MXPA97006188A - Inhibition of angiogenesis using interleucin - Google Patents

Abstract

The present invention relates to the use of interleukin 12 for the preparation of a medicament for the treatment of undesirable or uncontrolled angiogenesis, which is important in the treatment of diabetic retinopathy and degeneration.

Description

INHIBITION OF ANGIOGENESIS USING INTERLEUCINE 12 The present invention relates to the prevention of diseases mediated by undesirable angiogenesis. More particularly, the present invention relates to the use of Interleukin 12 (IL-12) for the manufacture of medicaments for preventing undesirable angiogenesis, particularly for the treatment of diseases dependent or associated with angiogenesis. Interleukin 12 (IL-12), formerly called killer cell stimulator factor (Kobayashi M., et al., J. Exp. Med. 170: 827-845, 1989) and cytotoxic lymphocyte maturation factor (Stern). AS, et al., Proc. Nati, Acad, Aci, USA 87: 6808-6812, 1990), has potent antitumor and antimetastatic activity in several murine tumor models (Brunda MJ, et al., J. Exp. Med. 178: 1223-1230, 1993; Nastala CL, et al., J. Immunol., 153: 1697-1706, 1994). Although the mechanism through which IL-12 exerts its antitumor effects is not completely understood, it has been shown that IL-12 induces a variety of biological effects on natural killer and T cells in vitro (Manetti, R., et al., J. Exp. Med. 179: 1273-1283, 1994; Wu C.

Y., et al., J. Immunol. 151: 1938-1949, 1993; Tripp C. S., et al.,: Proc. Nati Acad. Sci. USA 90: 3725-3729, 1993; Seder R.

FEF: 25363 A., et al., Proc. Nati Acad. Sci. USA 90: 10188-10192, 1993; Bloom E. T., et al., J. Immunol. 152: 4242-4254, 1994; Cesano A., et al., J. Immunol. 151: 2943-2957, 1993; Chan S. H., et al., J. Im unol. 148: 92-98, 1992). The activation of cytotoxic T lymphocytes by IL-12 is considered crucial in its antitumor activity (Brunda M. J., et al., J. Exp. Med. 178: 1223-1230, 1993). The anti-tumor effect in IL-12 is partially maintained in mice with severe combined immune deficiency (SCID) and nudity, both of which are deficient in T cells, and in euthymic mice deficient in CD8 * (Brunda MJ, et al., J. Exp. Med. 178: 1223-1230, 1993; O 'Toole M., et al., J. Immunol., 150: 294A, 1993). These results demonstrate that IL-12 has potent anti-tumor and anti-metastatic effects in vivo against murine tumors and also demonstrates the critical role of CD8 + T cells in mediating the antitumor effects against subcutaneous tumors. The present invention provides the use of Interleukin 12 for the preparation of effective medicaments for inhibiting undesirable angiogenesis. It has been observed that IL-12 inhibits the growth of a broad spectrum of tumors in vivo, but has no direct effect on tumor cells in vitro. In addition, in mice deficient in T cells the antitumor activity of IL-12 is not completely abrogated, suggesting that IL-12 has anti-angiogenic properties. IL-12 induces a strong inhibition of neovascularization. This effect is not mediated by a specific cell type of the immune system. Interferon gamma (IFN-α) seems to play a critical role in mediating the antiangiogenic effects of IL-12. The surprising recognition of the antiangiogenic properties of IL-12 is central to the appropriate design of treatment protocols including their co-administration with other inhibitors of neovascularization. Accordingly, the present invention provides the use of Interleukin 12 for the manufacture of medicaments for the treatment of diseases mediated by undesirable or uncontrolled angiogenesis, especially for the treatment of diseases wherein the disease mediated by undesired or uncontrolled angiogenesis. is neovascularization, particularly retinal / choroidal neovascularization. Another object of the present invention is to provide the above use wherein retinal / choroidal neovascularization is associated with diabetic retinopathy or where retinal / choroidal neovascularization is associated with macular degeneration. Another object of the present invention is to provide the use of Interleukin 12 for the manufacture of medicaments for the treatment of diseases mediated by uncontrolled angiogenesis., wherein the disease mediated by undesired or uncontrolled angiogenesis is corneal neovascularization. Still another object of the present invention is to provide the use of Interleukin 12 for the manufacture of a medicament for the treatment of diabetic retinopathy and for the treatment of macular degeneration. In addition, the invention comprises the use of Interleukin 12 for the manufacture of medicaments for the treatment of diseases mediated by undesirable or uncontrolled angiogenesis, wherein the diseases originate from solid tumors or from tumors that are born in the blood and their metastases . Still another object of the present invention is to provide the use of Interleukin 12 for the preparation of a medicament for the treatment of all forms of proliferative vitreoretinopathy, whether or not associated with diabetes. The above medicaments may contain one or more additional angiogenesis inhibitors. Also part of this invention is Interleukin 12 and the use of Interleukin 12 for the treatment of a disease as mentioned above. In addition, the invention comprises Interleukin 12 or the use of Interleukin 12 in combination with one or more additional angiogenesis inhibitors, for the treatment of the above diseases.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Effect of recombinant murine IL-12 on mouse corneal neovascularization induced by bFGF. These photographs represent the corneas of C57BL / 6 and SCID mice treated either with vehicle (control) or IL-12, 5 days after the implantation of the basic growth factor pills of the fibroblasts (P). There are new prominent vessels in the control corneas, while almost no vascular response is observed after treatment with IL-12. (Note that SCID mice have pre-existing iris vessels which are visible through the cornea since their iris is hypopigmented, so the vessels seen in the panel treated with IL-12 are in the plane of the iris and are not are corneal vessels induced by the growth factor pill of the basic fibroblasts). Figure 2. Angiogenic response 5 days after the implantation of the basic fibroblast growth factor pills in C57BL / 6 mice. The treatment consisted of either vehicle (21 corneas), IL-12 (30 corneas) or a monomeric mixture of IL-12 (10 corneas) as described below. The length of the vessels in mm and the number of clock hours were represented as the mean ± SD. Figure 3. Effects of IFN-α antibodies on the inhibition induced by IL-12 of mouse corneal neovascularization. Male C57BL / 6 mice were treated either with a single intraperitoneal injection of rat antibodies XMG1.2 IFN-? or rat IgG as described below. The length of the vessels and the clock hours of the neovascularization were measured at 5 days. This experiment was repeated twice separately with similar results. The data are presented as the mean ± SDM of at least 13 corneas. Figure 4. The effect of IFN-α treatment on mouse corneal neovascularization induced by the growth factor of basic fibroblasts. Male C57BL / 6 mice were treated either with intraperitoneal bolus injections of IFN-α. starting on the day of implantation of the pill or by continuous infusion of IFN beginning 3 days before the implantation of the pill. The length of the vessels and the clock hours were measured on day 5 after the implantation of the growth factor pill of the basic fibroblasts and were represented as the mean ± SD of 10 corneas in each group. Figure 5. Effect of IL-12 and AGM-1470 on the growth of Lewis lung carcinoma. Male C57BL / 6 mice were inoculated with Lewis lung carcinoma on day 0 and treatment with either saline, IL-12 or AGM-1470 started after the tumor became measurable. The treatment protocol and measurement procedures are described below. The results are representative of a single experiment of 4 animals in each group. Figure 6. Effect of IL-12 and AGM-1470 on metastasis of the spontaneous lung surface of Lewis lung carcinoma. The treatment protocol and evaluation procedure are described below. The results are representative of a single experiment of 4 animals in each group. Angiogenesis is essential for tumors and metastasis to extend beyond a few millimeters in diameter (Folkman J., N. Engl. J. Med. 285: 182-1186, 1971). Strategies to prevent the development of new blood vessels in tumors and metastases have been effective in suppressing the growth of these tumors (Millauer B., et al., Nature 367: 576-579, 1994; Kim K. J., et al., Nature 362: 841-844, 1993). To determine whether IL-12 has angiogenic properties, IL-12 was evaluated in a mouse corneal neovascularization model induced by the growth factor of basic fibroblasts. The results show that IL-12 is a potent inhibitor of angiogenesis in vivo and that this effect is mediated by IFN-α. Angiogenesis is the generation of new blood vessels in a tissue or organ. Under normal physiological conditions, humans or animals only experience angiogenesis in very specific and restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometrium and placenta. The control of angiogenesis is a highly regulated system of angiogenic stimulators and inhibitors. The control of angiogenesis has been found altered in certain disease states and, in many cases, the pathological damage associated with the disease is related to uncontrollable angiogenesis. It is thought that both controlled and uncontrolled angiogenesis proceed in a similar manner. The endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which lighten the blood vessels, then project through the basement membrane. Angiogenic stimulants induce endothelial cells to migrate through the eroded basement membrane. The cells that migrate form a "bud" out of the blood vessels of origin, where the endothelial cells undergo mitocis and proliferate. The endothelial buds fuse together to form capillary rings, creating the new blood vessel. In the disease state, the prevention of angiogenesis could remove the damage caused by the invasion of the new microvascular system. Persistent angiogenesis occurs in a multiplicity of disease states, tumor growth (both as a primary tumor and metastasis) and abnormal growth of endothelial cells, and supports the pathological damage observed in those conditions. The diverse pathological states created due to unregulated angiogenesis have been grouped as diseases dependent on angiogenesis or associated with angiogenesis. Therapies aimed at the control of angiogenic processes could lead to the abrogation or mitigation of these diseases. An example of disease mediated by angiogenesis is ocular neovascular disease. This disease is characterized by the invasion of new blood vessels in structures of the eye such as the retina or cornea. This is the most common cause of blindness and is implicated in approximately twenty eye diseases. Age-related macular degeneration, associated visual problems are caused by an internal growth of choroidal capillaries through defects in Bruch's membrane with proliferation of fibrovascular tissue beneath the retinal pigment epithelium. Angiogenic damage is also associated with diabetic retinopathy, premature retinopathy, rejection of cornea graft, neovascular glaucoma and retrolental fibroplasia. Other diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, excessive use of contact lenses, atopic keratitis, upper limbic keratitis, dry pterygium keratitis, sjogrens, acne rosacea, filectenulosis, syphilis , Mycobacterial infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoal infections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, Systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis, Johnson's disease of Steven, perifigoid radial keratotomy, and rejection of corneal graft.

Diseases associated with retinal / choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, ozone cell anemia, sarcoid, syphilis, elastic pseudoxanthoma, Pagets disease, venereal occlusion, arterial occlusion, obstructive disease of the carotid, uveitis / chronic vitritis, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, Eales disease, Bechets disease, infections that cause retinitis or choroiditis, presumed ocular histoplasmosis, Bests disease, myopia, stings optics, Stargarts disease, partial planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post laser complications. Other diseases include, but are not limited to, diseases associated with rubeosis (neovascularization of the angle) and diseases caused by abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy. Another disease in which angiogenesis is thought to be involved is rheumatoid arthritis. The blood vessels in the synovial lining of the joints undergo angiogenesis. In addition to forming new vascular networks, endothelial cells release factors and species that react with oxygen that lead to tissue growth and cartilage destruction. The factors involved in angiogenesis can actively contribute to, and help maintain, the chronically inflamed state of rheumatoid arthritis. Factors associated with angiogenesis may also have a role in osteoarthritis. The activation of chondrocytes by angiogenic related factors contributes to the destruction of the joint. At a later stage, angiogenic factors could promote the formation of new bones. The therapeutic intervention that prevents bone destruction could stop the progress of the disease and provide relief to people suffering from arthritis. Chronic inflammation may also involve pathological angiogenesis. Disease states such as ulcerative colitis and Crohn's disease show histological changes with the internal growth of new blood vessels in inflamed tissues. Bartonellosis, a bacterial infection found in South America, can result in a chronic stage characterized by the proliferation of vascular endothelial cells. Another pathological role associated with angiogenesis is found in atherosclerosis. Plaques formed within the lumen of blood vessels have been shown to have angiogenic stimulating activity.

One of the most frequent angiogenic diseases of childhood is the hemangioma. In most cases, the tumors are benign and are reversed without intervention. In the most severe cases, the tumors progress to large and infiltrating cavernous forms and create clinical complications. The systemic forms of hemangiomas, hemangiomatosis, have a high mortality rate. There are hemangiomas resistant to therapy that can not be treated with the therapeutic agents currently in use. Angiogenesis is also responsible for the damage found in hereditary diseases such as Osler-Weber-Rendu disease, or hereditary hemorrhagic telangiectasia. This is a hereditary disease characterized by multiple small angiomas, tumors of the blood or lymphatic vessels. Angiomas are found on the skin and mucous membranes, often accompanied by epistaxis (nosebleeds) or gastrointestinal hemorrhage and sometimes with pulmonary or hepatic arteriovenous fistula. Angiogenesis is prominent in the formation of solid tumors and metastases. Angiogenic factors have been found associated with several solid tumors such as rhabdomyosarcomas, retinoblastoma, Ewing's sarcoma, neuroblastoma and osteosarcoma. A tumor can not expand without a blood supply to provide nutrients and remove cellular waste. Tumors in which angiogenesis is important include solid tumors, and benign tumors such as acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas. The prevention of angiogenesis could stop the growth of these tumors and the resulting damage to the animal due to the presence of the tumor. Angiogenesis has been associated with tumors that are born in the blood such as leukemias, any of several acute or chronic neoplastic diseases of the bone marrow in which the unrestricted proliferation of white blood cells occurs, usually accompanied by anemia, blood coagulation damaged and growth of lymph nodes, liver and spleen. It is believed that angiogenesis plays a role in abnormalities in the bone marrow that give rise to tumors similar to leukemia. Angiogenesis is important in two stages of tumor metastasis. The first stage in which the stimulation of angiogenesis is important is in the vascularization of the tumor which allows the tumor cells to enter the bloodstream and circulate through the body. After the tumor cells have left the primary site, and have been installed in the secondary metastatic site, angiogenesis must occur before the new tumor can grow and expand. Therefore, the prevention of angiogenesis could lead to the prevention of tumor metastasis and possibly contain neoplastic growth at the primary site. The knowledge of the role of angiogenesis in the maintenance and metastasis of tumors has led to a prognostic indicator for breast cancer. The amount of neovascularization found in the primary tumor was determined by counting the density of microvessels in the area of the most intense neovascularization in invasive breast carcinoma. It was found that a high level of microvessel density correlates with tumor recurrence. The control of angiogenesis by therapeutic means could possibly lead to the cessation of tumor recurrence. Angiogenesis is also involved in normal physiological processes such as reproduction and wound healing. Angiogenesis is an important step in ovulation and also in the implantation of the blastula after fertilization. The prevention of angiogenesis could be used to induce amenorrhea, to block ovulation or to prevent implantation by the blastula. In wound healing, excessive repair of fibroplasia can be a damaging side effect of surgical procedures and can be caused or exacerbated by angiogenesis. Adhesions are a frequent complication of surgery and lead to problems such as small bowel obstruction. Various types of compounds have been used to prevent angiogenesis. Taylor et al., Have used prolamin to inhibit angiogenesis, see Taylor et al., Nature 297: 307 (1982). The toxicity of prolamine limits its practical use as a therapeutic agent. Folkman et al. Have described the use of heparin and steroids to control angiogenesis. See Folkman et al., Science 221: 719 (1983) and U.S. Patent Nos. 5,001,116 and 4,994,443. It has been found that steroids, such as tetrahydrocortisol, which lacks gluco and corticoid mineral activity, inhibit angiogenesis. Other factors found endogenously in animals, such as a 4 kDa glycoprotein of bovine vitreous humor and a cartilage-derived factor, have been used to inhibit angiogenesis. Cellular factors such as interferon inhibit angiogenesis. For example, interferon α or human interferon β have been shown to inhibit tumor-induced angiogenesis in mouse dermis stimulated by human neoplastic cells. Interferon ß is also a potent inhibitor of angiogenesis induced by allogeneic spleen cells. See, Sidky et al., Cancer Research 47: 5155-5161 (1987). It was reported that human recombinant interferon (alpha / A) was used successfully in the treatment of pulmonary hemangiomatosis, an illness induced by angiogenesis. See, White et al., N. Engl. J. Med. 320: 1197-1200, 1971. In accordance with the present invention, there are provided compositions and methods that are effective to inhibit undesirable angiogenesis in a mammal, both human and non-human. These compositions are easily administered by different routes including parenteral and can be given in doses that are safe and provide angiogenic inhibition in internal sites. The present invention provides a method of treating mammalian diseases mediated by undesired and uncontrolled angiogenesis by administering a composition comprising Interleukin 12 in a dose sufficient to inhibit angiogenesis. The present invention is especially useful for treating certain ocular neovascular diseases such as macular degeneration. The compositions contemplated as part of the present invention can preferably be given parenterally to the patient and thus stop the progression of the disease. Other diseases that can be treated using the present invention are diabetic retinopathy, neovascular glaucoma and retrolental fibroplasia.

Interleukin 12 can be prepared by methods known in the art, for example described in European Patent Application No. 433827, in International Patent Applications WO 9005147 and WO 9205256, in Kobayashi M., et al., J. Exp. Med. 170: 827-845, 1989 and Stern AS, et al., Proc. Nati, acad, aci. USA 87: 6808-6812, 1990. Interleukin 12 can be produced by known conventional chemical synthesis, recombinant methods or can be purified from natural sources. The term "Interleukin 12" also comprises polypeptides similar to those of the pure and / or recombinant protein but whose modifications are naturally provided or deliberately designed. This invention provides evidence that the inhibition of angiogenesis is a novel biological activity of IL-12. This inhibition of neovascularization was found and occurred at IL-12 concentrations that also result in an optimal antitumor effect (Brunda M.J., et al., J. Exp. Med. 178: 1223-1230, 1993). IL-12 is a specific species that agrees with the lack of inhibition of angiogenesis when IL-12 is used in chick chorioallantoic membrane assay. The mouse corneal neovascularization model was therefore the trial of choice to evaluate the antiangiogenic properties of IL-12. Using this model in strains of mice with different immunological backgrounds, no single cell type of the immune system (natural killer cells or T cells) could be recognized as the mediator of the antiangiogenic effects of IL-12. The mouse corneal neovascularization assay used in this study is a model derived from the basic fibroblast growth factor of angiogenesis. Therefore, it can be argued that IL-12 specifically inhibits angiogenesis induced by basic fibroblast growth factor. However, IL-12 was similarly inhibitory when the basic fibroblast growth factor pill was placed by a pill containing vascular endothelial growth factor (160 ng / pill). Treatment with IL-12 induces a sustained elevation of IFN-? in the bloodstream of the mice (Gately M. K., et al., Int.Immunol.6: 157-167, 1994). The administration of IFN-α antibodies prevents the inhibition of neovascularization induced by IL-12. In addition, treatment with either bolus injections or continuous infusion of IFN-? resulted in the inhibition of neovascularization. These findings suggest that IFN-? it is a necessary and sufficient mediator of the antiangiogenic activity of IL-12. In support of an important role of the IFN-? in the anti-tumor activity of IL-12 is the observation that the treatment of euthymic mice with IFN-α antibodies results in the loss of anti-tumor efficacy of IL-12 (Nastala C.L., et al., J. Immunol., 153: 1697-1706, 1994). The IFN-? has been used in murine tumor models (Brunda M.J., et al., Int.J. Cancer.40: 807-810, 1987) but the clinical use of IFN-? As an anticancer agent it has not been very successful. The pharmacokinetics of IFN-? may have contributed to disappointing results with this drug in clinical trials. After administration of the intravenous bolus, IFN-? had a relatively short half-life (hours) (Rutenfranz I., et al., J. Interferon Res. 8: 573-580, 1988) and subcutaneous injection did not result in detectable levels of serum drug (Cross SE, et al. al., J Interferon Res 45: 606-609, 1993). The observation that, in comparison with the bolus injection, the continuous intraperitoneal infusion of IFN-? achieved greater inhibition of angiogenesis, suggesting a pharmacokinetic difference between the two methods of administration. However, it can not be excluded that the three additional days of treatment with IFN-? continuous before implantation of the pill of the basic fibroblast growth factor may have a beneficial effect on the results of the experiment.

Since the IFN-? or the serum obtained from the animals treated with IL-12 had a significant effect on the proliferation of the endothelial cells in vitro, currently it is not clear how the IFN-? exerts its effect on blood vessels. The literature on IFN-? as an antiangiogenic agent is controversial and is mainly based on in vitro observations (Sato N., et al., J. Invest, Dermatol 95: 85S-89S, 1990, Saegusa Y., et al., J. Cell. Physiol 142: 488-495, 1990, Friesel R., et al., J. Cell. Biol. 104: 689-696, 1987; Saiki I. Et al., Int. J. Cancer 51: 641-645, 1992; , Kobayashi S., et al., Immunopharmacol. 27: 23-30, 1994). Since the IFN-? is involved in the regulation of numerous genes (Sen G. C, et al., J. Biol. Chem., 267: 5017-5020, 1992) it seems reasonable to assume that the actions downstream of the IFN-? they may be involved in anti-angiogenic effects. Experiments with mice with Lewis lung carcinoma confirm the potent antitumor activities of both IL-12 and the angiogenesis inhibitor AGM-1470 as a single agent (Brunda MJ, et al., J. Exp. Med. 178: 1223 -1230, 1993; Ingber D., et al., Nature 348: 555-557, 1990). The observation that simultaneous treatment with IL-12 and AGM-1470 has additive effects in the Lewis lung carcinoma model suggests that these agents act on endothelial cells through different pathways. Combinations of anti-angiogenic agents can improve this strategy to treat malignant diseases. The invention clearly demonstrates that IL-12 is a potent inhibitor of angiogenesis in vivo, an effect that seems to be mediated by the induction of a sustained release of IFN-α. The pharmaceutically acceptable formulations of IL-12 in relation to this invention can be made using formulation methods known to those skilled in the art. These formulations can be administered by the standard routes. In general, the formulations may be administered parenterally (eg, intravenous, subcutaneous or intramuscular) with the topical, transdermal, oral and rectal routes being contemplated. In addition, the formulations can be incorporated into biodegradable polymers that allow sustained release of IL-12, the polymers are implanted in the vicinity of where the release of the drug is desired, for example, at the site of a tumor. The biodegradable polymers and their use are described, for example, in detail in Bre et al., L. Neurosurg. 74: 441-446 (1991). The dose of IL-12 will depend on the condition being treated, the particular compound and other clinical factors such as the weight and condition of the human or animal and the route of administration of IL-12. It should be understood that the present invention has application for both human and veterinary use. For parenteral administration to humans, a dose of between about 0.1 to 20 mg / kg of 1 to 5 times a week, preferably between about 0.5 and 10 mg / kg of 1 to 3 times a week, and of more preferably between about 1 to 10 mg / kg of 1 to 3 times a week, is generally sufficient. Suitable formulations include those suitable for parenteral administration (including subcutaneous, intramuscular, intravenous, intradermal, intratracheal and epidural). The formulations can be conveniently presented in unit dosage forms and can be prepared by conventional pharmaceutical techniques. Such techniques include the step of associating IL-12 and pharmaceutical carriers or excipients. In general, the formulations are prepared by uniformly and intimately associating IL-12 with liquid carriers. Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injectable solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and sterile aqueous and non-aqueous suspensions which may include suspending agents and thickening agents. The formulations can be presented in single dose or multiple dose containers, for example, sealed vials and flasks, and can be stored under freeze-dried (lyophilized) conditions that require only the addition of the sterile liquid carrier, eg, water for injection, immediately before use. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as set forth above, or an appropriate fraction thereof, of the administered ingredient. Diseases associated with corneal neovascularization that can be treated according to the present invention include but are not limited to, diabetic retinopathy, premature retinopathy, rejection of cornea graft, neovascular glaucoma and retrolental fibroplasia, epidemic keratoconjunctivitis, vitamin A deficiency, excessive use of contact lenses, atopic keratitis, upper limbic keratitis, dry pterygium keratitis, sjogrens, acne rosacea, filectenulosis, syphilis, mycobacterial infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infections, Herpes zoster, protozoal infections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal degeneration, marginal keratolysis, trauma, rheumatoid arthritis, systemic lupus, polyarteritis, Wegeners sarcoidosis, Scleritis, Steven's Johnson's disease, perifigoid radial keratotomy, and graft rejection corneal Diseases associated with retinal / choroidal neovascularization that can be treated according to the present invention include, but are not limited to, diabetic retinopathy, macular degeneration, ozone cell anemia, sarcoid, syphilis, elastic pseudoxanthoma, Pagets, venereal occlusion, arterial occlusion, obstructive carotid disease, chronic uveitis / vitritis, mycobacterial infections, Lyme disease, systemic lupus erythematosus, prematurity retinopathy, Eales disease, Bechets disease, infections that cause retinitis or choroiditis, histoplasmosis Presumed ocular, Bests disease, myopia, optical stings, Stargarts disease, partial planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post laser complications. Other diseases include, but are not limited to, diseases associated with rubeosis (neovascularization of the angle) and diseases caused by abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy, associated or not with diabetes. Another disease that can be treated according to the present invention is rheumatoid arthritis. It is believed that the blood vessels in the synovial lining of the joints undergo angiogenesis. In addition to forming new vascular networks, endothelial cells release factors and species that react with oxygen that lead to tissue growth and cartilage destruction. The factors involved in angiogenesis can actively contribute to, and help maintain, the chronically inflamed state of rheumatoid arthritis. Another disease that can be treated according to the present invention are hemangiomas, Osier-Weber-Rendu disease, or hereditary hemorrhagic telangiectasia, solid or originating tumors in the blood and acquired immunodeficiency syndrome. A model of corneal neovascularization induced by basic fibroblast growth factor in mice was used to evaluate the effect of IL-12 on angiogenesis in vivo. Different mouse strains were treated with 1 mg of IL-12 per day intraperitoneally for 5 consecutive days. The degree of neovascularization was measured using the length of the vessels and the number of corneal clock hours of the formation of new blood vessels in response to a pill containing basic fibroblast growth factor. The antitumor activities of IL-12 and the angiogenesis inhibitor AGM-1470 were evaluated in mice with Lewis lung carcinoma. In vitro proliferation, studies were carried out on bovine capillary endothelial cells, endothelial cells of mouse pancreas and a cell lineage of mouse hemangioendothelioma. Corneal neovascularization in immunocompetent C57BL / 6 mice, mice with severe combined immune deficiency (SCID) and beige mice deficient in natural killer cells was almost completely inhibited as a result of treatment with IL-12. This potent suppression of angiogenesis was prevented by the administration of IFN-? Neutralizing antibodies. In addition, the IFN-? given either as an intraperitoneal bolus injection or as a continuous infusion of osmotic pump implanted intraperitoneally reproduced the antiangiogenic effects observed during treatment with IL-12. Treatment with IL-12 and AGM-1470 had an additive antitumor effect in mice with Lewis lung carcinoma suggesting a different anti-angiogenic mechanism of action. The invention is best illustrated by the following examples, which should not be construed in any way as limitations imposed on the scope thereof. On the contrary, it should be clearly understood that this resource can have several other modalities, modifications and equivalents thereof, which, after reading the present description, can be suggestive by themselves for those skilled in the art without departing from the spirit of the present invention and / or the scope of the appended claims.

Examples 1. materials Recombinant murine IL-12 (IL-12), recombinant murine gamma interferon (IFN-α), and IgGl blocking antibodies XMG1.1 IFN-? of rat were from Hoffmann-La Roche, Nutley, NJ, AGM-1470 (TNP-470) and the basic fibroblast growth factor was obtained from Takeda Industries, Osaka, Japan. All materials were obtained from Sigma, St Louis, Mo. A monomeric mixture of IL-12 was made by reducing IL-12 with dithiothreitol (10 mM) and iodoacetamide (50 mM). Subsequently, the mixture was dialyzed for 3 hours (molecular weight cut-off point 6-8000 D, dialysis membrane Spectra / Por, Houston, Tx) to eliminate the reducing compounds. The presence of monomers and absence of dimers in the mixture was confirmed by SDS-PAGE. 2. Cells and Culture Conditions In this study bovine capillary endothelial cells, a primary culture of mouse pancreatic islet endothelial cells and a mouse hemangioendothelioma cell lineage were used. Monolayer cultures were performed in minimal essential medium of Dulbecco's Modified Eagle (DMEM) supplemented with 100 U / ml penicillin, 100 mg / ml streptomycin, 2 mM L-glutamine ("complete medium"), and 10% bovine serum. (GIBCO BRL, Grand Island, NY) in an atmosphere of 10% C02. The bovine capillary endothelial cells were maintained in culture in the presence of 4 ng / ml of the basic fibroblast growth factor (bFGF), while the mouse pancreatic islet endothelial cells were grown in the presence of 6 ng / ml factor. basic fibroblastic growth and 10% NUSERUM IV culture supplement (Becton Dickinson Labware, Bedford, MA). The experiments involved bovine and mouse endothelial cells and were performed between 10 and 15 passes. 3. Mice C57BL / 6, SCID (C57BL / 6 / SCID / szj), and Beige (C57BL / 6 / bgj) mice were obtained from Jackson Laboratories, Bar Harbor, ME. Nude mice (NCR Nu / sed, Swiss white background) were obtained from Massachusetts General Hospital, Boston, MA). All animal studies were conducted in 6-8 week old male mice. 4. In Vitro Tests To evaluate effects on endothelial cell proliferation, bovine capillary endothelial cells, mouse pancreatic islet endothelial cells and hemangioendothelioma cells were cultured at a density of 10,000-12,500 cells / well in a 24-well plate. Twenty-four hours later, the cells were incubated in complete medium supplemented with 1 ng / ml of basic fibroblast growth factor and 5% of bovine serum and challenged with the compound to be tested. 72 hours later, the cells were harvested by trypsinization and counted with a Coulter counter.

. In Vivo Testing To study the effect of IL-12 and IFN-? on in vivo angiogenesis, a modification of a mouse corneal angiogenesis assay described above was used (Polawski IJ, et al., Am. J. Pathol., 143: 507-517, 1993; Muthukkaruppan V., et al., Science 205: 1416-1418, 1979). In summary, corneal microcavities were made in both eyes reaching 1 mm into the limbus and a pill containing basic fibroblast growth factor (~80 ng), sucralfate and hydron was implanted in both eyes. The vascular response was measured as the maximum length of the vessel and the number of clock hours and neovascularization was evaluated daily. The data presented in this study were obtained on the fifth day after the implantation of the basic fibroblast growth factor pill, which was found to be the maximum angiogenic response day. An osmotic pump (Alzet 2002, Alza Corporation, Palo Alto, CA) was implanted intraperitoneally in experiments designed to ensure continuous infusion of either saline or IFN-α. The mice were allowed to recover from laparatomy 3 days before the implantation of the basic fibroblast growth factor pills. After finishing the experiment the remaining pump volumes were checked to ensure proper function and release. Serum from mice treated with IL-12 was obtained by cardiac puncture 24 hours after the fifth day of injection of IL-12. 6. Experiments with tumor Male C57BL / 6 mice were inoculated with 106 of Lewis lung carcinoma. Treatment with either saline, IL-12, AGM-1470 or IL-12 plus AGM-1470 was started after the tumor volume reached 75 mm3. IL-12 was given at a dose of 1 mg / day intraperitoneally for 4 consecutive days. 2 days after resting this cycle was repeated again. AGM-1470 was administered subcutaneously on a day and a day and not at a dose of 30 mg / kg. Serial gauge measurements of perpendicular diameters were used to calculate tumor volumes in mm3 using the formula: longest diameter x shortest diameter2 x 0.52. three weeks after the inoculation, the tumors and lungs were dissected and weighed. The metastasis of the pulmonary surface was cut under a dissecting microscope. 7. Statistic analysis The statistical significance of the differences between the groups was calculated by applying the 2-tailed Student test. The results were presented as the mean ± standard error of the mean. 8. Effect of IL-12 on corneal neovascularization in mice.

Male C57BL / 6 mice were treated either with IL-12 (1 mg in 0.1 ml vehicle intraperitoneally / day for 5 consecutive days, starting on the day of implantation of the pill) or vehicle (1% syngeneic mouse serum in saline solution buffered with phosphate). During the treatment, no obvious toxicity was found. C57BL / 6 mice treated with IL-12 had almost no corneal neovascularization in response to the basic fibroblast growth factor pill, while the vehicle-treated mice had blood vessels which reached the pill within 5 days after implantation of the pill (p <0.0001; Figures 1 and 2). The results were obtained from three independent experiments. When IL-12 was reduced to a monomer mixture and the mice were treated daily with 1 mg of this mixture intraperitoneally for 5 days the inhibitory effect in vivo on neovascularization was lost (Figure 2). To investigate which cells of the system can mediate the inhibitory effect of IL-12 on angiogenesis, the mouse corneal neovascularization assay was used in strains of mice with an aberrant immune system. First, SCID mice deficient in T cells were studied. IL-12 retained its inhibition of angiogenesis in SCID mice (vessel length: 0.98 ± 0.06 mm versus 0.22 ± 0.02 mm (p = 0.0002) and clock hours: 4.6 ± 0.4 h versus 3 ± 0.3 h (p = 0.011) for the mice treated with vehicle and with IL-12 respectively). The degree of inhibition resembled that observed in euthymic C57BL / 6 mice. A similar pattern of inhibition was observed when treated with IL-12 beige mice, deficient in natural killer cells (vessel length: 0.7 + 0.05 mm versus no new vessels (p <0.0001) and clock hours: 3.6 ± 0.3 h versus without new vessels (p <0.0001) for mice treated with vehicle or IL-12 respectively). It was found that nude mice have spontaneous corneal neovascularization which masks the development of new blood vessels induced by the basic fibroblast growth factor. A consistent finding, however, that suggests some degree of inhibition of angiogenesis, was the lack of capillary growth in the pill in the animals treated with IL-12, while the vehicle-treated mice had vessels that clearly grew to the pill. 9. Effects of IL-12 and IFN-? on the proliferation of endothelial cells in vitro IL-12 (range of 0.001-100 ng / ml) had no effect on the proliferation of bovine or mouse endothelial cells or hemangioendothelioma cells. The serum obtained from either C57BL / 6, SCID or nude mice after being treated with IL-12 for 5 days had no inhibitory effect on the proliferation of any type of endothelial cells. The IFN-? (range of 0.0001-200 ng / ml) had only a minimal effect (16% inhibition compared to control cell numbers) on the proliferation of mouse pancreatic islet endothelial cells and had no effect on proliferation of the cells. capillary endothelial cells of bovine.

. The role of IFN-? as a mediator of the activity of IL-12 in vivo Treatment of C57BL / 6 mice with a single injection of IFN-α antibodies (1 mg / mouse intraperitoneally on the day of implantation of the pill administered 2 hours before the first injection with IL-12) completely abolished the antiangiogenic properties of IL-12 (p <0.0001). Control injections with rat IgG (1 mg / mouse intraperitoneally on the day of pill implantation) did not affect the inhibition of neovascularization by IL-12 (Figure 3). To investigate whether treatment with iFN-? results in a similar inhibition of angiogenesis as seen with IL-12, C57BL / 6 mice were treated with daily intraperitoneal injections of IFN-α. (250,000 U / day for 5 consecutive days). In these mice, significant inhibition (p = 0.0007) of vessel length was observed (Figure 4) while no obvious toxicity was found. To maintain a constant level of IFN-α, an osmotic mini-pump was implanted intraperitoneally, which released IFN-? at a final dose of 130.00 U / day for the duration of the experiment. The basic fibroblast growth factor pills were implanted 72 hours after the implantation of the pump. The implantation of pumps loaded with saline did not affect the development of new blood vessels in the control animals. The degree of neovascularization in these animals was comparable to that of control animals without a pump. However, animals with pumps containing IFN-? they had complete inhibition of the growth of new vessels in the cornea (Figure 4, p = 0.0002 for vessel length and p = 0.0004 for clock hours compared to controls). Both the control animals and the mice treated with IFN-? lost weight in the recovery period after laparotomy. After the implantation of the pills, the control animals gained weight while the mice treated with IFN-? they had stable weights and they were lethargic. 11. Effect of treatment with IL-12 and AGM-1470 on Lewis lung carcinoma Treatment with either IL-12 or AGM-1470 was effective in inhibiting primary tumor growth and spontaneous lung metastases in C57BL / 6 mice inoculated with Lewis lung carcinoma compared to control animals treated with saline. Simultaneous treatment of mice with Lewis lung carcinoma with IL-12 and AGM-1470 resulted in smaller primary tumor volumes (Figure 5) and less spontaneous pulmonary metastases (Figure 6) than was observed in the treated animals with IL-12 or AGM-1470 as sole agents. No obvious toxicity was found during treatment in any of the groups.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (14)

1. The use of Interleukin 12 for the manufacture of a medicament for the treatment of diseases mediated by undesired or uncontrolled angiogenesis.

2. The use according to claim 1, wherein the disease mediated by unwanted or uncontrolled angiogenesis is neovascularization.

3. The use according to claim 2, wherein the disease mediated by unwanted or uncontrolled angiogenesis is retinal / choroidal neovascularization.

4. The use according to claim 3, wherein retinal / choroidal neovascularization is associated with diabetic retinopathy.

5. The use according to claim 3, wherein retinal / choroidal neovascularization is associated with macular degeneration.

6. The use according to claim 1, wherein the disease mediated by undesired or uncontrolled angiogenesis is corneal neovascularization.

7. The use of Interleukin 12 for the manufacture of a drug for the treatment of diabetic retinopathy.

8. The use of Interleukin 12 for the manufacture of a medicament for the treatment of ocular degeneration.

9. The use according to claim 1, wherein the diseases mediated by undesirable or uncontrolled angiogenesis are diseases which have their origin in solid tumors or tumors that originate in the blood and their metastases.

10. The use according to claim 1, wherein the disease mediated by undesirable or uncontrolled angiogenesis is proliferative vitreoretinopathy.

11. The use of Interleukin 12 according to claims 1 to 10 together with one or more other angiogenesis inhibitors.

12. The use of Interleukin 12 in the treatment of a disease as defined in any of claims 1 to 11.

13. Interleukin 12, characterized in that it is for the treatment of diseases according to any of claims 1 to 11.

14. Interleukin 12 in combination with one or more additional angiogenesis inhibitors, characterized in that it is for the treatment of the diseases according to any of claims 1 to 11.