EP0809511A1

EP0809511A1 - Inhibition of angiogenesis using interleukin-12

Info

Publication number: EP0809511A1
Application number: EP96904008A
Authority: EP
Inventors: Moses Judah Folkman; Gary Arthur Truitt; Emile Eugene Voest
Original assignee: F Hoffmann La Roche AG; Childrens Medical Center Corp
Current assignee: F Hoffmann La Roche AG; Childrens Medical Center Corp
Priority date: 1995-02-16
Filing date: 1996-02-07
Publication date: 1997-12-03
Also published as: MX9706188A; AU4788396A; FI973356A; TR199700801T1; BR9607427A; FI973356A0; JPH11500119A; WO1996025171A1; CA2212370A1

Abstract

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

Description

INHIBITION OF ANGIOGENESIS USING INTERLEUKIN 12

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.

Interleukin 12 (IL-12), formerly called natural killer cell stimulatory factor (Kobayashi M., et al., J. Exp. Med. 170:827-845,

1989) and cytotoxic lymphocyte maturation factor (Stern A.S., et al., Proc. Natl. Acad. Sci. USA 87:6808-6812, 1990), has potent anti- tumor and antimetastatic activity in several murine tumor models (Brunda M. J., et al., J. Exp. Med. 178:1223-1230, 1993; Nastala C. L., et al., J. Immunol. 153:1697-1706, 1994). Although the mechanism through which IL-12 exerts its anti-tumor 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. Natl.

Acad. Sci. USA 90:3725-3729, 1993; Seder R. A., et al., Proc. Natl. 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. Immunol. 148:92-98, 1992). Activation of cytotoxic T lymphocytes by IL-12 is considered crucial in its anti-tumor activity (Brunda M. J., et al., J. Exp. Med. 178:1223-1230, 1993). The IL-12 anti-tumor effect is partially maintained in severe combined immune deficient (SCID) and 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,

1993; O'Toole M., et al., J. Immunol. 150:294A, 1993). These results demonstrate that IL-12 has potent in vivo anittumor and Wh/Ul 8.12.95 antimethastatic effects against murine tumors and demonstrate as well 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 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. In addition, in T cell deficient mice 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-γ) appears to play a critical role as a mediator of the antiangiogenic effects of IL- 12. The surprising recognition of antiangiogenic properties of IL- 12 is central to the proper design of treatment protocols including its co-administration with other inhibitors of neovascularization.

Consequently, 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.

It is another object of the present invention 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.

It is yet another object of the present invention 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.

Further, 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.

It is yet another object of the present invention to provide the use of Interleukin-12 for the preparation of a medicament for the treatment of all forms of proliferative vitreoretionopathy, 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.

Further, 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 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.)

Figure 2. 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.

Figure 3. Effects of IFN-γ-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-γ 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.

Figure 4. The effect of treatment with IFN-γ on basic fibroblast growth factor-induced mouse corneal neovascularization. Male C57BIJ6 mice were either treated with intraperitoneal bolus injections of IFN-γ 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.

Figure 5. 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-γ.

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.

Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. 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 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.

One example of a disease mediated by angiogenesis is 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. In 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. Other diseases associated with 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.

Other 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. 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. 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.

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.

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,

Ewing sarcoma, neuroblastoma, and osteosarcoma. A tumor cannot expand without a blood supply to provide nutrients and remove cellular wastes. 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.

Several kinds of compounds have been used to prevent angiogenesis. 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.

Other factors found endogenously in animals, such as a 4 kDa glycoprotein from 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 has been shown to inhibit tumor-induced angiogenesis in mouse dermis stimulated by human neoplastic cells. Interferon β is also a potent inhibitor of angiogenesis induced by allogeneic spleen cells. See Sidky et al., Cancer

Research 47:5155-5161 (1987). Human recombinant α interferon (alpha A) was reported to be successfully used in the treatment of pulmonary hemangiomatosis, an angiogenesis-induced disease. See, White et al., N. Engl. J. Med. 320:1197-1200, 1971.

In accordance with the present invention, 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).

Treatment with IL-12 induces a sustained elevation of IFN-γ in the bloodstream of mice (Gately M. , et al., Int. Immunol. 6:157- 167, 1994). The administration IFN-γ antibodies prevented the IL-

12-induced inhibition of neovascularization. In addition, treatment with either bolus injections or continuous infusion of IFN-γ resulted in inhibition of neovascularization. These findings suggest that IFN-γ is a necessary and sufficient mediator of the antiangiogenic activity of IL- 12. In support of an important role of

IFN-γ in the anti-tumor activity of IL-12 is the observation that treatment of euthymic mice with IFN-γ-antibodies resulted in loss of anti-tumor efficacy of IL-12 (Nastala C. L., et al., J. Immunol. 153:1697-1706, 199). IFN-γ 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-γ as an anti-cancer agent has not been very successful. The pharmacokinetics of IFN-γ may have contributed to the disappointing results with this drug in clinical trials. After intravenous bolus administration, IFN-γ 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). The observation that, in comparison with bolus injections, continuous intraperitoneal infusion of IFN-γ achieved enhanced inhibition of angiogenesis, suggests a pharmacokinetic difference between the two methods of administration. However, it cannot be excluded that the additional 3 days of continuous IFN-γ treatment before implantation of the basic fibroblast growth factor pellet may have had a beneficial effect on the outcome of the experiment.

Since either IFN-γ or serum obtained from IL-12-treated animals had a significant effect on endothelial cell proliferation in vitro, it is presently unclear how IFN-γ exerts its effect on blood vessels. The literature on IFN-γ 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.

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., Immunophar acol. 27:23-30, 1994). Since IFN-γ 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-γ may be involved in the antiangiogenic effects.

The experiments with Lewis lung carcinoma bearing mice confirm the potent anti-tumor activities of both IL-12 and the angiogenesis inhibitor AGM-1470 as single agents (Brunda M. J., 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 antiangiogenic agents may enhance this strategy to treat malignancies. The invention clearly demonstrates that IL-12 is a potent inhibitor of angiogenesis in vivo, an effect which appears to be mediated by inducing a sustained release of IFN-γ.

Pharmaceutically acceptable 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.

Neurosurg. 74:441-446 (1991). 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. For parenteral administration to humans, a dosage of between approximately 0.1 to 20 mg/kg 1 to 5 times a week, preferably between approximately 0.5 and 10 mg kg 1 to 3 times a week, and most preferably between approximately 1 to 10 mg/kg 1 to 3 times a week, is generally sufficient.

The formulations include those suitable for parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intratracheal, and epidural) administration. 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). In general, 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.

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.

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, systemic lupus, polyarteritis, Wegeners sarcoidosis, Scleritis, Steven's Johnson disease, periphigoid radial keratotomy, and corneal graph rejection.

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,

Pagets disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infections, Ly e'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.

Other 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. Corneal neovascularization in immune competent C57BL/6 mice, severe combined immune deficient (SCID) mice and natural killer cell deficient, beige mice was almost completely inhibited as a result of treatment with IL- 12. This potent suppression of angiogenesis was prevented by the administration of IFN-γ neutralizing antibodies. In addition, IFN- γ given either as intraperitoneal bolus injections or as a continuous infusion from an implanted osmotic pump intraperitoneally reproduced the antiangiogenic effects observed during treatment with IL-12. Treatment with IL-12 and AGM-1470 had an additive anti-tumor effect in Lewis lung carcinoma-bearing mice suggesting a different antiangiogenic mechanism of action.

This invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to 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

Interferon gamma (IFN-γ), and rat IgGl XMG1.2 IFN-γ blocking antibodies were of Hoffmann-La Roche, Nutley, NJ. AGM-1470 (TNP-470) and 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.

2. Cells and Culture Conditions 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

100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine ("full medium"), and 10 % bovine serum (GIBCO BRL, Grand Island, NY.) in an atmosphere of 10% CO2- Bovine capillary endothelial cells were maintained in culture in the presence of 4 ng/ml basic fibroblast growth factor (bFGF) whereas mouse pancreatic islet endothelial cells were grown in the presence of 6 ng/ml basic fibroblast growth factor and 10% NUSERUM IV culture supplement (Becton Dickinson Labware, Bedford, MA). Experiments involving bovine and mouse endothelial cells were performed between passage 10 and 15.

3. 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

Hospital, Boston, MA). All animal studies were conducted on male, 6-8 weeks old mice.

4. In Vitro Assays

To evaluate effects on endothelial cell proliferation, 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.

5. In Vivo Assays To study the effect of IL-12 and IFN-γ on angiogenesis in vivo, a modification of a previously described mouse corneal angiogenesis assay was used (Polakowski I. J., et al., Am. J. Pathol. 143:507-517, 1993; Muthukkaruppan V., et al., Science 205:1416-1418, 1979). In brief, corneal micropockets were made in both eyes reaching within 1 mm of the limbus and a pellet containing basic fibroblast growth factor (-80 ng), sucralfate and hydron was implanted in both eyes. The vascular response measured as the maximal vessel length and number of clock hours of neovascularization was assessed daily. Data presented in this study were obtained on the fifth day after implantation of the basic fibroblast growth factor pellet, which was found to be the day of maximal angiogenic response.

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-γ. 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.

6. Tumor experiments

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.

7. Statistical analysis

The statistical significance of differences between groups was calculated by applying Student's 2-tailed t-test. Results are presented as the mean ± standard error of the mean.

8. Effect of IL-12 on mouse corneal neovascularization.

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).

To investigate which cells of the immune system might 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. We first studied T cell-deficient SCID mice. 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 vehicle and IL-12 treated mice respectively). The extent of inhibition resembled that observed in the euthymic C57BL/6 mice. A similar pattern of inhibition was observed when natural killer cell-deficient, beige mice were treated with IL-12 (vessel length: 0.7±0.05 mm versus no new vessels (p< 0.0001) and clock hours: 3.6±0.3 h versus no new vessels (p< 0.0001) for vehicle or IL-12 treated mice respectively). Nude mice were found to have spontaneous corneal neovascularization which masked the development of basic fibroblast growth factor-induced new blood vessels. A consistent finding however, suggestive of some degree of inhibition of angiogenesis, was the lack of capillaries growing into the pellet in the IL-12-treated animals, whereas the vehicle- treated mice had vessels clearly growing into the pellet.

9. Effects of IL-12 and IFN-γ on endothelial cell proliferation in vitro.

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-γ (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.

10. The role of IFN-g as a mediator of IL-12 activity in vivo.

Treatment of C57BIJ6 mice with a single injection of IFN-g- antibodies (1 mg/mouse intraperitoneally on the day of pellet implantation administered 2 hours before the first injection with IL-12) totally abolished the antiangiogenic properties of IL-12 (p<0.0001). Control injections with rat IgG (1 mg/mouse intraperitoneally on the day of pellet implantation) did not affect the inhibition of neovascularization by IL-12 (Figure 3).

To investigate whether treatment with IFN-g resulted in similar inhibition of angiogenesis as seen with IL-12, C57BL/6 mice were treated with daily intraperitoneal injections of IFN-γ (250,000 U/day for 5 consecutive days). In these mice, significant

(p=0.0007) inhibition of vessel length was observed (Figure 4) whereas no obvious toxicity was encountered. To maintain a constant level of IFN-γ, an osmotic mini-pump was implanted intraperitoneally which released IFN-γ to a final dose of 130,000 U/day for the duration of the experiment. The basic fibroblast growth factor-pellets were implanted 72 hours after implantation of the pump. Implantation of pumps loaded with saline did not affect the development of new blood vessels in control animals. The extent of neovascularization in these animals was comparable to control animals without a pump. However, animals with pumps containing IFN-g had complete inhibition of new vessel growth in the cornea (Figure 4; p=0.0002 for vessel length and p=0.0004 for clock hours as compared with controls). Both the control animals and the IFN-γ-treated mice lost weight in the recovery period after the laparotomy. After implantation of the pellets the control animals gained weight whereas the IFN-γ-treated mice had stable weights and 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 as compared with control animals treated with saline.

Simultaneous treatment of Lewis lung carcinoma-bearing mice with IL-12 and AGM-1470 resulted in smaller primary tumor volumes (Figure 5) and less spontaneous lung metastases (Figure 6) than seen in animals treated with IL-12 or AGM-1470 as single agents. No obvious toxicity was encountered during treatment in either of the groups.

Claims

Q___ ___

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

2. A use of claim 1, wherein the disease mediated by undesired or uncontrolled angiogenesis is neovascularization.

3. A use of claim 2, wherein the disease mediated by undesired or uncontrolled angiogenesis is retinal/choroidal neovascularization.

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

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

6. The use of 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 medicament for the treatment of diabetic retinopathy.

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

9. The use of claim 1, wherein the diseases mediated by undesired or uncontrolled angiogenesis are diseases which stem from solid tumors or blood-born tumors and their metastases.

10. The use of claim 1, wherein the disease mediated by undesired or uncontrolled angiogenesis is proliferative vitreoretionopathy.

II. 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 one of claims 1 to 11.

13. Interleukin 12 for the treatment of diseases as mentioned in any one of claims 1 to 11.

14. Interleukin- 12 in combination with one or more additional angiogenesis inhibitors.for the treatment of diseases as mentioned in any of claims 1 to 11.

15. The invention as hereinbefore described.

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