However, it is often not possible, and thus, several classes of medications have been used. These include methylxanthines (for bronchodilation), adrenergic stimulants (stimulation of ß-adrenergic receptors), bronchodilators), glucocorticoids (decrease inflammation in the lung), chromones (sub-regulate mast cells, decrease inflammation in the lung), and anticholinergics (bronchodilators) (McFadden, ER, Jr., and Austen, KF, "Lung disease caused by immunologic and evironmental injury "(Lung disease caused by immunological and environmental injury), in: Harrison's Principles of Internal Medicine, McGraw Hill, New York, pp. 1512-1 51 9). Desensitization or immunotherapy with extracts of suspected allergens has also been suggested in order to reduce inflammation in asthma (McFadden and Austen, op.cit.; Jacquemin, M.G. , and Saint-Remy, JM 1 995, "Specific down-regulation of anti-allergen IgE and IgG antibodies in humans associated with injections of allergen-specific antibody complexes" (Specific sub-regulation of anti-allergen IgG and IgE antibodies in humans associated with injections of specific-allergen antibody complexes), Ther. Immunol. 2:41 -52).

ACTUAL TREATMENTS-I NMUNOLOGY The treatment regimens described above have had varying degrees of success. Due to the success rate, the research is far from perfect in many cases, which is why they continue to develop better treatments. A promising area of research is concerned with affecting the immune system. Due to the use of genetic engineering and / or chemical stimulation, it is possible to modify and / or stimulate the immune responses, so that the body's own immune system treats the disease, for example, antibodies that destroy cancer cells. This type of treatment moves away from those described above because it uses a biological process to fight against the disease. However, the treatment is still a direct treatment meaning that the antibodies created directly attack the cancer cells. The present invention can be used for treatments, which involve a radical departure from normal treatments, since the present invention does not involve directly affecting cancer cells, damaged or inflamed. Others have recognized that, at least theoretically, it is possible to treat cancer or an inflammation associated with angiogenesis by inhibiting angiogenesis. A typical example of current thinking related to this is discussed within PCT Publication WO 95/25543, published September 28, 1995. This published application describes inhibiting angiogenesis by administering an antibody, which binds to a antigen that is believed to be present on the surface of angiogenic endothelial cells. Specifically, the application describes administering an antibody which binds to avß3, which is a membrane receptor that is believed to mediate cell-cell and cell-extracellular matrix interactions generally referred to as cases of cell adhesion. By blocking this receptor, the treatment hopes to inhibit angiogenesis and thereby treat cancer and inflammation.

BRIEF DESCRIPTION OF THE INVENTION A method for selectively delivering agents to angiogenic endothelial cells is described. The method involves injecting, preferably into the circulatory system and more preferably intra-arterially, cationic liposomes (or polynucleotide / lipid complexes), which comprise cationic lipids and a compound that promotes or inhibits angiogenesis and / or includes an detectable brand. After administration, the cationic liposomes are selectively associated with angiogenic endothelial cells meaning that they associate with angiogenic endothelial cells at a rate of five times or greater (preferably ten times or more) instead of associating with corresponding quiescent endothelial cells who do not experience angiogenesis. When liposomes (or polynucleotide / lipid complexes) are associated with angiogenic endothelial cells, they are captured by the endothelial cell and have their desired effect. The substance can destroy the endothelial cell, promote further angiogenesis, promote coagulation and / or label the endothelial cell, so that it can be detected by an appropriate means. The substance which affects the angiogenic endothelial cell may be a nucleotide sequence, such as DNA, which encodes a protein, which when expressed, promotes or inhibits angiogenesis. The nucleotide sequence is preferably contained within a vector operably linked to a promoter, said promoter is preferably active only in angiogenic endothelial cells or can be activated in those cells by the administration of a compound thereby making it possible to turn on or off the gene by activation of the promoter. Additional details and color photographs describing the present invention are provided in a document by the inventors -Thurston et al. , "Cationic Liposomes Target Angiogenic Endothelial Cells in Tumors and I nflammation in Mice" (Cationic liposomes focused on angiogenic endothelial cells in tumors and inflammation in mice), J. Clin. Invest. , April 1, 1998. An inhibitor / lipid complex is described, which is comprised of cationic lipids and an angiogenesis inhibitor, wherein the complex is characterized as having, in the blood, greater affinity for angiogenic endothelial cells as compare with corresponding normal endothelial cells. The complex preferably includes a detectable label. The label may be present on either side of the complex and linked to the cationic lipid, the inhibitor, or both or neither. The invention also includes a nucleotide / cationic lipid complex comprised of cationic lipids and a nucleotide sequence. The sequence encodes a protein which affects angiogenesis. The complex is characterized by having, in the blood, higher affinity for angiogenic endothelial cells as compared to corresponding normal endothelial cells. The sequence is preferably a DNA sequence, which is operably linked to a promoter, said promoter is selectively activated within an angiogenic endothelial cell. Suitable promoters include FLT-1 gene promoters and FLK-1 gene promoters, as well as the von Willibrand Factor gene promoters. The nucleotide sequence can be an antisense sequence, which selectively interrupts the expression of genetic material within angiogenic endothelial cells preferably as compared to normal endothelial cells. The invention also includes a method for diagnosing an angiogenesis site. The method comprises administering complexes comprised of cationic lipids and a detectable label, wherein the complexes have, in the blood, greater affinity for angiogenic endothelial cells as compared to corresponding normal endothelial cells. The administered complexes are allowed to selectively associate with angiogenic endothelial cells. Subsequently, the detectable mark is detected, thereby determining an angiogenesis site based on the accumulation of the mark on the site. The brand can be a fluorescent label, a histochemical label, an immunohistochemical label, a radioactive label or any other suitable label. The method may include isolating tissue at the mark accumulation site and subsequently analyzing the isolated tissue. The method for diagnosing an angiogenesis site comprises a method by which a malignant tumor or malignant tissue is precisely identified and removed. This method comprises administering complexes comprised of cationic lipids and a label, which is detectable by the human eye. The mark can emit light, which is detectable directly with the human eye after the light of a particular wavelength is displayed on the mark. The complexes with detectable label are linked to the angiogenic endothelial cells. The patient, which is any mammal but is generally a human, is then operated on. When the patient is surgically opened, the tumor is exposed to light that causes the marks to fluoresce. All fluorescent tissue is removed. Thus, the method makes it possible to accurately locate and remove any tissue containing angiogenic endothelial cells. The invention includes a composition for selectively affecting angiogenic endothelial cells, comprising cationic lipids and a substance that affects angiogenesis, wherein the composition has, in the blood, higher affinity for angiogenic endothelial cells as compared to corresponding normal endothelial cells, wherein the composition is selectively associated with angiogenic endothelial cells of an angiogenic blood vessel for a time and in such a manner that the composition enters the angiogenic endothelial cells. This composition is formulated, preferably, for administration by injection into the circulatory system of a mammal. The composition has, preferably, in the blood, five times or greater and more preferably ten times or greater affinity for angiogenic endothelial cells as compared to corresponding normal endothelial cells. The composition is comprised, preferably of 5 mol% or more cationic lipids and the substance, which affects angiogenesis, is preferably an angiogenesis inhibitor but can be an angiogenesis promoter when the composition is used to heal wounds. An objective of the invention is to provide a method for selectively affecting angiogenic endothelial cells, thereby inhibiting or promoting angiogenesis. Another object of the invention is to provide a method for diagnosing an angiogenesis site by administering cationic liposomes containing a detectable label, said liposomes are designed for the purpose of selectively associating with angiogenic endothelial cells and not associating with corresponding endothelial cells that do not undergo angiogenesis. Another object of the invention is to provide cationic liposomes, said liposomes are comprised of cationic lipids and compounds, which are specifically intended and designed to either inhibit or promote angiogenesis, said compounds can be water soluble or easily dispersible in water or compatible with lipid or incorporated in the lipid layers. Another object of the invention is to provide a method for selectively affecting angiogenic endothelial cells in a manner that results in local intravascular blood coagulation, which completely blocks or blocks the flow of blood in a blood vessel. Another objective is to provide a method for analyzing angiogenic endothelial cells by labeling cells with a detectable label and thereby making it possible to separate the angiogenic endothelial cells away from surrounding cells for subsequent culture and / or analysis. Yet another object of the invention is to provide a method for destroying an unwanted tumor by delivering a toxic compound to angiogenic endothelial cells of the tumor, said compound destroys the angiogenic endothelial cells, and subsequently, destroys the tumor cells. Another object of the invention is to provide a method for selectively affecting angiogenic endothelial cells by delivering a complex of cationic lipids / DNA to angiogenic endothelial cells, wherein the DNA is linked to a promoter which is selectively activated within an environment, which it is preferably associated only with angiogenic endothelial cells, that is, the promoter is activated in quiescent endothelial cells. A feature of the invention is that the cationic liposomes of the invention selectively associate with angiogenic endothelial cells with a much greater preference (five times or greater, and preferably ten times or more) that are associated with corresponding endothelial cells not involved in angiogenesis. . An advantage of the invention is that the cationic liposomes of the invention can be used to accurately deliver small amounts of compounds toxic to endothelial cells, said cells being affected in a manner (eg, dead) so that the blood vessel is destroyed or rendered inoperative such as by a blood clot and the nutrient supplied to the surrounding tissues (such as tumor cells) is cut thereby destroying the tissue (eg, destroying a solid tumor). Another advantage of the invention is that the cationic liposomes of the invention can be used to inhibit angiogenesis associated with malignant or benign tumors associated with ongoing angiogenesis. Yet another advantage of the invention is that cationic liposomes can be used to provide delivery of targeted site compounds, which promote angiogenesis and thereby enhance wound healing. An important feature of the invention is that various classes of diseases and / or abnormalities are treated without directly treating the tissue involved in the abnormality, for example, by inhibiting angiogenesis, the blood supply to a tumor is cut off and the tumor dies without directly treating the tumor cells in any way. These and other objects, advantages and features of the present invention will become apparent to those skilled in the art upon reading the description herein provided in connection with the accompanying figures.IPTION OF THE FIGURES Figures 1-10 are presented here both in a color version and in a black and white version. This is done because the invention is best understood via color photos, such photos may not be acceptable under the practice of current PCT applications. The color photographs for Figures 1-10 are in US Application Serial No. 08 / 820,337, filed March 12, 1997, which is incorporated herein by reference. In addition, color photographs that clearly show the higher affinity of the complexes labeled of the invention by angiogenic endothelial cells relative to corresponding normal endothelial cells, are shown in Thurston, et al. , "Cationic Liposomes Target Angiogenic Endothelial Ceells Tumors and Inflammation in Mice" (Cationic Liposomes Focused on Angiogenic Endothelial Cells in Tumors and Inflammation in Mice), J. Clin. Invest. , April 1, 1 998 (incorporated by reference). Figure 1 is a fluorescence micrograph showing the uptake of DDAB: cholesterol-DNA complexes labeled with fluorescent red CM-Dil in angiogenic blood vessels of a follicle in a normal mouse ovary (Scale bar: 60 μm); Figure 2 is a fluorescence micrograph showing the uptake of DDAB: cholesterol-DNA complexes labeled with red fluorescent CM-Dil in angiogenic blood vessels in a section of a pancreatic tumor in a mouse RIP1 -TAG_5_- blood vessels stained with green with a fluorescent lectin (scale bar: 40 μm); Figure 3 is a low-magnification fluorescence micrograph showing little or no uptake of DOTAP: cholesterol-DNA complexes labeled with Texas Red (yellow-orange) in blood vessels of a normal mouse pancreatic islet (scale bar: 150 μm); Figure 4 is a low magnification fluorescence micrograph showing the uptake of DOTAP: cholesterol-DNA complexes labeled with Texas Red (yellow-orange) in blood vessels of a pancreatic tumor in an RI P 1 -TAG_2_mouse (Bar scale: 1 50 μm); Figure 5 is a confocal micrograph showing little or no uptake of DOTAP: cholesterol liposomes labeled with Texas Red (red-orange) in a normal pancreatic islet, vessels stained (green) with fluorescent lectin (bar scale: 50) μm); Figure 6 shows a confocal micrograph of uptake of DOTAP: cholesterol liposomes labeled with Texas Red (red-orange) in a pancreatic tumor in an RI P 1 -TAG_2_mouse. The vessels were stained by perfusion of fluorescent lectin Lycopersicon esculentum (green) after the liposomes were injected intravenously (scale bar: 50 μm); Figure 7 shows a confocal micrograph of uptake of DOTAP: cholesterol liposomes labeled with Texas Red (red-orange) in a pancreatic tumor in a RIP1 -TAG_2_mouse. The vessels were stained by perfusion of fluorescent lectin Lycopersicon esculentum (green) after the liposomes were injected intravenously (scale bar: 50 μm); Figure 8 shows a confocal micrograph of uptake of DOTAP: cholesterol liposomes labeled with Texas Red (red-orange) in a pancreatic tumor in a RI P 1 -TAG_2_mouse. The vessels were stained by perfusion of fluorescent lectin Lycopersicon esculentum (green) after the liposomes were injected intravenously. The possible vessel growth sites have intense uptake (scale bar: 50 μm); Figure 9 is a micrograph showing low uptake of DOTAP: cholesterol liposomes labeled with Texas Red (red-orange) in normal blood vessels in the trachea of a pathogen-free mouse, the vessels stained green with a fluorescent lectin ( scale bar: 50 μm); Figure 10 shows a confocal micrograph of uptake of DOTAP: cholesterol liposomes labeled with Texas Red (red-orange) in angiogenic blood vessels in the trachea of a mouse with Mycoplasma pulmonis infection (scale bar: 50 μm); Figure 11 is a graph showing the amount of uptake of DOTAP liposomes: cholesterol with Texas Red by blood vessels of pathogen-free mouse tracheas (normal) and infected with Mycoplasma pulmonis assessed by measuring the fluorescence intensity of liposomes. hours after the intravenous injection. The measurements were made with a Zeiss LSM 41 0 confocal microscope. The infected mice were inoculated intranasally with M. organisms. pneumonia and examined 4 weeks later. The asterisk designates the statistically significant difference (P <0.05, mean + SE, n = 4 mice per group); Figure 12 is an electron transmission micrograph showing DOTAP: cholesterol liposomes associated with an endothelial cell in the trachea of a mouse infected with M. pulmonis (scale bar: 50 μm); and Figure 13 is an electron transmission micrograph showing DOTAP: cholesterol liposomes taken up by an endothelial cell in the trachea of a mouse infected with M. pulmonis (scale bar: 80 μm).

DETAILED DESCRIPTION OF PREFERRED MODALI DADES Prior to describing the present method for selectively affecting / labeling angiogenic endothelial cells and the liposomes used in the method, it will be understood that this invention is not limited to the particular liposomes, methods or active substances already described. that such may, of course, vary. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. . Thus, for example, the reference to "a liposome" includes mixtures and large numbers of such liposomes, the reference to "an agent" includes a large number of agents and mixtures thereof, and the reference to "the method" includes one or more methods or steps of the type described herein. The publications discussed herein are provided solely for description before the filing date of the present application. In the present nothing should be construed as an admission that the present invention is not qualified to precede such publication by virtue of the prior invention. Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any method and material, similar or equivalent to those described herein, may be used in the practice or purity of the present invention, preferred methods and materials are described herein. All publications cited herein are incorporated herein by reference for the purpose of disclosing and describing specific aspects of the invention by which the publication is cited.

DEFINITIONS The terms "treatment", "treating", "treating" and the like are used herein to mean generally obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of partially or completely preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. "Treatment", as used herein, covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject, which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibit the symptom of the disease, that is, stop its development; or (c) alleviating the symptom of the disease, that is, causing the regression of the disease or symptom. The term "angiogenesis" refers to a process of tissue vascularization that involves the development of new vessels. Angiogenesis occurs via one of three mechanisms: (1) neovascularization, where the endothelial cells migrate out of the pre-existing vessels beginning the formation of the new vessels; (2) vasculogenesis, where the vessels arise from de novo precursor cells; or (3) vascular expansion, where small existing vessels enlarge in diameter to form larger vessels (Blood, C.H. and Zetter, B.R., 1990, Biochem. Biophys. Acta. 1032: 89-118). Angiogenesis is an important process in normal processes of neonatal growth and in the female reproductive system during the corpus luteum growth cycle (see Moses, M.A. et al., 1990, Science 248: 1408-1 0). Under normal conditions, all processes involving the new formation or remodeling of new or existing blood vessels is a self-limiting process, and the expansion of specific cell types is controlled and adjusted. Angiogenesis is also involved in wound healing and in the pathogenesis of a large number of clinical diseases including tissue inflammation, arthritis, asthma, tumor growth, diabetic retinopathy and other conditions. The clinical manifestations associated with angiogenesis are referred to as angigogenic diseases (Folkman, J. and Klagsbrun, M., 1987, Science 235: 442-7). Many experiments have suggested that tissues can produce angiogenic factors, which promote angiogenesis under conditions of poor blood supply both during normal and pathological conditions. These factors and compounds differ in cellular specificity and in the mechanisms by which they induce the growth of new blood vessels. These factors work through a variety of mechanisms. For example, they can induce the migration and proliferation of endothelial cells or stimulate the production of collagenase (see Klagsbrun, M, and D'Amore, PA, 1 991, "Regulators of angiogenesis" m Ann. Rev. Physiol 53: 217-39). There is a number of bioassays which allow the direct determination of angiogenic activities (Wilting, J. et al., 1 991, "A modified chorioallantoic membrane (CAM) assay for qualitative and quantitative studies of growth factors. , PBS, angiogenin, and bFGF "(Modified chorioallantoic membrane assay for qualitative and quantitative study of growth factors Studies on the effects of carriers, PBS, angiogenin and bFGF), Anat. Embrol. (Berl) 183: 259- 71). It has been proposed that angiogenic inhibitors may be useful in the treatment of diseases. For example, interfering with angiogenesis can restrict the growth of a tumor. Various means have been proposed for inhibiting angiogenesis including (1) inhibiting the release of angiogenic factors, (2) neutralizing angiogenic factors using such media as monoclonal antibodies, and (3) inhibiting endothelial cell responses (Folkman, J., et al. 1992, Seminars in Cancer Biology 3: 89-96), through the use of anti-angiogenic factors, molecules known to inhibit angiogenesis. Several endothelial cell inhibitors have been described, such as collagenase inhibitor, basement membrane change inhibitors, angiostatic steroids, fungal derivative inhibitors, platelet factor 4, thrombospondin, arthritis drugs, such as penicillamine, and alpha-interferon. , among others (see Folknam, J., et al., 1992, Seminars in Cancer Biology 3: 89-96, for examples see: Stepien, H., et al., 1996, "Inhibitory effects of fumagillin and its analogue TNP. -470 on the function, morphology, and angiogenesis of an oestrogen-induced prolactinoma in Fischer 344 rats "(Inhibitory effects of fumagillin and its analog TNP-470 on the function, morphology and angiogenesis of an estrogen-induced prolactinoma in Fischer 344 rats) , J. Endocrinol, 1 50: 99-1 06, Maione, TE, et al., 1 990, "Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides", (I nhibition of angiogenesis by platelet factor 4 human recomb inante and related peptides), Science 247: 77-9). The term "endothelial cells" means those cells that form the endothelium, the monolayer of simple squamous cells which delineate the inner surface of the circulatory system. These cells retain a capacity for cell division, although they proliferate very slowly under normal conditions, experiencing cell division perhaps only once a year. The proliferation of endothelial cells can be demonstrated by using [3H] thymidine to label cells in the S phase. In normal vessels, the proportion of endothelial cells that becomes marked is especially high at branch points in arteries, where turbulence and Wear seems to stimulate change. (Goss, R.J., 1 978, The Physiology of Growth, Academic Press, New York, pp. 120-137). Normal endothelial cells are quiescent, that is, they are not dividing and as such, they are distinguishable from angiogenic endothelial cells as discussed below. Endothelial cells also have the ability to migrate, an important process in angiogenesis. Endothelial cells form new capillaries when there is a need for them, such as during wound repair or when there is a perceived need for them as in tumor formation. The formation of new vessels is called angiogenesis, and involves molecules (angiogenic factors) which can be mitogenic or chemoattractants for endothelial cells (Klagsburn, supra). During angiogenesis, endothelial cells can migrate out of an existing capillary to begin the formation of a new vessel, ie, the cells of a vessel migrate in a manner that allows the extension of that vessel (Speidel, CC, Sm. J Anat. 52: 1-79). In vitro studies have documented both the proliferation and migration of endothelial cells; endothelial cells placed in culture can proliferate and spontaneously develop capillaries (Folkman, J., and Haudenschild, C, 1980, Nature 288: 551-56). The terms "angiogenic endothelial cells" and "endothelial cells undergoing angiogenesis" and the like are used interchangeably herein to mean endothelial cells (as defined above) that undergo angiogenesis (as defined above). Thus, angiogenic endothelial cells are endothelial cells which are proliferating at a rate beyond the normal condition of experiencing cell division sparingly once a year. The rate of normal proliferation differentiation of endothelial cells can be 2x, 5x or 10x or more than the noraml proliferation and can vary greatly depending on factors such as the age and condition of the patient, the type of tumor involved, the type of wound, etc. Provided that the difference in the degree of proliferation between normal endothelial cells and angiogenic endothelial cells is measurable and considered biologically significant, then the two types of cells are distinguishable by the present invention, ie, the differentiable angiogenic endothelial cells of quiescent endothelial cells. , normal, corresponding, in terms of preferential binding of cationic liposomes.

The terms "corresponding endothelial cells", "normal or quiescent endothelial cells" and the like are used in order to refer to normal, quiescent endothelial cells contained within the same type of tissue (under normal conditions) when some of the cells Endothelial cells are undergoing angiogenesis and some of the endothelial cells are quiescent. In connection with the present invention, angiogenic endothelial cells are preferentially focused and focused with a preference, which is five times, preferably ten times greater than the corresponding quiescent endothelial cell approach. The term "lipid" is used in its conventional sense as a generic term encompassing fats, lipids, the alcohol-soluble constituents of protoplasm, which are insoluble in water. Lipids make up fats, fatty oils, essential oils, waxes, steroids, sterols, phospholipids, glycolipids, sulpholipids, aminolipids, chromolipids (lipochromes), and fatty acids. The term encompasses both lipids that occur naturally and synthetically produced. Preferred lipids in connection with the present invention are: phospholipids, including phosphatidylcholines and phosphatidylethanolamines, and sphingomyelins. Where there are fatty acids, could be 1 2-24 carbons in length, containing up to 6 unsaturations (double bonds), and linked to the skeleton either by its acyl or ether bonds. Where there is more than one fatty acid linked to the skeleton, the fatty acids may be different (asymmetric), or there may be only 1 fatty acid chain present, for example, lysolecithins. Mixed formulations are also possible, particularly when the non-cationic lipids are derived from natural sources, such as lecithins (phosphatidyl cholines) purified from egg yolk, heart, brain or bovine liver or soy. Steroids and sterols, particularly cholesterol, and sterols substituted in position 3b. The term "cationic lipid" is used herein to encompass any lipid in the invention (as defined above) which is cationic. The lipid will be determined to be cationic when the lipid has a positive charge (at physiological pH) as measurable by instrumentation used at the time of measurement. Where there are fatty acids present in the cationic lipid, they could be 12-24 carbons in length, containing up to 6 unsaturations (double bonds), and linked to the skeleton either by their acyl or ether bonds; there could also be a single chain of fatty acid linked to the ekelet. Where there is more than one fatty acid linked to the skeleton, the fatty acids could be different (asymmetric). Mixed formulations are also possible. The term "liposome" encompasses any compartment enclosed by a lipid bilayer. Liposomes are also referred to as lipid vesicles. In order to form a liposome, the lipid molecules comprise elongated non-polar (hydrophobic) and polar (hydrophilic) portions. The hydrophobic and hydrophilic portions of the molecule are preferably placed at two ends of an elongated molecular structure. When such lipids are dispersed in water, they can spontaneously form bilayer membranes referred to as sheets. The sheets are composed of two monolayer sheets of lipid molecules with their non-polar (hydrophobic) surfaces facing each other and their polar surfaces (hydrophilic) facing the aqueous medium. The membranes formed by the lipids enclose a portion of the aqueous phase in a manner similar to that of a cellular membrane encompassing the contents of a cell. Thus, the bilayer of a liposome has simulations with a cell membrane without the protein components present in a cell membrane. As used in connection with the present invention, the term liposome includes multilamellar liposomes, which generally have a diameter in the range of 1 to 10 micrometers and are comprised anywhere from two to hundreds of concentric lipid bilayers alternating with layers of an aqueous phase, and also include unilamellar vesicles, which are comprised of a lipid layer simple, and generally have a diameter of 20 to 1000 nanometers, said vesicles can be produced by subjecting multilamellar liposomes to ultrasound. Preferred liposomes would be small unilamellar vesicles (SUVs), which have a single lipid bilayer, and a diameter in the range of 25-200 nm. Preferred complexes of polynucleotides (including DNA, RNA and analogs of synthetic polynucleotides) liposomes, would be prepared from the preferred liposomes. The complexes would be prepared so that 1 μg of polynucleotide is present per 1 -50 nmole of cationic lipid. When the expression of a DNA gene cartridge is the desired end product, the optimal ratio of polynucleotide to cationic lipid is determined empirically, by preparing a series of formulations in which a standard amount of DNA is mixed with different amounts of cationic liposome. within the range described above. These formulations are then administered in vivo, and the formulation that gives the highest expression can be determined. Cationic liposomes can be defined functionally by having a zeta potential of more than 0 mV. The term "cationic liposome", as used herein, is intended to encompass any liposome as defined above, which is cationic. It is determined that the liposome is cationic when present at physiological pH. It should be noted that the liposome by itself is the entity which is being determined as cationic, meaning that the liposome, which has a measurable positive charge within its physiological pH can, within an in vivo environment, bind to other substances . These other substances may be negatively charged, resulting in the formation of a structure which has no positive charge. The loading and / or structure of a liposome of the present invention within an in vivo environment has not been determined accurately. However, according to the invention, a cationic liposome of the invention will be produced using at least some lipids, which are cationic by themselves. The liposome does not need to be completely comprised of cationic lipids but must be comprised of a sufficient amount of a cationic lipid, so that when the liposome is formed and placed in an in vivo environment at physiological pH, the liposome initially has a charge positive. The term "nucleotide sequence complex / cationic lipids" refers to a combination of a nucleotide sequence, which may be an RNA or DNA sequence, which is combined with at least cationic lipids as defined above, and may include neutral lipids. When the DNA and cationic lipid sequences are combined, they will spontaneously form complexes which are not classical liposomes. The present invention is specifically directed towards the formation of specific complexes of nucleotide / cationic lipid sequences, wherein the nucleotide sequence is specifically designed to affect angiogenic endothelial cells. For example, the nucleotide sequence can encode a protein, which kills angiogenic endothelial cells. The sequence is preferably operably linked to a promoter, which is selectively activated only within the environment of an angiogenic endothelial cell, ie, not activated within a corresponding quiescent endothelial cell. In addition, the complex may include a sequence, which is an antisense sequence, which blocks the expression of genetic material within an angiogenic endothelial cell, and thereby severely interrupts the operation of, and / or kills the angiogenic endothelial cell. The DNA could be plasmid or linear. When a gene product is desired (either an RNA transcript by itself, or translated into a protein), an expression cartridge, which is comprised of a DNA promoter sequence, and a DNA sequence encoding it is necessary. a product of gene. Nucleotides with ligations other than phosphodiester are used in particular in antisense uses. The term "is associated with" refers to the action of cationic liposomes of the invention, which remain in proximity sufficiently close to angigogenic endothelial cells for sufficiently long periods, so that the liposome and / or its contents enter the endothelial cell . The liposomes of the invention can be associated with angiogenic endothelial cells under a variety of circumstances, but, most preferably, they are associated with the angiogenic endothelial cell when they are in in vivo conditions. Thus, the liposome can be modified by the binding, ligation or association of other molecules or materials present in the bloodstream before association with the angiogenic endothelial cell. A variety of forces may be responsible for the association of liposomes with angiogenic endothelial cells, such as nonspecific interactions, which occur between any two unrelated molecules, i.e., other macromolecules such as human serum albumin and human transferrin. These intramolecular forces can be classified into four general areas, which are (1) electrostatic; (2) hydrogen bonding; (3) hydrophobic; and (4) Van der Waals. Electrostatic forces are due to the attraction between oppositely charged ionic groups, such as between oppositely charged groups in a cationic liposome and groups present on or in the angiogenic endothelial cell. The attractive force (F) is inversely proportional to the square of the distance (d) between the charges. The hydrogen bonding forces are provided by the formation of reversible hydrogen bonds between hydrophilic groups. The liposomes of the invention may include hydrophilic groups, such as -COOH and similar groups may be present on the surface of endothelial cells such as -OH, '-N H2 groups. These forces are enormously dependent on the close clustering of two molecules carrying these groups. The hydrophobic forces operate in the same way that drops of oil in water merge to form a single large drop. Accordingly, non-polar hydrophobic groups, such as those present in the liposomes of the invention, tend to associate in an aqueous environment and may tend to associate with hydrophobic groups present on the surface of endothelial cells. Finally, Van der Waals forces are created between molecules, which depend on interactions between the outer electron clouds. The terms "selectively associate" and "selectively focus" and the like are used herein to describe a property of cationic liposomes of the invention, which causes cationic liposomes to associate with angiogenic endothelial cells to a greater degree than Cationic liposomes associate with corresponding normal endothelial cells not involved in angiogenesis. According to the invention, the selective or preferential association means that the liposome will associate to a degree of five times or more with endothelial cells undergoing angiogenesis as compared to corresponding normal endothelial cells that do not undergo angiogenesis. More preferably, the preferable or selective association indicates a selectivity of ten times or more between angiogenic endothelial cells and corresponding normal endothelial cells. The term "cancer" refers to a disease of inappropriate cell proliferation. This disorder is very evident clinically when the volume of tumor tissue compromises the function of vital organs. Concepts that describe normal tissue growth are applicable to malignant tissue because normal and malignant tissues may share similar growth characteristics, both at the cell level alone and at the tissue level. Cancer is as much a disorder of disordered tissue growth regulation as it is of disordered cell growth regulation. Duplication time refers to the time required for a tissue or tumor to double its size or number of cells. The time of duplication of a clinically apparent tumor is, in gel, considerably greater than the cell cycle time of the cells of which the tumor is composed. However, unlike a tumor, normal liver, heart or lungs in an adult do not have a doubling time since the organs are in stable state, so that the rates of cell production and cell death are equal (Stockdale, F., 1996, "Cancer growth and chemotherapy", in: Scientific American Medicine, vol.3, Scientific American Press, New York, pp. 12-18). The growth characteristics of tumors are such that the production of new cells exceeds cell death; A neoplastic case tends to produce an increase in the proportion of post cells that undergo self-renewal and a corresponding decrease in the proportion that progresses to maturation (McCulloch, EA, et al., 1982, "The contribution of blast cell properties to outcome variation in acute myeloblastic leukemia (AML) (The contribution of blast cell properties to overcome variation in acute myeloblastic leukemia), Blood 59: 601-608). For each tumor population, there is a doubling time and a specific growth curve can be established (Stockdale, F., supra). The growth pattern in tumors can be described by a gomperzian curve (Steel, GG, 1977, Growth kinetics of tumors, Oxford University Press, I nc., New York, p.40), which It indicates that during the development of a tumor the growth rate is initially very fast and then decreases progressively as the size increases.

GENERAL ASPECTS OF THE INVENTION The appended figures provide a clear visual representation of the highly selective manner in which the cationic liposomes of the invention are targeted to angiogenic endothelial cells. A basic embodiment of the invention involves a method to selectively affect angiogenic endothelial cells by admising (preferably by intravascular injection, more preferably intra-arterial injection) a formulation which comprises a pharmaceutically acceptable carrier and cationic liposomes, which contain a substance or DNA / cationic complexes. The substance can be a compound which inhibits angiogenesis, a compound which promotes angiogenesis and / or a detectable label. The cationic liposomes within the injected formulation are then allowed to enter the angiogenic endothelial cells (by endocytosis), which delineate the walls of the angiogenic blood vessels. Cationic liposomes associate with angiogenic endothelial cells for a sufficient period and in such a manner that the liposomes themselves and / or the contents of the liposomes enter the angiogenic endothelial cell. Subsequently, the compound entering the cell can inhibit or promote angiogenesis or simply provide a label, allowing detection of the site of angiogenesis. The selectivity of the angiogenic endothelial cell approach can be better understood by referring to the appended figures. Figure 1 shows a portion of a mouse ovary having a large round follicle (in yellow) placed therein. Because angiogenesis is occurring within a normal mouse ovary, cationic liposomes containing a detectable label are associated with angigogenic endothelial cells of the growing blood vessels of the follicle (red-orange). However, within Figure 1 it is not possible to clearly determine that the mark is associated only with angiogenic endothelial cells or if it is associated with all the tissue within the ovary and follicle. Figure 2 is a fluorescence micrograph showing a section of a pancreatic tumor of a mouse, which was injected intravenously with cationic (red-orange) liposomes of the invention containing a detectable label. Angiogenesis occurs easily within tumors. Thus, this photo provides some indication that the cationic (red-orange) liposomes of the invention specifically associate with angiogenic endothelial cells (green). However, these results do not dramatically demonstrate the specificity of the invention. A comparison of Figures 3 and 4 demonstrate the ability of the invention to locate an angiogenesis site. Figure 3 is a photograph showing blood vessels within normal pancreatic tissue of a mouse. There is much less labeling of normal endothelial cells than the corresponding angiogenic endothelial cells. This is clearly demonstrated by comparing Figure 3 with Figure 4, which is a picture of a pancreatic tumor inside a mouse. Figure 4 clearly shows a high degree of accumulation of the mark (yellow-orange) contained within the cationic liposomes in the area of a tumor. The dramatic difference between Figures 3 and 4 indicate the utility of the present invention to clearly and accurately mark the site of a tumor. However, because much of the tag is associated with the angiogenic blood vessels in Figure 4, it may not be possible to fully appreciate the specificity of the cationic liposomes to preferentially target angiogenic endothelial cells. Figure 5 is a picture of blood vessels (green) in a normal pancreatic mouse islet. The small amount of red-orange coloration indicates the limited association of cationic liposomes with normal endothelial cells lining the blood vessels of the pancreatic tissue.

The specificity of the cationic liposomes containing a detectable label is shown more clearly when comparing Figure 5 with the Figure 6. Figure 6 clearly shows a much greater degree of tag accumulation in the endothelial cells of the angiogenic blood vessels of the tumor within the pancreas of a mouse. The similar ability of cationic liposomes to target angiogenic endothelial cells is dramatically shown within Figures 7 and 8. Figure 7 clearly shows that the fluorescent label is associated only with the blood vessels, ie, the mark is not leaking or migrating to the surrounding tissue. The specificity is shown very dramatically in Figure 8, which clearly focuses on labeled cationic liposomes detected within angiogenic endothelial cells that show that the label is specific for those cells and does not leak or migrate into the surrounding tissue. Figures 9 and 10 demonstrate the same effect described above but with a different model of angiogenesis. Figures 1 to 8 were all directed to either normal or cancerous tissue. Figures 9 and 10, respectively, show normal and inflamed tissue of the trachea of a mouse. More specifically, Figure 9 shows the normal blood vessels of a trachea, i.e., a pathogen-free mouse trachea. Figure 10 shows the blood vessels of a trachea with the occurrence of infection-induced angiogenesis. The higher concentration of the detectable label in Figure 10 is apparent indicating that the cationic liposomes of the invention selectively associate with angiogenic endothelial cells - specifically associating with endothelial cells of the trachea, which have been induced to angiogenesis by an infection. Figure 11 is a graph depicting the difference in the specificity of cationic liposomes between their ability to associate with angiogenic endothelial cells and corresponding normal endothelial cells that do not undergo angiogenesis. As shown within Figure 11, the cationic liposomes of the invention (by this experiment) have shown an approximately 10x greater affinity for angiogenic endothelial cells as compared to corresponding endothelial cells that do not undergo angiogenesis.

Finally, Figures 12 and 13 show how the cationic liposomes of the invention enter the angiogenic endothelial cells. In Figure 12, cationic liposomes have contacted the surface of the angiogenic endothelial cell. Within Figure 13, cationic liposomes have entered angiogenic endothelial cells by endocytosis and are present within the cell. Having described in words and shown via the figures the specificity of the cationic liposomes of the invention, those skilled in the art will be able to produce a variety of different cationic liposomes containing a variety of different substances, in order to make use of the invention . However, for completeness, the following is a description of cationic liposomes and their manufacturing methods followed by a description of substances which either inhibit or promote angiogenesis.

LI POSOMAS Liposomes can be easily formed by placing lipids (as defined above), which include cationic lipids (as defined above) in an aqueous solution and stirring the solution for a period of several seconds to hours. The simple procedure spontaneously produces large, multilamellar liposomes or vesicles with diameters in the range from about 1 to 10 microns. These liposomes are comprised of two to several hundred concentric lipid bilayers, which may alternate with layers of the aqueous phase, in which the lipids were present. A substance, such as a compound which inhibits angiogenesis, promotes angiogenesis or provides a detectable label, may be included within the aqueous phase. The substance is preferably soluble in water or can, at least, be easily dispersed in water. The thickness of the aqueous layer and thus the total amount of the aqueous phase trapped within the liposome, depends on the balance of electrostatic repulsion forces between charged lipids and attractive Van der Waals forces between bilayers as a whole. Thus, the aqueous separation (and hence the volume of trapped aqueous material) increases with an increasing proportion of lipids charged on the membrane and with decreasing concentrations of electrolytes (charged ions) in the aqueous phase. The small liposomes or vesicles formed are unilamellar and have a size in the range of about 20 to 100 nanometers and can be produced by subjecting multilamellar vesicles to ultrasound. Larger unilamellar liposomes having a size in the range of about 0.1 to 1 μm in diameter can be obtained when the lipid is solubilized in an organic solvent or a detergent and the solubilized agent is removed by evaporation or dialysis, respectively. The fusion of smaller unilamellar liposomes by methods that require particular lipids or severe conditions of dehydration-hydration, can produce unilamellar vessels as large or larger than the cells. In order to form cationic liposomes of the invention it is necessary that the liposomes be produced using at least some cationic lipid. However, the cationic liposomes of the invention need not be completely comprised of cationic lipids. For example, using neutral lipids in an amount of about 45% and cationic lipids in an amount of about 55%, will produce cationic lipids which are useful in connection with the invention and preferentially target angiogenic endothelial cells. The combination of cationic liposomes with a substance which affects angiogenesis and / or a label includes the preparation of liposomes, wherein the liposomes are prepared according to standard technology, whereby, for example, solutions of sodium chloride are mixed. -. { 2- (9 (Z) -octadecenyloxy) ethyl} -2- (8 (Z) -heptadecenyl) 3- (2-hydroxyethyl) imidazolinium (DOTAP), cholesterol, and DH PE Red Texas, evaporate to dryness and the lipid film is subsequently rehydrated in 5% dextrose to produce vesicles multilamellar. These vesicles are extruded through polycarbonate membrane filters to produce unilamellar vesicles. The liposomes and the substance to be combined, eg, plasmid DNA, are mixed together in specific proportions in a 5% dextrose solution or other physiologically acceptable excipient. Useful cationic lipids include, DDAB, dimethyldioctadecyl ammonium bromide [available from Avanti Polar Lipids and Sigma Chemical Company], 1, 2-diacyl-3-trimethylammonium-propanes (including but not limited to, dioleoyl (DOTAP), dimipstoyl, dipalmitoyl , disearoyl) [all of these are available from Avanti Polar Lipids] DOTMA, N- [1 - [2,3-bis (oleolyloxy)] propyl] -N, N, N-trimethylammonium chloride, DOGS, dioctadecylamidoglycylspermine [available] from Promega Corporation], DC-cholesterol, 3b- [N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol DOSPA, 2,3-dioleoyloxy-N- (2- (sperminecarboxamide) -ethyl) -N trifluoroacetate, N-dimethyl-1 -propanaminium, 1,2-diacyl-sn-glycerol-3-ethylphosphocholines (including but not limited to dioleoyl (DOEPC), dilauroyl, dimyristoyl, diplamitoyl, distearoyl, palmitoyl-oleoyl) [all of these are available from Avanti Polar Lipids], b-alanyl cholesterol, C , cetyl trimethyl ammonium bromide diC14-amidine, Nt-butyl-N'-tetradecyl-3-tetradecyl laminopropionamidine, 14Dea2, O chloride, O'-ditetradecanolyl-N- (trimethylammonioacetyl) diethanolamine, (N, N, N ', N'-tetramethyl-N, N'-bis (2-hydroxylethyl) -2,3-iodide -dioleoilox-1, 4-butanediaminium [available from Promega Corporation], 1 - [2-acyloxy) ethyl] 2-alkyl (alkenyl) -3- (2-hydroxyethyl) imidazolinium chloride derivatives, such as 1 - [2- ( 9 (Z) -octadecenoyloxy) ethyl] -2- (8 (Z) -heptadecenyl-3- (2-hydroxyethyl) imidazolinium (DOTIM), 1 - [2- (hexadecanoyloxy) ethyl] -2-pentadecyl- chloride 3- (2-hydroxyethyl) imidazoline (DPTIM), 1- (2-tetradecanoyloxy) ethyl] -2-tridecyl-3- (2-hydroxyethyl) imidazolium chloride (DMTIM) - these 3 lipids are described in Solodin et al. , Biochem 43, 135737-13544, 1995.

This is from Tim Heath's lab in Wisconsin; Megabios has acquired patents for some of its lipid inventions. Derivatives of 2,3-dialkyloxypropyl ammonium quaternary compound, containing a portion of hydroxyalkyl in the quaternary amine, such as: 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI); 1,2-dioleyloxypropyl-3-dimethyl-1-hydroxyethyl ammonium bromide (DORI E); 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide (DORIE-HP); 1,2-dioleyloxypropyl-3-dimethyl-hydroxybutyl ammonium bromide (DORIE-HB); 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentyl ammonium bromide (DORIE-Hpe); 1, 2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRI E); 1, 2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DPRIE); 1, 2-diesteryloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DSRI E) -those lipids were developed by Vical, Felgner et al. , J. Biol. Chem. 269, 2550-2561, 1994. Cationic liposomes are prepared from the cationic lipids by themselves, or in admixture with other lipids, particularly neutral lipids, such as: CHOLESTEROL 1, 2-diacyl-sn-glycerol-3-phosphoethanolamines, (including but not limited to dioleoyl (DOPE); a large family of derivatives is available from Avanti Polar Lipids); 1,2-diacyl-sn-glycero-3-phosphocholines (a large family of derivatives is available from Avanti Polar Lipids); N. B. One could include asymmetric fatty acids, both synthetic and natural, and mixed formulations, for the diacyl derivatives above. Liposomes of the type described above and of other types which will occur to those skilled in the art, can be used in the present invention, the liposomes containing a substance, which either promotes or inhibits angiogenesis and / or includes a detectable brand. An example of liposomes of the invention are cationic liposomes containing a lipid-soluble or water-soluble substance, which inhibits angiogenesis. However, the lipid-soluble compounds may be in the lipid bilayer. The following provides a description of angiogenesis inhibitors. However, it should be noted that others will occur to those skilled in the art and / or will be developed after the present invention, and that such angiogenesis inhibitors could easily be used in connection with the present invention.

AGENTS I NHI BIORES OF ANGIOGENES IS Heparin is an angiogenesis enhancer, and heparin antagonists can block the angiogenic response. Protamine, a heparin ligation protein, shows anti-angiogenic properties (Taylor, S. and Folkman, J., 1982, "Protamine is an inhibitor of angiogenesis", (Protamine is an inhibitor of angiogenesis), Nature, 297 , 307), but it is not clinically useful because it is known to cause anaphylactic reactions on human exposure. Another anti-angiogenic agent is the heparin ligation protein, Major Basic Protein, which is also highly toxic and thus is not practical for human use. However, because of the high degree of focus selectivity obtained with the present invention, these and other compounds which inhibit angiogenesis but are thought to be too toxic for therapeutic use in humans may be useful because they can be used in very small amounts. Platelet factor 4 (PF4) exhibits both heparin ligation activity and anti-angiogenic properties, and since it is not as toxic as the other heparin antagonists, it may be clinically useful. Chemical modifications of PF4, as described in US Patent 5,112,946, enhance the anti-angiogenic properties of PF4. These modifications include the production of modified PF4 analogs through their free amino groups with fluorescein-isothiocyanate, PF4 mutants with specifically altered structural composition of the protein, and the production of PF4 fragments that retain the anti-angiogenic properties. In particular, a synthetic peptide of 13 amino acids corresponding to the carboxy terminus of PF4 has exhibited potent angiostatic activity. It has been shown that a variety of steroids to inhibit angiogenesis. This anti-angiogenic activity is enhanced by the addition of heparin or related molecules (Folkman, J., Weisz, PB, et al., 1989, "Control of angiogenesis with synthetic heparin substitutes", (Control of angiogenesis with synthetic heparin substitutes. ), Science 243, 1490-3). The so-called "angiostatic steroids," such as tetrahydrocortisone, have the ability to block angiogenesis in vivo. Specifically, 6-fluoro-17,21-dihydroxy-16β-methyl-pregna-4,9- (1 1) -diene-3,20-dione has been used as a potent angiostatic spheroid. It has been found that drugs that modulate collagen metabolism inhibit angiogenesis. The proline amino acid analogues specifically inhibit collagen synthesis and inhibit angiogenesis in vivo. Specifically, L-azetidine-2-carboxylic acid (LACA), cis-hydroxyproline (CHP), D, L-3,4-dehydroproline (DHP), and thioproline (TP) each exhibit anti-angiogenic activity in order to decrease activity (Ingber, D., and Folkman, J., 1988, "I nhibition of angiogenesis through modulation of collagen metabolism", (Inhibition of angiogenesis through the modulation of collagen metabolism), 1988, Lab. Invest. 59, 44-51). Each of these analogues also potentiate the anti-angiogenic effects of angiostatic steroids and heparin. Human thrombospondin, a glycoprotein found in the alpha granules of platelets, inhibits angiogenesis in trimer or monomer or fragment form, as described in US Pat. No. 5,192,744. Each works in the glycosylated form and is predicted to work in the non-glycosylated form. The inhibitory properties of angiogenesis are present following the deletion of the heparin binding domain associated with the amino terminus and the platelet binding domain found at the carboxyl terminus of the monomeric protein. Peptides that exhibit laminin activity block angiogenesis and prevent the formation of an excess of blood vessels in tissues. The specific peptides with such activity are: 1) tyrosine-isoleucine-glycine-serine-arginine; 2) proline-aspartin-serine-glycine-arginine; and 3) cysteine-aspartate-proline-glycine-tyrosine-isoleucine-glycine-serine-arginine. It is predicted that these peptides maintain their anti-angiogenic activity in a cyclic fashion. Other examples of substances that inhibit angiogenesis include extracts of cartilage tissue showing activity of collagenase, the protein derived from retinal pigment endothelial cells (Arch. Ophtamol., 1 03, 1 870 (1988), anti-cancer factor induced Cultured cartilage cells (Takigawa, M. and Suzuki, f., 1988, "Establishment of clonal cell lines producing cartilage-derived anti-tumor factor (CAFT)", (Establishment of clonal cell lines producing anti-tumor factor derived from cartilage (CAFT)), Protein, Nucleic Acid and Enzyme, 33, 1803-7), anti-inflammatory drugs such as indomethacin (Peterson, HI, 1 986, "Tumor angiogenesis inhibition by prostaglandin sythease inhibitors", (Inhibition of tumor angiogenesis by inhibitors of prostaglandin synthase), Anticancer Res., 6, 251-3), ribonuclease inhibitors (Shapiro, R., and Vallee, BL, 1987, "Human placental ribonuclease inhibitor abolishes both angiogenic and ribonucleolytic activities of angiogenin "(The human placental ribonuclease inhibitor ablated both the angiogenic and ribonucleolytic activities of angiogenin), PNAS 84, 2238-41), sulfuric polysaccharide complexes and glycan peptide (eg, JPA-S63 (1988) - 1 19500), gold preparations for arthritis, herbimycin A (JPA-S6381 988) -295509) and fumagillin or fumagillol derivatives. A number of fumagillol derivatives have angiogenesis inhibiting properties, as described in US Patent 5,202, 352. The above references are incorporated by reference to describe and disclose angiogenesis inhibitors.

ANGIOGENIC FACTORS A number of biological compounds stimulate angiogenesis. It has been shown that angiogenin is a potent angiogenic factor in chicken CAM or rabbit cornea. Angiotrophin, an isolated factor in peripheral blood monocytes, is another angiogenic compound that has been proposed to play a role in normal wound healing (Biochemistry 27, 6282, (1988)). Other factors involved in wound healing, such as fibrin, also induce vascularization.

Another class of mediators of angiogenesis are angiogenic polypeptide factors, such as growth factors, which include basic and acidic fibroblast growth factors (FGFs), transforming growth factor alpha (TGF-a), and growth factor. platelet derivative (PDGF). It has been shown that each of these molecules induces angiogenesis in vivo. Other similar molecules that exhibit angiogenic activity are vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF-a), transformation growth beta factor (TGF-β), and ligation growth factors of heparin (HBGFs). Other angiogenic factors have been described in addition to angiogenic polypeptide factors. Prostaglandins E-i and E2, which are angiogenic factors derived from lipids, are attractants of well-known inflammatory cells with angiogenic properties (J. Nati. Cancer Inst. 69, 475-482 (1982)). Nicotinamide elicits an angiogenic response when tested in chicken cornea or in a chicken CAM assay (Science 236, 843-845 (1987)).

DETECTABLE MARKS The cationic liposomes of the invention can be used to deliver detectable labels of any kind. The labels are either soluble in the lipid used to make the liposomes, or are soluble or at least dispersible, within water or an aqueous solution, such as an aqueous saline solution or aqueous dextrose solution. The label can be a radioactive label, fluorescent label, histochemical or immunohistochemically detectable substance, or detectable dye. The label may be present in any appropriate amount and may be included in, or complexed with, the liposome by itself or together with a substance which inhibits or promotes angiogenesis.

DOSAGE The amount of angiogenic inhibitor or promoter administered to a patient (which can be any animal with a circulatory system with endothelial cells which undergo angiogenesis) will vary depending on a wide range of factors. For example, it would be necessary to provide substantially higher doses to humans than to smaller animals. The amount of angiogenic inhibitor or promoter will depend on the size, age, sex, weight and condition of the patient, as well as the potency of the substance being administered. Having indicated that there is considerable variability in terms of dosage, it is believed that those skilled in the art can, using the present disclosure, easily determine appropriate dosage by first administering extremely small amounts and increasing the dose in increments until the desired results are obtained. . Although the amount of the dose will vary greatly based on the factors as described above, in general, the present invention makes it possible to administer substantially smaller amounts of any substance as compared to delivery systems, which target the surrounding tissue, for example, they focus the tumor cells by themselves.

COMPLEXES OF SEQUENCE OF N UCLEOTI DOS / LI PIDOS CATIONICOS When the nucleotide sequences including DNA and RNA sequences are combined with lipids, both form complexes. By choosing particular amounts of nucleotide and lipid sequences and choosing particular lipids, it is possible to form complexes which do not aggregate together in vitro. The general information that relates to the formation of such complexes is described within the PCT publication WO 93/12240, published June 24, 1993, which is incorporated herein by reference, to specifically disclose and describe the formation of nucleotide / lipid sequence complexes. In connection with the present invention, the nucleotide sequences are specifically designed to affect angiogenic endothelial cells and not affect other cells, and specifically not affect other corresponding endothelial cells, ie, quiescent endothelial cells. The DNA sequences used in connection with the present invention are operably linked to promoters and those promoters are specifically designed so that the expression of the nucleotide sequence is obtained only within the environment of an angiogenic endothelial cell. First, the promoter can be an activatable promoter, which can be activated after the sequence has been delivered to an angiogenic endothelial cell. More preferably, the promoter is designed so that it is activated within the specific environment of an angiogenic endothelial cell. There are a number of phenomena that occur naturally within the environment of an angiogenic endothelial cell, which do not occur within the environment of a quiescent endothelial cell. By taking advantage of the differences between the two cell types, the promoter is specifically designed so that it is activated only in the presence of an angiogenic endothelial cell. The transcription of DNA cartridges could be restricted to a simple or narrow range of cell types using a specific gene promoter. Endothelial cells selectively express several proteins for which genes and their promoters have been elucidated. The promoters of the flt-1 and flk-1 genes of vascular endothelial growth factor (VEGF) receptors, the von Willibrand factor gene promoter (VWF), and the tie family gene promoters have been shown to direct selective expression in endothelial cells when they bind to reporter gene constructs. The following publications are cited to disclose and describe promoters, which are activated in angiogenic endothelial cells. Hatva, E., et al, 1996, "Vascular growth factors and receptors in capillary hemangioblastomas and hemangiopericytomas", (vascular growth factors and receptors in hemangiopericytomas and capillary hemangioblastomas), Am. J. Path. 148: 763-75; Strawn, L. M. , et al. , 1 996"Flk-1 as a target for tumor growth inhibition", (Flk-1 as a target for inhibition of tumor growth), Cancer Res. 56: 3540-5; Milllauer, B., et al. , 1996, "Dominant-negative inhibition of Flk-1 suppresses the growth of many tumor types in vivo" (dominant negative inhibition of Flk-1 suppresses the growth of many types of tumors in vivo), Cancer Res. 56: 1615-20; Sato, T. N., et al. , 1 996, "Distinct roles of receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation", (Various roles of receptor tyrosine kinases Tie-1 and Tie-2 in the formation of blood vessels), Nature 376: 70-4; Ozaki, K., et al. , 1 996, "Use of won Willebrand factor promoter to transduce suicidal gene to human endothelial cells, H UVEC" (Use of the von Willebrand factor promoter to transduce suicide gene to human endothelial cells, HUVEC), Human Gene Therapy: 13 1483- 90; Ronicke, V., et al. , 1 996, "Characterization of endothelial-specific murine vascular endothelial growth factor receptor-2 (Flk-1) protnoter", (Characterization of murine-specific vascular endothelial growth factor receptor-2 (Flk-1) promoter endothelium), Circulation Res. 79: 277-85; Shima, D.T. , et al. , 1996, "The mouse gene for vascular endothelial growth factor, Genomic structure, definition of the transcriptional unit, and characterization of transcriptional and post-transcriptional regulatory sequences", (The mouse gene for vascular endothelial growth factor. of the transcriptional unit and characterization of transcriptional and posttranscription regulatory sequences), J. Biol. Chem. 271: 3877-8; Morishita, K., et al, 1995, "A novel promoter for vascular endothelial growth factor receptor (Flt-1) that confers endothelial-specific gene expression" (A novel promoter for vascular endothelial growth factor receptor (Flt-1 ) that confers the expression of endothelium specific gene), J. Biol. Chem. 270: 27948-53; Patterson, C, et al. , 1995, "Cloning and functional analysis of the promoter for KDR / flk-1, a receptor for vascular endothelial growth factor", (Functional analysis and promoter cloning for KDR / flk-1, a receptor for vascular endothelial growth factor) , J. Biol. Chem. 270: 231 1 1 -8; Korhonen, J., et al. , 1995, "Endothelial-specific gene expression directed by the gene promoter alive," (Expression of specific endothelial gene directed by the gene promoter in vivo), Blood 86: 1 828-35. N. B. Ozaki's reference describes another useful approach - that of expressing herpes simplex virus (TK) thymidine kinase in endothelial cells, and subsequent treatment with the ganciclovir prodrug. Alternatively, the nucleotide sequence may be an antisense sequence which will be ligated to sequences which must be expressed within an angiogenic endothelial cell, thereby blocking the expression of sequences that occur naturally from an angiogenic endothelial cell, which are necessary for the survival of that cell.

FORMATION OF BLOCKS Another aspect of the invention, which can be performed using liposomes or nucleotide / lipid sequence complexes, involves the formation of blood clots. Specifically, the liposome or complex of the invention is designed to have an effect on angiogenic endothelial cells, resulting in the formation of blood clots in the angiogenic blood vessels. Blood clots prevent the flow of nutrients and oxygen to the rest of the vessel, resulting in the death of the vessel and the surrounding tissue.

The basic concept of forming clots within the tumor vasculature in order to eliminate an unwanted tumor has been done using antibodies to focus the tumor vasculature. The present invention could achieve improved results using cationic lipids, said lipids contain an agent which promotes the thrombogenic cascades. For example, the cationic liposomes of the invention could be constructed to encompass human tissue factor (TF), which is the major initiation receptor of the thrombotic process (cascades of blood coagulation). Tumor cells are dependent on the supply of blood. Local disruption of the tumor vasculature will produce an avalanche of tumor cell death. The tumor vascular endothelium is in direct contact with the blood. However, the cells by themselves are out of the bloodstream and for the most part, poorly accessible to many materials injected into the circulatory system. This aspect, as well as other aspects of the invention, work particularly well in that the cells being focused are the angiogenic endothelial cells, which do not transform themselves, that is, they are cells which are unlikely to acquire mutations, which make them resistant to therapy. The tumor cells undergo considerable mutations, and such mutations frequently make the cells resistant to therapy. The results with respect to decreasing tumor size using antibody-directed approach, has been shown by others as follows: Burrows, F.J. , and P. E. Thorpe. Eradication of large tumors in mice with an immunotoxin directed against tumor vasculature, (Eradication of large tumors in mice with an immunotoxin directed against the tumor vasculature), Proc. Nati Acad. Sci. USA 90: 8996-9000, 1993, Huang, X., G. Molema, S. King, L. Watkins, T.S. Edgington, and PE Thorpe, Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature, (Tumor infarction in mice by targeted approach with tissue factor antibody to tumor vasculature), Science 275: 574-550, 1997. In order to perform clot formation with the present invention, it is preferable to form a DNA / cationic lipid complex. The complex will include DNA, which encodes a protein, such as human tissue factor, said protein is a major initiation receptor for thrombotic cascades (blood coagulation). The gene encoding TF is preferably operably linked to a promoter, said promoter is activated in the environment of an angiogenic endothelial cell and is not activated within the environment of a quiescent endothelial cell. Thus, the cationic lipids of the complex will cause the complex to associate with angiogenic endothelial cells. Subsequently, the complex will be carried within the angiogenic endothelial cell and the DNA of the complex will be expressed. The expressed protein will initiate the blood coagulation cascade. When blood clots form within the vessel, the supply of nutrients and additional oxygen to the surrounding tumor cells will be cut off. Subsequently, the tumor cells will die. Variations in the human tissue factor, such as truncated human tissue factor (tTF) can also be used to initiate coagulation. The genetic material encoding tTF and other factors is known (see the reference by Huang, et al., Cited above and the publication cited therein).

MENTAL EXPERI MODELS OF ANGIOGENESIS The present invention was facilitated by the use of rodent models for angiogenesis. Chronic inflammatory diseases, such as asthma and bronchitis, induce remodeling of vasculature and tissue in the mucosa of the respiratory tract. To learn about the pathogenesis of chronic inflammation of the airways, a model was used where chronic inflammation and tissue remodeling occurs in tracheas of rats and mice. Angiogenesis develops in the mucosa of the airways as the result of infection with Mycoplasma pulmonis. In this model, the organisms of Mycoplasma pulmonis cause a persistent infection in the tracheal and bronchial epithelium. The mucosa of the respiratory tract of rats infected with M. pulmonis has several distinct abnormalities: 1) thickening of the epithelium and lamina propria; 2) changes in the cellular composition of the epithelium; 3) angiogenesis; 4) increased sensitivity of the angiogenic vessels for the inflammatory mediator substance P in terms of plasma leakage; 5) leakage induced by substance P of capillaries as well as venules; and 6) increased number of receptors for substance P (NK1 receptors) in capillary endothelial cells. In this model, angiogenesis is driven by chronic inflammation, and blood vessels are more susceptible to inflammatory mediators.

Studies using lectin perfusion to stain the surface of luminal endothelial cells revealed the degree of angiogenesis in rats after infection with M. pulmonis. Numerous capillary-like vessels are present in the tracheal mucosa of infected rats, and these vessels flee following intravenous injection of the inflammatory mediator substance P. In mice, M. pulmonis causes acute lung inflammation that has its peak 6-9 days after of inoculation followed by persistent infection of the respiratory tract. The response of mice to M. pulmonis infection is very species-dependent: for example, C3H species show higher mortality and greater reduction of cytokine tumor necrosis factor alpha than C57 BL species. Some aspects of mucosal remodeling, such as epithelial hyperplasia, have been described in the airways of mice infected with M. pulmonis. In C57BL / 6 mice infected with nasal inoculation of M. pulmonis, the number of tracheal vessels increases dramatically, apparently via growth of new capillaries. In this species, the vasculature of tracheal mucosa already. It is not flat, and small vessels grow perpendicular to the plane of the mucosa. Numerous apparent vascular buds are found in regions of increased vascularity. Thus, the infection of C57BL / 6 mice by M. pulmonis produces chronic inflammation of the respiratory tract with endothelial proliferation, vascular remodeling and angiogenesis. In contrast, in infected C3H / HeNCr mice by nasal inoculation of M. pulmonis, the number of vascular endothelial cells in the tracheal mucosa increases but the number of vessels does not.

The increased vascularity is not due to an increase in the length or number of vessels but to an increase in the diameter of the vessels, and this increase in the size of the vessels is due to a doubling of the number of endothelial cells. The size of individual endothelial cells in infected tracheas does not increase significantly. Circulating antibody levels for M. pulmonis are similar in the two mouse species. Thus, the infection of C3H / HeNCr mice by M. pulmonis produces chronic respiratory infection with vascular remodeling and endothelial proliferation but not a significant increase in the number of vessels, while in C57BL / 6 mice it produces endothelial proliferation and new glasses. In a second model, angiogenesis occurs in tumors that result from the transgenic expression of the SV40 viral oncogene. The "RIP-Tag" transgenic mouse model provides the opportunity to study phenotypic changes in angiogenic endothelial cells in a well-characterized progression of normal tissue to tumors. In the transgenic mouse model "RI P-Tag", the SV-40 virus oncogene, angiene T large (Tag), is driven by a region of the rat insulin promoter (RIP). When inserted into the murine genome, this construct induces Tag expression specifically in pancreatic beta cell beta cells, which are located in approximately 400 islets scattered throughout the pancreas. All the islets of the pancreas in these mice express Tag, however, the islets normally develop up to about 6 weeks of age. After this point, approximately 50% of the islands become hyperplastic.

However, of these hyperplastic islets, a small fraction (< 5%) develops in tumors for approximately 10 weeks. This bottleneck in tumorigenesis seems to be overcome when an islet acquires the ability to induce angiogenesis. Hence, this phase of tumorigenesis has been called "angiogenic change". A similar angiogenic change also seems to exist in other models of murine tumorigenesis, as well as in several human tumors. Thus, the RIP-Tag model provides a well-characterized framework for examining the progression of angiogenesis in tumors.

EXAMPLES The following examples are set forth in order to provide those of ordinary skill in the art with a disclosure and complete description of how to make cationic liposomes and perform the methodology for using such liposomes, and are not intended to limit the scope of what is contemplated. like the invention. Efforts have been made to ensure accuracy with respect to the numbers used (eg, quantities, temperature, etc.) but some experimental errors and deviations must be considered. Unless indicated otherwise, the parts are parts by weight, the molecular weight is the weight average molecular weight; the temperature is in degrees Celsius; and the pressure is at or near atmospheric. It should be noted that each of the Examples below represents a number of experiments, which were performed with the procedures and results being summarized. Those skilled in the art will appreciate that not every experiment gave positive results. However, it is believed that the following leads precisely to the results obtained.

EXAMPLE 1 Distribution of cationic lipids in normal mice The liposomes and / or the plasmid DNA were labeled and the cellular distribution of the labeled complexes were determined at various times after the intravenous injection. The experiments were performed on pathogen-free mice (20-25 g body weight) of both sexes. The small cationic unilamellar vesicle liposomes were prepared from cationic lipid DDAB or DOTAP and the neutral lipid DOPE or cholesterol, labeled with Texas Red or the fluorescent red carbocyanin dye Dil or CM-Dil, and in some cases complexed with DNA of plasmid containing a reporter gene, such as luciferase or β-galactosidase. The endothelial cells were labeled using the fluorescein of lectin from the fluorescent plant Lycopersicon esculentum. Monocytes / macrophages were labeled using fluorescent beads (Duke, 500 nm). Cell nuclei were labeled with DAPI, YO-PRO or dye 33342 from Hoechst. Liposomes or fluorescent DNA-liposome complexes containing 1 0-60 μg of DNA up to 300 μl were injected into non-anesthetized mice via the tail vein. In some experiments, 500 nm fluorescent beads were injected after the complexes. From 5 minutes to 24 hours later, the animals were anesthetized with sodium pentobarbital and then perfused through the left ventricle with fixative (1% paraformaldehyde in phosphate buffered saline) followed by the fluorescent lectin to mark the endothelial surface of the vasculature After perfusion, the tissues were removed and prepared either as complete mounts or cut into sections using a Vibratome or tissue cutter. In addition, some specimens were processed for electron microscopy. The tissues were examined by epifluorescence microscopy or by confocal microscopy. In addition, some specimens were examined by electron transmission microscopy.

Results: In mice examined from 5 minutes up to 24 hours after injection, liposomes or liposome-DNA complexes labeled with CM-Dil or Dil were very abundant in the lungs.

In addition, they were very numerous in endothelial cells of alveolar capillaries. The fluorescence in alveolar capillaries was uniformly distributed in all the lobes of both lungs. further, some fluorescence of CM-Dil or Dil was in intravascular macrophages / monocytes. Following the lung, the liver and spleen had the most liposomes or labeled complexes. In these organs, the fluorescence of CM-Dil or Dil co-localized with the fluorescent beads. In the liver, the fluorescence of CM-Dil or Dil and beads were in Kupffer cells. In the spleen, they were in macrophages. The ovary also had blood vessels heavily labeled with liposomes or complexes labeled with CM-Dil or Dil. Specifically, it was observed that endothelial cells in angiogenic blood vessels of large follicles and corpus luteum of mouse ovary avidly took DDAB: cholesterol (liposomes or) -DNA complexes labeled with CM-Dil or Dil after intravenous injection. These observations were documented photographically (Figure 1). Other ovarian blood vessels contained relatively few labeled complexes. These results were used to infer that angiogenic endothelial cells preferentially take liposomes and liposome-DNA complexes, i.e., that the cationic liposomes used in the experiments were much more likely to associate with endothelial cells undergoing angiogenesis as compared to corresponding endothelial cells who do not experience angiogenesis. Liposomes or labeled complexes were also very abundant in endothelial cells of high endothelial venules (HEV) of lymph nodes and Peyer's patches of the small intestine, while they spread in capillary endothelial cells of these lymphoid organs. Liposomes or labeled complexes were also numerous in capillary endothelial cells of the anterior pituitary, myocardium, diaphragm, adrenal cortex and adipose tissue. The liposomes or labeled complexes were abundant in monocytes / macrophages attached to the venules of the bladder, uterus and fallopian tube. Some venules contained large numbers of marked monocytes / macrophages. In addition, a small proportion of the endothelial cells of arterioles, capillaries and venules were marked in these organs.few labeled liposomes or complexes were associated with capillary endothelial cells of the posterior pituitary, renal medulla, intestinal hairs (ileum), pancreas, and adrenal medulla. Almost no liposomes or labeled complexes were found in endothelial cells in the brain, thyroid gland, renal cortex, pancreatic islets, trachea, or brochios, with the exception of an occasional monocyte / macrophage. Conclusions: The formulation of DDAB liposomes: cholesterol or liposome-DNA complexes labeled with CM-Dil or Dil used in these studies focused on three main cell types: endothelial cells, macrophages and monocytes. The uptake of liposomes or complexes was organ and vessel specific. The majority were captured by capillary endothelial cells of the lung and macrophages of the liver and spleen. Capillary endothelial cells of the ovary, anterior pituitary, heart, diaphragm, adrenal cortex, and adipose tissue were also focused. The blood vessels that took liposomes or complexes in the ovary were sites of angiogenesis. In addition, HEV of lymph nodes and Peyer's intestinal patches were focused. The focus of endothelial cells or macrophages of other organs was less frequent and more variable. Blood vessels of the brain, thyroid, renal cortex, trachea and bronchi were not focused. In addition, the experiments documented that liposomes or complexes did not leak out of the vasculature in most organs. Although they were found in extravascular cells of the spleen, which have blood vessels with a discontinuous endothelium, they did not extravasate in other organs.

Finally, the avid uptake of cationic liposomes and liposome-DNA complexes by blood vessels of large ovarian follicles and corpus luteum indicates that the endothelial cells of the angiogenic blood vessels were preferential uptake sites.

EXAMPLE 2 Uptake of DDAB complexes: cholesterol (liposome or) -DNA in RIP-TAG_5_mice The results of the experiments in Example 1 indicated that the angiogenic blood vessels in the ovarian follicles and corpus luteum avidly captured cationic liposomes and liposome complexes. DNA Accordingly, an experiment was conducted to determine whether the endothelial cells of angiogenic blood vessels of tumors avidly take cationic liposomes or liposome-DNA complexes. The model of transgenic RIP-tag5 of tumors was used, as described under the Model Section of Experimental Mice, Hanahan, D. Heritable formation of pancreatic beta-cell tumors in transgenic mice expressing recombinant insulin / simian virus 40 oncogenes, ( Hereditable of pancreatic beta cell tumors in transgenic mice expressing recombinant insulin / simian virus oncogenes 40). Nature 315: 1 15-22, 1985; Hanahan, D., and J. Folkman. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. (Patterns and mechanisms of emergence of angiogenic change during tumorigenesis). Cell 86: 353-64, 1996. In this model, designated RI P-Tag, the SV-40 virus oncogene, large T antigen (Tag), is driven by a region of the rat insulin promoter (RIP). When inserted into the murine genome, this construct induces the expression of angiotensin T specifically in beta cells of pancreatic islets. An important attribute of this model is that several stages of tumor development, and therefore several stages of angiogenesis, are present concurrently in each RI mouse P-TAG_5_. Although all 300-400 islets express the T antigen, the islands initially develop normally. However, at 6 weeks of age, approximately half are hyperplastic, and of these, a small proportion develop into tumors for 10 weeks. Tumorgenesis seems to coincide with the onset of angiogenesis. This conversion has been designated the "angiogenic change", Folkman, J., k. Watson, D. Ingber, and D. Hanahan, Induction of angiogenesis during the transition from hyperplasia to neoplasia, (Induction of angiogenesis during the transition from hyperplasia to neoplasia), Nature 339: 58-61, 1989., and J. Folkman, Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. (Patterns and mechanisms of emergence of angiogenic change during tumorigenesis), Cell 86: 353-64, 1 996. A similar angiogenic change seems to exist in other murine models of tumorigenesis, as well as in several human tumors, Hanahan, D., and J. Folkman, Patterns and emerging mechanisms of the angiogenic swith during tumorigenesis, (Patterns and mechanisms of emergence of angiogenic change during tumorigenesis), Cell 86: 353-64, 1 996. Methods and materials were used as for Example 1 . Specifically, DDAB: cholesterol liposomes labeled with CM-Dil or Dil were intravenously injected into a tumor-bearing RIP-TAG_5_mouse, and DDAB: cholesterol-DNA complexes labeled with CM-Dil or Dil were injected intravenously into another RIP-TAG_5_mouse. The distribution of the liposomes or complexes in angiogenic blood vessels of pancreatic islet cell tumors was examined 24 h after injection and compared with that in pancreatic islet vessels of normal mice. Results: Two novel observations were made: (1) the liposomes or complexes were taken by endothelial cells of angiogenic blood vessels without leaking through the endoteio, and (2) the endosomal uptake of the liposomes or complexes was higher in vessel endothelial cells Angiogenic blood cells than in endothelial cells of pancreatic islet noramles. (Figure 2 is from a tissue specimen). Conclusions: This experiment gave consistent results with the preferential uptake of DDAB liposomes: cholesterol or liposome-DNA complexes by tumor angiogenic vessels. Before repeating the experiment (1), the fluorescence intensity of the liposome-DNA complexes was increased, (2) the methods for locating cationic liposome and liposome-DNA complex sites in tumors of RI P mice were improved -Tag; and (3) greater familiarity with the structure and function of angiogenic blood vessels was obtained in pancreatic islet cell tumors in RI P-TAG_5_mice.ptake of DOTAP complexes: cholesterol-DNA in RI P-TAG_2_mice Purpose: The fluorescence intensity of the omasome-DNA complexes has been increased by using Texas-DHPE Red instead of Dil; the method for preparing the pancreas of RIP-TAG_2_mice was improved to locate sites of uptake of fluorescent complexes of cationic-DNA liposomes; and the structure and function of angiogenic blood vessels in pancreatic islet cell tumors in RI P-TAG_2_mice have been studied. With these improvements, experiments of the type described in Example 2 were performed to determine how many cationic liposomes and lipid-DNA complexes were captured. Methods: Small unilamellar vesicle liposomes of DOTAP: cationic cholesterol, labeled with Texas Red-DHPE, were prepared. The liposome-DNA complexes were prepared in a total lipid: DNA ratio of 24: 1 (nmol / μg) in 5% glucose, using 60 μg of plasmid DNA in 300 μ. The complexes (300 μl) were injected into the tail veins of transgenic RI P1 -TAG_2_C57BL / 6 mice without anesthetizing and normal C57BL / 6 mice without anesthetizing. For four hours after the injection of the complexes, the mice were anesthetized by intraperitoneal injection of Nembutal 50 mg / kg. The vasculature was fixed by perfusion of 1% paraformaldehyde through the ascending aorta, and the luminal surface of the vasculature was stained by perfusion of green fluorescent Ictin, Thurston, G., P. Baluk, A. Hirata and D. M . McDonald, Permeability-unrelated changes revealed at endothelial cell borders in inflamed venules by lectin binding (Changes unrelated to permeability revealed at endothelial cell borders in inflamed venules by lectin ligation, Am J Physiol 271: H2547-2562, 1 996. Full tissue assemblies or sections of Vibratome were mounted in Vectashield, and the vessels were examined using a Zeiss Axiophot fluorescence microscope or a Zeiss LSM 41 0 confocal microscope equipped with a krypton-argon laser and optimized photomultiplier tubes. on Kodak Ektachrome film (ASA 400) or as digital confocal image files Results: This experiment clearly showed the avid uptake of DOTAP: cholesterol-DNA complexes tagged with Texas Red by angiogenic endothelial cells in pancreatic tumors of RIP1 mice -TAG_2_. The uptake by tumor vessels far exceeded the uptake of complex coughs by the corresponding endothelial cells of normal pancreatic islets (compare Figures 3 and 4). The tumors were easily distinguished from adjacent tissues due to the heavy labeling of their blood vessels with the red fluorescent liposome complexes. The geometry of the vasculature of the tumors was variable, varying from the typical pattern of normal islets to a dense, tortuous, anastomosing network of sinusoidal vessels conspicuously larger and more densely packed than in normal islets. In the latter case, the vasculature resembles that of the corpus luteum. The intensity of marking of tumor vessels was poorly related to the size of the tumor. Larger tumors had the highest score. Some blood vessels in small or medium sized tumors had focal, plump, and aneurysm-like protrusions. These sites were particularly conspicuous due to the presence of unusually numerous points marked with Texas Red, which are presumed to be endosomes. The Red Texas marking of these sites was greater than that of adjacent vessels. It seems that these structures could be hair buds. The structures were not found in large tumors that had a complex, dense vasculature, where the vessels were uniformly and heavily weighted. There is no evidence of extravasation of complexes marked with Texas Red in tumors. In addition, no complex marked with Texas Red was seen within the piles of extravascular erythrocytes in the tumors. Heavy labeling of the tumor vasculature resembled that of the ovarian corpus luteum during the early stage of its development.ptake of cationic liposomes and liposome-DNA complexes by angigogenic blood vessels in tumors and chronic inflammation Purpose: Experiments of the type described in Example 3 were performed to extend the observations to other models of angiogenesis. These experiments also addressed the question of whether DNA had to be present for cationic liposomes to focus on angiogenic blood vessels. Four animal models of angiogenesis were examined with respect to whether there is a preferential uptake of DOTAP liposomes: cholesterol or liposome-DNA complexes by angiogenic blood vessels. Models: tumor model RI P 1 -TAG_2_. Mice were produced Transgenic C57BL / 6 and the phenotype was obtained at birth for PCR analysis. The mouse model was described before. HPV tumor model. Transgenic HPV mice (human papilloma virus) were produced and the birth phenotype was obtained for PCR analysis. Non-transgenic littermates were used as controls. In this model, the oncogene of the human papilloma virus is driven by a region of the keratin promoter 14. When inserted into the murine genome, this construct induces the expression of HPV specifically in epidermal cells. All the transgenic mice developed dysplasia accompanied by angiogenesis in the skin of the upper thorax or ears, and a small proportion develops tumors. Infection model with Mycoplasma pulmonis in mice. This infection results in chronic inflammation of the airways accompanied by angiogenesis in the airway mucosa. After anesthesia (87 mg / kg ketamine and 13 mg / kg xylazine injected intraperitoneally), male and female 8-week-old, pathogen-free mice or C57BL / 6 mice (both from Charles River) were inoculated intranasally with 3x104 colony-forming units of Mycoplasma pulmonis (species 5782C-UAB CT7), in a volume of 50 μl. The pathogen-free mice served as controls and were inoculated with sterile broth. The infected and control mice were separately caged under barrier conditions. Levels of antibody serum were measured for M. pulmonis at the end of the experiment (Microbiological Associates, Bethesda MD). Mice were studied 1 to 8 weeks after infection. Infection model with Mycoplasma pulmonis in rats. As in mice, this infection causes chronic respiratory disease, a characteristic of which is angiogenesis in the airway mucosa. After anesthesia (40 mg / kg of ketamine and 8 mg / kg of xylazine injected intraperitoneally), 8-week-old, pathogen-free (Charles River) male Wistar rats were inoculated intranasally daily for three consecutive days with Mycoplasma. pneumonia of the species 5782C4 in a volume of 200 μl. Pathogen-free rats inoculated with broth served as controls. The infected and control rats were separately caged under barrier conditions. Levels of antibody serum were measured for M. pulmonis and other pathogens at the end of the experiment (Microbiological Associates, Bethesda MD). Methods: DOTAP cationic liposomes were prepared. cholesterol, labeled with DHPE-Texas Red as described under Example 3. The liposomes were injected into a vein of the tail of mice at a dose of 360 nmol of total lipid in a volume of 100 μl in 5% glucose. The rats were infected via the femoral vein. The liposome-DNA complexes were prepared at a total lipid: DNA ratio of 24: 1 in 5% glucose, using 60 μg of plasmid DNA in 200-300 μl. Liposomes or complexes (200-300 μl) were injected into a tail vein of RIP-TAG_2_, HPV or infected with M. pulmonls mice. Mice free of non-transgenic pathogens were used as controls. In 20 minutes or 4 hours after the injection, the mice or rats were anesthetized by intraperitoneal injection of Nembutal 50 mg / kg. The vasculature was fixed by perfusion of 1% paraformaldehyde through the ascending aortaminal surface of the vasculature was stained by green fluorescent lectin perfusion, Thurston, G., P. Baluk, A. Hirata, and D. M. McDonald. The changes related to permeability were revealed in borders of endothelial cells in inflamed venules by lectin ligation. Am J Physiol 271: H2547-2562, 1996. The whole-tissue assemblies or sections of Vibratome were mounted in Vectashield, and the vessels were examined with a Zeiss fluorescence microscope or confocal microscope. The amount of uptake of liposomes or fluorescent complexes was quantified by confocal microscopy. Briefly, a series of 12 confocal images separated by 2.5 μm was collected in the focal axis (z) in the rostral region of the trachea in the Texas Red and fluorescein channels using 20x NA 0.6 (Zeiss) lenses and standardized size adjustments of the tiny confocal hole, gain of photomultiplier tube and laser energy. The projections were generated from the series of images showing the vessels (fluorescein-L. esculentum) and liposomes (Texas Red) separately. Using the confocal counting program, approximately 200 μm2 regions were defined in area in the vessel images, then the average fluorescence of the corresponding regions of the liposome image was measured. Support intensity was determined by measuring fluorescence in selected regions adjacent to the vessels. Measurements were made in 25 vessels per trachea and 4 tracheas per group (n = 4). The significance of the differences was assessed using the Student's t test. The tissues prepared for electron transmission microscopy were processed as previously described, McDonald, DM Endothelial gaps and permeability of venules of rat tracheas exposed to inflammatory stimuli, (Endothelial orifices and permeability of rat tracheal venules exposed to inflammatory stimuli), Am. J. Physiol. 266: L61-L83, 1 994. Briefly, perfusion of primary fixative (3% glutaraldehyde in 75 mM cacodylate buffer, pH 7. 1, plus 1% sucrose, 4% PVP, 0.05% CaCl 2, and 0.075% H2O2) for 5 min at room temperature, was followed by perfusion of a secondary fixative (3% glutaraldehyde in 75 mM cacodylate buffer, pH 7.1, containing 0.05% CaCl2, 1% sucrose, and 4% PVP) for 5 min. The tissues were allowed to settle in situ for 1 h at room temperature, then they were removed and left overnight in secondary fixative at 4 ° C. The tissues were cut with a razor blade or sliced with a tissue cutter, post-fixed in osmium (2% OsO4 in 1 00 mM cacodylate buffer, pH 7.4, for 1 8 h at 4 ° C), washed in H2O (1 8 h at 4 ° C), and stained en bloc with uranyl acetate (aqueous, 37 ° C for 48 hours). The tissue was then dehydrated through acetone, inflitred and embedded in epoxy resin. Ultra-thin sections were cut with an ultra-humillome, mounted on single-aperture specimen grids, and examined with an EM-1 or Zeiss electron microscope. Results: The experiments revealed that DOTAP: cholesterol liposomes labeled with Texas Red, in the absence of DNA, were selectively targeted to tumor angiogenic endothelial cells in RIP1 -TAG_2_mice, similar to the previous findings with DOTAP-cholesterol complexes. DNA marked with Texas Red and DDAB complexes: cholesterol-DNA labeled with Dil. This and subsequent experiments in transgenic RIP 1 -TAG_2_mice confirmed that the uptake of cationic liposomes by angiogenic blood vessels of hyperplastic islets and tumors far exceeded that of the corresponding normal vessels (Figures 5, 6, 7 and 8). In some vessels of hyperplastic islets and small tumors, liposomes were taken by endothelial cells in focal regions (Figure 8), whereas in larger tumors, uptake was more generalized (Figure 6). It was thought that focal regions of uptake are possible sites for new vessel growth (Figure 8). Because this property of cationic liposomes or liposome-DNA complexes had the potential practical use of selectively delivering substances to angiogenic endothelial cells, it is considered desirable to determine if this property of angiogenic endothelial cells in tumors was shared by endothelial cells in other sites of pathological angiogenesis. This questión was conducted in experiments where the uptake of DOTAP liposomes labeled with Texas Red by angiogenic endothelial cells was examined in the trachea of mice with Mycoplasma pulmonis infection, which causes chronic inflammation of the respiratory tract, a characteristic of which is angiogenesis (compare figures 9 and 1 0). It was found that angiogenic endothelial cells in regions of chronic inflammation were sites of unusually high uptake of cationic liposomes (Figure 10). Specifically, the vessels in the tracheae of mice infected with M. pulmonis had an unusually large amount of uptake. The confocal microscopic measurements of angiogenic blood vessels showed that the infected mice had 20 to 30 times more uptake than the controls (Figure 11). Some angiogenic vessels had 1 00 times as much uptake. Electron microscopy and confocal studies of angiogenic endothelial cells in mice infected with M. pulmonis suggested that cationic liposomes associate first with (Figure 12) and then internalize into endosomes (Figure 13). Similarly, cationic liposomes were avidly taken by angiogenic blood vessels in ovarian follicles and corpus luteum in mice, dysplastic skin from transgenic HPV mice and tracheae from rats with angiogenesis due to infection with M. pulmonis. Conclusions: These experiments confirmed that cationic liposomes and liposome-DNA complexes are preferentially focused on the angiogenic endothelial cells of tumors and sites of chronic inflammation. The present invention is shown and described herein in which the most practical and preferred embodiments are considered. However, it is recognized that deviations can be made therefrom, which are within the scope of the invention, and that obvious modifications will occur to a person skilled in the art upon reading this disclosure.

BIBLIOGRAPHY The following documents are cited by letter and number throughout the text above. Its contents are incorporated herein expressly by reference: A. US Patent No. 4,897,355 B. US Patent No. 4,393,448 C. US Patent No. 5,328,470 D. WO93 / 12240 E. WO / 91/06309 F. Huang, et al. Cancer Res.52, 5135 (1992). G. Rosenecker et al., PNAS 93, 7236 (1996) H. Abdi, et al., In Vitro Cell Dev. Biol. Anim.31,310 (1995). I. Chonn et al., Curr. Opin. Biotechnol.6, 698 (1995). J. Green et al., Adv. Exp. Med. Biol.383, 83 (1995). K. Hung, et al., Gene 159, 65 (1995). L. Ledley, et al., Hum. Gene Ther.6, 1129 (1995). M. Mori, et al., Cancer Chemother. Pharmacol.35, 447 (1995). N. Volm, et al., Et al, Curr. Opin. Oncol.7, 429 (1995). O. Litzinger, et al., Biochim. Biophys. Acta 1190, 99 (1994). P. Northfelt, et al., Drugs 48, 569 (1994). Q. Oku, et al., Crit. Rev. Ther. Drug Carrier Sys. 11, 231 (1994). R. Gabizon, et al., Ann Biol. Clin (Paris) 51, 811 (1993). S. Huang, et al, Am. J. Pathol. 143, 10 (1994). T. Mori, et al., Pharm. Res. 10, 507 (1993).

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Literature cited in the Examples: 1 Arfors. K.-E. , G. Rutili. and E. SyensjÜ. Microvascular transport of macromolecules in normal and inflammatory conditions. (Microvascular transport of macromolecules under normal and inflammatory conditions). Acta Physiol. Scand. Suppl. 463: 93-103, 1 979. 2. Baluk, P., and D. M. MeDonald. The b2-adrenergic receptor agonist formsoterol reduces microvascular leakage by inhibiting endothelial gap formation. (Formoterol b2-adrenergic receptor agonist reduces microvascular leak by inhibiting the formation of endothelial orifices). Am. J. Physiol. 266: L461-L468, 1 994. 3. Bowden, J. J., A. Garland, P. Baiuk, P. Lefevre, E. Grady, S. R. Vigna, N. W. Bunnett, and D. M. McDonald. Direct observation of substance P-induced internalization of neurokinin 1 (NK 1) receptors at sites of inflammation. (Direct observation of hormone induced induction by substance P of neurokinin 1 (NK1) receptors at sites of inflammation). Proc. Nati Acad. Sci. USA 9 1: 8964-8968, 1 994. 4. Brigham, K., B. Meyríck, B. Christman. M. Magnuson, G. King, and L. C. Berry. In vivo transfection of murine lungs with a functioning prokaryotic gene using a liposome vehicle. (Live transfection of murine lungs with a functioning prokaryotic gene using a liposome vehicle). Am. J. Med. Sci. 298: 278-281, 1989.

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(Hereditary formation of pancreatic beta cell tumors in transgenic mice expressing recombinant insulin / simian virus oncogenes 40) Nature 31 5: 1 15-22, 1 985. 9. Hanahan, D., and J. Folkman Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. (Patterns and emerging mechanisms of angiogenic displacement during tumorigenesis) Cell 86: 353-64, 1 996. 1 0. Lundberg, J. M., and A. Saria. Capsaicin-induced desensitization of airway mucosa to cigarette smoke, mechanical and chemical irritants.

(Desensitization induced by capsaicin from airway mucosa to cigarette smoke, mechanical irritants and chemicals). Nature 302: 251-253,1983. 1 1. Majno, O., and G. E. Palade. Studies on inflammation. 1 . The effect of histam ine and serotonin on vascular permeability: an electron microscopic study. (The effect of histamine and serotonin on vascular permeability: a microscopic study of electrons). J. Biophys. Biochem. Cytol. 1 1: 571-604, 1 96 1. 12. McDonald, D.M. Neurogenic inflammation in the rat trachea. 1 . Changes in venules, leucocytes, and epithelial cells. (Neurogenic inflammation in the trachea of rats 1. Cam bios in venules, leukocytes and epithelial cells). J. Neurocytology 17: 583-603, 1988. 13. McDonald, D.M. The ultrastructure and permeability of tracheobronchial blood vessels in health and disease. (The ultrastructure and permeability of tracheobronchial blood vessels in health and disease). Eur. J. Respir. Dis. 3 (Suppl 12): 572s-585s, 1 990. 14. McDonald, D.M. Endothelial gaps and permeability of venules of rat tracheas exposed to inflammatory stimuli. (Endothelial orifices and permeability of rat tracheal venules exposed to inflammatory stimuli). Am. J. Physiol. 266: L61-L83, 1 994. 1 5. McDonald. D. M., and A. Goldfein. The relation of lutein cell Golgi apparatus morphology to ovarian progesterone secretion during the rat estrous cycle. (The relationship of the morphology of the Golgi apparatus of lutein cells with ovarian progesterone secretion during the rat skeletal cycle) Anat. Rec. 151: 385, 1965. 16. McDonald, D. M., K. Seiki, M. Prizant, and A. Goldfein. Ovarian progesterone secretion in relation to the lutein cell Golgi apparatus during rat estrous cycle. (Secretion of ovarian progesterone in relation to the Golgi apparatus of lutein cells during the estrous cycle of rats) Endocrinology 85: 236-243, 1 969. 17. Nabel. E. G., D. Gordon, Z. Y. Yang, L. Xu. H. San, G. Plautz, B. Y. Wu, X. Gao, L. Huang, and O. J. Nabel. Gene transfer in vivo with DNA-liposome completes lack of autoimmunity and gonadal localization. (Gene transfer in vivo with complete DNA liposomes lacks autoimmunity and gonadal location). Hum. Gene Ther. 3: 649-56, 1992. 1 8. Philip, R., D. Liggitt, M. Philip, P. Dazin, and R. J. Debs. In vivo gene delivery: Efficient transfection of T lymphocytes in adult mice. (Delivery of genes in vivo: efficient transfection of T lymphocytes in adult mice) J. Biol. Chem. 268: 16087, 1 993. 19. Thurston, G., P. Baluk, A. Hirata, and D. M. McDonald. Permeability-related changes revealed at endothelial cell borders in inflamed venules by lectin binding. (Changes related to permeability revealed in limits of endothelial cells in inflamed venules by lectin ligation). Am J Physiol 271: H2547-2562, 1 996.

. Tsukada, K., T. Matsushima, and N. Yamanaka Neovascularization of the corpus luteum of rats during the estrus cycle. (Neovascularization of the corpus luteum of rats during the estrus cycle). Pathol Int 46: 408-416, 1 996. twenty-one . Umeno, E., J. A. Nadel, and D. M. McDonald. Neurogenic inflammation of the rat trachea: fate of neutrophils that adhere to venules. (Neurogenic Inflammation of Rat Trachea: Death of Neutrophils Adhering to Venules) J. Appl. Physiol. 69: 2131-2136, 1990. 22. Zhu, N., D. Liggitt, Y. Liu, and R. Debs. Systemic gene expression after intravenous DNA delivery into adult mice. (Systemic gene expression after intravenous delivery of DNA in adult mice). Science 261: 209-21 1, 1 993. 23. Bowden, J. J., T. R. Schoeb, J. R. Lindsey, and D. M. McDonald. Dexamethasone and oxytetracycline reverse the potentiation of neurogenic inflammation in airways of rats with Mycoplasma pulmonis infection. (Dexamethasone and oxytetracycline reverse the potentiation of neurogenic inflammation in airways of rats with Mycoplasma pulmonis infection). Am J Respir Crit Care Med 1 50: 1 391-401, 1994. 24. McDonald, D. M. Infections intensify neurogenie plasma extravasation in the airway mucosa. (Infections intensify the extravasation of neurogenic plasma in the airway mucosa). Am Rev Respir Dis 146: S40-4, 1992.

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Claims (10)

1 . An inhibitor / lipid complex, comprising: cationic lipids; and an angigenesis inhibitor, wherein the complex is characterized by having, in the blood, higher affinity for angiogenic endothelial cells as compared to corresponding normal endothelial cells.

2. The complex of claim 1, further comprising a detectable label.

3. A nucleotide / cationic lipid complex, comprising: cationic lipids; and a nucleotide sequence, said sequence affecting angiogenesis, wherein the complex is characterized by having, in the blood, greater affinity for angiogenic endothelial cells as compared to corresponding normal endothelial cells.

4. The complex of claim 3, wherein the nucleotide sequence is a DNA sequence operably linked to a promoter, which is selectively activated within an angiogenic endothelial cell, said promoter being optionally selected from the group consisting of a promoter. of gene FLT-1, a promoter of gene FLK-1 and a gene promoter of Factor von Willbrand.

5. The complex of claim 3, wherein the nucleotide sequence is an antisense sequence, said sequence selectively disrupting the expression of genetic material within angiogenic endothelial cells.

6. A method for diagnosing an angiogenesis site, comprising: administering to a mammal complexes comprising cationic lipids and a detectable label wherein the complexes have, in the blood, higher affinity for angiogenic endothelial cells as compared to corresponding normal endothelial cells; allow the complexes to be selectively associated with angiogenic endothelial cells; and detect the brand and thereby determine an angiogenesis site based on an accumulation of the brand on the site. The method of claim 6, wherein the mammal is a human and the detectable tag is selected from the group consisting of fluorescent tags, histochemical tags, immunohistochemical tags and radioactive tags, further comprising the method: isolating tissue at the site of the accumulation of the brand; and analyze the isolated tissue. The method of claim 6, further comprising: surgically opening the mammal to expose an area that is believed to contain a high concentration of angiogenic endothelial cells; exposing the area to a wavelength of light which causes the detectable mark to emit fluorescent light rays; Remove the tissue identified by the fluorescent mark. 9. A composition for selectively affecting angiogenic endothelial cells, comprising: cationic lipids and a substance that affects angiogenesis, wherein the composition has, in the blood, higher affinity for angiogenic endothelial cells as compared to corresponding normal endothelial cells; wherein the composition is selectively associated with angiogenic endothelial cells of an angiogenic blood vessel for a time and in a manner such that the composition enters the angiogenic endothelial cells. The composition of claim 9, wherein the composition is formulated for administration by injection into the circulatory system of the mammal and further, wherein the composition has, in the blood, five times or more affinity for angiogenic endothelial cells as compared with corresponding normal endothelial cells; wherein the composition is comprised of 5 mol% or more of cationic lipids; wherein the substance which affects angiogenesis is an inhibitor of angiogenesis.