WO1997041844A1 - Combinations of angiostatic compounds - Google Patents

Abstract

The present invention is directed to compositions containing combinations of angiostatic compounds and methods for their use in preventing pathological neovascularization.

Description

COMBINATIONS OF ANGIOSTATIC COMPOUNDS

Background ofthe invention

The present invention relates to certain compounds useful in preventing and

treating neovascularization. Specifically, the invention is directed to compositions

containing two or more combinations of angiostatic agents and methods of using

combinations of these angiostatic agents to treat neovascularization.

Angiogenesis is a term used to describe the development of new blood vessels or

neovascularization (L. Diaz-Flores et al., Angiogenesis: an Update, Histology and

Histopathology. volume 9, pages 807-843 (1994)). Though angiogenesis is a normal

process for the development or maintenance of the vasculature, pathological conditions

(i.e.. angiogenesis dependent diseases) arise where blood vessel growth is actually

harmful. Such pathologies include diabetic retinopathies, proliferative

vitreoretinopathies, psoriasis, arthritis and solid tumor development. The progression of

angiogenesis occurs in several phases which include: elaboration of the angiogenic

signal; dissolution of the blood vessel basement membrane; endothelial cell proliferation;

endothelial cell migration; and formation and differentiation of capillary tubules and

loops. Each of these phases is a potential target for pharmacological intervention.

Tumor growth is dependent on neovascularization. For solid tumors to grow

beyond the size of a pea, they must become vascularized. They do so by secreting their own angiogenic factor(s) which recruit new blood vessels to provide essential nutrients

and oxygen.

Angiogenesis is also associated with important diseases of ocular tissue especially

in older patients and diabetics. Any abnormal growth of blood vessels in the eye can

scatter and block the incident light prior to reaching the retina. Neovascularization can

occur at almost any site in the eye and significantly alter ocular tissue function. Some of

the most threatening ocular neovascular diseases are those which involve the retina. For

example, many diabetic patients develop a retinopathy which is characterized by the

formation of leaky, new blood vessels on the anterior surface of the retina and in the

vitreous causing proliferative vitreoretinopathy. A subset of patients with age related

macular degeneration develop subretinal neovascularization which leads to their eventual

blindness.

Current therapy for the treatment of ocular neovascular disease is not very

effective. Retinal neovascularization is often treated with multiple laser burns to the

retina to remove the pathological vasculature. Panretinal photocoagulation, however,

destroys normal retinal tissue. Patients with neovascular diseases of the anterior chamber

(e.g. corneal neovascularization, iritis rubeosis) are treated with potent topical ocular

glucocorticoids. These therapies are only partially effective and generally only slow

neovascularization and the progress of the overall disease. In addition, they can cause

severe side effects if used over a relatively long period of time.

Other attempts have been made to provide therapies for the prevention or

treatment of pathological angiogenesis. For example, angiostatic steroids functioning to

inhibit angiogenesis in the presence of heparin or specific heparin fragments in the chicken embryo model of neovascularization are disclosed in Crum, et al., A New Class of

Steroids Inhibits Angiogenesis in the Presence of Heparin or a Heparin Fragment,

Science, volume 230, pages 1375-1378 (1985). Other groups of angiostatic steroids

useful in inhibiting angiogenesis are disclosed in commonly assigned WIPO Publication

No. WO 93/10141 (Clark et al.) and United States Patent No. 5,371,078 (Clark et al.), as

well as WO 95/18621 (Prioa et al.).

Glucocorticoids, as mentioned above, have also been shown to inhibit

angiogenesis. However, the use of glucocorticoid therapy in general is complicated by

the inherent problems associated with steroid applications. Such problems include

elevated intraocular pressure (Kitazawa, Increased Intraocular Pressure Induced by

Corticosteroids, American Journal of Ophthalmology, volume 82, pages 492-495 (1976)),

and the development of posterior subcapsular cataracts.

Suramin, a complex molecule, has been described as a growth factor antagonist

and possessing angiosuppressive action (Takano, Angiosuppressive and Antiproliferative

Actions of Suramin: A Growth Factor Antagonist, Growth Factors. Peptides and

Receptors. Ed. T.W. Moody, Plenum Press, New York, pages 255-264 (1993)). Suramin

and its derivatives have also been disclosed in WIPO Publication No. WO 90/15816, as

inhibitors of fibroblast growth factor (an angiogenesis factor) as well as apparent

angiostatic agents.

Fumagillin and analogs of fumagillin have been reported to possess angiostatic

properties (Ingber et al., Synthetic Analogues of Fumagillin that Inhibit Angiogenesis and

Suppress Tumor Growth, Nature, volume 348, pages 555-557 (1990)). Several European

patent applications have disclosed fumagillin analogs including: European Patent Application Nos. 0 354 787 Al, 0 386 667 Al and 0 470 569 Al, all assigned to Takeda

Chemical (Japan) or Fujisawa Pharmaceutical (Japan).

Anti-estrogens, or estrogen antagonists, have also been reported to possess

angiostatic activity. Antiestrogens have been shown to alter the activity of a number of

growth factors that are important in the control of cellular proliferation (Freiss et al.

Antisteroidal and anti-growth factor activities of antiestrogens, Journal of Steroid

Biochemistry and Molecular Biology, volume 37, pages 777-781 (1990)). They are

known to affect biochemical functions and to inhibit angiogenesis in a dose dependent

manner (Gagliardi et al., Inhibition of Angiogenesis by Antiestrogens, Cancer Research.

volume 53, pages 533-535 (1993)).

Still other therapies have been proposed, including, the use of protamine (S.

Taylor, Protamine is an Inhibitor of Angiogenesis, Nature, volume 297, pages 307-312

(1982)), and the use of Vitamin D₃ analogs (Oikawa et al., Inhibition of Angiogenesis by

Vitamin D₃ Analogues, European Journal of Pharmacology, volume 178, pages 247-250 (1990)). The use of a variety of pharmaceutical proteins has also been proposed for

treating angiogenesis. Such therapies have included: monoclonal antibodies directed to

fibroblast growth factor, disclosed in WO 91/06668; platelet factor 4, disclosed in WO

93/02192; and thrombospondin fragments, disclosed in WO 93/16716.

Summary ofthe Invention

The present invention involves the angiostatic therapy of a combination of two or

more molecules selected from a set of angiostatic compounds. As such, the present invention is directed to methods of using combinations of angiostatic compounds for the

prevention and/or treatment of neovascularization in human patients. The present

invention is also directed to compositions containing combinations of angiostatic

compounds. In particular, the compositions are useful for controlling ocular neovascularization.

The combination therapy of the present invention is believed to have the

advantage of providing effective, multi-mechanistic angiostatic therapy which is more

efficacious with fewer side effects.

Detailed Description of the Invention

The compositions and methods of the present invention involve various

combinations of angiostatic compounds. Table 1 contains a list of different angiostatic

compounds useful in the present invention:

Table 1

Classes of Angiostatic Agents (examples where specified)

Anti-mitotics (5-flurouracil, mytomycin-C, taxol) Estrogen metabolites (2-methoxyestradiol)

Matrix metalloproteinase inhibitors (betimastat, BB-2516, TIMPs, minocycline, GM6001 )

Plasminogen activator/urokinase inhibitors

Urokinase receptor antagonists Platelet factor 4 and analogs Heparinases

Cartilage-derived inhibitor of angiogenesis

Thrombospondin and related analogs (TSP-1)

Angiostatin, vasculostatin

Proliferin-related protein

Fumagillin analogs (TNP-470)

Tecogalan

Pentosan polysulfate

Thalidomide and related analogs

CM101

Tyrosine kinase inhibitors (SU101)

Anti-sense oligonucleotides (specific for bFGF and VEGF)

Suramin and suramin analogs

Angiostatic steriods α_vB₃ and α_vβ₅ integrin antagonists

Cytotoxic antibodies against endothelial cell antigens (anti-endoglin)

Interferon (α-IFN)

VEGF and bFGF antagonists (VEGF receptor-chimeric proteins) flk-1 and fit- 1 antagonists

IL- 1 , and TNF antagonists

Additionally, compounds of formula (I) below are also useful angiostatic compounds of

the present invention:

wherein:

n is 1 or 2;

R is H, C,-C₆ alkyl or C₃-C₆ cycloalkyl;

Y is H, C,-C₆ alkyl, C₃-C₆ cycloalkyl, O, NR, C(R)₂ , CH(OH) or S(O)_n. n' is 0 to 2;

R' is H, C(O)R, C(O)N(R)₂, PO₃ ^' , SO₃ ^" , or HO₂C(CH₂)₂(C=O)-~ ;

R" is H or C,-C₆ alkyl;

R³ is H, C,-C₆ alkyl, (CH₂)_q(OH), — (C=O)O(CH₂)_qCH₃ or

; provided that R' can not be H, when R is q is 1 to 10; and

Z, if present, is H, C_rC₆ alkyl, C₃-C₆ cycloalkyl, or selected from the group consisting of:

wherein:

D is O or NR; and

E and E' are independently H, F or Cl. The compounds ofthe present invention also include pharmaceutically acceptable salts of

the compounds of formula (I) and the compounds included in the angiostatic classes listed on Table 1.

The initiation of new blood vessel formation may arise quite differently in various

tissues or as a result of different diseases. Many substances have been found to induce

neovascularization; see, Folkman, et al., Angiogenic Factors, Science, volume 235, pages 442-447 (1987). It is believed, however, that once initiated, the process of

neovascularization is similar in all tissues regardless ofthe associated disease; see, Furcht,

Critical Factors Controlling Angiogenesis: Cell Products, Cell Matrix, and Growth

Factors, Laboratory Investigation, volume 55, No. 5, pages 505-509 (1986).

There are many theories associated with the cause of neovascularization, and there

may be different inducers depending on the disease or surgery involved; see, BenEzra,

Neovasculogenic Ability of Prostaglandins, Growth Factors, and Synthetic Chemoattractants, American Journal of Ophthalmology, volume 86, No. 4, pages 455-

461, (October, 1978). Regardless of the cause or the associated disease, it is believed that

angiostatic agents work by inhibiting one or more steps in the process of

neovascularization.

As stated above, angiogenesis is brought about through a number of biochemical

and cellular mechanisms involving several steps, each of which is a potential target for

pharmacological intervention. This process generally consists of the following steps:

breakdown of blood vessel basement membranes, endothelial cell activation, migration,

proliferation and formation of capillary tubules. In response to an angiogenic signal,

endothelial cells become "activated" and release proteases and other degradative enzymes which dissolve the basement membranes surrounding the cells. The endothelial cells can

now migrate towards the stimulus elongating and aligning themselves to create a sprout.

Rapid proliferation lengthens the columns of cells and branches will appear at the top of

the column Branches from adjacent columns fuse and in this way a network of new

capillaries is formed. Thus, inhibition of this complex process of angiogenesis is possible

at several stages These include interception of the activation signal, inhibition of

basement membrane breakdown, inhibition of cell migration, inhibition of cell

proliferation as well as interference with the formation of capillary tubules.

Different compounds may affect different stages of the angiogenic process. For

example, antibodies to growth factors such as bFGF (Hori et al., Suppression oj solid

tumor growth by immunoneutralizing monoclonal antibody against human basic

fibroblast growth factor, Cancer Research, volume 51 , pages 6180-6184 (1991)) and

VEGF (Kim et al., Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo, Nature, volume 362, pages 841-844 (1993)) can

interfere with the activation signal; fumagillin-type and suramin-type compounds inhibit

endothelial cell migration; chroman deπvatives and fumagillin-type compounds (Ingber et

al., Synthetic analogs of fumagillin that inhibit angiogenesis and suppress tumour growth, Nature, volume 348, pages 555-557 (1990)) and suramin-type compounds (C Stein,

Suramin A novel antineoplastic agent with multiple potential mechanisms of action,

Cancer Research, volume 53, pages 2239-2248 (1993)) inhibit cell proliferation, and

angiostatic steroids are currently thought to exert their anti-angiogenic effects by

inhibiting basement membrane breakdown (Ashino-Fuse et al., Medroxyprogesterone

Acetate, An Anti-Cancer And Anti-Angiogemc Steroid, fnhibits The Plasminogen Activator In Bovine Endothelial Cells, volume 44, pages 859-864 (1989)) or by inhibiting

PAI-1 synthesis (Blei et al., Mechanism of Action of Angiostatic Steriods: Suppression of

Plasminogen Activator Activity Via Stimulation of Plasminogen Activator Inhibitor

Synthesis, Journal of Cellular Physiology, volume 155, pages 568-578 (1993)). Therefore, therapeutic intervention is possible at several points in the process.

While applicants do not wish to be bound by any theory, it is believed that the

inhibition of multiple cellular/biological mechanisms associated with angiogenesis will

more effectively inhibit neovascularization. Therefore, the use of combinations of

compounds affecting different mechanisms of angiogenesis would be more effective in

preventing neovascularization than a single therapeutic approach. For example,

combinations of anti-proliferative compounds such as chroman derivatives and angiostatic steroids which affect basement membrane breakdown would be more effective

in preventing neovascularization than either drug alone. Therefore, the present invention

sets forth the use of combinations of different angiostatic agents to provide a more

effective therapeutic approach to inhibiting neovascularization. As used herein, the term

"angiostatic agent or compound" refers to any compound which inhibits one or more

processes of angiogenesis such that angiogenesis is inhibited or retarded.

Preferred compounds of formula (I), also known as "chroman derivatives,"

include the following:

2-(6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-benzo[l,2-b]pyran-2-yl)ethyl 2-(6- methoxy-2-naphthyl)propionate ("Compound A");

2-(5-hydroxy-2,4,6,7-tetramethyl-2,3-dihydro-benzo[l,2-b]furan-2-yl)methyl 2-(6- methoxy-2-naphthyl)propionate ("Compound B");

N-(2-(6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-benzo[l,2-b]pyran-2-yl)methyl) 2- (6-methoxy-2-naphthyl)propionamide ("Compound C");

2-(5-hydroxy-2,4,6,7-tetramethyl-2,3-dihydro-benzo[l ,2-b]furan-2-yl)ethyl 2-(6- methoxy-2-naphthyl)propionate ("Compound D");

l-[2-(5-hydroxy-2,4,6,7-tetramethyl-2,3-dihydro-benzo[l,2-b]furan-2-yl)2-ethyl]-4-[4,4'- fluorobenzhydryljpiperzine ("Compound E")

2-(6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-benzo[l,2-b]pyran-2-yl)ethanol ("Compound F")

Vitamin E succinate (VES) ("Compound G")

2-(6-hydroxy-2,5,7,8-tetramethyl-2,3-dihydro-2H-benzo[l,2-b]pyran-2-yl)ethyl 2-(3- fluoro-4-phenyl-phenyl)propionate ("Compound H").

"Angiostatic steroids" are compounds containing the 6-6-6-5-ring steroid

backbone and possessing angiostatic activity. Preferred angiostatic steroids of the present

invention have been disclosed in United States Patent No. 5,371 ,078 (Clark et al.) and

WIPO Publication WO 93/10141 (Clark et al.); the entire contents of these publications

are incorporated herein by reference. Preferred angiostatic steriods are: 21 -Nor-5β-pregnan-3c<, 17α,20-triol

-Nor-5^β-pregn- 17(20)en-3α, 16-diol-3-acetate- 16-(O-methyl)malonate

21 -Nor-5^β-pregnan-3α, 17α,20-triol-3 -acetate

21-Nor-5α-pregnan-3α,17α,20-triol-3-phosphate

4-Androsten-3-one-17β-carboxylic acid

10

21 -Nor-5β-pregn- 17(20)en-3α, 16-diol

15 21 -Nor-5β-pregnan-3α, 17 β,20-triol

20-Acetamido-21-nor-5^β-pregnan-3α,17α-diol-3-acetate

10

3β-Azido-5β- pregnan -1 1 β,17α,21-triol-20- one-21 -acetate

17α-Ethynyl-5( 10)-estren- 17β-ol-3-one

21 -Nor-5α-pregnan-3α, 17β,20-triol

α-Methyl-5β-pregnan-3α, 11 β, 17α,21 -tetrol-20-one-21 -methyl ether

17α-Ethynyl- 1 ,3,5(10)-estratrien-3 , 17β-diol

4,9( 11 )-Pregnadien- 17α,21 -diol-3,20-dione-21 -acetate

4,9( 1 1 )-Pregnadien- 17α,21 -diol-3 ,20-dione

"Suramin-type compounds" are compounds which mimic the anti-angiogenic

action of suramin. Suramin and suramin-type compounds are known to those skilled in

the art. Examples of suramin-type compounds are: suramin,

3-hydroxy-2,7-naphthalenesulfonic acid, 4,5-dihydroxy-2, 7-naphthalenedi sulfonic acid,

2,2^'-[(l,8-dihydroxy-3,6-disulfo-2,7-napthylene)bis(azo]di-benzenearsonic acid, 5 4,4^'-bis[[4-(o-hydroxyanilino)-6-(m-sulfoanilino)-s-triazin-2-yl]amino]-

2,2 ^'stilbenedisulfonic acid,

4,5-dihydroxy-3-[(p-nitrophenyl)azo]-2,7-naphthalenedisulfonic acid, 4,5-dihydroxy-3,6-bis[(4-sulfo-l -naphthyl)azo]-2,7-naphthalene-disulfonic acid, 3-[(5-chloro-2-hydroxyphenyl)azo]-4,5-dihydroxy-2,7-naphthalene-disulfonic I o acid,

4,5 ^'-dihydroxy-3,6 ^' [3,3 ^'-dimethoxy-4,4 ^'-biphenylylene)bis(azo)-di- 1 - naphthalenesulfonic acid,

3,6-[(2,3-dimethyl-5-oxo-l-phenyl-3-pyrazolin-4-yl)azo]-4,5-dihydroxy-2,7- naphthalenedisulfonic acid, 15 5,5 ^'-[ureylenebis[2-sulfo-p-phenylene_azo]bis[6-amino-4-hydroxy-2- naphthalenesulfonic acid,

4-[(o-arsonophenyl)azo]3-hydroxy-2,7-naphthalenedisulfonic acid, 4.5-dihydroxy-3-(phenylazo)-2,7-naphthaienedisulfonic acid, 4-acetamido-5-hydroxy-6-(phenylazo)- 1 ,7-naphthaIenedisulfonic acid, 20 2-[p-f(l -hydroxy-4-sulfo-2-naphthyl)azo]phenyl]-6-methyl-7- benzothiazolesulfonic acid,

4-[(2,4-dimethyiphenyl)azo]-3-hydroxy-2,7-napthalenedisulfonic acid, 3-[(4-Sulfophenyl)azo]-4,5-dihydroxy-2,7-naphthalenedisulfonic acid, 3-[(4-nitrophenyl)azo]-4-amino-5-hydroxy-2,7-naphthalene-disulfonic acid, 25 l-nitro-4,6,8-naphthalenetrisulfonic acid, l -amino-4,6,8-naphthalenetrisulfonic acid and pharmaceutically acceptable salts thereof.

The most preferred suramin-type compounds include: suramin and 4,4^'-bis[[4-(o- hydroxyanilino)-6-(m-sulfoanilino)-s-triazin-2-yl]amino]-2,2^'stilbenedisulfonic acid. 30 "Fumagillin-type compounds" are oxaspiro[2,5]octane derivatives, such as those described in European Patent Application Nos. 0 354 787 A 1 , 0 386 667 A 1 and 0 387 650 Al , the entire contents of these three publications are incoφorated herein by reference to the extent they disclose angiostatic fumagillin-type compounds.

"Anti -estrogen compounds" are those compounds which at least partially bind estrogen receptors. A number of anti-estrogens have been shown to inhibit angiogenesis. Anti-estrogen compounds of the present invention include: clomiphene, tamoxifen, nafoxidine, ICI 164,984 and ICI 182,780.

Other angiostatic compounds contained in the Table 1 are known in the art to possess angiostatic activity. Of those classes listed, 5-flurouracil, mitomycin-C, taxol, 2- methoxyestradiol, betimastat, BB-2516, TIMPs, minocycline, GM6001 , PF4, CDI, TSP- 1 , TNP-470, SU101 , anti-endoglin, α-IFN and VEGF receptor-chimeric proteins are preferred for use in combinations of angiostatic compounds ofthe present invention.

The following are the most preferred types of angiostatic compounds to be included in combinations of angiostatic compounds ofthe present invention: compounds of formula (I), anti-mitotics, angiostatic steroids, fumagillin- type compounds, suramin-type compounds, estrogen metabolites, matrix metalloproteinase inhibitors and thalidomide.

The following are the most preferred combinations of compounds to be used in compositions and methods ofthe present invention:

1 ) A compound of formula (I) and an angiostatic steroid 2) A compound of formula (I) and a suramin-type compound

3) A compound of formula (I) and a fumagillin-type compound

4) A compound of formula (1) and an anti-mitotic

5) An angiostatic steroid and a fumagillin-type compound

6) An anti-mitotic and an angiostatic steriod 7) A compound of formula (I) and an estrogen derivative

8) An angiostatic steroid and an estrogen metabolite

9) A compound of formula (I) and a matrix metalloprotease inhibitor

The combination of angiostatic compounds of the present invention are useful in

inhibiting pathological neovascularization in human patients. As used herein, the term "pathological neovascularization" refers to those conditions where the formation of blood

vessels (neovascularization) is harmful to the patient. Examples of pathological

neovascularization dependent diseases include: head trauma, spinal trauma, systemic or

traumatic shock, stroke, hemorrhagic shock, cancer, arthritis, arteriosclerosis,

angiofibroma, arteriovenous malformations, corneal graft neovascularization,

inappropriate wound healing, diabetic retinopathy, granulations, burns, hemangioma,

hemophilic joints, hypertrophic scars, ocular neovascularization, nonunion fractures,

Osier-Weber Syndrome, psoriasis, pyogenic granuloma, retrolental fibroplasia,

scleroderma, trachoma, vascular adhesions, and solid tumor growth.

In particular, the compositions of the present invention are useful in preventing

and treating any ocular neovascularization, including, but not limited to: retinal diseases

(diabetic retinopathy, chronic glaucoma, retinal detachment, sickle cell retinopathy and

subretinal neovascularization due to age related macular degeneration); rubeosis iritis;

proliferative vitreoretinopathy; inflammatory diseases; chronic uveitis; neoplasms

(retinoblastoma, pseudoglioma and melanoma); Fuchs' heterochromic iridocyclitis; neovascular glaucoma; corneal neovascularization (inflammatory, transplantation and

developmental hypoplasia of the iris); neovascularization following a combined

vitrectomy and lensectomy; vascular diseases (retinal ischemia, choroidal vascular

insufficiency, choroidal thrombosis and carotid artery ischemia); neovascularization of

the optic nerve; and neovascularization due to penetration of the eye or contusive ocular

injury. Additionally the combinations of angiostatic agents are useful in treating

pterygium (primary and recurrent), glaucoma filtration surgery bleb failure,

hyperkeratosis, cheloid formation and polyp formation.

The use of the compositions of the present invention to ameliorate complications

arising from glaucoma filtration surgery is a particularly important aspect of the

invention. Glaucoma filtration surgery involves the surgical creation of a fistula with a

conjuctival flap which allows the direct drainage of aqueous humor from the anterior

chamber into the conjuctival tissue thereby lowering the elevated intraocular pressure

associated with glaucoma. However, in many patients, the filtration "bleb" becomes

scarred or healed over so that aqueous drainage can no longer occur. It has been noted

that failing filtration blebs may become vascularized prior to failure. This vascuiarization

may feed the fibroblasts which migrate, and proliferate, and block the bleb, or the

vascuiarization itself may also result in physical blockage of the bleb. It is therefore

likely that inhibition of filtration bleb neovascularization may inhibit filtration bleb

failure.

The angiostatic compounds may be contained in various types of pharmaceutical

compositions, either together as a single composition or in separate compositions, in

accordance with formulation techniques known to those skilled in the art. For example,

the compounds may be included in tablets, capsules, solutions, suspensions and other

dosage forms adapted for oral administration; solutions and suspensions adapted for

parenteral use; solutions, suspensions or gels for topical ocular administration; solutions

and suspensions adapted for intra-vitreal or intra-cameral use; and suppositories for rectal

use. Solutions, suspensions and other dosage forms adapted for topical application to the involved tissues, such as tissue irrigating solutions, are particularly preferred for treatment

of acute conditions associated with surgery or other forms of trauma.

The present invention is particularly directed to the provision of compositions

adapted for treatment of ophthalmic tissues. Various types of vehicles may be used. The

vehicles will generally be aqueous in nature. Aqueous solutions are generally preferred,

based on ease of formulation, as well as a patient's ability to easily administer such

compositions by means of instilling one to two drops of the solutions in the affected eyes.

However, the compounds of the present invention may also be readily incorporated into

other types of compositions, such as suspensions, viscous or semi-viscous gels or other

types of solid or semi-solid compositions. Suspensions may be preferred for compounds

of the present invention which are relatively insoluble in water. The ophthalmic

compositions ofthe present invention may also include various other ingredients, such as

buffers, preservatives, co-solvents and viscosity building agents.

An appropriate buffer system (e.g., sodium phosphate, sodium acetate or sodium

borate) may be added to prevent pH drift under storage conditions.

Ophthalmic products are typically packaged in multidose form. Preservatives are

thus required to prevent microbial contamination during use. Suitable preservatives

include: benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl

paraben, phenylethyl alcohol, edetate disodium, sorbic acid, polyquaternium-l , or other

agents known to those skilled in the art. Such preservatives are typically employed at a

level of from 0.001 to 1.0 percent by weight, based on the total weight ofthe composition

(wt.%). The route of administration (e.g., topical, parenteral or oral) and the dosage

regimen will be determined by skilled clinicians, based on factors such as the exact nature

of the condition being treated, the severity of the condition, the age and general physical

condition of the patient, and so on.

As indicated above, use of compounds of the present invention to prevent or

reduce angiogenesis in ophthalmic tissues is a particularly important aspect of the present

invention. The compounds may also be used as an adjunct to ophthalmic surgery, such as

by vitreal or subconjunctival injection following ophthalmic surgery. The compounds

may be used for acute treatment of temporary conditions, or may be administered

chronically, especially in the case of degenerative disease. The compounds may also be

used prophylactically, especially prior to ocular surgery or noninvasive ophthalmic

procedures, or other types of surgery.

The use of physiologically balanced irrigating solutions as pharmaceutical

vehicles for the angiostic compounds is preferred when the compositions are administered

intraocularly. As used herein, the term "physiologically balanced irrigating solution"

means a solution which is adapted to maintain the physical structure and function of

tissues during invasive or noninvasive medical procedures. This type of solution will

typically contain electrolytes, such as sodium, potassium, calcium, magnesium and/or

chloride; an energy source, such as dextrose; and a buffer to maintain the pH of the

solution at or near physiological levels. Various solutions of this type are known (e.g.,

Lactated Ringers Solution). BSS® Sterile Irrigating Solution and BSS Plus® Sterile

Intraocular Irrigating Solution (Alcon Laboratories, Inc., Fort Worth, Texas, USA) are

examples of physiologically balanced intraocular irrigating solutions. The latter type of solution is described in United States Patent No. 4,550,022 (Garabedian, et al.), the entire

contents of which are hereby incoφorated in the present specification by reference.

The specific type of formulation selected will depend on various factors, such as

the compound or its salt being used, the dosage frequency, and the disease being treated.

Topical aqueous solutions, suspensions, ointments, creams and gels are the preferred

dosage forms for the treatment of pterygium, hyperkeratosis, and cheloid and polyp

formation. Topical ophthalmic formulations are suitable for preventing glaucoma

filtration bleb failure or scar formation associated with ophthalmic surgery.

In general, the doses used for the above described puφoses will vary, but will be

in an effective amount to inhibit or reduce neovascularization. As used herein, the term

"pharmaceutically effective amount" to inhibit or reduce neovascularization, is that

amount of a combination of two or more compounds of the present invention which inhibits formation of new blood vessels or reduces the number of blood vessels which are

involved in the pathological condition. The compounds will normally be contained in

these formulations in an amount from about 0.01 to about 10.0 weight/percent. Preferable

concentrations range from about 0.1 to about 5.0 weight/percent. Thus, for topical

administration, these formulations are delivered to the disease site one to six times a day,

depending on the routine discretion of the skilled clinician. Systemic administration, for

example, in the form of tablets or suppositories is useful for the treatment of polyp

formation. Tablets containing 10- 1000 mg of a compound can be taken 2-3 times per day

depending on the discretion of the skilled clinician. The compositions of the present invention are further illustrated by the following

examples. The term "angiostatic compound" refers to any compound of the present

invention, as described above.

Example 1

Topical combination compositions useful for controlling ocular neovascularization:

Example 2

A preferred topical composition useful for controlling neovascularzation:

The above formulation is prepared by first placing a portion of the purified water

into a beaker and heating to 90°C. The hydroxypropylmethylcellulose (HPMC) is then

added to the heated water and mixed by means of vigorous vortex stirring until all of the

HPMC is dispersed. The resulting mixture is then allowed to cool while undergoing mixing

in order to hydrate the HPMC. The resulting solution is then sterilized by means of

autoclaving in a vessel having a liquid inlet and a hydrophobic, sterile air vent filter.

The sodium chloride and the edetate disodium are then added to a second portion of

the purified water and dissolved. The benzalkonium chloride is then added to the solution,

and the pH of the solution is adjusted to 7.4 with 0.1M NaOH/HCl. The solution is then

sterilized by means of filtration. The angiostatic compounds are sterilized by either dry heat or ethylene oxide. If

ethylene oxide sterilization is selected, aeration for at least 72 hours at 50°C. is necessary.

The sterilized angiogenic compound is weighed aseptically and placed into a pressurized

ballmill container. The tyloxapol, in sterilized aqueous solution form, is then added to the

ballmill container. Sterilized glass balls are then added to the container and the contents of

the container are milled aseptically at 225 φm for 16 hours, or until all particles are in the

range of approximately 5 microns.

Under aseptic conditions, the micronized drug suspension formed by means of the

preceding step is then poured into the HPMC solution with mixing. The ballmill container

and balls contained therein are then rinsed with a portion of the solution containing the

sodium chloride, the edetate disodium and benzalkonium chloride. The rinse is then added

aseptically to the HPMC solution. The final volume of the solution is then adjusted with

purified water and, if necessary, the pH of the solution is adjusted to pH 7.4 with

NaOH/HCl.

Example 3

Formulation for oral administration:

Tablet:

10-1000 mg of two angiostatic compounds with inactive ingredients such as starch, lactose and magnesium stearate can be formulated according to procedures known to those skilled in the art of tablet formulation. Example 4

Formulation for sterile intraocular injection:

Example 5

Preferred formulation for a topical ocular solution:

Example fr

A preferred formulation for oral administration:

Tablet:

5-100 mg of Compound A and 10-1000 mg of another Angiostatic Compound with inactive ingredients such as starch, lactose and magnesium stearate can be formulated according to procedures known to those skilled in the art of tablet formulation. Example 7

Formulations for topical dermatological use:

Cream: 1 mg/g each of two angiostatic compounds in cream base of purifed water, emulsifying wax, propylene glycol, stearic acid, isopropyl palmitate, synthetic beeswax, polysorbate 60, potassium sorbate, sorbic acid, propyl gallate, citric acid, and sodium hydroxide.

Ointment: 1 mg/g each of two angiostatic compounds in base of mineral oil and polyethylene.

Example 8

Formulation for suppository:

10-500 mg each of two angiostatic compounds with the following inactive ingredients: glycerin, butylateal hydroxytoluene, butylated hydroxy anisole, edetic acid, polyethylene glycol, and sodium chloride.

Some of the compounds of the present invention may contain a nonsteroidal anti-

inflammatory agent (NSAIA) component or a calcium channel blocker (flunarizine)

component. These individual moieties may add additional pharmaceutical benefit to the

angiostatic efficacy of compounds of formula (I).

The compounds of formula (I) are synthesized by known methods in the art.

Compounds containing a non-steroidal anti-inflammatory agent (flurbiprofen or

naproxen) can be made by methods illustrated in Scheme 1 and 2, and Examples 11-14. Compounds containing a flunarizine moiety may be made by methods disclosed in

commonly assigned PCT Patent Publication No. WO/9515958, the entire contents of

which are hereby incoφorated by reference. Other compounds of formula (I) are

commercially available from: Sigma Chemical Co. (St. Louis, Missouri) and Aldrich

Chemical Co. (Milwaukee, Wisconsin).

Scheme 1

A-OH + H-X-(CH₂)_n-Y-(CH₂)_m-Z — > A-X-(CH₂)„-Y-(CH₂)_m-Z (eq 1 )

II III I

A-OH — > A-CI + H-X-(CH₂)„-Y-(CH₂)_m-Z — >^* A-X-(CH₂)„-Y-(CH₂)_n,-Z (eq.2) II IV III I

A-OH — > A-0^"M⁺ + L-(CH₂)„-Y-(CH₂)_m-Z — >A-0-(CH₂)_n-Y-(CH₂)_m-Z (eq. 3)

II V L=C1, BR, I, OMs, OTs I

VI

A-OH — > A-0^"M⁺ + Br-CH₂-C(0)OEt — ^ A-0-CH,-C(0)OEt + II V VII

H-NR-(CH₂)„-Y-(CH₂)_m-Z —> A-NR-(CH₂)„-Y-(CH₂)_τn-Z (eq 4)

VIII I

The conversion of the carboxylic acid containing nonsteroidal anti-inflammatory agents (II) to esters or amides (I) may be carried out by the following methods:

(i) As illustrated in equation 1 above, carboxylic acids (II) may be reacted with the appropriate amine or alcohol derivative (III) in the presence of a coupling reagent, such as dicyclohexylcarbodiimide or l -(3-dimethylaminopropyl)-3-ethyl carbodiimide HCl, and 4-dimethylamine pyridine or 1 -hydroxybenzotriazole, in an inert organic solvent, such as acetonitrile or tetrahydrofuran, and at a temperature from 0°C to 50°C. (ii) As illustrated in equation 2 above, carboxylic acids (II) may be converted to acid chlorides (IV) by reacting them with a reagent such as thionyl chloride or oxalyl chloride, in the presence of an inert solid or neat, at a temperature from 0°C to 80°C. The resulting acid chloride (IV) may be reacted with the desired amine or alcohol (III) in an inert solvent such as tetrahydrofuran, in the presence of pyridine or a tertiary amine, such as triethylamine.

(iii) As illustrated in equation 3 above, esters (I) may be formed by reacting carboxylate anions (V), formed by reacting the carboxylic acid (II) with a base such as sodium hydride, with a halide (iodide, bromide, chloride) or sulfonate (mesylate, tosylate) (VI), in a solvent such as acetonitrile or dimethylformamide, at a temperature from 0°C to

100°C.

(iv) As illustrated in equation 4 above, amides (I) may be prepared by reacting carboxylate anions (V), formed by reacting carboxylic acid (II) with a base such as sodium hydride, with ethyl bromoacetate. The resulting ester (VII) is reacted with the desired amine (VIII), neat or in an inert solvent, such as acetonitrile or dimethylformamide, at a temperature from 0°C to 100°C.

The intermediate compounds (X) of Scheme 2 below, which can be used as

compounds (III) and (VIII), were prepared using the general methods described in Cohen,

et al., Lewis Acid Mediated Nucleophilic Substitution Reactions of 2-Alkoxy-3,4-dihydro-

2H-l-ben∑opyrans: Regiochemistry and Utility in the Synthesis of 3,4-Dihydro-2H-l -

benzopyran-2-carboxylic Acids, Journal of Organic Chemistry, volume 54, pages 3282-

3292, (1989). The nitrile (IX) can be reduced using a reagent such as lithium aluminum

hydride to afford the amine (X), which may be isolated as the hydrochloride salt.

The use of certain protecting groups and deprotection steps may be necessary, as

will be appreciated by those skilled in the art. Scheme 2

Compounds of formula (I) may exist as mixtures of stereoisomers. The

preparation of the individual stereoisomers may be effected by preparing and resolving

the acids (II), by known methods, and then using a single stereoisomer as starting

material. Compounds (III), (VI) and (VIII) may be prepared as single stereoisomers from

compounds of formula (XI_a._b), shown in Table 5 below, using known methods:

Table 5

wherein:

W is (CH₂)_p-Q; p is 0-1;

Q is CH₂OH or CO₂H;

R' is H, C(O)R, C(O)NR₂, PO₃ ^" , or SO₃ ^" ; and

R" is H or C,-C₆ alkyl. The alcohols (XI_a._b) may be resolved by forming esters with optically active carboxylic acids, separating the diastereomers, and then hydrolyzing the resolved diastereomers. The corresponding carboxylic acids (XI_a._b) may be resolved by forming an ester with an optically active alcohol, separating the diastereomers, and then hydrolyzing the resolved diastereomers. Or, the carboxylic acids (XI_a-b) may be resolved by forming an amine salt with an optically active amine. Separation by recrystallization and neutralization of the resolved carboxylic acid salt may be utilized to provide the resolved carboxylic acid. Resolution of the esters and amides (I) may also be effected using chromatographic techniques known to those skilled in the art. The amines of formula (I), where Y is NR, may be converted to amine salts by reacting the amine with acids of sufficient strength to produce an organic or inorganic salt. The pharmaceutically acceptable anions include: acetate, bromide, chloride, citrate, maleate, fumarate, mesylate, phosphate, sulfate and tartrate.

Methods of synthesizing the compounds formula (I) are further illustrated by the

following examples:

Example 11

Synthesis of N-[(6-hydroxv-2.5.7.8-tetramethvl-3.4-dihvdro-2H-l -benzo[1.2-b]pyran-2- vnmethyl] 2-(6-methoxv-2-naphthvnpropionamide

The intermediate, (6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro-2H- 1 -benzo[ 1 ,2-b]pyran-2- yl)methylamine, was first synthesized:

A 1 molar (M) ethereal solution of lithium aluminum hydride (Aldrich, 32.4 mL, 32.43 mmol) was added slowly over a 5 minute period to a chilled, (4-6°C) stirring solution of (2-cyano-6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-l-benzo[l,2-b]pyran in tetrahydrofuran (50 mL). After 2 hours, the reaction mixture was quenched by the slow sequential addition of 10% aqueous tetrahydrofuran (30 mL), 15% sodium hydroxide (10 mL) and then water (20 mL), while stirring. The resulting suspension was filtered through celite, and the celite pad was washed with ethyl ether (400 mL). The organic layer was separated, dried (Na₂SO₄), and concentrated in vacuo. resulting in a residue. A 1 M ethereal solution of hydrochloride was then added to a solution of the residue in ethyl ether (100 mL), a solid formed, and the solid was then collected by filtration and washed with ethyl ether to give 2.31 g (65.4% yield) of a white solid. The product was used crude in the next reaction.

1H-NMR (DMSO-d₆/TMS): 1.15 (s, 3H), 1.75 (t, 2H), 1.99 (s, 6H), 2.01 (s, 3H), 2.54 (t, 2H), 2.98 (s, 2H).

MS (CI): 236 (m+l).

The hydrochloride salt of (6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-l-benzo[l,2- b]pyran-2-yl)mefhylamine (0.30 g, 1.10 mmole) and 6-methoxy-α-methyl naphthaleneacetic acid (Aldrich, 0.28 g, 1.21 mmole) were stirred in the presence of dimethylaminopyridine (Aldrich, 0.26 g, 2.20 mmole) and l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (Janssen Chimica-Spectrum, 0.21 g, 1.10 mmole), in tetrahydrofuran (4.0 mL) under an atmosphere of nitrogen. After stirring 17 hours at ambient temperature, the reaction mixture was diluted with ethyl acetate (70 mL), washed with water (2x 15 mL), followed by brine (15 mL) and then dried (sodium sulfate). The mixture was concentrated in vacuo and the residue subjected to flash chromatography

(silica gel, 100-50:0-50, v:v, hexanes:ethyl acetate). The appropriate fractions were concentrated in vacuo. and the resulting crystalline foam suspension was then washed in hexanes to give 0.28 g (58.3% yield) of N-[(5-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro-

2H-l-benzopyran-2-yl)methyl]-2-(6-methoxy-2-naphthyl)propionamide as a white amoφhous solid.

Η-NMR (CDC1₃) d 1.03-1.08 (d,3H), 1.57-1.64 (m, 6H), 1.70 (t, 2H,), 2.04-2.05 (m, 6H,), 2.48-2.51 (m, 2H), 3.16-3.58 (m, 2H), 3.74 (q, IH), 3.91 (s, 3H), 4.91 (br s, IH), 5.751 (t, IH), 7.01-7.19 (m, 2H), 7.29-7.40 (t, IH), 7.52-7.81 (m, 3H).

Elemental Analysis: Calculated for C₂₈H₃₃NO₄ Calculated: C, 75.14; H, 7.43; N, 3.13. Found: C, 75.04; H, 7.50; N, 2.97. Melting point: 67-70°C.

Example 12

Synthesis of 2-(6-hvdroxv-2.5.7.8-tetramethyl-3.4-dihvdro-2H-henzo[L2-h]pyran- 2ynethyl 2-(6-methoxv-2-naphthvnpropionate

A solution of 1 ,3-dicyclohexylcarbodiimide (Aldrich, 0.89 g, 4.31 mmol) in acetonitrile (25 mL), was added dropwise to a stirring slurry of (+)-6-methoxy-a-methyl-2- naphthaleneacetic acid (Aldrich, 0.90 g, 3.91 mmol), 2-hydroxy-2,5,7,8-tetramethyl-3,4- dihydro-2H-benzo[l,2-b]pyran-2yl)ethanol (0.98 g, 3.91 mmol, USP 5,266,709 column 45) and 1 -hydroxybenzotriazole hydrate (Aldrich, 0.59 g, 4.31 mmol), in acetonitrile (50 mL). After stirring for 18 hours, the reaction mixture was concentrated in vacuo. The residue was partitioned between water (30 mL) and methylene chloride (30 mL). The layers were separated, and the aqueous layer was extracted with methylene chloride (2 x 20 mL). The combined organic extracts were washed with water (20 mL), then dried (magnesium sulfate) and concentrated in vacuo. Flash chromatography (silica gel, 2:8, v:v, ethyl acetate: hexanes) of the residue afforded a white solid upon the concentration of the appropriate fractions. The white solid was recrystallized from an ethyl acetate- hexanes mixture to give 0.60 g (33.1% yield) of 2-(6-hydroxy-2,5,7,8-tetramethyl-3,4- dihydro-2H-benzo[ 1 ,2-b]pyran-2yl)ethyl 2-(6-methoxy-2-naphthyl)propionate, a mixture of diastereomers, as a white solid.

1H NMR (CDC1₃) d 1.1 (d, 3H), 1.6-1.5 (m, 3H), 1.6 (m, 2H), 1.9 (m,2H). 2.0 (s, 6H), 2.1 (s, 3H), 2.4 (t, 2H), 3.8 (q, 2H), 3.9 (s, 3H), 4.2 (s, IH), 4.1-4.4 (m, 2H), 7.1-7.7 (m,6H).

Elemental Analysis: Calculated for C₂₉H₃₄O₅ Calculated: C, 75.30; H, 7.41. Found: C, 75.24; H, 7.46. Melting Point: 99.5-101.5°C. Example 13

Synthesis of 2-(5-hvdroxy-2.4.6.7-tetramethyl-3.4-dihydro-benzo[1.2-h]furan-2ynethyl 2- (6-methoxy-2-naphthynpropionate

A solution of 2-(5-hydroxy-2,4,6,7-tetramethyl-2,3-dihydrobenzo[l,2-b]furan-2- yl)ethanol (1.30 g, 5.51 mmol) and 6-methoxy-α-methyl naphthaleneacetic acid (Aldrich,

1.39 g, 6.06 mmol) was stirred in the presence of dimethylaminopyridine (0.67 g, 5.51 mmol) and l-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (1.06 g, 5.51 mmol), in tetrahydrofuran (25 mL). The reaction mixture was stirred at ambient temperature under nitrogen for 24 hours, diluted with ethyl acetate (150 mL), washed with water (2x 40 mL) and then brine (30 mL). The organic extract was dried (sodium sulfate) and concentrated in vacuo. The residue was subjected to flash chromatography

(silica gel, 100-50:0-50, v:v, hexanes:ethyl acetate), and the appropriate fractions were combined to give 1.84 g (74.5% yield) of a foam residue. Fractional crystallization and recrystallization from methylene chloride-hexanes gave 0.40 g (13.0% yield) of white solid.

Η-NMR (CDCLJ: 1.34 (s, 3H), 1.54-1.57 (d, 3), 1.99 (t, 2H), 2.01 (s, 3H), 2.05 (s, 3H), 2.10 (s, 3), 2.73-2.81 (d, 1 ), 2.90-2.97 (d, 1), 3.77-3.89 (q, IH), 3.91 (s, 3H), 4.102 (s, IH, 4.165-4.29 (m, 2H), 7.10-7.16 (m, 2H), 7.35-7.40 (m, IH), 7.64-7.70 (m, 2H).

Elemental Analysis: Calculated for C₂₈H₃₂O₅ 0.1 mole CH₂C1₂. Calculated: C, 73.84; H, 7.10. Found: C, 73.85, 73.83; H, 7.12. Melting point: 129.5- 131°C.

Example 14

Synthesis of2-⁽6-hydroxv-2.5.7.8-tetramethvl-3.4-dihydro-2H-benzo [1.2-b]pyran- 2yhethyl 2-('3-fIuoro-4-phenyl-phenvnpropionate

The intermediate, 2-(6-benzyloxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-benzo[l ,2-b]pyran- 2yl)ethyl 2-(3-fluoro-4-phenyl-phenyl)propionate, was first synthesized:

A solution of flubiprofen (Sigma, 2.0 g, 8.2 mmol), 2-(6-benzyloxy-2,5,7,8-tetramethyl- 3,4-dihydro-2H-benzo[l,2-b]pyran-2-yl)ethanol (2.4 g, 8.2 mmol) 1- hydroxybenzotriazole hydrate (Aldrich, 2.4 g, 13.9 mmol) and l-(3- dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (Aldrich, 2.8 g, 12.3 mmol), in acetonitrile (40 ml), was stirred at ambient temperature. After 72 hours, the reaction mixture was concentrated in vacuo and the residue partitioned between water and methylene chloride. A solid formed which was removed by filtration and discarded. The layers were separated and the aqueous layer was extracted with methylene chloride (2 x 25 ml). The combined organic extracts were then dried (magnesium sulfate) and concentrated in vacuo. The residue was chromatographed (silica gel, 2:8, v:v, ethyl acetate:hexane). Concentration of the appropriate fractions afforded 3.0 g (64% yield, mixture of stereoisomers) ofthe product as a clear oil.

1H NMR (CDCLJ d: 1.23-1.27 (m, 3H), 1.53-1.57 (m, 3H), 1.75 (m, 2H), 1.95 (m, 2H), 2.08 (s, 3H), 2.14 (s, 3H), 2.21 (s, 3H), 2.55 (t, 3H), 3.75 (m, 2H), 4.3 (m, IH), 4.65 (s, 2H), 7.1-7.7 (m, 13H).

A solution of 2-(6-benzyloxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-benzo

[l ,2-b]pyran-2yl)ethyl 2-(3-fluoro-4-phenyl-phenyl)propionate in ethyl acetate was treated with 10% palladium on charcoal (Aldrich, 0.5 g). The resulting mixture was hydrogenated on a Parr Apparatus [initial pressure 60 pounds/inch (psi)]. After 18 hours, the reaction mixture was filtered, and the resulting solution concentrated in vacuo. The residue was subjected to flash chromatography (silica gel, 2:8, v:v, ethyl acetate:hexane). Concentration of the appropriate fractions afforded a clear oil. Hexane was added to the oil and a white solid formed upon standing. The white solid was collected by filtration to afford 0.91 g (36% yield) of 2-6-hydroxy-2,5,7,8-tetra methyl -,4-dihydro-H-benzo[ 1,2- b]pyran-yl)ethyl 2-(3-fluoro-4-phenyl-phenyl) propionate as a mixture of stereoisomers.

Η NMR (CDClj d: 1.22-1.23 (m, 3H), 1.51-1.55 (m, 3H), 1.65-1.8 (m, 2H), 1.85-2.00 (m, 2H), 2.08 (s, 6H), 2.14 (s, 3H), 2.57 (t, 2H), 3.75 (q, IH), 4.1-4.5 (m, 2H), 7.10-7.65 (m, 8H).

Elemental Analysis: Calculated for C₃₀H₃₃FO₄. Calculated: C,75.60; H, 6.98. Found: C,75.69; H.,7.01. Melting point: 85-87°C.

Other angiostatic compounds of the present invention are known to those skilled

in the art. These compounds may be obtained by commercial sources, or synthesized by

methods described in the respective publications incoφorated herein or listed above.

The invention in its broader aspects is not limited to the specific details shown and

described above. Departures may be made from such details within the scope of the

accompanying claims without departing from the principles of the invention and without

sacrificing its advantages.

Claims

What is claimed is:

1. A method of treating pathological neovascularization which comprises

administering to a human a pharmaceutically effective amount of a combination of two or

more angiostatic compounds.

2. A method according to Claim 1 wherein the angiostatic compounds are selected from the group consisting of: anti-mitotics, estrogen metabolites, matrix

metalloproteinase inhibitors, plasminogen activator/urokinase inhibitors, urokinase

receptor antagonists, platelet factor 4 and analogs, heparinases, cartilage-derived inhibitor

of angiogenesis, thrombospondin and related analogs, angiostatin, vasculostatin, proliferin-related protein, fumagillin-type compounds, tecogalan, pentosan polysulfate,

thalidomide and related analogs, CM101 , tyrosine kinase inhibitors, anti-sense

oligonucleotides, suramin-type compounds, angiostatic steriods, α_vβ₃ and α_vβ₅ integrin

antagonists, cytotoxic antibodies against endothelial cell antigens, interferon, VEGF and

bFGF antagonists, flk-1 and flt-1 antagonists, IL-1 and TFN antagonists, and a compound

according to formula (I):

wherein:

n is 1 or 2;

R is H, C,-C₆ alkyl or C₃-C₆ cycloalkyl; Y is H, C,-C₆ alkyl, C₃-C₆ cycloalkyl, O, NR, C(R)₂ , CH(OH) or S(O)_n. ; n' is 0 to 2;

R' is H, C(O)R, C(O)N(R)₂, PO₃ ^" , SO₃ ^' or HO₂C(CH₂)₂(C=O)— ;

R" is H or C,-C₆ alkyl;

R³ is H, C,-C₆ alkyl, (CH₂)_q(OH), — (C=O)O(CH₂)_qCH₃ or

; provided that R' can not be H, when R³ is ³ ; q is 1 to 10; and

Z, if present, is H, C_rC₆ alkyl, C₃-C₆ cycloalkyl, or selected from the group consisting of:

wherein: D is O or NR; and

E and E' are independently H, F or Cl; and pharmaceutically acceptable salts thereof.

3. A method according to Claim 1 , wherein: R is H, R' is H; R" is CH₃; R³ is CH₃; and Y is C,-C₂ alkyl.

4. A method according to Claim 1, wherein one of the compounds is selected from the group consisting of:

5. A method according to Claim 1, wherein the compounds are combined in a single composition comprising a topical ophthalmic formulation.

6. A method according to Claim 2, wherein the compounds are combined in a single composition comprising a topical ophthalmic formulation.

7. A method according to Claim 1, wherein the compounds are combined in a single composition comprising a surgical irrigating solution.

8. A method according to Claim 2, wherein one ofthe compounds are combined in a single composition comprising a surgical irrigating solution.

9. A method according to Claim 1 , wherein the compounds comprise an angiostatic steroid and a compound of formula (I).

10. A method according to Claim 1 , wherein the compounds comprise a suramin-type compound and a compound of formula (I).

11. A method according to Claim 1 , wherein the compounds comprise a fumagillin-type compound and a compound of formula (I).

12. A method according to Claim 1 , wherein the compounds comprise an angiostatic steroid and a fumagillin-type compound.

13. A method according to Claim 1 , wherein the compounds comprise an angiostatic steroid and a suramin-type compound.

14. A method according to Claim 1 , wherein the compounds comprise an anti-

mitotic and compound of formula (I). 15. A composition for treating pathological neovascularization which

comprises a pharmaceutically effective amount of a combination of two or more

angiostatic compounds in a pharmaceutically acceptable vehicle.

16. A composition according to Claim 15 wherein the angiostatic compounds

are selected from the group consisting of: anti-mitotics, estrogen metabolites, matrix

metalloproteinase inhibitors, plasminogen activator/urokinase inhibitors, urokinase

receptor antagonists, platelet factor 4 and analogs, heparinases, cartilage-derived inhibitor of angiogenesis, thrombospondin and related analogs, angiostatin, vasculostatin,

proliferin-related protein, fumagillin-type compounds, tecogalan, pentosan polysulfate,

thalidomide and related analogs, CM101 , tyrosine kinase inhibitors, anti-sense

oligonucleotides, suramin-type compounds, angiostatic steriods, α_vβ₃ and α₅β₃ integrin antagonists, cytotoxic antibodies against endothelial cell antigens, interferon, VEGF and

bFGF antagonists, flk-1 and flt-1 antagonists, IL-1 and TFN antagonists, and a compound

according to formula (I):

wherein:

n is 1 or 2;

R is H, C_rC₆ alkyl or C₃-C₆ cycloalkyl;

Y is H, C,-C₆ alkyl, C₃-C₆ cycloalkyl, O, NR, C(R)₂ , CH(OH) or S(O)_n. ; n' is 0 to 2;

R' is H, C(O)R, C(O)N(R)₂, PO₃ ^" , SO₃ ^' or HO₂C(CH₂)₂(C=O)~; R" is H or C,-C₆ alkyl;

R >3^J i •s H, C,-C₆ alkyl, (CH₂),(OH), — (C=O)O(CH₂)_qCH₃ or

; provided that R' can not be H, when R 3 is ' — — ' ° ? , q is 1 to 10; and

Z, if present, is H, C,-C₆ alkyl, C₃-C₆ cycloalkyl, or selected from the group consisting of:

wherein:

D is O or NR; and

E and E' are independently H, F or Cl; and pharmaceutically acceptable salts thereof.

17. A composition according to Claim 15, wherein: R is H, R' is H; R" is CH₃;

R , 3^J is CH₃; and Y is C,-C₂ alkyl.

18. A composition according to Claim 15, wherein one ofthe compounds is selected from the group consisting of:

.

19. A composition according to Claim 15, wherein the composition is a topical ophthalmic formulation. 20. A composition according to Claim 16, wherein the composition is a topical ophthalmic formulation.

21. A composition according to Claim 15, wherein the composition is a surgical irrigating solution.

22. A composition according to Claim 16, wherein the composition is a surgical irrigating solution.

23. A composition according to Claim 15, wherein the compounds comprise an angiostatic steroid and a compound of formula (I).

24. A composition according to Claim 15, wherein the compounds comprise a suramin-type compound and a compound of formula (I).

25. A composition according to Claim 15, wherein the compounds comprise a fumagillin-type compound and a compound of formula (I).

26. A composition according to Claim 15, wherein the compounds comprise an angiostatic steroid and a fumagillin-type compound.

27. A composition according to Claim 15, wherein the compounds comprise an angiostatic steroid and a suramin-type compound.

29. A composition according to Claim 15, wherein the compounds comprise

an anti-mitotic and compound of formula (I).