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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/208—IL-12
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
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  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

• the present invention relates to the prevention of diseases mediated by unwanted angiogenesis. More particularly, the present invention relates to the use of Interleukin-12 (IL-12) for the manufacture of medicaments for preventing unwanted angiogenesis, particularly for the treatment of angiogenesis dependent or associated diseases.
• IL-12 Interleukin-12
• Interleukin 12 formerly called natural killer cell stimulatory factor (Kobayashi M., et al., J. Exp. Med. 170:827-845,
• IL-12 IL-12 induces a variety of biological effects on natural killer and T cells in vitro (Manetti R., et al., J.
• SCID severe combined immune deficient
• nude mice both of which are T cell-deficient, and in CD8*-depleted euthymic mice (Brunda M. J., et al., J. Exp. Med. 178:1223-1230,
• the present invention provides the use of Interleukin-12 for the preparation of medicaments effective in inhibiting unwanted angiogenesis.
• IL-12 was observed to inhibit the growth of a wide spectrum of tumors in vivo, but had no direct effect on tumor cells in vitro.
• the anti-tumor activity of IL-12 is not completely abrogated, suggesting that IL-12 has antiangiogenic 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- ⁇ ) appears to play a critical role as a mediator of the antiangiogenic effects of IL- 12.
• IFN- ⁇ Interferon gamma
• the present invention provides the use of Interleukin-12 for the manufacture of medicaments for the treatment of diseases mediated by undesired or uncontrolled angiogenesis, especially for the treatment of diseases wherein the disease mediated by undesired or uncontrolled angiogenesis is neovascularization, particularly retinal/choroidal neovascularization. It is another object of the present invention to provide the above use wherein the retinal/choroidal neovascularization is associated with diabetic retinopathy or wherein the retinal/choroidal neovascularization is associated with macular degeneration.
• the invention comprises the use of Interleukin-12 for the manufacture of medicaments for the treatment of diseases mediated by undesired or uncontrolled angiogenesis wherein the diseases stem from solid tumors or blood-born tumors and their metastases.
• the above medicaments may contain one or more additional angiogenesis inhibitors.
• Interleukin-12 Also part of this invention is Interleukin-12 and the use of Interleukin 12 for the treatment of a disease as mentioned above.
• 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.
• FIG. 1 Effect of recombinant murine IL-12 on bFGF-induced mouse corneal neovascularization. These photos represent corneas of either vehicle (control) or IL-12-treated C57BL/6 and SCID mice, 5 days after implantation of the basic fibroblast growth factor pellet (P). There are prominent new vessels in the control corneas, whereas almost no vascular response is seen after treatment with IL-12. (Note that SCID mice have preexistent iris vessels which are visible through the cornea since their iris is hypopigmented. Thus, the vessels seen in the IL-12 treated panel are in the plane of the iris and are not corneal vessels induced by the basic fibroblast growth factor pellet.)
• FIG. 1 Angiogenic response 5 days after implantation of the basic fibroblast growth factor pellets in C57BL 6 mice. Treatment consisted of either vehicle (21 corneas), IL-12 (30 corneas) or a monomeric mixture of IL-12 (10 corneas) as described below. Vessel length in mm and number of clock hours are presented as mean ⁇ SEM.
• FIG. 3 Effects of IFN- ⁇ -antibodies on IL-12-induced inhibition of mouse corneal neovascularization.
• Male C57BL/6 mice were treated with either single intraperitoneal injections of rat XMG1.2 IFN- ⁇ antibodies or rat IgG as described below. Vessel length and clock hours of neovascularization were measured on day 5. This experiment was repeated on two separate occasions with similar results. Data are presented as the mean ⁇ SEM of at least 13 corneas.
• FIG. 4 The effect of treatment with IFN- ⁇ on basic fibroblast growth factor-induced mouse corneal neovascularization.
• Male C57BIJ6 mice were either treated with intraperitoneal bolus injections of IFN- ⁇ starting on the day of pellet implantation or by continuous infusion of IFN starting 3 days before implantation of the pellet.
• Vessel length and clock hours were measured on day 5 after implantation of the basic fibroblast growth factor pellet and are presented as mean ⁇ SEM of 10 corneas in each group.
• FIG. 1 Effect of IL-12 and AGM-1470 on 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 was started after the tumor became measurable. Treatment protocol and measurement procedures are described below. Results are representative of a single experiment of 4 animals in each group.
• Figure 6 Effect of IL-12 and AGM-1470 on spontaneous lung surface metastases of Lewis lung carcinoma. Treatment protocol and counting procedure are described below. Results are representative of a single experiment of 4 animals in each group.
• Angiogenesis is fundamental for tumors and metastases to enlarge 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 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 antiangiogenic properties, IL-12 was evaluated in a model of basic fibroblast growth factor-induced mouse corneal neovascularization. The results show that IL-12 is a potent inhibitor of angiogenesis in vivo and that this effect is mediated by IFN- ⁇ .
• Angiogenesis is the generation of new blood vessels into a tissue or organ. Under normal physiological conditions, humans or animals only undergo 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 to be altered in certain disease states and, in many cases, the pathological damage associated with the disease is related to the uncontrolled angiogenesis.
• Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a "sprout" off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel. In the disease state, prevention of angiogenesis could avert the damage caused by the invasion of the new microvascular system.
• Persistent angiogenesis occurs in a multiplicity of disease states, tumor growth (both as primary tumor and metastasis) and abnormal growth by endothelial cells, and supports the pathological damage seen in these conditions.
• the diverse pathological states created due to unregulated angiogenesis have been grouped together as angiogenesis dependent or angiogenesis associated diseases. Therapies directed at control of the angiogenic processes could lead to the abrogation or mitigation of these diseases.
• ocular neovascular disease This disease is characterized by invasion of new blood vessels into the structures of the eye such as the retina or cornea. It is the most common cause of blindness and is involved in approximately twenty eye diseases.
• age-related macular degeneration the associated visual problems are caused by an in 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, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma and retrolental fibroplasia.
• corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's mariginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis, Steven's Johnson disease, periphigoid radial keratotomy, and corneal graph rejection.
• Diseases associated with retinal/choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagets disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, Eales disease, Bechets disease, infections causing a retinitis or choroiditis, presumed ocular histoplasmosis, Bests disease, myopia, optic pits, Stargarts disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications.
• diseases include, but are not limited to, diseases associated with rubeosis (neovascularization of the angle) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy.
• rubeosis neovascularization of the angle
• diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy.
• Another disease in which angiogenesis is believed to be involved is rheumatoid arthritis.
• the blood vessels in the synovial lining of the joints undergo angiogenesis.
• the endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction.
• the factors involved in angiogenesis may 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 the chondrocytes by angiogenic- related factors contributes to the destruction of the joint. At a later stage, the angiogenic factors would promote new bone formation.
• Therapeutic intervention that prevents the bone destruction could halt the progress of the disease and provide relief for persons suffering with arthritis.
• Chronic inflammation may also involve pathological angiogenesis.
• Such disease states as ulcerative colitis and Crohn's disease show histological changes with the ingrowth of new blood vessels into the inflamed tissues. Bartonellosis, a bacterial infection found in South America, can result in a chronic stage that is characterized by proliferation of vascular endothelial cells.
• Another pathological role associated with angiogenesis is found in atherosclerosis. The plaques formed within the lumen of blood vessels have been shown to have angiogenic stimulatory activity.
• hemangioma One of the most frequent angiogenic diseases of childhood is the hemangioma. In most cases, the tumors are benign and regress without intervention. In more severe cases, the tumors progress to large cavernous and infiltrative forms and create clinical complications. Systemic forms of hemangiomas, the hemangiomatoses, have a high mortality rate. Therapy-resistant hemangiomas exist that cannot be treated with therapeutics currently in use. Angiogenesis is also responsible for damage found in hereditary diseases such as Osier- Weber-Rendu disease, or hereditary hemorrhagic telangiectasia. This is an inherited disease characterized by multiple small angiomas, tumors of blood or lymph vessels. The angiomas are found in the skin and mucous membranes, often accompanied by epistaxis (nosebleeds) or gastrointestinal bleeding and sometimes with pulmonary or hepatic arteriovenous fistula.
• Angiogenesis is prominent in solid tumor formation and metastasis. Angiogenic factors have been found associated with several solid tumors such as rhabdomyosarcomas, retinoblastoma,
• Tumors in which angiogenesis is important include solid tumors, and benign tumors such as acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas. Prevention of angiogenesis could halt the growth of these tumors and the resultant damage to the animal due to the presence of the tumor.
• Angiogenesis has been associated with blood-born tumors such as leukemias, any of various acute or chronic neoplastic diseases of the bone marrow in which unrestrained proliferation of white blood cells occurs, usually accompanied by anemia, impaired blood clotting, and enlargement of the lymph nodes, liver, and spleen. It is believed that angiogenesis plays a role in the abnormalities in the bone marrow that give rise to leukemia-like tumors.
• Angiogenesis is important in two stages of tumor metastasis.
• the first stage where angiogenesis stimulation is important is in the vascularization of the tumor which allows tumor cells to enter the blood stream and to circulate throughout the body. After the tumor cells have left the primary site, and have settled into the secondary, metastatic site, angiogenesis must occur before the new tumor can grow and expand. Therefore, prevention of angiogenesis could lead to the prevention of metastasis of tumors and possibly contain the neoplastic growth at the primary site.
• 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 microvessel density in the area of the most intense neovascularization in invasive breast carcinoma. A high level of microvessel density was found to correlate with tumor recurrence. Control of angiogenesis by therapeutic means could possibly lead to cessation of the recurrence of the tumors.
• Angiogenesis is also involved in normal physiological processes such as reproduction and wound healing. Angiogenesis is an important step in ovulation and also in implantation of the blastula after fertilization. Prevention of angiogenesis could be used to induce amenorrhea, to block ovulation or to prevent implantation by the blastula. In wound healing, excessive repair or fibroplasia can be a detrimental side effect of surgical procedures and may be caused or exacerbated by angiogenesis. Adhesions are a frequent complication of surgery and lead to problems such as small bowel obstruction.
• Taylor et al.. have used prolamine to inhibit angiogenesis, see Taylor et al., Nature 297:307 (1982).
• the toxicity of prolamine limits its practical use as a therapeutic.
• Folkman et al.. have disclosed 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.
• Steroids, such as tetrahydrocortisol which lack gluco and mineral corticoid activity, have been found to inhibit angiogenesis.
• interferon inhibits angiogenesis.
• interferon ⁇ or human interferon ⁇ has been shown to inhibit tumor-induced angiogenesis in mouse dermis stimulated by human neoplastic cells.
• Interferon ⁇ is also a potent inhibitor of angiogenesis induced by allogeneic spleen cells. See Sidky et al., Cancer
• compositions and methods are provided that are effective in inhibiting unwanted angiogenesis in an animal, both human and non-human. These compositions are easily administered by different routes including parenteral and can be given in dosages that are safe and provide angiogenic inhibition at 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 dosage sufficient to inhibit angiogenesis.
• the present invention is especially useful for treating certain ocular neovascular diseases such as macular degeneration.
• the compositions which are contemplated as part of the present invention preferably can be given parenterally to the patient and thereby halt 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 may be prepared by methods known in the art, e. g. 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 A. S., et al., Proc. Natl. Acad. Sci. USA 87:6808-6812, 1990.
• Interleukin- 12 may be produced by known conventional chemical synthesis, recombinant methods or may be purified form natural sources.
• the term "Interleukin-12" also comprises polypeptides similar to those of the purified and/or recombinant protein but which modifications are naturally provided or deliberately engineered.
• This invention provides evidence that inhibition of angiogenesis is a new biological activity of IL-12. This inhibition of neovascularization was profound and occurred at concentrations of
• IL-12 which also result in an optimum anti -tumor effect (Brunda M. J., et al., J. Exp. Med. 178:1223-1230, 1993).
• IL-12 is species specific which is in agreement with the lack of inhibition of angiogenesis when IL-12 was used in the chick chorioallantoic membrane assay.
• the mouse corneal neovascularization model was therefore the assay of choice to evaluate the antiangiogenic properties of IL-12. Using this model in strains of mice with different immunological backgrounds, no individual cell type of the immune system (natural killer or T cells) could be recognized as the mediator of the anti-angiogenic effects of IL-12.
• the mouse corneal neovascularization assay used in this study is a basic fibroblast growth factor-driven model of angiogenesis. It may therefore be argued that IL-12 specifically inhibits basic fibroblast growth factor-induced angiogenesis. However, IL-12 was equally inhibitory when the basic fibroblast growth factor pellet was replaced by a pellet containing vascular endothelial growth factor (160 ng/pellet).
• IFN- ⁇ in the anti-tumor activity of IL-12 is the observation that treatment of euthymic mice with IFN- ⁇ -antibodies resulted in loss of anti-tumor efficacy of IL-12 (Nastala C. L., et al., J. Immunol. 153:1697-1706, 199).
• IFN- ⁇ has been used in murine tumor models (Brunda M. J., et al., Int. J. Cancer 40:807-810, 1987) but the chnical use of IFN- ⁇ as an anti-cancer agent has not been very successful. The pharmacokinetics of IFN- ⁇ may have contributed to the disappointing results with this drug in clinical trials.
• IFN- ⁇ After intravenous bolus administration, IFN- ⁇ has a relatively short half life (hours) (Rutenfranz L, et al., J. Interferon Res. 8:573-580, 1988) and subcutaneous injections do not result in detectable levels of the drug in serum (Cross S. E., et al., J Interferon Res 45:606-609, 1993).
• IFN- ⁇ Since either IFN- ⁇ or serum obtained from IL-12-treated animals had a significant effect on endothelial cell proliferation in vitro, it is presently unclear how IFN- ⁇ exerts its effect on blood vessels.
• the literature on IFN- ⁇ as an antiangiogenic agent is controversial and mainly based on observations in vitro (Sato N., et al., J. Invest. Dermatol. 95:85S-89S, 1990; Saegusa Y., et al., J. Cell.
• IFN- ⁇ is involved in the regulation of numerous genes (Sen G. C, et al., J. Biol. Che . 267:5017-5020, 1992) it seems reasonable to assume that actions downstream of IFN- ⁇ may be involved in the antiangiogenic effects.
• formulations of IL-12 in connection with this invention can be made using formulation methods known to those of ordinary skill in the art. These formulations can be administered by standard routes. In general, the formulations may be administered parenterally (e.g., intravenous, subcutaneous or intramuscular) with topical, transdermal, oral, or rectal routes also being contemplated. In addition, the formulations may be incorporated into biodegradable polymers allowing for sustained release of IL-12, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor. The biodegradable polymers and their use are described, for example, in detail in Brem et al., J.
• the dosage of IL-12 will depend on the condition being treated, the particular compound, and other clinical factors such as weight and condition of the human or animal and the route of administration of IL-12. It is to be understood that the present invention has application for both human and veterinary use.
• the formulations include those suitable for parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intratracheal, and epidural) administration.
• parenteral including subcutaneous, intramuscular, intravenous, intradermal, intratracheal, and epidural
• the formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques.
• Such techniques include the step of bringing into association the IL-12 and the pharmaceutical carrier(s) or excipient(s).
• the formulations are prepared by uniformly and intimately bringing into association the IL-12 with liquid carriers.
• Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
• the formulations may be presented in unit-dose or multi-dose containers, for example, seated ampoules and vials, and may be stored in a freeze-dried (lyophilized) conditions requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
• sterile liquid carrier for example, water for injections
• Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient.
• corneal neovascularization Diseases associated with corneal neovascularization that can be treated according to the present invention include but are not limited to, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma and retrolental fibroplasia, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical bums, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's mariginal degeneration, mariginal keratolysis, trauma, rheumatoid arthritis
• 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, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum,
• diseases include, but are not limited to, diseases associated with rubeosis (neovascularization of the angle) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy, whether or not associated with diabetes.
• Another disease which 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, the endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction. The factors involved in angiogenesis may 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, sohd or blood borne tumors and acquired immune deficiency syndrome.
• a model of basic fibroblast growth factor-induced corneal neovascularization in mice was used to evaluate the effects of IL-12 on angiogenesis in vivo.
• Different strains of mice were treated with 1 mg IL-12 per day intraperitoneally for 5 consecutive days.
• Extent of neovascularization was measured using vessel length and number of corneal clock hours of new blood vessel formation in response to a basic fibroblast growth factor containing pellet.
• the anti-tumor activities of IL-12 and the angiogenesis inhibitor AGM- 1470 were evaluated in Lewis lung carcinoma-bearing mice.
• In vitro proliferation studies were performed on bovine capillary endothelial cells, mouse pancreas endothelial cells, and a mouse hemangioendothelioma cell line.
• IL-12 Recombinant murine IL-12 (IL-12), recombinant murine
• Interferon gamma IFN- ⁇
• rat IgGl XMG1.2 IFN- ⁇ blocking antibodies were of Hoffmann-La Roche, Nutley, NJ.
• AGM-1470 TNP-470
• basic fibroblast growth factor were obtained from Takeda Industries, Osaka, Japan. All other materials were purchased 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). Hereafter the mixture was dialyzed for 3 hours (molecular weight cut off point 6-8000 D, Spectra/Por dialysis membrane, Houston, Tx) to eliminate the reducing compounds. The presence of monomers and absence of dimers in the mixture was confirmed by SDS- PAGE.
• Bovine capillary endotheUal cells a primary culture of mouse pancreatic islet endothelial cells and a mouse hemangioendothelioma cell line were used in this study. Monolayer culturing was performed in Dulbecco's modified Eagle's minimal essential medium (DMEM) supplemented with
• bovine and mouse endothelial cells were performed between passage 10 and 15.
• mice Male C57BIJ6, SCID (C57BIJ6/SCID/szj), and Beige (C57BL/6/bgj) mice were purchased from the Jackson Laboratories, Bar Harbor, ME. Nude mice (NCR Nu/sed, Swiss white background) were obtained from the Massachusetts General
• bovine capillary endothelial cells, mouse pancreatic islet endotheUal cells and hemangioendothelioma cells were plated at a density of 10,000 - 12,500 cells/well in a 24 well plate. Twenty-four hours later, cells were incubated in full medium supplemented with 1 ng/ml basic fibroblast growth factor and 5% bovine serum and challenged with the compound to be tested. After 72 hours, cells were harvested by trypsinization and counted with a Coulter counter.
• 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- ⁇ . Mice were allowed to recover from the laparotomy for 3 days before implantation of the basic fibroblast growth factor pellets. After termination of the experiment the remaining pump volumes were checked to ensure adequate function and delivery.
• Serum of IL-12-treated mice was obtained by cardiac puncture 24 hours after the fifth daily injection of IL-12.
• mice Male C57BL 6 mice were inoculated with 10 ⁇ Lewis lung carcinoma cells. Treatment with either saline, IL-12, AGM-1470 or IL-12 plus AGM-1470 was initiated after the tumor volume reached 75 m ⁇ . IL-12 was given at a dose of 1 mg/day intraperitoneally for 5 consecutive days. After 2 days of rest this cycle was repeated again. AGM-1470 was administered subcutaneously every other day at a dose of 30 mg/kg. Serial caliper measurements of perpendicular diameters were used to calculate tumor volumes in mm ⁇ using the formula: longest diameter x shortest diameter ⁇ x 0.52. Three weeks after inoculation, tumors and lungs were resected and weighed. Lung surface metastases were counted under a dissecting microscope.
• mice Male C57BL/6 mice were treated with either IL-12 (1 mg in 0.1 ml vehicle intraperitoneally/day for 5 consecutive days, starting on the day of pellet implantation) or vehicle (1% syngeneic mouse serum in phosphate-buffered saline). During treatment, no obvious toxicity was encountered. C57BL/6 mice treated with IL-12 had almost no corneal neovascularization in response to the basic fibroblast growth factor pellet whereas mice treated with vehicle had blood vessels which reached the pellet within 5 days after implantation of the pellet (p ⁇ 0.0001; Figures 1 and 2). Results were obtained from three independent experiments. When IL-12 was reduced to a monomeric mixture and the mice were treated daily with 1 mg of this mixture intraperitoneally for 5 days the in vivo inhibitory effect on neovascularization was lost (Figure 2).
• mice with an aberrant immune system were treated with IL-12.
• the extent of inhibition resembled that observed in the euthymic C57BL/6 mice.
• IL-12 (range 0.001-100 ng/ml) had no effect on the proliferation of either bovine or mouse endothelial cells or hemangioendothelioma cells. Serum obtained from either C57BL/6, SCID or nude mice after they were treated with IL-12 for 5 days had no inhibitory effect on proUferation of either type of endothelial cells.
• IFN- ⁇ (range 0.0001-200 ng/ml) had only a minimal effect (16 % inhibition as compared with control cell numbers) on mouse pancreatic islet endothelial cell proliferation and no effect on bovine capillary endothelial cell proliferation.
• IFN-g as a mediator of IL-12 activity in vivo.
• mice were treated with daily intraperitoneal injections of IFN- ⁇ (250,000 U/day for 5 consecutive days). In these mice, significant
• 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 as compared with control animals treated with saline.

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• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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• 1996
  • 1996-02-07 AU AU47883/96A patent/AU4788396A/en not_active Abandoned
  • 1996-02-07 BR BR9607427A patent/BR9607427A/pt not_active Application Discontinuation
  • 1996-02-07 EP EP96904008A patent/EP0809511A1/en not_active Withdrawn
  • 1996-02-07 WO PCT/EP1996/000507 patent/WO1996025171A1/en not_active Application Discontinuation
  • 1996-02-07 JP JP8524631A patent/JPH11500119A/ja active Pending
  • 1996-02-07 TR TR97/00801T patent/TR199700801T1/xx unknown
  • 1996-02-07 CA CA002212370A patent/CA2212370A1/en not_active Abandoned
• 1997
  • 1997-08-15 FI FI973356A patent/FI973356A0/fi unknown

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