HK1142528A - Methods for treating neovascular ocular diseases - Google Patents

Description

Method for treating neovascular ocular diseases

The present invention relates to compositions and methods for inhibiting undesired angiogenesis of ocular tissues, and thus for preventing and/or treating ocular diseases involving angiogenic processes. More particularly, the invention relates to compositions and methods for preventing and/or treating neovascularization of ocular tissue using a substance that inhibits VEGF in combination with a second therapy.

Angiogenesis, also known as neovascularization, is the fundamental process by which new blood vessels are formed. Under normal physiological conditions, angiogenesis is highly regulated and essential in reproduction, embryonic development, tissue repair and wound healing (for review see Carmeliet, 2005, Nature, 438, 932-. However, angiogenesis also occurs in a variety of pathological conditions, including tumor growth and metastasis; inflammatory disorders such as rheumatoid arthritis, psoriasis, osteoarthritis, inflammatory bowel disease, crohn's disease, ulcerative colitis, and other inflammatory disorders; and ocular neovascularization such as that found in diabetic retinopathy, age-related macular degeneration (AMD), and a variety of other ocular diseases (see, e.g., Folkman, 1995, nat. med., 1, 27-31). Indeed, angiogenesis occurs in response to a variety of pro-angiogenic stimuli, such as growth factors, cytokines, and other physiological molecules, as well as other factors such as hypoxia and low pH (Folkman and Shing, 1992, JBC, 267, 10931). The angiogenic cascade of new blood vessel development requires the cooperation of a variety of molecules that regulate essential cellular processes such as extracellular matrix (ECM) remodeling, invasion, migration, proliferation, differentiation and tubule formation (Brooks, 1996, eur.j. cancer, 32A, 2423). After the initiation phase, pro-angiogenic molecules such as VEGF, bFGF, PDGF, and others activate endothelial cells by stimulating their cell surface receptors (e.g., VEGFR1/Flt-1 and VEGFR 2/FIk-I/KDR; reviewed in Ferrara, 2004, endocr. Rev., 25, 581-611). These activated cells undergo a cell proliferation process that increases expression of cell adhesion molecules, increases secretion of proteolytic enzymes, and increases cell migration and invasion. A number of different molecules have been implicated in promoting proliferation and invasion, including integrins for adhesion, selectins and members of the immunoglobulin gene superfamily as well as proteolytic enzymes, such as matrix metalloproteinases and serine proteases for degradation of the extracellular matrix (Brooks, 1996, eur.j. cancer, 32A, 2423). Ultimately, a complex cascade of biochemical signals derived from cell surface receptors interact with extracellular matrix components and soluble factors, leading to lumen formation and differentiation into mature blood vessels.

Although little is known about the molecular mechanisms of choroidal and/or retinal neovascularization, the particular angiogenic process has been found to be the major cause of severe visual loss in patients with AMD and patients with other retinopathies such as diabetic retinopathy or retinopathy of prematurity.

Age-related macular degeneration is the leading cause of blindness in developed countries, with about 1500 million patients in the united states. AMD is characterized by progressive macular degenerative disease. There are two forms of AMD: new blood vessels and non-new blood vessels. The non-neovascular form of AMD is more common and results in slow degeneration of the macula, with progressive loss of vision over years. The neovascular form of the disease is the leading cause of severe loss of vision, and it is due to abnormal proliferation of blood vessels behind the retina leading to bleeding and fibrosis, with consequent abnormal vision. The main objective of current therapeutic attempts and clinical trials is to stop the growth of neovascular membranes in AMD, for example using angiogenesis inhibitors, laser photocoagulation and/or photodynamic therapy (PDT) (see e.g. WO 2004034889). However, only a portion of the eye meets the criteria for such therapeutic intervention, and treatment with these means has a high recurrence rate and poor efficacy. Thus, despite advances in therapy, AMD remains the most common cause of visual impairment in developed countries.

Another major cause of blindness in adults aged 20-74 years is Diabetic Retinopathy (DR). 700 thousands of people in the United states have diabetes. Although the management of diabetic retinopathy has progressed due to milestone clinical trials, the risk of complications such as visual acuity loss, night vision loss, and peripheral vision loss remains significant, and treatment sometimes fails. Diabetic retinopathy is characterized by abnormal neovascularization in the retinal vasculature with edema and rupture of the blood-retinal barrier (BRB), resulting in hemorrhage, macular edema, tissue damage, and retinal scarring. Unfortunately, current treatment options (e.g., laser photocoagulation) are not entirely satisfactory, and the disease often continues to progress.

There is increasing evidence that inhibition of different molecules involved in the angiogenic cascade provides a means of blocking diseaseThe potential for a key mediating step in disease progression to treat these neovascularization-associated disorders, including the likely etiology of neovascularization-associated disorders of tumor and ocular tissues (see, e.g., Shibuya, 2003, Nippon Yakurigaku Zasshi, 122, 498-. Examples of angiogenesis inhibitors, including inhibitors of their associated receptors, are well known in the art and include, for example, ZD6474(Tuccillo et al, 2005, Clin Cancer Res., 11, 1268-; soluble Tie2 and VEGF-1 receptors (see Hangai et al, 2001, Hum Gene ther., 12, 1311-; pigment Epithelium Derived Factor (PEDF) (Rasmussen et al, 2001, Hum Gene ther., 12, 2029-; tissue inhibitors of metalloproteinase-3 (Takahashi et al, 2000, Am J Ophthalmol., 130, 774-; VEGF inhibiting aptamers, e.g.(Pegatanib, Pfizer); antibodies or fragments thereof, e.g. anti-VEGF antibodies such as bevacizumab (bGene Tec (Genentech)), or a fragment thereof, for example, ranibizumab (ranibizumab) ((II)Gene tack); soluble fins-like tyrosine kinase 1(sFlt1) polypeptides or polynucleotides (Harris et al, Clin Cancer Res.2001, 7(7) (1992-7; U.S. Pat. No. 5,861,484); PTK/ZK, which inhibits VEGF signaling by blocking tyrosine kinases (Maier et al, 2005, Graefes arch. clin. exp. ophthalmol., 243, 593-one 600); KRN633(Nakamura et al, 2004, Molcancer ther.,3, 1639-1649); integrin inhibitors (e.g., α v β 3 and α 5 β 1); VEGF-(Regeneron); and alpha 2-antiplasmin (Matsuno et al, Blood 2003; 120: 3621-. Most of these angiogenesis inhibitors are directed to block the activation step induced by vascular endothelial cell growth factor (VEGF) mediated by the original growth factor. VEGF has therefore been considered as an attractive target for anticancer therapy, particularly in combination therapy with chemotherapy, radiotherapy or other anti-angiogenic agents (see Ferrara, 2005, Oncology, 69, 11-16). Similarly, various studies have shown that neovascularization in various vascular beds of certain animal models can be retrograde or prevented using various types of anti-VEGF agents (e.g., Gragoudas et al, 2004, N.Engl. J. Med., 351, 2805-; Rothen et al, 2005, Ophthalmol Clin North Am., 18, 561-.

However, despite these encouraging results, there is currently no standard and effective therapy for treating neovascularization and vascular hyperpermeability of ocular tissues. Indeed, due to the emergence of resistance to anti-VEGF therapy, interest in treating cancer by anti-angiogenic therapies that inhibit the Vascular Endothelial Growth Factor (VEGF) pathway has been diminished.

Thus, there is a need for improved methods of treating neovascular eye diseases that inhibit or eliminate the ability of various forms of neovascularization, including that of the retina and/or choroid, and to treat related disorders. Future efforts are aimed at identifying new therapies to improve the efficacy of anti-angiogenic therapies. The present invention fulfills these needs and further provides other related advantages.

The present invention is directed to improved compositions and methods for treating ocular neovascularization using a compound that inhibits VEGF in combination with a second therapy. In one aspect of the invention, the invention provides compositions and methods for preventing and treating the following diseases: choroidal and/or retinal neovascularization and associated ocular disorders, and more particularly AMD, CNV, retinopathy of prematurity, traumatic eye injury, diabetic retinopathy, certain inflammatory ocular disorders (e.g., shotgun retinochoroidopathy or multifocal choroiditis), and the like.

According to a first embodiment of the invention, there is provided a combination comprising therapeutically effective amounts of (i) at least one anti-angiogenic compound and (ii) at least one corticosteroid administered simultaneously or sequentially or alternately at staggered times.

The combination of the invention is of particular interest for the treatment and/or prevention of ocular diseases associated with neovascularization.

According to a particular embodiment, the combination further comprises an ophthalmically compatible solvent component.

The term "a" or "an" as used throughout this application means "at least one", "at least a first", "one or more", or "a plurality" of the recited compounds or steps, unless the context indicates otherwise. More specifically, "at least one" and "one or more" mean one or a number greater than one, with one, two or three being particularly preferred.

The term "and/or" as used herein includes the following meanings: "and", "or" and "all or any other combination of elements linked by the term".

The term "about" as used herein means within 20%, preferably within 10% and more preferably within 5% of a given value or range.

The terms "comprises," "comprising," and "including," when used herein to define products, compositions, and methods, are intended to mean that the products, compositions, and methods include the recited compounds or steps, but do not exclude other compounds or steps.

The term "patient" refers to a vertebrate, particularly a member of a mammalian species and includes, but is not limited to, livestock, pets, primates, including humans. The term "patient" is in no way limited to a particular disease state, and includes patients who have developed the disease and patients who have not yet developed the disease.

The term "treatment" as used herein includes prophylaxis and/or therapy. Thus, the compositions and methods of the present invention are not limited to therapeutic use and may be used for prophylactic use. Thus, "treatment" of a condition, disorder or condition includes: (i) preventing or delaying the appearance of clinical symptoms of a developing condition, disorder or condition in an individual who may be suffering from such condition, disorder or condition, or who is susceptible to such condition, disorder or condition but has not yet experienced or manifested clinical or subclinical symptoms of such condition, disorder or condition, (ii) inhibiting the condition, disorder or condition, i.e., arresting or reducing the development of the disease or at least one clinical or subclinical symptom of the disease, or (iii) alleviating the disease, i.e., causing regression of the condition, disorder or condition or at least one clinical or subclinical symptom thereof.

The term "corticosteroid" refers to any naturally occurring or synthetic compound characterized by a hydrogenated cyclopentanoyl perhydro-phenanthrene ring system and having immunosuppressive and/or anti-inflammatory activity. Naturally occurring corticosteroids are typically produced by the adrenal cortex. The synthetic corticosteroid may be halogenated.

Non-limiting examples of corticosteroids are 1' - α, 17- α, 21-trihydroxypregn-4-ene-3, 20-dione; 11- β, 16- α, 17, 21-tetrahydroxypregn-4-ene-3, 20-dione; 11- β, 16- α, 17, 21-tetrahydroxypregna-1, 4-diene-3, 20-dione; 11- β, 17- α, 21-trihydroxy-6- α -methylpregn-4-ene-3, 20-dione; 11-dehydrocorticosterone; 11-deoxycorticosterol; 11-hydroxy-1, 4-androstadiene-3, 17-dione; 11-ketotestosterone; 14-hydroxyandrost-4-ene-3, 6, 17-trione; 15, 17-dihydroxyprogesterone; 16-methyl hydrocortisone; 17, 21-dihydroxy-16- α -methylpregna-1, 4, 9(11) -triene-3, 20-dione; 17- α -hydroxypregn-4-ene-3, 20-dione; 17-alpha-hydroxypregnanolone; 17-hydroxy-16-beta-methyl-5-beta-pregn-9 (11) -ene-3, 20-dione; 17-hydroxy-4, 6, 8(14) -gestriene-3, 20-dione; 17-hydroxypregna-4, 9(11) -diene-3, 20-dione; 18-hydroxycorticosterone; 18-hydroxy cortisone; 18-oxocortisol; 21-acetoxypregnenolone; 21-deoxyaldosterone; 21-deoxycorticosterone; 2-deoxyecdysone; 2-methyl cortisone; 3-dehydroecdysone; 4-pregnene-17-alpha, 20-beta, 21-triol-3, 11-dione; 6, 17, 20-trihydroxypregn-4-en-3-one; 6-alpha-hydroxycortinol; 6-alpha-methylprednisolone, 6-alpha-methylprednisolone 21-acetate, 6-alpha-methylprednisolone 21-sodium hemisuccinate, 6-beta-hydroxycortinol, 6-alpha, 9-alpha-difluoroprednisolone 21-acetate 17-butyrate and 6-hydroxycorticosterone; 6-hydroxydexamethasone; 6-hydroxy prednisolone; 9-flucortisone; alclometasone dipropionate; aldosterone; alestrol; alphaderm; amadone; amcinonide; anagesterone; androstenedione; anecortave acetate; beclomethasone; beclomethasone dipropionate; betamethasone 17-valerate; betamethasone sodium acetate; betamethasone sodium phosphate; betamethasone valerate; (ii) a dehydroepiandrosterone; budesonide; (ii) carpoterone; chlormadinone; prednisone; prednisone acetate; cholesterol; ciclesonide; clobetasol; clobetasol propionate; clobetasol; (ii) a chlorocortolone; (ii) clocortolone pivalate; progesterone chloride; (ii) prednisolone; corticosterone; cortisol; cortisol acetate; cortisol butyrate; (ii) hydrocortisone cypionate; cortisol octanoate; cortisol sodium phosphate; cortisol sodium succinate; cortisol valerate; cortisone; cortisone acetate; (ii) a kovar; trutodo pine; daturalones (daturaolone); deflazacort, 21-deoxycorticosterol, dehydroepiandrosterone; demegestone; deoxycorticosterone; delosozone; a delphinidin derivative; (ii) donepezil; desoximetasone; dexafen; dexamethasone; dexamethasone 21-acetate; dexamethasone acetate; dexamethasone sodium phosphate; dichloro pine; diflorasone; diflunisal diacetate; diflucortolone; difluprednate; dihydroelathericina; a polyprednisole ester; a polypetaxole; ecdysone; ecdysterone; emoxolone; emthylosol; glycyrrhetinic acid; fluzacort; flucinolone; (ii) chlorofluoronenide; fludrocortisone; fludrocortisone acetate; fluoprogesterone; flumethasone; flumethasone pivalate; (ii) flumonade; flunisolide; the skin is relaxed; fluocinolone acetonide; fluocinonide; (ii) a fluke butyl ester; 9-flucortisone; fluocortolone; fluoro hydroxy androstenedione; fluorometholone; fluorometholone acetate; fluoxymesterone; fluoropolygon acetate; fluprednidene; fluprednidone; fluoro-hydrogen shrinkage; fluticasone; fluticasone propionate; formylboxolone; 2, fulvestrant; formocortah; pregnenolone; glyderinine; halcinonide; halobetasol propionate; halometasone; prednisone halide; a haloprogesterone; a hydrocortisone ester; hydrocortisone cypionate; hydrocortisone; hydrocortisone 21-butyrate; hydrocortisone ethylene acrylate; hydrocortisone acetate; hydrocortisone butyrate propionate; hydrocortisone butyrate; hydrocortisone cypionate; hydrocortisone hemisuccinate; probutanoic acid (probutate) hydrocortisone; hydrocortisone sodium phosphate; hydrocortisone sodium succinate; hydrocortisone valerate; hydroxyprogesterone; achyranthes bidentata sterone; isoflupredone; isoflupredlone acetate; isoprednisone; loteprednol etabonate; (ii) meclosone; prednisolone valerate (mecortiolon); medroxyprogesterone; medroxyprogesterone; (ii) medroxyprogesterone; megestrol; megestrol acetate; melengestrol; methylprednisolone; 1, meperidinone; methylprednisolone; methylprednisolone ethylen-acrylate; methylprednisolone acetate; methylprednisolone hemisuccinate; methylprednisolone sodium succinate; methyltestosterone; metrobolone; mometasone; mometasone furoate; mometasone furoate monohydrate; nisone; nomegestrol; norgestimate; (ii) a ketene dimer; hydroxymethyltestosterone; (ii) a palatasone; (ii) a parehasone acetate; pinosterone (ponasterone); prednisolone ester; prednisolone ester; prednisolone; prednisolone 21-diethylaminoacetate; prednisolone 21-hemisuccinate; prednisolone acetate; prednisolone farnesoate; prednisolone hemisuccinate; prednisolone-21 (β -D-glucuronide); prednisolone methylsulfonylbenzoate; prednisolone sodium phosphate; selectorine prednisolone; prednisolone tert-butyl ethyl ester; prednisolone tetrahydrophthalate; prednisone; prednisolone valerate; prednisolone; pregnenolone; pracinonide; (ii) trilonene; progesterone; promegestrol; rhapontigenin (rhapontisterone); rimexolone; luoxibolone; amaranthone (rubrosterone); stilzophyllin; alternative cortisone; tropanone; triamcinolone acetonide; triamcinolone acetonide; triamcinolone acetonide 21-palmitate; triamcinolone acetonide; triamcinolone diacetate; triamcinolone acetonide hexa; trimegestone; tacrolidone; and wortmannin.

The terms "anti-angiogenic compound", "angiogenesis inhibitor" and "anti-angiogenic compound" are used interchangeably herein to refer to a compound that inhibits angiogenesis (i.e., the growth of new blood vessels).

According to a particular embodiment, the anti-angiogenic compound is an immunosuppressive compound.

According to a preferred embodiment, the immunosuppressive compound is selected from the group consisting of calcineurin inhibitors and mTOR inhibitors.

According to another particular embodiment, the anti-angiogenic compound is a compound that inhibits VEGF.

As used herein, "compound" refers to any substance, chemical, or substrate, whether organic or inorganic, or any protein, including antibodies and functional fragments thereof, peptides, polypeptides, peptoids, nucleic acids, oligonucleotides, and the like. The compounds for use in the present invention include those described herein in any of their pharmaceutically acceptable forms, including isomers, e.g., diastereomers and enantiomers, salts, esters, solvates, and polymorphs thereof, as well as racemic mixtures and pure isomers of the compounds described herein

As used herein, "a compound that inhibits VEGF" refers to a compound that inhibits the activity or production of vascular endothelial cell growth factor (VEGF). It relates to compounds, for example, capable of binding to VEGF, including small organic molecules, antibodies or antibody fragments specific for VEGF, peptides, cyclic peptides, nucleic acids, antisense nucleic acids, RNAi and ribozymes that inhibit VEGF expression at the nucleic acid level. Examples of compounds that inhibit VEGF are nucleic acid ligands for VEGF, such as those described in US 6,168,778 or US 6,147,204, EYE001 (previously referred to as NX1838), which is a modified pegylated aptamer that binds with high affinity to the major soluble human VEGF isoform; VEGF polypeptides (e.g., US 6,270,933 and WO 99/47677); oligonucleotides that inhibit VEGF expression at the nucleic acid level, such as antisense RNAs (e.g., US5,710,136; US5,661,135; US5,641,756; US5,639,872 and US5,639,736). Other examples of VEGF signalling inhibitors known in the art (see description of the invention) include, for example, ZD6474(Tuccillo et al, 2005, Clin Cancer res., 11, 1268-76); COX-2 inhibitors, Tie2 receptor inhibitors, angiogenin inhibitors, and neuropilin inhibitors; pigment epithelial cell derived factor (PEDF), endostatin and angiostatin, soluble fins-like tyrosine kinase 1(sFlt1) polypeptides or polynucleotides (Harris et al, 2001, Clin Cancer Res., 7, 1992-1997; US5,861,484); PTK787/ZK 222584; KRN633(Maier et al, 2004, Mol Cancer ther., 3, 1639-; VEGF-(reigerland corporation); and α 2-antiplasmin (Matsuno et al, 2003, Blood, 120, 3621-. For reviews of VEGF and its inhibitors see, e.g., Campochiaro, 2004, Expert Opin Biol ther, 4, 1395-1402; ferrara, 2004, endocr.rev., 25, 581-; the contents of which are incorporated herein by reference. According to a preferred embodiment, the compound inhibiting VEGF is an antibody or antibody fragment or aptamer to VEGF or a related family member such as (VEGF B.IC, D; PDGF). Preferred examples are anti-VEGF antibodies, such as Avastin^TM(also known as bevacizumab,gene tache) or fragments thereof, e.g. Lucentis^TM(also known as rhuFAb V2 or AMD-Fab; ranibizumab, Gene Takk.) and other anti-VEGF compounds, e.g. VEGF inhibiting aptamers, e.g. Macugen^TM(also known as Peganib sodium, anti-VEGF aptamer or EYE001, Peyer). According to a more preferred embodiment, the compound inhibiting VEGF may further be an immunosuppressive compound, and is more preferably selected from the group consisting of calcineurin inhibitors and mTOR inhibitors.

"antibody" as used herein includes polyclonal and monoclonal antibody preparations, CDR-grafted antibody preparations, and preparations comprising hybrid antibodies, engineered antibodies, F (AB) 'that exhibit immunological binding properties with the parent molecule of the antibody'₂Fragments, F (AB) molecules, Fv fragments, single domain antibodies, chimeric antibodies, and functional fragments thereof. Antibodies may also be humanized. The term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody or functional fragment thereof derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) and an antibody or functional fragment thereof not produced by the method.

Non-limiting examples of calcineurin inhibitors are tacrolimus (also known as FK-506-tenze pharmaceuticals (Fujisawa Pharma, co)), LX211 (also known as ISAtx247-Iso Teknika, Inc), ascomycin, pimecrolimus, and cyclosporines and derivatives thereof, including those listed in the above compound definitions. According to a most preferred embodiment, the calcineurin inhibitor of the present invention is cyclosporin a. See, e.g., Wilasrusmee et al, 2005(int. Angiol.; 24, 372-379) for an illustration of the anti-angiogenic properties of cyclosporines.

Non-limiting examples of mTOR inhibitors are rapamycin (also known as sirolimus, wheaten), temsirolimus (temsirolimus) (CCI-779, wheaten), everolimus (RAD001, norward pharmaceutical) and AP23573(Ariad pharmaceutical) and their derivatives, including those listed in the above compound definitions.

According to another embodiment, the combination further comprises an effective amount of a biocompatible polymer or fibrin glue component to delay the release of the compound that inhibits VEGF and/or the corticosteroid, particularly into the interior of the eye after administration of the combination into the eye. According to another particular embodiment, the combination further comprises an ophthalmically compatible solvent component in an amount effective to solubilize the polymer or fibrin glue components effective, when the combination is administered into the eye, to provide a delayed release of the compound that inhibits VEGF and/or the corticosteroid in the eye relative to intraocular administration of a substantially identical composition without the polymer or fibrin glue components.

In another aspect of the invention, the combination of the invention may further comprise a compound selected from the group consisting of: estrogens (e.g., estradiol), androgens (e.g., testosterone), retinoic acid derivatives (e.g., 9-cis-retinoic acid, 13-trans-retinoic acid, all-trans-retinoic acid), vitamin D derivatives (e.g., calcipotriol, calcipotriene), non-steroidal anti-inflammatory drugs, selective 5-hydroxytryptamine reuptake inhibitors (SSR 1; e.g., fluoxetine, sertraline, paroxetine), tricyclic antidepressants (TCA; e.g., maprotiline, amoxapine), phenoxyphenols (e.g., triclosan), antihistamines (e.g., loratadine, epinastine), phosphodiesterase inhibitors (e.g., ibudilast), anti-infectives, protein kinase C inhibitors, MAP kinase inhibitors, anti-apoptotic drugs, growth factors, vitamins, unsaturated fatty acids, and/or ocular anti-infectives, for the treatment of ocular disorders as described herein (see, e.g., compounds disclosed in US 2003/0119786; WO 2004/073614; WO 2005/051293; US 2004/0220153; WO 2005/027839; WO 2005/037203; WO 03/0060026). In another embodiment of the invention, mixtures of these agents may be used. Ophthalmic anti-infective agents that may be used include, but are not limited to, penicillins (ampicillin, azlocillin, carbenicillin, dicloxacillin, methicillin, nafcillin, oxacillin, penicillin G, piperacillin, and ticarcillin), cephalosporins (cefamandole, cefazolin, cefotaxime, cefsulodin, ceftazidime, ceftriaxone, cephalothin, and moxalactam), aminoglycosides (amikacin, gentamicin, netilmicin, tobramycin, and neomycin), other drugs such as aztreonam, bacitracin, ciprofloxacin, clindamycin, chloramphenicol, fannomycin, fusidic acid, imipenem, metronidazole, teicoplanin, and vancomycin), antifungal drugs (amphotericin B, clotrimazole, econazole, fluconazole, flucytosine, itraconazole, ketoconazole, miconazole, natamycin, oxiconazole, and terconazole), antiviral drugs (acyclovir, ethyldeoxyuridine, foscarnet, ganciclovir, idoxuridine, trifluridine, vidarabine and (S) -1- (3-hydroxy-2-phosphonomethoxypropyl) cytosine (HPMPC)), antitumor drugs (cell cycle non-specific drugs such as alkylating agents (chlorambucil, cyclophosphamide, nitrogen mustard, melphalan and busulfan), anthracyclines (doxorubicin, daunomycin and actinomycin D), cisplatin and nitrosourea), antimetabolites such as antimyrimidines (cytarabine, fluorouracil and 5-azacytidine), antifolates (methotrexate), antipurines (mercaptopurine and thioguanine), bleomycin, vinca alkaloids (vincristine and vinblastine), podophyllotoxins (etoposide (VP-16)) and nitrosoureas (carmustine, (BCNU)), and proteolytic enzyme inhibitors such as inhibitors of plasminogen activator. The dosages for topical and subconjunctival administration of the above drugs, as well as the Intravitreal dosage and Intravitreal half-life can be reviewed in Intravitreal Surgery Principles and Practice, Peyman G a and Shulman, J editions, second edition, 1994, Appleton-longge, relevant sections of which are specifically incorporated herein by reference.

According to another embodiment, said combination further comprises a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil and the like. Saline and aqueous dextrose, polyethylene glycol (PEG) and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, sodium stearate, glyceryl monostearate, glycerol, propylene glycol, water, and the like. The combination product may also contain minor amounts of wetting or emulsifying agents or pH buffering agents or viscosity increasing agents, if desired. Examples of suitable pharmaceutical carriers are described in e.w. martin, "Remington's pharmaceutical sciences. In a preferred embodiment, the combination product is formulated according to conventional methods suitable for pharmaceutical compositions for injection into the eye. Typically, the combination product for injection is a solution in sterile isotonic aqueous buffer. The combination product may also contain a solubilizer, if necessary. In general, the ingredients may be provided separately or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet labeled with a metered amount of active agent. When the combination product is for administration by infusion, it may be dispensed from an infusion bottle containing sterile pharmaceutical grade water or saline. When the combination product is administered by injection, an ampoule of sterile water or saline for injection may be provided so that the ingredients may be mixed prior to administration.

According to the invention, the compound that inhibits VEGF (e.g. cyclosporin a) is present in the combination product in an amount of less than or equal to about 10%, preferably less than or equal to about 5%, more preferably less than or equal to about 2%, even more preferably less than or equal to about 1%. In an advantageous embodiment, it is less than or equal to about 0.5%, preferably less than or equal to about 0.1%, more preferably less than or equal to about 0.05% and even more preferably less than or equal to 0.01%. According to particular embodiments, the compound that inhibits VEGF is cyclosporin a and its concentration in the combination product is between about 0.001% and about 0.05% (e.g., 0.049%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, and 0.001%).

According to the present invention, when the combination product is administered for the treatment of an anterior ocular disease, the corticosteroid in the combination product is present in an amount of about 0.01% to about 4%, more preferably it is present in an amount of about 0.01% to about 1.0% (e.g. 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05% and 0.01%). In particular embodiments, it is present in an amount of about 0.01% to about 0.12%. In an advantageous embodiment, it is about 0.012%.

According to the invention, when the combination is administered for the treatment of a disease of the posterior part of the eye, the corticosteroid in the combination is present in an amount of from 0.05mg to about 2mg, more particularly from about 0.05mg to about 1mg and even more particularly from about 0.05 to about 0.5 mg.

For corticosteroid doses, the recommended doses are as follows:

ophthalmic corticosteroids	Minimum approved concentration for ocular administration	Minimum standard recommended dose
			Clocontrone valerate	0.1％	N/A
Hydrocortisone	1.0％	0.5 μ g/day 3 times
			Dexamethasone	0.1％	0.05 μ g/day 4-6 times
Fluorometholone	0.1％	0.05 μ g/day 2-4 times
			Loteprednol etabonate	0.2％	0.1 μ g/4 times daily
Methoxypyr	1.0％	0.5. mu.g/up to every 4 hours
			Prednisolone acetate	0.12％	0.06 μ g/day 2-4 times
Rimexolone	1.0％	0.5 μ g/4 times daily

(N/A is unusable)

For corticosteroids, other standard recommended doses are provided, for example, in the Merck Manual of Diagnosis & Therapy (17 th edition, MH Beers et al, Merck & Co.), and the Physicians' Desk Reference 2003 (57 th edition, Medical Economics Staff et al, Medical Economics Co., 2002). In one embodiment, the dose of corticosteroid administered is a dose equivalent to the dose of prednisolone as defined herein. For example, a low dose of a corticosteroid can be considered to be a dose equivalent to a low dose of prednisolone.

According to the invention, the concentration of corticosteroid may be the lowest approved concentration (see table above) or 95% or less of the lowest approved concentration. For example, a low concentration of a corticosteroid of the present invention may be 90%, 85%, 80%, 70%, 60%, 50%, 25%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% of the lowest approved concentration.

For example, for ocular administration, low concentrations of clocortolone valerate are between 0.01% and 0.1% (e.g., 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05% and 0.01%), low concentrations of hydrocortisone are between 0.01% and 1.0% (e.g., 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05% and 0.01%), low concentrations of dexamethasone are between 0.01% and 0.1% (e.g., 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05% and 0.01%), low concentrations of fluorometholone are between 0.01% and 0.1% (e.1%, e.01%, 0.05%, 0.0.05%, 0.0.0.01%, low concentrations of methoprene (e.05%, 0.0.1%, 0.05%, 0.0.0.1%, 0.0.1%, 0.05%, 0.0.0.0.1%, 0.1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and 0.01%), a low concentration of rimexolone is between 0.01% and 1.0% (e.g., 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and 0.01%), and a low concentration of prednisolone is between 0.01% and 0.12% (e.g., 0.12%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, and 0.01%).

According to a particular embodiment, the combination product of the invention comprises 0.012% of prednisolone acetate and 0.05% of cyclosporine.

According to another embodiment, the invention relates to a method of inhibiting, treating or preventing neovascularization of ocular tissue and associated diseases or disorders in a patient in need of such treatment, comprising the step of administering to said patient a combination of the invention.

According to one embodiment, administration of (i) at least one anti-angiogenic compound and (ii) at least one corticosteroid produces a synergistic effect in the inhibition, treatment, or prevention of neovascularization of ocular tissue and related disorders.

According to a particular embodiment, administration of (i) at least one compound that inhibits VEGF and (ii) at least one corticosteroid produces a synergistic effect in inhibiting, treating or preventing neovascularization of ocular tissue and related disorders.

According to another particular embodiment, administration of (i) at least one calcineurin inhibitor or/and mTOR inhibitor and (ii) at least one corticosteroid produces a synergistic effect in the inhibition, treatment or prevention of neovascularization of ocular tissues and related disorders.

According to a preferred embodiment, administration of (i) at least one cyclosporin, even more preferably cyclosporin a, and (ii) at least one corticosteroid produces a synergistic effect in the inhibition, treatment or prevention of neovascularization of ocular tissues and related disorders.

As used herein, "patient" refers to any animal having ocular tissue that may be afflicted with neovascularization. Preferred animals are mammals, including but not limited to humans and other primates. The term also includes livestock, such as cattle, pigs, sheep, horses, dogs and cats. The term "patient" is in no way limited to a particular disease state, and includes patients who have developed the disease and patients who have not yet developed the disease. According to a particular embodiment of the invention, the patient treated with the combination of the invention does not undergo cell transplantation, and more particularly does not suffer from Graft Versus Host Disease (GVHD).

In accordance with the present invention, the methods can be used to inhibit, prevent and treat a variety of diseases and disorders characterized by the production of ocular neovascularization and related disorders. According to the present invention, ocular neovascularization and its associated disorders (or diseases or conditions) are, for example, macular edema, ischemic retinopathy, intraocular neovascularization, age-related macular degeneration (AMD) (and more particularly exudative AMD), corneal neovascularization, retinal neovascularization, choroidal neovascularization, retinopathy of prematurity, traumatic eye injury, diabetic macular edema, diabetic retinal ischemia, diabetic retinal edema, proliferative diabetic retinopathy, shotgun's disease, multifocal choroiditis, and any neovascularization associated with any pathological state of the eye.

According to the invention, compound (i) and compound (ii) of the combination product can be administered simultaneously or sequentially or alternately with time. Simultaneous administration refers to co-administration. In such a case, the two essential compounds [ (i) and (ii) ] may be mixed prior to administration to form a composition, or may be administered to the patient simultaneously. It is also possible to apply them sequentially, i.e. one after the other, irrespective of which component of the combination of the invention is applied first. Finally, it is also possible to apply a pattern of intermittent applications that are applied alternately with staggered times or that are stopped and restarted at certain time intervals, which may be regular or irregular. It has been indicated that the route of administration and the site of administration of the two components may be different. The time interval of administration is not critical and can be defined by the skilled person. Intervals of 10 minutes to 72 hours, advantageously 30 minutes to 48 hours, preferably 1 to 24 hours and very preferably 1 to 6 hours, may be recommended; but the intervals may be longer and more than a month.

Administration of the combination product for ophthalmic applications is preferably by intraocular injection, but other modes of administration may also be effective. Typically, ophthalmic compositions are delivered to an individual intraocularly (via a chemical delivery system or invasive device). However, the invention is not limited to intraocular delivery, but also includes topical administration (extraocular application) or systemic administration (e.g., by oral or other parenteral routes, such as subcutaneous administration). Parenteral administration is used in appropriate circumstances as is well-established for the physician. Preferably, the ophthalmic compositions are administered in unit dosage forms suitable for single administration of precise dosages.

As noted above, in situ delivery to the intraocular area may be accomplished by injection, intubation, or other invasive devices designed to introduce precisely metered amounts of the desired ophthalmic composition into a particular chamber or tissue within the eye (e.g., the posterior chamber or retina). Can be injected intraocularly into the vitreous (intravitreal) or subconjunctival (subconjunctival), or into the sclera behind the eye (retrobulbar), or under the Tenon's capsule (sub-Tenon), and can be in depot form. Other routes of intraocular administration are also contemplated, as well as sites and forms of injection, all of which are encompassed by the present invention. In a preferred embodiment, the combination product of the invention is delivered by subretinal injection.

In one embodiment, the ophthalmic composition is injected intraocularly (e.g., into the vitreous or subretinal space) to treat or prevent an ocular disorder. When the ophthalmic composition is administered by intraocular injection, the active ingredient should be concentrated to minimize the injection volume. For example, such volumes may require compensatory drainage of vitreous humor to prevent elevated intraocular pressure and leakage of injectate through open areas formed by delivery needles. More preferably, the injection volume is between about 1.0mL and 0.05 mL. Most preferably, the injection volume is about 0.1 mL.

For injection, concentrations of less than about 20mg/mL can be injected, and any amount can be effective, depending on the factors described above. Preferably, the dosage administered is about 10 mg/mL. Sample concentrations include, but are not limited to, about 5 μ g/mL to about 50 μ g/mL; about 25 μ g/mL to about 100 μ g/mL; about 100 μ g/mL to about 200 μ g/mL; about 200 μ g/mL to about 500 μ g/mL; about 500 μ g/mL to about 750 μ g/mL; about 500. mu.g/mL to at most 1mg/mL, etc. Preferably 50 mg/mL. The concentrations of compounds (i) and (ii) may further differ for one of said combination products. In a preferred embodiment, up to 100 micrograms of compound (ii) is administered.

Intraocular injections can be performed by a variety of methods well known in the art. For example, one may use, for exampleThe eye is washed and the compound of the invention in a suitable carrier is injected with a precision gauge needle (e.g., 27 gauge needle) at a location in the eye such that the compound is positioned at the posterior pole toward the ventral surface. It may be necessary to apply positive pressure prior to injection to prepare the eye for injection. In some cases, it is necessary to perform a vitrectomy in advance. Local anesthesia or general anesthesia may be necessary.

Suitable syringes for carrying out the method of the invention may be fitted with 21-40 gauge needles and are preferably of small volume, for example 1.5mL or more preferably 0.1 mL. Although the needle and syringe may be of the type in which the needle is separable from the syringe, the preferred arrangement is for the syringe/needle to be of unitary construction. This will obviously limit the possibility of needle detachment from the syringe. Tamper evident types are also preferred. Thus, the combination of the present invention may be provided in the form of a single unit dose, or in the form of separate unit doses containing respective portions of the combination, both in a pre-prepared syringe ready for direct administration.

A suitable syringe type is, for example, manufactured by Becton Dickinson and Company under the trade nameThe syringe of (1). In this type of syringe, the substance is forced through the needle into the eye by pressure exerted on the wall of the flexible reservoir containing the needle, rather than by the plunger. As the name implies, the reservoir and needle structure form a single unit.

Topical application of the ophthalmic combination product of the present invention for the treatment or prevention of ocular disorders may be an ointment, a gel or eye drops. The topical ophthalmic composition may further be an aqueous formulation capable of gelling in situ. Such formulations contain a gelling agent in a concentration effective to promote gelling upon contact with the eye or with tear fluid present outside the eye. Suitable gelling agents include, but are not limited to, thermosetting polymers such as tetra-substituted ethylene diamine block copolymers of ethylene oxide and propylene oxide (e.g., poloxamers); polycarbophil; and polysaccharides such as gellan, carrageenan (e.g., kappa-carrageenan and iota-carrageenan), chitosan, and alginate gums.

The phrase "capable of gelling in situ" as used herein includes not only low viscosity liquids that form gels upon contact with the eye or with tear fluid outside the eye, but also more viscous liquids, such as semifluids and thixotropic gels, which exhibit a significantly increased viscosity or gel stiffness when applied to the eye.

For the preparation of topical ophthalmic compositions for the treatment of ocular disorders, a therapeutically effective amount of the combination product of the present invention is placed in an ophthalmic carrier according to techniques known in the art, e.g. US5,041,434 discloses a topical ophthalmic formulation comprising a steroid, while US 4,271,143 and US 4,407,792 describe a sustained release ophthalmic formulation of an ophthalmic drug and a high molecular weight polymer for the formation of a high viscosity gel. Further GB 2007091 describes an ophthalmic composition in the form of a gel comprising an aqueous solution of a carboxyvinyl polymer, a water-soluble alkaline substance and an ophthalmic drug. Alternatively, US 4,615,697 discloses controlled release compositions and methods of use based on bioadhesives and therapeutic agents, such as anti-inflammatory agents.

The amount of the combination used in the present methods and the concentration of the compound in the topical ophthalmic combination depends on the diluent, delivery system or device, the clinical condition of the patient, the side effects and stability of the compound in the formulation. Thus, the physician applies a suitable formulation comprising suitable concentrations of compounds (i) and/or (ii) and selects the amount of formulation to administer, based on clinical experience with the patient or similar patients.

Since the combination product comprises two or more active agents, the active agents may be administered as a mixture, a mixture in the same ophthalmic composition, separate formulations, sustained release formulations, liposomes, microcapsules or any of the preceding embodiments.

The combination product may also be administered in the form of a sustained release formulation (using carrier formulations such as microspheres, microcapsules, liposomes, etc.), a topically applied ointment or solution, an intravenous solution or suspension, or an intraocular injection, according to methods known to those skilled in the art for the treatment or prevention of ocular disorders. By "sustained release", "timed release" or "controlled release" is meant that the therapeutically active component is released from the formulation at a controlled rate so as to maintain a therapeutically beneficial level of the component (but below toxic levels) over a period of time ranging, for example, from about 12 to about 24 hours, thereby providing a dosage form for, for example, 12 hours or 24 hours. The timed release drug delivery system may be administered intraocularly to achieve a sustained release effect of the combination product over a period of time. The combination product may be in the form of a carrier, such as a microcapsule or a macrocapsule or a biocompatible polymer matrix, such as polycaprolactone, polyglycolic acid, polylactic acid, polyanhydride, polylactic acid-co-glycolide, polyamino acids, polyethylene oxide, acrylic-capped polyethylene oxide, polyamide, polyethylene, polyacrylonitrile, polyphosphazene, polyorthoester, Sucrose Acetate Isobutyrate (SAIB), and other polymers, such as U.S. Pat. nos. 6,667,371; 6,613,355, respectively; 6,596,296; 6,413,536, respectively; 5,968,543; 4,079,038, respectively; 4,093,709, respectively; 4,131,648, respectively; 4,138,344, respectively; 4,180,646, respectively; 4,304,767; 4,946,931, the entire contents of which are incorporated herein by reference, or lipids that can be formulated as microspheres or liposomes. Microscopic or macroscopic ophthalmic compositions may be administered through a needle, or may be implanted by intraocular sutures, such as intravitreal or sub-retinal sutures. The sustained release properties may be provided by a variety of carrier formulations (coated or uncoated microspheres, coated or uncoated capsules, lipid or polymer components, single or multi-compartment structures, combinations of the above, and the like). The formulation and loading of microspheres, microcapsules, liposomes, etc., and their intraocular implantation are standard techniques known to those skilled in the art.

The invention also provides a method of treating or preventing an ocular disorder associated with neovascularization, said method comprising the step of administering a combination of the invention in a biocompatible, biodegradable matrix, for example in the form of a gel or polymer, which is preferably suitable for insertion into the retina or the anterior or posterior cavity of the eye as an implant. In the case of delivery of the combination product as an implant, it may be added as a liquid to any known biocompatible, biodegradable matrix or formed, for example, in the form of micelles (using known chemical methods) or microparticles.

Sustained release delivery systems include any of a variety of biopolymers (biologically based systems), systems employing liposomes, colloids, resins and other polymeric delivery systems or compartmentalized reservoirs which may be employed with the compositions described herein to provide a sustained or long-term source of the therapeutic compound.

In any sustained release device prepared, the compounds (i) and/or (ii) are preferably present in an amount of about 10% to 90% by weight of the implant. More preferably, the compounds (i) and/or (ii) comprise from about 50% to about 80% by weight of the implant. In a preferred embodiment, the compounds (i) and/or (ii) constitute about 50% by weight of the implant. In a particularly preferred embodiment, the compounds (i) and/or (ii) constitute about 70% by weight of the implant.

In one form, all of the implants of the present method are formulated with compounds (i) and/or (ii) embedded in a bioerodible polymeric matrix. The release of the compound is achieved by the following process: the polymer erodes and the previously embedded compound is exposed to the vitreous, and the compound dissolves and is released. The release kinetics obtained by this form of drug release are different from those obtained by formulations that release the drug by swelling of the polymer (e.g., hydrogels, such as methylcellulose). In the latter case, the active compound is not released by erosion of the polymer, but by swelling of the polymer, the active compound is released from the exposed pathways as a liquid diffusion. Parameters that determine release kinetics include the size of the active compound particles, the water solubility of the active compound, the ratio of active compound to polymer, the method of preparation, the exposed surface area, and the erosion rate of the polymer.

Exemplary particularly attractive biocompatible, non-biodegradable polymers include polyurethanes or polyureas, particularly polyurethanes, polymers that can be crosslinked to produce non-biodegradable polymers such as crosslinked polyvinyl acetate and the like. Ethylene-vinyl ester copolymers are also of particular interest, having an ester content of from 4% to 80%, such as ethylene-vinyl acetate (EVA) copolymers, ethylene-vinyl caproate copolymers, ethylene-vinyl propionate copolymers, ethylene-vinyl butyrate copolymers, ethylene-vinyl valerate copolymers, ethylene-vinyl pivalate copolymers, ethylene-vinyl diethylacetate copolymers, ethylene-vinyl 3-methylbutyrate copolymers, ethylene-vinyl 3, 3-dimethylbutyrate copolymers and ethylene-vinyl benzoate copolymers.

Other exemplary naturally occurring or synthetic non-biodegradable polymeric materials include polymethyl methacrylate, polybutyl methacrylate, plasticized polyvinyl chloride, plasticized polyamide fiber, plasticized soft polyamide fiber, plasticized polyethylene terephthalate, natural rubber, silicone, polyisoprene, polyisobutylene, polybutadiene, polyethylene, polytetrafluoroethylene, polyvinylidene chloride, polyacrylonitrile, crosslinked polyvinylpyrrolidone, poly (chlorotrifluoroethylene), chlorinated polyethylene, poly (4, 4' -isopropylidenediphenylene carbonate), dichloroethylene-acrylonitrile copolymer, chloroethylene-diethyl fumarate copolymer, silicone rubber (particularly pharmaceutical grade), polydimethylsiloxane, ethylene-propylene rubber, silicone-carbonate copolymer, poly (ethylene-co-propylene copolymer), poly (ethylene-co-, Vinylidene chloride-vinyl chloride copolymers, vinyl chloride-acrylonitrile copolymers, vinylidene chloride-acrylonitrile copolymers, polyolefins, poly (vinyl-olefins), polystyrene, poly (haloolefins), polyvinyls, polyacrylates, polymethacrylates, polyoxides, polyesters, polyamides and polycarbonates.

The diffusion of the active compounds (i) and/or (ii) from the implant can also be controlled by the structure of the implant. For example, diffusion of compounds (i) and/or (ii) from the implant may be controlled by a membrane attached to a polymer layer containing the drug. The membrane layer is located between the polymer layer comprising compounds (i) and/or (ii) and the intended treatment site. The membrane may comprise any of the above-described biocompatible materials, other than compounds (i) and/or (ii) present in the polymer, compositions of the polymer comprising compounds (i) and/or (ii), desired diffusion rates, and the like.

It will be appreciated by those skilled in the art that the residence time in the intraocular environment of any ophthalmic combination product employed in the method of the present invention depends on, inter alia, the following factors: the physicochemical and/or pharmacological properties of the compound used in the formulation, the concentration of the compound used, the bioavailability of the compound, the disease to be treated, the mode of administration and the preferred time of treatment. This balance is usually struck depending on the desired time of action in the eye and the condition being treated.

According to the methods of the invention, the frequency of treatment is determined by the disease being treated, the deliverable concentration of compound (i) and/or (ii), and the method of delivery. If the combination product is delivered by intravitreal injection, the frequency of doses can be monthly. The preferred dosage frequency is once every three months. Dosage frequency can also be determined by observation, with the next dose delivered after significant clearance of the currently delivered combination product. Generally, an effective amount of a compound is an amount that provides subjective symptom relief or an objectively identifiable improvement that can be noted by a clinician or other qualified observer.

Ophthalmic combination products prepared for use in the methods of the present invention for preventing or treating ocular disorders preferably have a residence time of from several hours to several months and possibly even years, but where the residence time is the latter, a particular delivery system is required to achieve the duration described and/or, alternatively, repeated administration is required. Most preferred residence times (i.e. durations in the eye) for the combination product for use in the method of the invention are hours (i.e. 1 to 24 hours), days (i.e. 1, 2, 3, 4, 5,6 or 7 days) or weeks (i.e. 1, 2, 3, 4 weeks). Alternatively, the combined product may have a residence time of at least several months, e.g. 1 month, 2 months, 3 months, and residence times of up to 4, 5,6, 7 to 12 months or more.

The methods or uses of the invention can be performed alone or in combination with one or more conventional therapies (e.g., photodynamic therapy, laser surgery, laser photocoagulation, or one or more biological or pharmaceutical treatments, as desired. The use of multiple therapeutic approaches provides a broad base of intervention for the patient. In one embodiment, the method of the invention may be performed before or after a surgical intervention. In another embodiment, it may be performed before or after photodynamic therapy, laser surgery, laser photocoagulation. Those skilled in the art can readily formulate appropriate treatment regimens and parameters that can be applied.

The present invention further relates to a method of improving the treatment of a patient undergoing one or more of the above-listed conventional treatments, which method comprises co-treating said patient with a combination product of the invention.

According to another embodiment, the present invention relates to a method of inhibiting, treating, or preventing an angiogenesis-mediated ocular disease or condition in a patient, comprising administering to said patient an amount effective to inhibit, reduce, or prevent angiogenesis of a combination product comprising (i) at least one anti-angiogenic compound and (ii) at least one corticosteroid.

According to another embodiment, the invention relates to a method of inhibiting, treating, or preventing an angiogenesis-mediated ocular disease or condition in a patient, comprising administering to said patient an amount effective to inhibit, reduce, or prevent angiogenesis of a combination product comprising (i) at least one compound that inhibits VEGF and (ii) at least one corticosteroid.

According to another embodiment, the invention relates to a method of inhibiting, treating or preventing an angiogenesis-mediated ocular disease or condition in a patient, the method comprising administering to the patient an amount effective to inhibit, reduce or prevent angiogenesis of a combination comprising (i) at least one calcineurin inhibitor or/and mTOR inhibitor and (ii) at least one corticosteroid.

According to a preferred embodiment, the present invention relates to a method of inhibiting, treating or preventing an angiogenesis-mediated ocular disease or condition in a patient, comprising administering to said patient an angiogenesis-inhibiting, reducing or preventing effective amount of a combination product comprising (i) at least one cyclosporin, even more preferably cyclosporin a, and (ii) at least one corticosteroid.

According to another embodiment, the present invention relates to a method of inhibiting, treating, or preventing an angiogenesis-mediated ocular disease or condition in a patient, comprising co-administering to said patient an effective amount of (i) at least one anti-angiogenic compound and (ii) at least one corticosteroid to inhibit, reduce, or prevent angiogenesis.

According to another embodiment, the invention relates to a method of inhibiting, treating, or preventing an angiogenesis-mediated ocular disease or condition in a patient, comprising co-administering to said patient an angiogenesis-inhibiting, reducing, or preventing effective amount of (i) at least one compound that inhibits VEGF and (ii) at least one agent that causes increased degradation of excess aggregated matrix.

According to another embodiment, the invention relates to a method of inhibiting, treating or preventing an angiogenesis-mediated ocular disease or condition in a patient, comprising co-administering to said patient an effective amount of (i) at least one calcineurin inhibitor or/and mTOR inhibitor and (ii) at least one corticosteroid to inhibit, reduce or prevent angiogenesis.

According to a preferred embodiment, the present invention relates to a method of inhibiting, treating or preventing an angiogenesis-mediated ocular disease or condition in a patient, comprising co-administering to said patient an effective amount of (i) at least one cyclosporin, even more preferably cyclosporin a, and (ii) at least one corticosteroid to inhibit, reduce or prevent angiogenesis.

According to another embodiment, the present invention relates to a method of causing regression of neovascularization in a patient, comprising administering to said patient an effective amount of a combination product comprising (i) at least one anti-angiogenic compound and (ii) at least one substance that causes increased degradation of excess aggregated matrix.

According to another embodiment, the invention relates to a method of causing regression of neovascularization in a patient, comprising administering to said patient an effective amount of a combination product comprising (i) at least one compound that inhibits VEGF and (ii) at least one substance that causes increased degradation of excess aggregated matrix.

According to another embodiment, the invention relates to a method of causing regression of neovascularization in a patient, comprising administering to said patient an effective amount of a combination comprising (i) at least one calcineurin inhibitor or/and mTOR inhibitor and (ii) at least one agent that causes enhanced degradation of excess aggregated matrix.

According to a preferred embodiment, the present invention relates to a method of causing regression of neovascularization in a patient, comprising administering to said patient an effective amount of a combination product comprising (i) at least one cyclosporin, even more preferably cyclosporin a, and (ii) at least one substance which causes enhanced degradation of excess aggregated matrix.

According to another embodiment, the present invention relates to a method of causing regression of neovascularization in a patient, comprising co-administering to said patient (i) at least one anti-angiogenic compound and (ii) at least one agent that causes increased degradation of excess aggregated matrix.

According to another embodiment, the invention relates to a method of causing regression of neovascularization in a patient, comprising co-administering to said patient (i) at least one compound that inhibits VEGF and (ii) at least one substance that causes increased degradation of excess aggregated matrix.

According to another embodiment, the invention relates to a method of causing regression of neovascularization in a patient, comprising co-administering to said patient (i) at least one calcineurin inhibitor or/and mTOR inhibitor and (ii) at least one agent that causes increased degradation of excess aggregated matrix.

According to a preferred embodiment, the present invention relates to a method of causing regression of neovascularization in a patient, comprising co-administering to said patient (i) at least one cyclosporin, even more preferably cyclosporin a, and (ii) at least one substance that causes enhanced degradation of excess aggregated stroma.

As used herein, "causing regression of neovascularization" refers to reducing the amount of neovasculature, particularly in the eye, in an individual having neovascular disease, particularly ocular neovascular disease as defined above.

According to another embodiment, the present invention relates to the use of (i) at least one anti-angiogenic compound and (ii) at least one corticosteroid for the preparation of a composition for the prophylactic or therapeutic treatment of ocular neovascularization and disorders related thereto, and more particularly those disorders mentioned above, in a patient.

According to another embodiment, the present invention relates to the use of (i) at least one compound inhibiting VEGF and (ii) at least one corticosteroid for the preparation of a composition for the prophylactic or therapeutic treatment of ocular neovascularization and disorders related thereto, and more particularly those disorders mentioned above, in a patient.

According to another embodiment, the present invention relates to the use of (i) at least one calcineurin inhibitor or/and mTOR inhibitor and (ii) at least one corticosteroid for the preparation of a composition for the prophylactic or therapeutic treatment of ocular neoangiogenesis and disorders related thereto, and more particularly those mentioned above, in a patient.

According to a preferred embodiment, the present invention relates to the use of (i) at least one cyclosporin, even more preferably cyclosporin a, and (ii) at least one corticosteroid for the preparation of a composition for the prophylactic or therapeutic treatment of ocular neovascularisation and its associated disorders, and more particularly those mentioned above, in a patient.

In another aspect, the invention relates to a kit. One kit of the invention comprises a container containing (i) at least one anti-angiogenic compound and a container containing (ii) at least one corticosteroid, and instructions for scheduling the administration of the compounds. Another kit of the invention comprises a container for preparing a composition comprising (i) at least one compound that inhibits VEGF and a container comprising (ii) at least one corticosteroid, and instructions for scheduling the administration of the compounds. Another kit of the invention comprises a container containing (i) at least one calcineurin inhibitor or/and mTOR inhibitor and a container containing (ii) at least one corticosteroid for preparation, and instructions for scheduling administration of the compounds. Preferred kits of the invention comprise a container containing (i) at least one cyclosporin, even more preferably cyclosporin a, and a container containing (ii) at least one corticosteroid for preparation, together with instructions for scheduling administration of the compounds. The container may be a single container containing both compounds (i) and (ii) together, or it may be a plurality of containers or compartments containing divided doses of both compounds (i) and (ii), such as blister packs. The kit also has instructions for scheduling the administration of the combination product. The instructions will instruct the individual to consume the combination product or separate compounds at the appropriate time. For example, the time suitable for delivery of the combination product may be when symptoms occur. Alternatively, the time suitable for administration of the combination product may be a routine procedure, such as once a month or once a year. Compounds (i) and (ii) may be administered simultaneously or they may be administered separately at times sufficiently close to produce a synergistic response.

It will be appreciated by persons skilled in the art that the invention described herein is susceptible to variations and modifications other than those specifically described. The present invention includes all such variations and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

Each document, reference, patent application, or patent cited herein is hereby incorporated by reference in its entirety, to the extent that the reader is intended to read and consider the above-identified document as part of this document. For the sake of brevity only herein, none of the documents, references, patent applications or patents cited herein are repeated herein.

The present invention is not to be limited in scope by the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

The invention described herein may include one or more numerical ranges (e.g., sizes, concentrations, etc.). A range of values should be understood to include all values within the range, including the values defining the range as well as values near the range that result in the same or substantially the same result as values very near the boundary defining the range.

Example (b):

the effect of the combination product of the invention on VEGF-induced vascular leakage was evaluated in a model of blood-retinal barrier breakdown in rabbits.

The aim of this study was to determine the effect of the combination product of the invention (tested at various concentrations) in reducing vascular leakage in a model of VEGF-induced blood-retinal barrier breakdown in rabbits (Edelman et al, 2005, Experimental Eye Research, 80, 249-.

The combination tested was a mixture of Triamcinolone Acetonide (TA) and cyclosporin a (csa).

Male Fauve de Bourgogne (colored) rabbits (CEGAV-FR-61350 Saint Mars-d' Egrenne) of about 4 months of age and weighing between 2.0kg and 2.5kg were applied.

Design of research

72(56) colored rabbits were randomly divided into 7(7) groups (8 animals per group).

On day 0, test combinations and controls (50 μ Ι _) were administered to the right eye (left eye served as control and remained untreated) by single intravitreal injection.

On day 5, animals were treated by single intravitreal injection of 500ng rhVEGF165 (50. mu.L) into the right eye (test and control).

On the 7 th day, the day,

intravenous injection of fluorescein sodium 47 hours after VEGF challenge;

-measuring fluorescein leakage from the vitreous retinal part of the eyes using a non-invasive scanning ocular fluorometry 1 hour after fluorescein injection;

the ratio Rt of the vitreoretinal fluorescein content between the right-treated eye and the left-untreated eye is used to evaluate the change in permeability of the blood-retinal barrier.

Route of administration and method

Administration was performed in all groups on day 0. Animals were anesthetized by intramuscular injection of xylazine (7.5mg/kg) and ketamine (32 mg/kg). Test combinations and controls (50 μ L) were injected into the middle vitreous of the right eye using a suitable needle (26-G needle). After each eye was washed with povidone-iodine, an injection was made approximately 3mm posterior to the border of the upper temporal quadrant of the eye. Intravitreal injection under a working microscope in an enlarged eye (1 drop)Phenylephrine 10% and 1 dropTropicamide 0.5%), instilled for 15-20 minutes prior to injection, and contact lenses applied.

Induction of vascular leakage

On day 5, 500ng rhVEGF with carrier protein (diluted in PBS) was injected by using a 100- μ L Hamilton syringe₁₆₅A single 50. mu.L intravitreal injection into all groups of treated eyes induced an increase in retinal vascular permeability (Edelman et al, ARVO meeting 2003, Fort Lauderdale, FL-USA. invest. Ophthalmol. Vis. Sci.2003; 44: ARVOe-abstrate No. 328). The injection was performed under the microscope on animals anesthetized by intramuscular injection of a mixture of xylazine (7.5mg/kg) and ketamine (32 mg/kg). The pupil is preceded (about 15-20 minutes) by one dropAnd a drop ofEnlargement (see above).

Quantification of vascular leakage

On day 7, after 47 hours of induction, fluorescein sodium (10% in 0.9% saline solution, 50mg/kg) was injected intravenously through the ear margin of conscious animals. 1 hour after the injection of fluorescein, the ocular fluorescein was measured on both eyes using an FM-2Fluorotron Master eye photometer. 20 minutes before the test, rabbits were anesthetized with an intramuscular injection of 32mg/kg ketamine, 7.5mg/kg xylazine and one dropAnd a drop ofDilate the pupil (see above). A series of 148 step scans (step size 0.25mm) were taken along the optical axis from the cornea to the retina.

Termination of the study

At the end of the evaluation period (day 7), an excess was injected intravenouslyThe animals were sacrificed.

Seven treatment groups (8 rabbits per group) were as follows (doses and percentages are provided):

control, i.e. vector alone

TA 400. mu.G (i.e. TA 0.8%)

TA 135. mu.G (i.e. TA 0.27%)

TA 75 μ G (i.e. TA 0.15%)

CsA 15. mu.G (i.e. CsA 0.03%)

TA 75. mu.G + CsA 15. mu.G (ratio 5) (i.e. TA 0.15%/CsA 0.03%)

TA 135. mu.G + CsA 1.5. mu.G (ratio 90) (i.e. TA 0.27%/CsA 0.003%)

The results obtained are summarized in the following table:

treatment of	Inhibition of retinal vascular leakage
		CsA 15μg	Is free of
TA 75μg	13％
		TA 135μg	27％
TA 75μg+CsA 15μg	50％
		TA 135μg+CsA 1.5μg	73％
TA 400μg	96％

Thus, the inventors have shown that:

-dose-dependent inhibition of VEGF-induced retinal vascular leakage induced by intravitreal injection of triamcinolone acetonide; significant and almost complete protection was observed at the 400 μ g TA dose;

-CsA did not have any significant effect;

low subtherapeutic doses of triamcinolone acetonide (i.e. 75 μ g and 135 μ g) in combination with CsA showed a greater effect than TA alone in specific ratios (i.e. 5 and 90, corresponding to CsA doses of 15 μ g and 1.5 μ g, respectively).

Similarly, in a separate study, 75 μ g triamcinolone acetonide in combination with CsA showed a greater effect than TA alone (44% versus 10% inhibition of VEGF-induced retinal vascular leakage, respectively) at a ratio of 100 (i.e., corresponding to a CsA dose of 0.75 μ g).

Claims (15)

1. A combination comprising therapeutically effective amounts of (i) at least one anti-angiogenic compound and (ii) at least one corticosteroid administered simultaneously or sequentially or alternately at staggered times.

2. The combination of claim 1 wherein said anti-angiogenic compound is an immunosuppressive compound.

3. The combination of claim 2 wherein the immunosuppressive compound is selected from the group consisting of calcineurin inhibitors and mTOR inhibitors.

4. The combination of claim 1, wherein the anti-angiogenic compound is a compound that inhibits VEGF.

5. A combination product according to claim 3 wherein the calcineurin inhibitor is cyclosporin a.

6. The combination of any one of the preceding claims, wherein the combination further comprises an ophthalmically compatible solvent component.

7. The combination of claim 1, wherein the anti-angiogenic compound is present in an amount less than or equal to about 10%.

8. The combination of claim 1 wherein the anti-angiogenic compound is cyclosporin a and the concentration is between about 0.001% and about 0.05%.

9. The combination of claim 1, wherein the corticosteroid is present in an amount of about 0.01% to about 4%.

10. The combination product of claim 1 comprising 0.012% prednisolone acetate and 0.05% cyclosporine.

11. A method of treating an angiogenesis-mediated ocular disease or disorder in a patient, the method comprising administering to the patient an angiogenesis inhibiting, reducing, or preventing effective amount of a combination product comprising (i) at least one anti-angiogenic compound and (ii) at least one corticosteroid.

12. A method of treating an angiogenesis-mediated ocular disease or disorder in a patient, the method comprising administering to the patient an angiogenesis inhibiting, reducing, or preventing effective amount of a combination product comprising (i) at least one compound that inhibits VEGF and (ii) at least one corticosteroid.

13. A method of treating an angiogenesis-mediated ocular disease or disorder in a patient, the method comprising administering to said patient an angiogenesis inhibiting, reducing or preventing effective amount of a combination product comprising (i) at least one calcineurin inhibitor or/and mTOR inhibitor and (ii) at least one corticosteroid.

14. A method of treating an ocular neovascular disease in a patient, said method comprising the step of administering to said patient a therapeutically effective amount of a combination comprising (i) at least one cyclosporin and (ii) at least one corticosteroid in simultaneous or sequential administration or alternating administration with respect to time.

15. The method of claim 14 wherein said cyclosporin is cyclosporin a.