WO2005051357A1

WO2005051357A1 - Antiangiogenic agents

Info

Publication number: WO2005051357A1
Application number: PCT/US2004/038971
Authority: WO
Inventors: Albert W. Brzeczko; Nestor M. Vargas
Original assignee: Entremed, Inc.
Priority date: 2003-11-19
Filing date: 2004-11-19
Publication date: 2005-06-09

Abstract

There is disclosed an improved 2-methoxyestradiol, and analogs thereof, which is in an amorphous, noncrystalline form. This novel form of 2-methoxyestradiol, and analogs thereof, has greater bioavailability when administered orally and/or has greater solubility in water or aqueous solutions. A method of making the improved 2-methoxyestradiol, and analogs thereof, is also disclosed.

Description

ANTIANGIOGENIC AGENTS

FIELD OF THE INVENTION The present invention relates to treating disease states characterized by abnormal cell mitosis and to treating disease states characterized by abnormal angiogenesis and to treating disease states characterized by a combination of these events. More particularly, the present invention relates to an improved form of 2-methoxyestradiol (2ME₂), and analogs thereof, and their effect in treating various disease states; especially, diseases characterized by abnormal cell mitosis and/or abnormal angiogenesis. A method of making the improved form of 2ME₂, and analogs thereof, is also disclosed.

BACKGROUND OF THE INVENTION Angiogenesis is the generation of new blood vessels into a tissue or organ. Under normal physiological conditions, humans and animals undergo angiogenesis only in very specific, restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development, and formation of the corpus luteum, endometrium and placenta. Angiogenesis is controlled through 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, pathological damage associated with the diseases is related to 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. Endothelial cells, lining 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 a new blood vessel. Persistent, unregulated angiogenesis occurs in many disease states, tumor metastases, and abnormal growth by endothelial cells. The diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic-dependent or angiogenic-associated 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 ingrowth 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 fϊbroplasia. 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, and pterygium keratitis sicca. Other diseases associated with undesirable angiogenesis include Sjδgren's syndrome, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infection, Herpes zoster infections, protozoan infections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegener's sarcoidosis, scleritis, Stevens- Johnson's disease, pemphigoid, and radial keratotomy. Diseases associated with retinal/choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, pseudoxanthoma elasticum, Paget's disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, Mycobacteria infections, lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, Eales' disease, Behcet's disease, infections causing retinitis or choroiditis, presumed ocular histoplasmosis, Best's disease, myopia, optic pits, Stargardt's disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications. Other eye-related 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 prolific vitreoretinopathy. Another angiogenesis associated disease 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. Angiogenesis may also play 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 promote new bone growth. 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 diseases as ulcerative colitis and Crohn's disease show histological changes with the ingrowth of new blood vessels and the inflamed tissues. Bartonelosis, 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. The hypothesis that tumor growth is angiogenesis-dependent was first proposed in 1971. (Folkman, New Eng. J. Med., 285:1182-86 (1971)). In its simplest terms, this hypothesis states: "Once tumor 'take' has occurred, every increase in tumor cell population must be preceded by an increase in new capillaries converging on the tumor." Tumor 'take' is currently understood to indicate a prevascular phase of tumor growth in which a population of tumor cells occupying a few cubic millimeters volume, and not exceeding a few million cells, can survive on existing host microvessels. Expansion of tumor volume beyond this phase requires the induction of new capillary blood vessels. For example, pulmonary micrometastases in the early prevascular phase in mice would be undetectable except by high power microscopy on histological sections. Examples of the indirect evidence which support this concept include: (1) The growth rate of tumors implanted in subcutaneous transparent chambers in mice is slow and linear before neovascularization, and rapid and nearly exponential after neovascularization. (Algire, et al, J. Nat. Cancer Inst, 6:73-85 (1945)). (2) Tumors grown in isolated perfused organs where blood vessels do not proliferate are limited to 1-2 mm³ but expand rapidly to >1000 times this volume when they are transplanted to mice and become neovascularized. (Folkman, et al, Annals of Surgery, 164:491-502 (1966)). (3) Tumor growth in the avascular cornea proceeds slowly and at a linear rate, but switches to exponential growth after neovascularization.

(Gimbrone, Jr., et al, J. Nat. Cancer Inst., 52:421-27 (1974)). (4) Tumors suspended in the aqueous fluid of the anterior chamber of a rabbit eye remain viable, avascular, and limited in size to < 1 mm . Once they are implanted on the iris vascular bed, they become neovascularized and grow rapidly, reaching 16,000 times their original volume within 2 weeks. (Gimbrone, Jr., et al, J. Exp. Med., 136:261-76). (5) When tumors are implanted on a chick embryo chorioallantoic membrane, they grow slowly during an avascular phase of >72 hours, but do not exceed a mean diameter of 0.93 + 0.29 mm. Rapid tumor expansion occurs within 24 hours after the onset of neovascularization, and by day 7 these vascularized tumors reach a mean diameter of 8.0 + 2.5 mm. (Knighton, British J. Cancer, 35:347-56 (1977)). (6) Vascular casts of metastases in a rabbit liver reveal heterogeneity in size of the metastases, but show a relatively uniform cut-off point for the size at which vascularization is present. Tumors are generally avascular up to 1 mm in diameter, but are neovascularized beyond that diameter. (Lien, et al, Surgery, 68:334-40 (1970)). (7) In transgenic mice that develop carcinomas in the beta cells of the pancreatic islets, pre-vascular hyperplastic islets are limited in size to < 1 mm.

At 6-7 weeks of age, 4-10% of the islets become neovascularized, and from these islets arise large vascularized tumors of more than 1000 times the volume of the pre-vascular islets. (Folkman, etal, Nature, 339:58-61 (1989)). (8) A specific antibody against VEGF (vascular endothelial growth factor) reduces microvessel density and causes "significant or dramatic" inhibition of growth of three human tumors which rely on VEGF as their sole mediator of angiogenesis (in nude mice). The antibody does not inhibit growth of the tumor cells in vitro. (Kim, et al, Nature, 362:841-44 (1993)). (9) Anti-bFGF monoclonal antibody causes 70% inhibition of growth of a mouse tumor which is dependent upon secretion of bFGF as its only mediator of angiogenesis. The antibody does not inhibit growth of the tumor cells in vitro. (Hori, et al, Cancer Res., 51:6180-84 (1991)). (10) Intraperitoneal injection of bFGF enhances growth of a primary tumor and its metastases by stimulating growth of capillary endothelial cells in the tumor. The tumor cells themselves lack receptors for bFGF, and bFGF is not a mitogen for the tumor cells in vitro. (Gross, et al, Proc. Am. Assoc. Cancer Res., 31:79 (1990)). (11) A specific angiogenesis inhibitor (AGM-1470) inhibits tumor growth and metastases in vivo, but is much less active in inhibiting tumor cell proliferation in vitro. It inhibits vascular endothelial cell proliferation half- maximally at 4 logs lower concentration than it inhibits tumor cell proliferation. (Ingber, et al, Nature, 48:555-57 (1990)). There is also indirect clinical evidence that tumor growth is angiogenesis dependent. (12) Human retinoblastomas that are metastatic to the vitreous develop into avascular spheroids that are restricted to less than 1 mm³ despite the fact that they are viable and incorporate H-thymidine (when removed from an enucleated eye and analyzed in vitro). (13) Carcinoma of the ovary metastasizes to the peritoneal membrane as tiny avascular white seeds (1-3 mm ). These implants rarely grow larger until one or more of them becomes neovascularized. (14) Intensity of neovascularization in breast cancer (Weidner, et al, New Eng. J. Med., 324:1-8 (1991); Weidner, et al, JNat. Cancer Inst., 84:1875- 87 (1992)) and in prostate cancer (Weidner, et al, Am. J. Pathol, 143(2):401-09 (1993)) correlates highly with risk of future metastasis. (15) Metastasis from human cutaneous melanoma is rare prior to neovascularization. The onset of neovascularization leads to increased thickness of the lesion and an increased risk of metastasis. (Srivastava, et al, Am. J. Pathol, 133:419-23 (1988)). (16) In bladder cancer, the urinary level of an angiogenic protein, bFGF, is a more sensitive indicator of status and extent of disease than is cytology. (Nguyen, et al, J. Nat. Cancer Inst., 85:241-42 (1993)). Thus, it is clear that angiogenesis plays a major role in the metastasis of cancer. If this angiogenic activity could be repressed or eliminated, then the tumor, although present, would not grow. In the disease state, prevention of angiogenesis could avert the damage caused by the invasion of the new microvascular system. Therapies directed at control of the angiogenic processes could lead to the abrogation or mitigation of these diseases. Angiogenesis has been associated with a number of different types of cancer, including solid tumors and blood-borne tumors. Solid tumors with which angiogenesis has been associated include, but are not limited to, rhabdomyosarcomas, retinoblastoma, Ewing's sarcoma, neuroblastoma, and osteosarcoma. Angiogenesis is also associated with blood-borne 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 tumors and multiple myeloma diseases. One of the most frequent angiogenic diseases of childhood is the hemangioma. A hemangioma is a tumor composed of newly formed blood vessels. 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, 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 heredity diseases such as Osier- Weber-Rendu disease, or heredity 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 epitaxis (nose bleeds) or gastrointestinal bleeding and sometimes with pulmonary or hepatitic arteriovenous fistula. 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 compounds have been used to inhibit angiogenesis. Taylor, et al.

(Nature, 297:307 (1982)) have used protamine to inhibit angiogenesis. The toxicity of protamine limits its practical use as a therapeutic. Folkman, et al. (Science, 221:719 (1983), and U.S. Pat. Nos. 5,001,116 and 4,994,443) have disclosed the use of heparin and steroids to control angiogenesis. Steroids, such as tetrahydrocortisol, which lack gluccocorticoid and mineralocorticoid activity, have been found to be angiogenic inhibitors. 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 alpha or human interferon beta have been shown to inhibit tumor-induced angiogenesis in mouse dermis stimulated by human neoplastic cells. Interferon beta is also a potent inhibitor of angiogenesis induced by allogeneic spleen cells. (Sidky, et al, Cancer Res., 47:5155-61(1987)). Human recombinant interferon (alpha/A) was reported to be successfully used in the treatment of pulmonary hemangiomatosis, an angiogenesis-indύced disease. (White, et al, New Eng. J. Med., 320:1197-1200 (1989)). Other agents that have been used to inhibit angiogenesis include ascorbic acid ethers and related compounds. (Japanese Kokai Tokkyo Koho No.58-13 (1978)). Sulfated polysaccharide DS 4152 also inhibits angiogenesis. (Japanese Kokai Tokkyo Koho No. 63-119500). Additional anti-angiogenic compounds include Angiostatin® (U.S. Patent Nos. 5,639,725; 5,792,845; 5,885,795; 5,733,876; 5,776,704; 5,837,682; 5,861,372, and 5,854,221) and Endostatin (U.S. Patent No. 5,854,205). Another compound which has been shown to inhibit angiogenesis is thalidomide. (D'Amato, et al, Proc. Nail. Acad. Sci., 90:4082-85 (1994)). Thalidomide is a hypnosedative that has been successfully used to treat a number of diseases, such as rheumatoid arthritis (Gutierrez-Rodriguez, Arthritis Rheum., 27 (10):1118-21 (1984); Gutierrez-Rodriguez, et al, J. Rheumatol., 16(2): 158-63 (1989)), Behcet's disease (Handley, et al, Br. J. Dermatol, 127 Suppl, 40:67-8 (1992); Gunzler, Med. Hypotheses, 30(2): 105-9 (1989)), graft versus host rejection (Field, et al., Nature, 211(55): 1308-10 (1966); Heney, et al, Br. J. Haematol, 78 (l):23-7 (1991)), Mycobacteria diseases (Vicente, et al, Arch. Intern. Med., 153(4):534 (1993)), Herpes simplex and Herpes zoster infections (Naafs, et al, Int. J. Dermatol, 24(2):131-4 (1985)), chronic inflammation, ulcerative colitis (Meza, et al, Drug Ther, 23 (11): 74-80, 83 (1993); Powell, et al, Br. J. Dermatol, 113 Suppl 28: 141-4 (1985)), leprosy (Barnes, et al, Infect. Immun., 60(4): 1441-46 (1992)) and lupus (Burrows, BMJ, 307: 939-40 (1993)). Although thalidomide has minimal side effects in adults, it is a potent teratogen. Thus, there are concerns regarding its use in women of child-bearing age. Although minimal, there are a number of side effects that limit the desirability of thalidomide as a treatment. One such side effect is drowsiness. In a number of therapeutic studies, the initial dosage of thalidomide had to be reduced because patients became lethargic and had difficulty functioning normally. Another side effect limiting the use of thalidomide is peripheral neuropathy, in which individuals suffer from numbness and dysfunction in their extremities. Thus, improved methods and compositions are needed that are easily administered and capable of inhibiting angiogenesis and/or cell mitosis and for treating diseases associated therewith. What is also needed are safe and effective treatments that do not create unwanted side effects. 2-Methoxyestradiol is an endogenous metabolite of estradiol (E2). When administered orally, it exhibits anti-tumor and anti-proliferative activity with little toxicity. In vitro data suggests that 2-methoxyestradiol does not engage the estrogen receptor for its anti-proliferative activity and is not estrogenic over a wide range of concentrations, as assayed by estrogen dependant MCF-7 cell proliferation. 2-Methoxyestradiol is known to inhibit cell mitosis and angiogenesis and to treat various diseases associated therewith. See U.S. Pat. No. 5,504,074. However, 2-methoxyestradiol is produced in a crystalline form and has a relatively low solubility in water or aqueous solutions. Due to its relatively low solubility, it is difficult to achieve a therapeutic dose of 2-methoxyestradiol in the blood of a patient in need of such treatment. Therefore, there is a need for a form of 2-methoxyestradiol having greater bioavailable.

SUMMARY OF THE INVENTION The present invention relates to a form of 2-methoxyestradiol, and analogs thereof, that has greater bioavailable when administered orally and/or has greater solubility in water or aqueous solutions. The novel form of 2ME₂, and analogs thereof, is in an amorphous, non-crystalline form. The method by which the more bioavailable form of estradiol derivatives, especially 2-methoxyestradiol, and analogs thereof, is prepared is as follows. An estradiol derivative and at least one water-soluble polymer or copolymer are dissolved in a solvent or cosolvent system. This solution is then spray dried to form a solid powder having an amorphous, non-crystalline form and having improved solubility in water or aqueous solutions. The novel form of 2-methoxyestradiol, and analogs thereof, is useful in treating diseases associated with undesirable cell mitosis and/or undesirable angiogenesis. A mammalian disease characterized by undesirable cell mitosis, as defined herein, includes but is not limited to excessive or abnormal stimulation of endothelial cells (e.g., atherosclerosis), solid tumors and tumor metastasis, benign tumors, for example, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, vascular malfunctions, abnormal wound healing, inflammatory and immune disorders, Bechet's disease, gout or gouty arthritis, abnormal angiogenesis accompanying: rheumatoid arthritis, skin diseases, such as psoriasis, diabetic retinopathy and other ocular angiogenic diseases such as retinopathy of prematurity (retrolental fibroplasic), macular degeneration, corneal graft rejection, neovascular glaucoma and Osier Weber syndrome (Osier- Weber-Rendu disease). Other undesired angiogenesis involves normal processes including ovulation and implantation of a blastula. Accordingly, the compositions described above can be used to block ovulation and implantation of a blastula or to block menstruation (induce amenorrhea). The novel form of 2-methoxyestradiol, and analogs thereof, is especially useful in treating cancer. Accordingly, it is an object of the present invention to provide a novel form of 2-methoxyestradiol, and analogs thereof, that have greater bioavailable when administered orally. Another object of the present invention is to provide a novel form of 2- methoxyestradiol, and analogs thereof, that have greater solubility in water or aqueous solutions. A further object of the present invention is to provide a novel form of 2- methoxyestradiol, and analogs thereof, that are more effective in treating diseases associated with cell mitosis and/or angiogenesis. Yet another object of the present invention is to provide a novel form of 2- methoxyestradiol, and analogs thereof, that are more effective at treating cancer, especially solid tumors and metastatic tumors. These and other objects, features and advantages of the present invention will be apparent after reading the following detailed description of the preferred embodiments. DETAILED DESCRIPTION OF THE INVENTION The present invention comprises a novel form of 2-methoxyestradiol, and analogs thereof, that has greater bioavailability when administered orally and/or has greater solubility in water or aqueous solutions. This novel form of 2- methoxyestradiol, and analogs thereof, is in an amorphous, non-crystalline form. This novel form of 2-methoxyestradiol, and analogs thereof, also exhibits anti- mitotic, anti-angiogenic and/or anti-tumor properties.

Method of Preparation In accordance with the present invention, the novel form of 2- methoxyestradiol, and analogs thereof, is prepared by dissolving 2ME₂, or analogs thereof, and at least one water-soluble polymer or copolymer in a cosolvent system. The solution is then spray dried to form a solid powder having an amorphous, non-crystalline form. Water-soluble polymers or copolymers useful in the present invention include, but are not limited to polyvinylpyrrolidone. The polyvinylpyrrolidone can generally have a weight average molecular weight of approximately 15,000 to 90,000. Other water-soluble polymers or copolymers that are used in spray drying processes may also be used. The ratio of the amount of 2ME₂ to water- soluble polymer is approximately 3 : 1 to 1 :3 ; preferably, 1:1. The cosolvent system comprises any solvent or combination of two or more solvents that will dissolve both 2-methoxyestradiol and the water-soluble polymer(s) or copolymer(s). Useful solvents include, but are not limited to water, acetone, ethyl alcohol, and N-methyl-2-pyrrolidone ("NMP"). A preferred solvent system is a mixture of acetone and ethyl alcohol. The cosolvents may be heated and agitation may also be provided so that the 2ME₂, or analogs thereof, and water-soluble polymer(s) or copolymer(s) are completely dissolved in the cosolvents. Heating of the cosolvents should be relatively mild so as to avoid excessive evaporation of the cosolvents. Optionally, the solution of 2-methoxyestradiol and water-soluble polymer(s) or copolymer(s) may include additives that will improve the bioavailability of the novel form of 2-methoxyestradiol. Such additives include surfactants including, but not limited to, tocopheryl polyethylene glycol succinate ("TPGS"). Surfactants are also added to the solution of the present invention to enhance wetting, dispersion, dissolution, stability and/or solubilization of the novel form of 2-methoxyestradiol. The amount of additive used is that amount that is effective to render the novel form of 2-methoxyestradiol more bioavailable and/or to enhance wetting, dispersion, dissolution, stability and/or solubilization. Generally, amounts of surfactant that can be used to enhance bioavailability and/or to enhance wetting, dispersion, dissolution, and/or solubilization are approximately 1% to 25% by weight; preferably, approximately 4% to 15% by weight. Spray drying of the solution is carried out in a conventional two-fluid nozzle spray drier, such as Niro SD Micro¹"¹ spray dryer available from Niro Inc., Columbia, Maryland. The spray drier is adjusted to have a gas inlet temperature of approximately 105°- 135° C. and a gas outlet temperature of approximately 70°-90° C. The spray drying produces a solid powder that is collected at the bottom of the spray drier. The temperatures listed herein are specific to the Niro SD Micro*¹¹¹ spray dryer. Those skilled in the art will understand that different temperatures may be used for commercial scale equipment or equipment from other manufacturers. The solid powder is transferred from the spray drier to a fluid bed drier, such as the Niro MP l¹™ fluid bed. The solid powder is dried in the fluid bed drier to remove additional amounts of the cosolvent system not removed from the solid powder during processing in the spray drier. The fluid bed drier is adjusted to have an air inlet temperature of approximately 50°-90° C. The powder is dried in the fluid bed drier for a period of time sufficient to remove substantially all of the cosolvents from the powder; i.e., not more than 1% by weight cosolvents in the final product. Generally speaking, drying in the fluid bed drier takes approximately 10 minutes to 1 hour. The temperatures and times listed herein are specific to the Niro MP l^ta fluid bed. Those skilled in the art will understand that different temperatures and times may be used for commercial scale equipment or equipment from other manufacturers.

Experimental Data The invention relates to a form of estradiol derivatives, especially 2- methoxyestradiol, that is more bioavailable and/or has greater water or aqueous solubility. 2-Methoxyestradiol has the formula:

The more bioavailable form of 2ME₂ is prepared by first dissolving polyvinyl pyrrolidone (PVP) in ethyl alcohol. Acetone is added to the mixture. Then, 2ME₂ is added to the mixture of PVP, ethyl alcohol and acetone. The mixture is then heated to a temperature of 35°-40° C along with constant agitation. After the 2ME₂ is completely dissolved, the mixture is permitted to cool down to a temperature of 25°-30° C. The ratios and weights of the ingredients of the mixture are shown in the Table I below: TABLE I

The mixture is then spray dried in a Niro SD Micro*"¹ spray dryer having a gas inlet temperature of 105°- 135° C. and a gas outlet temperature of 70°-90° C. Powder is collected from the spray drier and is then placed in a Niro MP l^ta fluid bed drier. The fluid bed drier has a product retention screen of 100 mesh and an exhaust filter bag having pores of 3-20 microns. The fluid bed drier has an air inlet temperature of 50°-90° C. The powder is dried for 20 minutes. The dried powder is then collected from the fluid bed drier. The powder is in an amorphous, non-crystalline form and has greater bioavailability when administered orally and/or has greater solubility in water or aqueous solutions than 2-methoxyestradiol in its conventional crystalline form. The increased bioavailability of the present invention is dramatically demonstrated by the following data. The current clinical form of 2- methoxyestradiol being tested by EntreMed, Inc.; the prior art crystalline form of 2-methoxyestradiol, has a typical Cmax (nM) at 60 mg/km of 4.32. The amorphous form of 2-methoxyetsradiol in accordance with the present invention has a Cmax (nM) at 60 mg/km of 1195.47. Tables II, III and IV below show the composition of 16 different spray dryable formulas in accordance with the present invention.

TABLE II

TABLE III

TABLE IV

* Weight average, determined by light scattering Each of the foregoing 16 formulas was spray dried and dried in a fluid bed drier in accordance with the present invention. The solid amorphous powder for each of the formulas in Table II and III demonstrated enhanced bioavailablity.

Indications The invention can be used to treat any disease characterized by abnormal cell mitosis. Such diseases include, but are not limited to: abnormal stimulation of endothelial cells (e.g., atherosclerosis), solid tumors and tumor metastasis, benign tumors, for example, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, vascular malfunctions, abnormal wound healing, inflammatory and immune disorders, Bechet's disease, gout or gouty arthritis, abnormal angiogenesis accompanying: rheumatoid arthritis, skin diseases, such as psoriasis, diabetic retinopathy, and other ocular angiogenic diseases such as retinopathy of prematurity (retrolental fibroplasic), macular degeneration, corneal graft rejection, neuroscular glaucoma, liver diseases and Oster Webber syndrome (Osier- Weber Rendu disease). Diseases associated with corneal neovascularization can be treated according to the present invention. Such diseases include, but are not limited to, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma and retrolental fibroplasias, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, Sjogren's, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi's sarcoma, Mooren's ulcer, Temen's marginal degeneration, marginal keratolysis, trauma, rheumatoid arthritis, systemic lupus, polyarteritis, Wegener's sarcoidosis, Scleritis, Steven- Johnson disease, pemphigoid, radial keratotomy, and corneal graph rejection. Diseases associated with retinal/choroidal neovascularization can be treated according to the present invention. Such diseases include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, Eales' disease, Bechet's disease, infections causing a retinitis or choroiditis, presumed ocular histoplasmosis, Best's disease, myopia, optic pits, Stargart's 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 (neovasculariation 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 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, 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. Other diseases that can be treated according to the present invention are hemangiomas, Osler-Weber-Rendu disease, or hereditary hemorrhagic telangiectasia, solid or blood borne tumors and acquired immune deficiency syndrome. In addition, the invention can be used to treat a variety of post-menopausal symptoms, osteoporosis, cardiovascular disease, Alzheimer's disease, to reduce the incidence of strokes, and as an alternative to prior estrogen replacement therapies. The compounds of the present invention can work by estrogenic and non-estrogenic biochemical pathways. In addition to the compounds of the present invention, the pharmaceutical composition of this invention may also contain, or be co-administered (simultaneously or sequentially) with, one or more pharmacological agents of value in treating one or more disease conditions referred to hereinabove. A person skilled in the art will be able by reference to standard texts, such as Remington's Pharmaceutical Sciences 17th edition, to determine how the formulations are to be made and how these may be administered. The pharmaceutical composition may be used for the prophylaxis or treatment of conditions associated with angiogenesis or accelerated cell division or inflammation. In a still further aspect of the present invention there is provided a method of prophylaxis or treatment of a condition associated with angiogenesis or accelerated or increased amounts of cell division hypertrophic growth or inflammation, said method including administering to a patient in need of such prophylaxis or treatment an effective amount of a conjugated prodrug according to the present invention, as described above. It should be understood that prophylaxis or treatment of said condition includes amelioration of said condition. By "an effective amount" is meant a therapeutically or prophylactically effective amount. Such amounts can be readily determined by an appropriately skilled person, taking into account the condition to be treated, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable dose, mode and frequency of administration. Pharmaceutically acceptable salts of the compound of the formula may be prepared in any conventional manner for example from the free base and acid. In vivo hydrolysable esters, amides and carbamates may be prepared in any conventional manner. Administration The compositions described above can be provided as physiologically acceptable formulations using known techniques, and these formulations can be administered by standard routes. In general, the combinations may be administered by the topical, oral, rectal or parenteral (e.g., intravenous, subcutaneous or intramuscular) route. In addition, the combinations may be incorporated into biodegradable polymers allowing for sustained release, the polymers being implanted in the vicinity of where delivery is desired, for example, at the site of a tumor or within or near the eye. The dosage of the composition will depend on the condition being treated, the particular derivative used, and other clinical factors such as weight and condition of the patient and the route of administration of the compound. However, for oral administration to humans, a dosage of 0.01 to 100 mg/kg/day, preferably 0.01-20 mg/kg/day, is generally sufficient. The formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intraocular, intratracheal, and epidural) and inhalation 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 active ingredient and a pharmaceutical canier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid earners or finely divided solid carriers or both, and then, if necessary, shaping the product. Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil emulsion, etc. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide a slow or controlled release of the active ingredient therein. Formulations suitable for topical administration in the mouth include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the ingredient to be administered in a suitable liquid carrier. Formulations suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising the ingredient to be administered in a pharmaceutical acceptable carrier. A preferred topical delivery system is a transdermal patch containing the ingredient to be administered. Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate. Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing, in addition to the active ingredient, ingredients such as carriers as are known in the art to be appropriate. Formulation suitable for inhalation may be presented as mists, dusts, powders or spray formulations containing, in addition to the active ingredient, ingredients such as carriers as are known in the art to be appropriate. 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, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) conditions requiring only the addition of the sterile liquid canier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tables of the kind previously described. 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. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavoring agents. All of the publications mentioned herein are hereby incorporated by reference in their entireties. It should be understood, of course, that the foregoing relates only to certain disclosed embodiments of the present invention and that numerous modifications or alterations may be made therein without departing from the spirit and scope of the invention as set forth in the appended claims.

Claims

What is claimed is: 1. A method comprising the steps of: dissolving 2-methoxyestradiol, or analogs thereof, and at least one water-soluble polymer or copolymer in a cosolvent; and spray drying the solution to form a solid powder having an amorphous, non-crystalline form.

2. The method of Claim 1, wherein said water-soluble polymer or copolymer is polyvinylpyrrolidone.

3. The method of Claim 2, wherein said polyvinylpyrrolidone has a weight average molecular weight of approximately 15,000 to 90,000.

4. The method of Claim 1, wherein said cosolvent is selected from acetone, ethyl alcohol, N-methyl-2-pyrrolidone, or mixtures thereof.

5. The method of Claim 1, wherein said cosolvent is a mixture of acetone and ethyl alcohol.

6. The method of Claim 1, wherein the solution of 2-methoxyestradiol, polyvinylpyrrolidone and cosolvent further includes a surfactant in an amount sufficient to improve the bioavailability of the solid powder.

7. The method of Claim 6, wherein said surfactant is tocopheryl polyethylene glycol succinate.

8. The method of Claim 1, wherein said spray drier has a gas inlet temperature of approximately 105° to 135° and a gas outlet temperature of approximately 70° to 90°.

9. The method of Claim 1 further comprising the step of processing the solid powder is a fluid bed drier.

10. The method of Claim 9, wherein said fluid bed drier has an air inlet temperature of approximately 50° to 90°.

11. The method of Claim 9, wherein said solid powder is processed in said fluid bed drier for a period of time sufficient to remove substantially all of said cosolvent from said solid powder.

12. The method of Claim 9, wherein said solid powder is processed in said fluid bed drier for a period of time such that said solid powder has no more than 1% by weight cosolvent in said solid powder.

13. The method of Claim 9, wherein said solid powder is processed in said fluid bed drier for approximately 10 minutes to 1 hour.

14. A composition comprising 2-methoxyestradiol, or analogs thereof, in an amorphous, non-crystalline form.

15. The composition of Claim 14, wherein said 2-methoxyestradiol is substantially free of a solvent for said 2-methoxyestradiol.

16. The composition of Claim 14, wherein said 2-methoxyestradiol contains not more than 1% by weight of a solvent for said 2-methoxyestradiol.

17. The composition of Claim 14, wherein said 2-methoxyestradiol has improved bioavailability.