WO2003022266A1

WO2003022266A1 - Hypoestoxides, derivatives and agonists thereof for use as inhibitors of angiongenesis

Info

Publication number: WO2003022266A1
Application number: PCT/US2001/041880
Authority: WO
Inventors: Emmanuel A. Ojo-Amaize; Emeka J. Nchekwube; Howard B. Cottam; Donna N. Nchekwube
Original assignee: Paraquest, Inc.
Priority date: 2001-08-24
Filing date: 2001-08-24
Publication date: 2003-03-20

Abstract

A method for inhibiting angiogenesis in a mammal, the method comprising administering to a mammal an effective amount of a compound having the formula (I) and pharmaceutically acceptable salts thereof, wherein R is selected from the group consisting of (a) H (b) PO3 (c) PO3M, wherein M is selected from the group consisting of an alkali metal salt and an alkaline earth metal salt, (d) PO2M2, wherein each M is independently selected form the group consisting of alkali metal salts and alkaline earth metal salts (e) alkyl of 1 to 12 carbon atoms, which may be either substituted or unsubstituted, straight-chain or branched, having 0 to 6 double bonds (f) (CH2)n morpholine, wherein n = 1 to 4 (g) morpholinomethylphenyl (h) orthoaminophenyl (i) orthohydroxyphenyl (j) (CH2)mCOOR2, wherein m = 1 to 4, R2 is selected from the group consisting of H, an alkali metal salt, an alkaline earth metal salt, NH4?+, and N+(R¿3)4, wherein each R3 is independently selected from the group consisting of H and alkyl of 1 to 4 carbon atoms; (k) COR1, wherein R1 is selected from the group consisting of H, (CH2)pCH3, wherein p = 0 to 6, (CH2)mCOOR2, wherein m and R2 are previously defined, and (CH2)rSO3, wherein r = 1 to 4.

Description

HYPOESTOXIDES, DERIVATIVES AND AGONISTS THEREOF FOR USE AS INHIBITORS OF ANGIOGENESIS

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the use of diterpene compounds, in particular hypoestoxides, derivatives and agonists thereof for anti-angiogenic therapy. 2. Background Art

Angiogenesis, the sprouting of new blood vessels, plays a role in diverse diseases including diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, psoriasis, atherosclerosis, wound healing, solid tumors, hypertension, restenosis, and other diseases (Kim S. et al., Am. J. Pathology, 156: 1345-1362 (2000)). The complexity of the angiogenic process, which involves both positive and negative regulators, provides a number of targets for therapy. Many positive regulators, including growth factor receptors, matrix metalloproteinases and integrins, have been correlated, for example, with increased vascularity of tumors and poor prognosis for patient survival (Cherrington J.M. et al. Adv. Cancer Res. 79: 1-38 (2000)). Thus, these serve as ideal targets for anti-angiogenesis therapy. Indeed, the concept of angiogenesis inhibition has, in recent years, led to the idea of designing novel treatment strategies for atherosclerosis and restenosis (Isner, J.F. Circulation 99: 1653-1655 (1999)) whereby drugs that inhibit angiogenesis would restrict plaque growth and development at the vasa vasorum, reduce established plaque mass, and acutely stabilize the so-called vulnerable lesion. The clinical importance of plaque neovascularization is suggested by studies that show a higher prevalence of neovascularization in lesions with plaque rupture, mural hemorrhage, or unstable angina (Moulton, K.S. et al. Circulation 99: 1726-1732 (1999)).

Only a limited number of potent anti-angiogenic agents are presently known; TNP-470, as described by Isner, J.F. Circulation 99: 1653-1655 (1999), is one example. The development of new anti-angiogenic drugs is by necessity, therefore, a continuous process. Anti-angiogenic treatment could play a major role in the treatment of many common diseases, but medical science lacks a sufficient number of effective anti-angiogenic agents to make such treatment widespread. SUMMARY OF THE INVENTION

Applicants' invention rests on their finding that a select group of hypoestoxide analogs possess unexpected inhibitory activity against angiogenesis, and that such analogs may be used to treat conditions in which angiogenesis plays a role.

Described herein is a method for inhibiting angiogenesis comprising administering to a subject in need of anti-angiogenic therapy a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula

I:

I

and pharmaceutically acceptable salts thereof, wherein R is selected from the group consisting of a) H, b) PO₃ ⁼, c) PO₃M, wherein M is selected from the group consisting of an alkali metal salt and an alkaline earth metal salt, d) PO₂M₂, wherein each M is independently selected from the group consisting of alkali metal salts and alkaline earth metal salts, e) alkyl of 1 to 12 carbon atoms, which may be either substituted or unsubstituted, straight-chain or branched, having 0 to 6 double bonds, f) (CH2)_nmorpholine, wherein n = 1 to 4, g) morphoUnomethylphenyl, h) orthoaminophenyl, i) orthohydroxyphenyl, j) (CH₂)_mCOOR₂, wherein m = 1 to 4, R₂ is selected from the group consisting of H, an alkali metal salt, an alkaline earth metal salt, NH₄ ⁺, and

wherein each R₃ is independently selected from the group consisting of H and alkyl of 1 to 4 carbon atoms; k) CORi, wherein R_x is selected from the group consisting of H, (CH₂)_PCH3, wherein p = 0 to 6, (CH2)_mCOOR₂, wherein m and R₂ are as previously defined, (CH₂)qN⁺(R₃)₄, wherein q = 1 to 4 and R₃ is as previously defined, and (CH₂)_rSO₃ ^~, wherein r = 1 to 4.

The method further includes administering to a subject in need of anti- angiogenic therapy a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula IV:

IV

Compounds of Formulas I and IV are described in, for example, U.S. Patent No. 5,801,193, U.S. Patent No. 5,994, 328, and U.S. Patent No. 6,001,871, all of which are hereby incorporated by reference.

In another aspect, the invention provides a method of treating a subject to alleviate pathological effects of angiogenesis (including, for example, arthritis, atherosclerosis, restenosis, psoriasis, diabetic retinopathy, age-related macular degeneration, solid tumors such as glioblastoma multiforme, prostate, breast carcinomas, etc.)

According to a feature of the present invention, there is provided a therapeutic method for treating a subject having a angiogenic condition, comprising inhibiting angiogenesis by administering to the subject an effective amount of at least one compound having the formula of Formula I, a pro drug of Formula I, or of Formula IV.

BRIEF DESCRIPTION OF THE FIGURES Figures 1 and 2 show the effect of JO-4 on basic fibroblast growth factor

(bFGF)-induced angiogenesis in the chick chorioallantoic membrane (CAM). Figure 1 shows the number of blood-vessel branch points formed in the CAM at increasing levels of JO-4; Figure 2 shows the percent inhibition of blood vessel formation at increasing levels of JO-4. In both Figures, the IC50 value (the concentration at which JO-4 reduces blood vessel formation by 50%) is lOμM.

Figures 3 and 4 show the effect of JO-4 on cell adhesion and cell migration on vitronectin. Vitronectin is the substrate for endothelial cell integrin avb3 and is an important receptor for regulating angiogenesis in vivo. Although JO-4 does not inhibit cell adhesion (Figure 3) on vitronectin, JO-4 does inhibit endothelial cell migration (Figure 4) on vitronectin with an IC50 of lOμM.

Figures 5 and 6 show that JO-4 similarly does not inhibit endothelial cell attachment (Figure 5) on collagen, the substrate for integrin a2bl. However, JO-4 does inhibit cell migration (Figure 6) on collagen, with an IC50 of lOOμM.

Figures 7 and 8 show that JO-4 does not inhibit cell attachment (Figure 7) on fibronectin, the substrate for integrin a5bl, another integrin found to regulate angiogenesis. JO-4 does, however, inhibit cell migration (Figure 8) on fibronectin, with an IC50 of l00μM.

Figure 9 shows that JO-4 (listed as "IA-1") inhibits VEGF-induced human vascular endothelial cell proliferation with an IC50 of 28.6 μM. Tyrphostin AG1478, a potent inhibitor of angiogenesis had an IC50 of 37.3 μM in the same experiment. Figure 10 shows that JO-4 (labeled "Hypoestoxide") ameliorates collagen- induced arthritis (CIA) in mice. JO-4 ameliorated the collagen-induced hind paw edema by 58% and 46% on days 5 and 7, respectively. In the same experiment, indomethacin showed a 60% and 52% inhibition on corresponding days after induction of arthritis. These results demonstrate the in vivo efficacy of hypoestoxide when the agent was administered orally to type II collagen-induced arthritic mice. DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwise indicated, conventional laboratory animal handling techniques, cell biology, organic and medicinal chemical synthesis within the skill of the art. Such techniques are explained fully in the literature. See Aggarwal, B.B. and Puri, R.K. eds. Human cytokines: Their role in disease and therapy, Blackwell Science, Inc. (1993); Sun, L. and McMahon, G. "Inhibition of tumor angiogenesis by synthetic receptor tyrosine kinase inhibitors," Drug Discovery Today 5: 334-353 (2000); Shweiki, D. et al., "Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis," J. CJin. Invest. 91: 2235-2243 (1993); Gospodarowicz, D. et al., "Control of proliferation of human vascular endothelial cell, J. Cell Biol 77: 774-788 (1978); Griffioen, AW. and Molema, G., "Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases and chronic inflammation," Pharmacol. Rev., 52: 237-68 (2000); Rosen, L. "Antiangiogenic strategies and agents in clinical trials," Oncologist, 5 suppl 1: 20-27 (2000); Silverman, R.B. The Organic Chemistry of Drug Design and Drug Action, Academic Press, Inc. NY (1 92); Smith, M.B. Organic Synthesis, McGraw Hill, Inc. NY, (1994); Okogun, J.I. et al., Z. Naturforsch, 37c: 558-561 (1982); Adesomoju, A.A. et al; Phytochemistry, 22: 2535-2536 (1983). Those skilled in the art will understand those conventional techniques used to practice the instant teachings.

The following terminology will be used in accordance with the definitions set out below in describing the present invention. The term "vascular endothelial growth factor" (VEGF) refers to a cytokine released by fibroblasts, epithelial cells, several tumor cells, luteal cells, vascular smooth muscle cells, and leukocytes that potentiates vascular permeability and growth of new capillaries. VEGF is angiogenic in vitro, and it causes microvascular endothelial cells grown on 3D collagen gels to invade the underlying matrix and to form capillary-like tubules. In vivo, VEGF promotes vascular leakage, causes von Willebrand factor release, and synergizes with tumor necrosis factor-α to promote procoagulant activity on endothelial cells. VEGF induces expression of the only metalloproteinase that can initiate the degradation of interstitial collagen types I to III under normal physiological conditions. Monoclonal antibodies to VEGF inhibit the growth of rhabdomysarcoma, glioblastoma multiforme, and leiomyosarcoma cell lines in nude mice, but they have no effect on the growth rate of these tumor cells in vitro, implying a direct effect on reducing the vascular density in antibody-treated tumors (Kim, K.J. et al. Nature 362:841-844 (1993). In situ analysis of glioblastoma multiforme brain tumor specimens shows that VEGF production is specifically induced in a subset of glioblastoma cells distinguished by their proximity to necrotic foci (Aggarwal, B.B. and Puri, R.K. eds. Human Cytokines: Their role in disease and therapy, Blackwell Science, Inc. 1993). Because of these multiple effects, VEGF has been postulated to play a role in the pathogenesis of autoimmune disease, as well as in wound healing.

The term "angiogenesis" refers to the process of neovascularization, that is, the formation of new blood vessels, whether caused by VEGF release or otherwise. The current state of knowledge makes it clear that the cascade of angiogenic events depends on complex processes that include cell-cell interactions, various intracellular signaling pathways, and the appropriate extra-cellular micro environment. The result of this process is the migration and/or proliferation of endothelial and vascular smooth muscle cells. This migration and proliferation is of importance not only during normal growth, but also in pathological conditions.

As used herein, the term "angiogenic condition" means a disease of a living animal or one of its parts in which neovascularization is a component of the disease' s pathology. "Disease" in this context refers to any condition which impairs or interrupts normal functioning; it is not limited to a defective state of health. Angiogenic conditions include, for example, nasal polyps, sub-acute thyroiditis, arthritis, castleman's disease, retinal and chroidal neovascularization, glomerulonephritis, unicellular parasite infections, Alzheimer's disease, viral infections, restenosis, tumoural angiogenesis, atherosclerosis, asthma, diabetic vitreoretinopathy, myelodysplastic syndromes, chronic pancreatic diseases, pancreatic carcinoma, tendonitis, hemangioma, coronary vasculitis, Kawasaki disease, otitis media, corneal angiogenesis, uveitis, fibrocystic diseases, cystic fibrosis, and chronic pulmonary inflammation. One skilled in the art of medicine will be able to recognize additional angiogenic conditions.

The method of the invention can inhibit excessive vascular growth, and is therefore useful for impeding or arresting altogether the progression of the foregoing angiogenic conditions. The invention is particularly useful in treating angiogenic conditions which have as an additional element endothelial or vascular smooth muscle cell proliferation that is not necessarily associated with the unwanted neovascularization. For example, psoriasis may additionally involve endothelial cell proliferation that is independent of the endothelial cell proliferation associated with neovascularization. Likewise, a solid tumor which requires neovascularization for continued growth also may be a tumor of endothelial or vascular smooth muscle cells. In this case, the method of the invention can inhibit the tumor cells themselves from growing.

As used herein, the term "JO-4" means a compound which is a bicycle [9,3,1] pentadecane diterpene compound, as described in Z Naturforsc 37 c: 558-561 (1982) and in Heterocycles 20:2125-2128 (1983); these references refer to this compound as a "hypoestoxide." The chemical structure of JO-4 is illustrated in Formula II. π

JO-4 is a prodrug of JO-4A. In terms of Formula I, JO-4A is derived from Formula I when R is H. The structure of JO-4 A is illustrated in Formula III m

JO-4B has the same basic structure as the compound illustrated in Formula I, but lacks the alkyl group represented by "R:"

IV

It is understood that the compounds illustrated in Formulas I and IV include prodrugs, such as JO-4. The term "prodrug," as used herein, refers to a pharmacologically inactive compound that is converted to an active drug by a metabolic transformation. (Silverman, Richard B., The Organic Chemistry of Drug Design, Acad. Press (1992). There are numerous reasons why a prodrug strategy is used in drug design, the most common of which are to overcome problems associated with the compound, such as solubility, absorption and distribution, site specificity, instability, prolonged release, toxicity, poor patient acceptability, and formulation. Literature is widely available that describes how one can, without undue experimentation, determine how to deliver compounds to a locus to permit them to act on the locus; literature is also widely available that describes how one can calculate a therapeutically effective amount of a compound for inhibiting angiogenesis at the locus of action. The most common prodrug form for drugs containing alcohol or carboxylic acid functional groups is an ester. Using skills well known in the art, it is possible to alter the structure of the compound to improve its pharmacokinetic properties and, thereby, transform it into a useful drug for therapeutic administration to an animal. JO-4 is a prodrug for JO-4A in the presence of serum esterases in the in vivo setting, and in the in vitro setting if the culture medium contains added serum (which is most often the case). A preferred embodiment of the hypoestoxide compound for use in the method for inhibiting angiogenesis is the metabolite JO-4A, which is the free alcohol derivative of JO-4. JO-4 serves as an ester prodrug form for the delivery of JO-4 A, which is formed over time after administration of JO-4 to cells or animals. In similar fashion, many other ester prodrugs of JO-4 A provide delivery of JO-4 A. Such prodrug forms and methods for making them are well known in the art, as cited above. These prodrugs are known to yield the parent drugs of interest upon exposure to esterases commonly found in serum of animals and humans. It is understood that the prodrugs of JO-4A useful in the claimed method yield JO-4A and actively inhibit angiogenesis. The term "agonists" as used herein refers to substances that elicit the same response (i.e. inhibiting angiogenesis in subjects in need of such inhibition) as the compounds indicated in Formulas I and IV. Agonists of the compounds of Formula I include, but are not limited to, the prodrugs of JO-4A, such as JO-4 (Formula II). COMPOUNDS USED IN THE METHOD The compounds tested in the method of the invention included JO-4 (Formula

II) which is an ester of JO-4A, JO-4A (Formula III) and JO-4B (Formula IV). Compound Preparation

Preparation of JO-4A (Formula III). JO-4 crystals (82 mg, 0.22 mmol) were dissolved in a mixture of methanol (3 niL) and dioxane (3 mL) with warming and then cooled to room temperature. Fresh sodium methoxide powder was added to "pH 10." The mixture was stirred at room temperature overnight and the clear, orange- yellow reaction mixture was neutralized with Dowex -50 H+ resin, filtered and evaporated in vacuo to yield a pale yellow syrup which slowly crystallized in the freezer overnight. Yield = 65 mg, 90%. Preparation of JO-4B (Formula IV) (Method of E. J. Corey and G. Schmidt,

Tetrahedron Letters, 399-402, 1979). JO-4A (50 mg, 0.15 mmol) was dissolved in dichloromethane (1 mL) and cooled to 0° C. and 1.5 molar equivalents of pyridinium dichromate was added with efficient stirring. The reaction mixture was allowed to stir at room temperature for 6 hours and then diluted with ether, filtered and evaporated to yield an off-white sohd (30 mg, 60%>). The reaction yields a compound of Formula IV: IV

Esters of JO-4. As shown in Formula I, the compounds used in the method of the invention comprise esters of JO-4 A (Formula III), including JO-4 (Formula II), which was disclosed in Heterocycles 20:2125-2128 (1983) and in Z Naturoorsch 37c:558-561 (1982). Isolation of JO-4 from Hypoestes rosea.

The general procedure for isolation of pure JO-4 (Formula II) from dried Hypoestes rosea plant material involved solid/liquid extraction using boiling hexanes in a large Soxhlet apparatus. Hypoestes rosea is a shrub of the family Acantheceae. (Okugun, J. I. et al, Z Naturforsch 37c:558-561 (1982)). The crude extract obtained from the hexanes upon evaporation was subjected to flash silica gel column chromatography using a step gradient solvent system beginning with petroleum ether (30-60 bp) and stepping to 5% ethyl acetate, then to 10% and then 20%. At 30% ethyl acetate JO-4 was eluted from the column. The appropriate fractions were combined and concentrated to dryness, and petroleum ether or hexanes was added to obtain crystalline JO-4. One such procedure provided 240 mg pure JO-4 from 10 g crude extract from leaves. Preferably, the crude extract is first dissolved in a minimum of ethyl acetate, absorbed onto silica gel, and evaporated to a dry powder before loading onto a column prepacked in petroleum ether. Extraction of specific parts of the plant indicates that the leaves, as opposed to the stems, are the structures that contain the majority of the JO-4. The compounds of Formulas I and IV include pharmaceutically acceptable salts of those formulas. As used herein, the term "pharmaceutically acceptable salt" refers to salts which are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a mineral or organic acid or an inorganic base. Such salts are known as acid addition and base addition salts. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such pharmaceutically acceptable salts are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, thydroxybutyrate, glycollate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, napththalene-2 -sulfonate, mandelate and the like. Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.

It should be recognized that the particular ion forming a part of any salt of this invention is not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the ion does not contribute undesired qualities to the compound as a whole. The method of the invention includes administering to a mammal in need of anti-angiogenic treatment a therapeutically effective amount of at least one hypoestoxide compound having Formula I:

I

wherein R is selected from the group consisting of a) H, b) PO₃= c) PO₃M, wherein M is selected from the group consisting of an alkali metal salt and an alkaline earth metal salt, d) PO₂M₂, wherein each M is independently selected from the group consisting of alkali metal salts and alkaline earth metal salts, e) alkyl of 1 to 12 carbon atoms, which may be either substituted or unsubstituted, straight-chain or branched, having 0 to 6 double bonds, f) (CH₂)_nmorpholine, wherein n = 1 to 4, g) morphohnomethylphenyl, h) orthoaminophenyl, i) orthohydroxyphenyl, j) (CH_)_mCOOR₂, wherein m = 1 to 4, R₂ is selected from the group consisting of H, an alkali metal salt, an alkaline earth metal salt, NH , and TR_J)^ wherein each R₃ is independently selected from the group consisting of H and alkyl of 1 to 4 carbon atoms; k) CORi, wherein Ri is selected from the group consisting of H, (CH₂)_PCH₃, wherein p = 0 to 6, (CH₂)_mCOOR_2? wherein m and R₂ are as previously defined, (CH₂)qN⁺(R₃)₄, wherein q = 1 to 4 and R₃ is as previously defined, and (CH₂)_rSO₃ ^"", wherein r = 1 to 4; and pharmaceutically acceptable salts thereof.

The method of the invention further includes administering to a subject in need of anti-angiogenic therapy a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula IV: IV

In one presently preferred embodiment, compounds of Formula 1 are administered as JO-4 (Formula II) or JO-4A (Formula III).

As used herein, the term "effective amount" refers to that amount of a compound of the present invention which inhibits angiogenesis in the subject to which it is administered. The therapeutically effective amount of compounds of Formulas I and IV depends on the compound selected, the mode of administration employed, and the angiogenic condition desired to be treated. In general, satisfactory results would be obtained when administered orally or intravenously at a daily dosage of from about 0.001 mg to about 1000 mg per kg animal body weight, which may be divided into doses given 1 to 4 times a day or in sustained release form. If administered by injection, satisfactory results would generally be obtained when administered at a daily dosage of from about 0.001 mg to about 200 mg per kg animal body weight, preferably in the range of from about 50 mg to about 200 mg per kg, which may be given in divided doses 1 to 4 times a day or in sustained release form. Dosage forms suitable for oral administration comprise from about 0.001 mg to about 1000 mg of the compound admixed or in association with a solid or liquid carrier. Methods are well known in the art for determining therapeutically effective amounts of the compounds used in the method of the invention.

The compounds of Formulas I and IV may be administered in formulations suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intratracheal, and epidural) administration. In a presently preferred embodiment, these formulations contain less than 1% by weight, and more preferably about 0.2% by weight, of the compounds of Formula I and IV. These formulations may be prepared by conventional pharmaceutical techniques.

In general, the formulations are prepared by uniformly combining a compound of Formulas I or IV with a pharmaceutically acceptable liquid carrier, a finely divided solid carrier, or both, and then shaping the product if necessary. As used herein, "pharmaceutically acceptable carrier" refers to a carrier that is compatible with the other ingredients of the formulation and does not harm the subjects to which it is administered.

Suitable pharmaceutically acceptable carriers include, for example, water, alcohols, natural or hardened oils and waxes, calcium and sodium carbonates, calcium phosphate, kaolin, talc, and lactose.

A formulation may optionally contain one or more excipients, including the following: preservatives, such as ethyl-p-hydroxybenzoate; suspending agents such as methyl cellulose, tragacanth, and sodium alginate; wetting agents such as lecithin, polyoxyethylene stearate, and polyoxyethylene sorbitan mono-oleate; granulating and disintegrating agents such as starch and alginic acid; binding agents such as starch, gelatin, and acacia; lubricating agents such as magnesium stearate, stearic acid, and talc; and flavoring and coloring agents.

Formulations of the present invention suitable for oral administration may be presented in any of the following forms: discrete units such as capsules, cachets, or tablets each containing a predetermined amount of the active ingredient; powder or granules; solutions or suspensions in an aqueous liquid or a non-aqueous liquid; or, as oil-in-water liquid emulsions or water-in-oil emulsions, and any other form suitable for oral administration. Compounds of Formula I and IV are generally easiest to administer as solid preparations. For this reason, sohd tablets and solid-filled gelatin capsules are generally preferred, although other preparations may be more appropriate depending on the desired route of administration.

A tablet may be made by compressing or molding a compound of Formulas I or IV optionally with one or more excipients. For example, a compressed tablet may be prepared by compressing, in a suitable machine, a compound of Formulas I or IV in a free-flowing form such as a powder or granules, together with any of various excipients, such as a binder, lubricant, inert diluent, preservative, 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 compound therein. Liposomes may be used for this purpose; liposomes also facilitate movement of the compounds across tissue barriers. Formulations suitable for topical administration in the mouth include lozenges comprising a compound of Formulas I or IV and a flavored base, such as sucrose and acacia or tragacanth; pastilles comprising a compound of Formulas I or IV and an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising a compound of Formulas I or IV in a suitable liquid carrier. Formulations suitable for topical administration to the skin may be presented as ointments, creams, gels, and pastes comprising a compound of Formulas I or IV and a pharmaceutically acceptable carrier.

Formulations suitable for administration to the eye include eye drops and ointments. Eye drops may comprise a compound of Formulas I or IV and a carrier such as water and glycerin; ointments may comprise a compound of Formulas I or IV and a carrier such as mineral oil and petroleum jelly.

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 may comprise either solid or liquid preparations. Where the carrier is a solid, it may be a coarse powder having a particle size, for example, in the range of 20 to 500 microns. This powder 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. Where the carrier is a liquid, it may be administered as a nasal spray or as nasal drops, either of which may comprise aqueous or oily solutions of a compound of Formulas I or IV. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations. Such formulations comprise a compound of Formulas I or IV and a pharmaceutically acceptable carrier. Formulations suitable for parenteral administration include, for example, 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, for example, in unit-dose or multi-dose containers, sealed ampules and vials, and may be stored in freeze-dried (lyophilized) conditions requiring only the addition of the sterile liquid carrier immediately prior to use.

EXAMPLES

The following non-limiting Examples illustrate results obtained with the method of the present invention.

Example 1 Cell adhesion assays

Wells of 48 non-tissue culture treated culture dishes (Costar, Inc.) were coated with 5 μg/ml vitronectin, fibronectin, or collagen (obtained from Collaborative Biomedical Products, Bedford, MA.) for one hour at 37°C and blocked with 2% heat denatured bovine serum albumin in phosphate buffered saline for one hour. Fifty thousand cells in 25 μg/ml of an anti-α5βl function blocking antibody (JBS5),

25 μg/ml of an anti-αv β3 function blocking antibody (LM609), 25 μg/ml of an anti- α2βl function blocking antibody, 25 μg/ml of an anti-βl function blocking antibody (P4cl0) or 0-100μM JO-4 in adhesion buffer (Hepes buffered Hanks balanced salt solution, HBSS, (obtained from Irvine Scientific, Irvine, CA) containing 1% bovine serum albumin,2 mM MgCl2 , 2mM CaCl) were allowed to adhere to dishes for twenty minutes at 37°C. Each experiment was performed in triplicate, with triplicate samples per condition. The data are presented in Figure 3 as percent of adhesion exhibited by the positive control (adhesion medium alone) +/- standard error of the mean. Example 2

Migration assays

Migration assays were performed as follows: the lower side of 8 μM pore transwell inserts (Costar, Inc.) were coated with 5 μg/ml of fibronectin, vitronectin, Del-1, collagen or no protein for one hour and were blocked with 2% bovine serum albumin in phosphate buffered saline for one hour. Human umbilical vein endothelial cells (HUVECs) (25,000) were added to the upper chamber of inserts in migration buffer (Hepes buffered M199 medium containing 1% BSA, 1.8 mM CaCl2, 1.8 mM MgC12, and 25μg/ml of various antibodies or 0-100 μM JO-4. Cells were allowed to migrate from the upper to the lower chamber for four hours at 37°C. Non-migratory cells were removed from the upper chamber by wiping the upper surface with an absorbent tip. Cells that had migrated to the lower side of the transwell insert were then fixed for 15 minutes with 3.7% paraformaldehyde for 15 minutes and with a 2% crystal violet solution. After extensive water washing to remove excess crystal violet, the number of cells which had migrated (to the bottom of the insert) were counted in three representative high power (200X) fields per insert. No cells were found on the culture dish in which the insert was placed. The data are presented in Figure 4 as number of cells migrating +/- standard error of the mean.

Example 3 In ovo chick chorioallantoic membrane angiogenesis assays

Angiogenesis assays were performed essentially as described by Kim, S. et al. (Am. J. Pathol. 156: 1345-1362 (2000)) as follows: chorioallantoic membranes (CAMs) often-day old embryonated chicken eggs were stimulated with cortisone acetate pre-treated filter discs saturated in 1 μg/ml bFGF or saline. Twenty-five μg of function blocking anti- α5βlin saline, 0-500 μM JO-4 in 25μl, or 25 μl of saline were applied to the growth factor saturated filter disk 24 hours later. CAMs were harvested on the fourth day of stimulation by fixation with a drop of 3% Para formaldehyde in phosphate buffered saline prior to excision of the stimulated area. Blood vessel branch points in the 5mm disk were counted at 30 X magnification under fiber optic illumination in a blinded fashion. At least ten embryos were used per treatment group. Each experiment was performed a minimum of three times. Data were evaluated in terms of average number of blood vessel branch points per treatment group +/- standard error of the mean. Statistical analyses were performed using Student's t-test.

Example 4 VEGF-induced cell proliferation

This assay was performed essentially as described by Gospodarowicz, D., et al. (J. CellBiol. 1978, 77:774-788). HUVECs were incubated in M199 medium containing 20% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 4 mM L- glutamate, 15 mM Hepes, lOμg/ml heparin for 2 days at 37°C. Confluent cultures were suspended with 0.25 % trypsin. Trypsinization was stopped with addition of culture media and the cell suspension was distributed in microtiter dishes so that 10,000 cells were placed per well. JO-4 was dissolved in 0.4% DMSO in complete culture media. VEGF was added to all culture wells and different concentrations of JO-4 (lOOμM, lOμM, lμM, O.lμM, and 10 nM) were added to the JO-4 treated wells. As a positive VEGF antagonist control, tyrphostin AG1478 was added in wells without JO-4. All cultures were incubated for two days at 37°C. At the end of two days, cell proliferation was measured by fluorescent quantitation. Significant criteria for antagonism were set at > 50% of maximal stimulation or inhibition. The results are shown in Figure 9.

Example 5 Type II collagen-induced arthritis in mice (CIA)

Arthritic inflammation was induced in mice essentially as previously described by Terato, K. et a\.(Autoimmιmity 22: 137-147 (1995)). A group of 5 Balb/cByJ strain of mice, 6-8 weeks of age, were used for the induction of inflammation (arthritis) by monoclonal antibodies (mAbs) responding to type II collagen, plus LPS. A combination of 4 different mABS (D8, F10, DI-2G and A2) was administered intravenously at a total of 4 mg/mouse at day 0. This was followed by the intravenous administration of 25 μg of LPS 72 hours later (day 3). From day 3, 1 hr after LPS administration, 30 mg/kg hypoestoxide, 3 mg/kg indomethacin, and/or vehicle (2% tween 80/distilled water) were administered orally once daily for 3 consecutive days. At day 5, one or two paws (particularly the hind paws) began to appear red and swollen, and by day 7, arthritis symptoms of the hind paws were severely red and swollen. A plethysmometer (Ugo Basile Cat #7150) with water cell (12 mm diameter) was used for the measurement of volume of the two hind paws on day 0, 5, and 7. The percent inhibition of increase in volume relative to vehicle control was calculated by the following formula:

Inhibition (%) = [ l-(Tn - To)/ (Cn - Co)] x 100 Where:

Co (Cn): volume on day 0 (day n) in vehicle control group

To (Tn): volume on day 0 (day n) in test compound-treated group Inhibition of edema in both hind paws by more than 30% is considered as significant anti-inflammatory (anti-arthritis) activity. The results of this experiment are shown in Figure 10.

Example 6

Measurement of tumor volume in mice

B16-F1 melanoma cells growing exponentially in vitro were harvested by 15 minutes incubation with 0.25% trypsin-EDTA solution (Irvine Scientific, Irvine, Calif). The mice were given subcutaneous (s.c.) injections of 50,000 viable cells on the right side (Fidler, I.J. Cancer Research 35: 218-224 (1975)). Four days after injection, the mice in the experimental group were treated s.c. (1 x daily for 12 days) on the left side with 10 mg/kg or 2 mg/kg of JO-4 in 0.2 ml of physiological saline. The control, non-treated group received vehicle only. The tumors were measured daily with a microcaliper, and the tumor volume was calculated by the formula:

Length x width x height x 0.5236 (Jungwirth, A. et al., Proc. Natl. Acad. Sci. USA, 94: 5810-5813. (1997)). For statistical analysis, Student's t-test was used to calculate the level of significance.

TABLE 1 Effect of subcutaneous administration of JO-4 on the growth of subcutaneous implantation of BI6-F1 Melanoma in C57BL/6 Mice

TREATMENT NO. MICE/GROUP TUMOR VOLUME (mm³) % SURVIVAL

Control (no drug) 10 17,570 +/-3,280 0 JO-4 10 mg/kg 10 1,417 +/- 367 100 " 2 mg/kg 10 2,547 +/- 572 100

All of the references cited herein are hereby incorporated in their entireties by reference.

While there has been illustrated and described what are presently considered to be the preferred embodiments of the present invention, it will be understood by those skilled in the art that various other modifications may be made, and equivalents may be substituted, without departing from the true scope of the invention. Therefore, it is intended that the present invention not be limited to the particular embodiments disclosed, but that the invention include all embodiments falling within the scope of the appended claims.

Claims

What is claimed is:

1. A method for inhibiting angiogenesis in a mammal, the method comprising administering to a mammal an effective amount of a compound having the formula

and pharmaceutically acceptable salts thereof, wherein R is selected from the group consisting of a) H, b) PO₃ ⁼, c) PO₃M, wherein M is selected from the group consisting of an alkali metal salt and an alkaline earth metal salt, d) PO₂M₂, wherein each M is independently selected from the group consisting of alkali metal salts and alkaline earth metal salts, e) alkyl of 1 to 12 carbon atoms, which may be either substituted or unsubstituted, straight-chain or branched, having 0 to 6 double bonds, f) (CH₂)_nmorpholine, wherein n = 1 to 4, g) morpholinomethylphenyl, h) orthoaminophenyl, i) orthohydroxyphenyl, j) (CH₂)_mCOOR₂, wherein m = 1 to 4, R₂ is selected from the group consisting of H, an alkali metal salt, an alkahne earth metal salt, NH₄ ⁺, and N^Rs)^ wherein each R₃ is independently selected from the group consisting of H and alkyl of 1 to 4 carbon atoms; k) COR_l3 wherein Ri is selected from the group consisting of H, (CH₂)pCH₃, wherein p = 0 to 6, (CH₂)_mCOOR2, wherein m and R₂ are as previously defined, (CH₂)_nN⁺(R₃)₄, wherein q = 1 to 4 and R₃ is as previously defined, and (CH₂)_rSO₃ ^~, wherein r = 1 to 4.

2. The method of claim 1 , wherein R is H.

3. The method of claim 1, wherein R is COCH3.

4. The method of claim 1, wherein the effective amount is 0.001 mg to 1000 mg per kg of body weight of the mammal.

5. The method of claim 4, wherein the effective amount is 50 mg to 200 mg per kg of body weight of the mammal.

6. The method of claim 1, further comprising administering the compound in a pharmaceutically acceptable carrier.

7. A method for treating an angiogenic condition, the method comprising administering to a mammal in need of such treatment an effective amount of a compound having the formula

and pharmaceutically acceptable salts thereof, wherein R is selected from the group consisting of a) H, b) PO₃ ⁼, c) PO₃M, wherein M is selected from the group consisting of an alkali metal salt and an alkaline earth metal salt,

' d) PO₂M₂, wherein each M is independently selected from the group consisting of alkali metal salts and alkaline earth metal salts, e) alkyl of 1 to 12 carbon atoms, which may be either substituted or unsubstituted, straight-chain or branched, having 0 to 6 double bonds, f) (CH₂)_nmorpholine, wherein n = 1 to 4, g) morpholinomethylphenyl, h) orthoaminophenyl, i) orthohydroxyphenyl, j) (CH₂)_mCOOR2, wherein m = 1 to 4, R₂ is selected from the group consisting of H, an alkali metal salt, an alkaline earth metal salt, NH ⁺, and N⁺(Rs) , wherein each R₃ is independently selected from the group consisting of H and alkyl of 1 to 4 carbon atoms; k) CORi, wherein Ri is selected from the group consisting of H, (CH₂)_PCH₃, wherein p = 0 to 6, (CH₂)_mCOOR₂, wherein m and R are as previously defined, (CH₂)_qN⁺(R₃) , wherein q = 1 to 4 and R₃ is as previously defined, and (CH₂)_rSO₃ ^", wherein r = 1 to 4.

8. The method of claim 7, wherein the angiogenic condition is selected from the group consisting of nasal polyps, sub-acute thyroiditis, arthritis, castleman's disease, retinal and chroidal neovascularization, glomerulonephritis, unicellular parasite infections, Alzheimer's disease, viral infections, restenosis, tumoural angiogenesis, atherosclerosis, asthma, diabetic vitreoretinopathy, myelodysplastic syndromes, tendonitis, hemangioma, coronary vasculitis, Kawasaki disease, otitis media, corneal angiogenesis, glaucoma, retinitis pigmentosa, uveitis, endometriosis, adenomyosis, dys-functional uterine bleeding, uterine leiomyoma, fϊbrocystic diseases, cystic fibrosis, chronic pulmonary inflammation, psoriasis, obesity, and undesirable hair growth,

9. The method of claim 7, wherein R is H.

10. The method of claim 8, wherein R is H.

11. The method of claim 7, wherein R is COCH₃.

12. The method of claim 8, wherein R is COCH₃.

13. The method of claim 7, wherein the effective amount is 0.001 mg to 1000 mg per kg of body weight of the mammal.

14. The method of claim 8, wherein the effective amount is 0.001 mg to 1000 mg per kg of body weight of the mammal.

15. The method of claim 13, wherein the effective amount is 50 mg to 200 mg per kg of body weight of the mammal.

16. The method of claim 14, wherein the effective amount is 50 mg to 200 mg per kg of body weight of the mammal.

17. The method of claim 7, further comprising administering the compound in a pharmaceutically acceptable carrier.

18. The method of claim 8, further comprising administering the compound in a pharmaceutically acceptable carrier.

19. A method for treating an angiogenic condition, the method comprising administering to a mammal in need of such treatment an effective amount of a compound having the formula

20. The method of claim 19, wherein the effective amount is 0.001 mg to 1000 mg per kg of body weight of the mammal.

21. The method of claim 20, wherein the effective amount is 50 mg to 200 mg per kg of body weight of the mammal.

22. The method of claim 19, further comprising administering the compound in a pharmaceutically acceptable carrier.