WO2007038453A2

WO2007038453A2 - Use of an anti-vascular endothelial growth factor (vegf) agent to ameliorate inflammation

Info

Publication number: WO2007038453A2
Application number: PCT/US2006/037332
Authority: WO
Inventors: Gholam Ali Peyman
Original assignee: Advanced Ocular Systems Limited
Priority date: 2005-09-26
Filing date: 2006-09-26
Publication date: 2007-04-05
Also published as: WO2007038453A3

Abstract

Use of an anti-vascular endothelial growth factor (VEGF) agent for the manufacture of a medicament to ameliorate inflammation at a site in the body that may be the eye, a joint, the brain, etc. or to reduce corneal neovascularization. In one embodiment, one or more other agents, such as non-steroidal anti-inflammatory agents, steroids, etc., may be included with the anti-VEGF agent. The anti-VEGF agent may be bevacizumab, ranibizumab, sunitinib maleate, pegaptanib, etc.

Description

EiBUIEW-OF AN AGENTVO AMELIORATE INFLAMMATION

Field of the Invention

[0001] This invention relates generally to methods for treating or preventing, in a patient, an inflammatory ailment using at least an anti-vascular endothelial growth factor (VEGF) agent. More particularly, the invention relates to an ocular prophylaxis or treatment method comprising ocularly providing to a patient having or at risk for developing an ocular inflammatory ailment a formulation comprising an anti-VEGF agent under conditions sufficient to ameliorate a cause and/or effect of fluid leakage in the eye. The invention also relates to an ocular prophylaxis or treatment method comprising the step of: ocularly providing to a patient, having or at risk for developing an ocular disease from fluid leakage from new ocular blood vessels to a surrounding area, a formulation comprising an anti-vascular endothelial growth factor (VEGF) agent selected from the group comprising bevacizumab, ranibizumab, pegaptanib, sunitinib maleate, TNP470, integrin av antagonists, 2-methoxyestradioi, paclitaxel, anti-VEGF siRNA, or P38 mitogen activated protein kinase inhibitors under conditions sufficient to ameliorate a cause and/or effect of fluid leakage in the eye.

Background

[0002] Inflammation is a localized, protective response of vascularized tissue to sub-lethal tissue injury or destruction. The response functions to destroy, dilute, or sequester both the injurious agent and the injured tissue. Inflammation can be classified according to duration as either acute or chronic. In the acute form of an inflammatory response, classical signs are pain, heat, redness, swelling, and loss of function. Histologically, there are a complex series of events including dilatation of arterioles, capillaries and venules, with increased permeability and blood flow, exudation of fluids including plasma proteins, and leukocyte migration and accumulation at the site of injury. This reaction may trigger a systemic response such as fever, leukocytosis, protein catabolism, and altered hepatic synthesis of plasma proteins such as C-reactive protein. Chronic inflammation is characterized by macrophage and lymphocyte infiltration into the affected and surrounding tissue. P[J$0Di3f& ΛήlaTftBaϊ!b3 is a homeostatic response to tissue damage by a range of stimuli, including infection and trauma. For example, an inflammatory response helps to destroy or inactivate invading pathogens. In cases of autoimmune diseases such as rheumatoid arthritis, etc., inflammation is a response against self. The inflammatory process removes waste and debris and restores normal function, either through resolution or repair. Tissue structure is normal after resolution, whereas repair leads to a functional, but morphologically altered, organ. In acute inflammation, tissue damage is followed by resolution or healing by scar formation, whereas in chronic inflammation, damage and repair continue concurrently. The initial inflammatory response is usually acute, and may or may not evolve into chronic inflammation. However, chronic inflammation is not always preceded by an acute phase. Although usually beneficial to the organism, inflammation itself may lead to tissue damage, resulting in escalation of chronic inflammation. Inflammation underlies the pathology of virtually all rheumatologic diseases. The severity of disorders, such as arthritis, is classified according to the degree of inflammation and its destructive effects.

[0004] Anti-VEGF agents affect the process of angiogenesis, which is the growth of new blood vessels from pre-existing vasculature. It is a fundamental process required for embryogenesis, growth, tissue repair after injury, and the female reproductive cycle. It also contributes to the pathology of conditions such as cancer, age related macular degeneration, psoriasis, diabetic retinopathy, and chronic inflammatory diseases in joints or lungs. Angiogenesis is stimulated when hypoxic, diseased, or injured tissues produce and release angiogenic promoters such as VEGF, platelet derived growth factor (PDGF), or fibroblast growth factor (FGF)-I . These angiogenic factors stimulate the migration and proliferation of endothelial cells in existing vessels and, subsequently, the formation of capillary tubes and the recruitment of other cell types to generate and stabilize new blood vessels.

[0005] Angiogenic factors may be pro-inflammatory factors. Relatively minor irritation of internal tissues, such as that which can occur during surgery, does not lead to neovascularization, but encourages tissue adhesion and scarring. Agents that inhibit angiogenesis such as the previously disclosed TNP470, integrin av antagonists, 2-methoxyestradiol, paclitaxel, P38 mitogen activated protein kinase inhibitors, anti-VEGF siRNA, and sunitinib maleate (Sutent^®/SU11248) may inhibit

^!!i^;ritis, retinal vasculitis, optic nerve neuritis, papillitis, retinitis proliferation in diabetes, etc. Expression of adhesion molecules such as integrin avb3 and e-selectin are upregulated in new vessels, and new vessels appear sensitive to inflammogens. The angiogenic factor FGF-1 enhances antigen-induced synovitis in rabbits, but is not pro-inflammatory when administered alone. However, angiogenesis occurs in the absence of inflammation, such as during embryonic growth and in the female reproductive cycle.

[0006] Angiogenesis enhances tumour growth, and anti-angiogenic agents are used clinically as a treatment modality. Mechanisms by which new vessels enhance tumour growth include providing metabolic requirements of the tumour, generating growth factors by vascular cells, and inhibiting apoptosis. Inhibiting the function of growth factors such as VEGF can reduce or prevent pathological angiogenesis in tumours.

[0007] Angiogenesis may also contribute to thickening of airways in asthma and of lung parenchyma in pulmonary fibrosis, and to growth of sarcoid granulomas. Growth of granulation tissue into airspaces also may be angiogenesis-dependent in bronchi after lung transplant and in alveoli after acute lung injury or in other forms of pulmonary fibrosis. Angiogenesis may also contribute to growth of the synovial pannus in rheumatoid arthritis. Interposition of expanded, innervated synovium between articulating surfaces may contribute to pain on movement. In each of these situations, the expanded tissue may impair function.

[0008] The new blood vessels that result from angiogenesis have incomplete walls and are particularly susceptible to disruption and fluid extravasation. This has been proposed as a cause of pulmonary haemorrhage in inflammatory lung disease. Hemosiderin deposits and extravasated erythrocytes are commonly present in inflammatory synovitis, although the contribution of angiogenesis to synovial micro- haemorrhage is unknown, and its contribution to synovial inflammation remains unclear. The inflammatory potential is evident, however, in patients with haemophilia. ffφbW]/

occurs as an orderly series of events, beginning with production and release of angiogenic growth factors (proteins) that diffuse into nearby tissues. The angiogenic growth factors bind to specific receptors located on the endothelial cells of nearby pre-existing blood vessels. Once growth factors bind to their receptors, the endothelial cells are activated and begin to produce enzymes and other molecules that dissolve tiny holes in the sheath-like basement membrane that surrounds existing blood vessels. The endothelial cells begin to divide and proliferate, and they migrate through the holes of the existing vessel towards the diseased tissue or tumour. Specialized adhesion molecules or integrins (avb3, avbδ) help to pull the new blood vessels forward. Additional enzymes, termed matrix metalloproteinases (MMP), are produced and dissolve the tissue in front of the sprouting vessel tip in order to accommodate it. As the vessel extends, the tissue is remoulded around the vessel. Sprouting endothelial cells roll up to form a blood vessel tube and individual blood vessel tubes connect to form blood vessel loops that can circulate blood. The newly formed blood vessel tubes are stabilized by smooth muscle cells, pericytes, fibroblasts, and glial cells that provide structural support, permitting blood flow to begin.

[0010] VEGF is a specific angiogenesis growth factor that binds to receptors on blood vessels and stimulates the formation of new blood vessels. VEGF is a potent inducer of both endothelial cell proliferation and migration, and its biologic activities are largely specific for endothelial and vascular smooth muscle cells. Unlike basic fibroblast growth factor (bFGF), high levels of VEGF are not present in early surgical wounds. Rather, VEGF levels peak seven days after the wound is created, at which point VEGF appears to be a major stimulus for sustained induction of blood vessel growth and high levels of PDGF have been shown. There are abundant sources of VEGF in wounds. Many cell types produce VEGF, including keratinocytes, macrophages, fibroblasts, and endothelial cells. Thus, there is massive VEGF secretion, particularly in the setting of hypoxia, which is often observed in wounds.

[0011] Anti-VEGF agents inhibit the action of VEGF. As one example of an anti- VEGF agent, bevacizumab is a recombinant humanized monoclonal IgGI antibody that binds to and inhibits the biologic activity of human VEGF in in vitro and in vivo assay systems by preventing binding of VEGF with its receptor on the surface of vascular endothelial cells, thus preventing endothelial cell proliferation and new vessel 'ifolfflStieJn^'SBlvϋcϊiijifliab contains human framework regions and the complementarity- determining regions of a murine antibody that binds to VEGF; it has a molecular weight of about 149 kilodaltons. Bevacizumab, by binding to VEGF, blocks VEGF from binding to receptors and thus blocks angiogenesis. Bevacizumab is typically administered by intravenous infusion, diluted in 0.9% sodium chloride for injection from a 25 mg/ml preparation, for treatment of colorectal cancer.

[0012] Ranibizumab is a derivative of the full-length antibody bevacizumab (Fab fragment), and is further modified to increase its affinity for VEGF. Both bevacizumab and ranibizumab bind all biologically active isoforms and proteolytic fragments of VEGF, but there are differences. Monovalent binding of a Fab fragment such as ranibizumab to its target antigen would not force the target to dimerize, and hence is useful to manipulate cell receptor function, but its effective antigen binding capacity is lower than its full antibody counterpart. However, VEGF, which is the desired target, is a soluble factor and not a cellular receptor. Therefore, the increased effective binding by the full length antibody bevacizumab enhances inhibition of the VEGF signal and thus provides an enhanced anti-angiogenic effect. Bevacizumab has also been "humanized" to decrease any antigenic effect it may have on the patient, and bevacizumab has a higher molecular weight; this full-length antibody likely will not penetrate the retina to the same extent as the lower molecular weight fragment ranibizumab. However, the increased size of bevacizumab may decrease its clearance rate from the site of action.

[0013] Another anti-VEGF agent is Sunitinib maleate (Sutent®). Sunitinib maleate is an orally bioavailable indolinone with potential antineoplastic activity. It blocks the tyrosine kinase activities of vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor b (PDGFRb), and c-kit, thereby inhibiting angiogenesis and cell proliferation. This agent also inhibits the phosphorylation of Fms-related tyrosine kinase 3 (FLT3), another receptor for tyrosine kinase expressed by some leukemic cells (NCI04). A systemic dose for cancer treatment is between 12.5 mg/day to 50 mg/day. "MV_n ^■ .^■/^''' " AMm^™1on 3g.7 -tfc1 IP-a. vailable anti-inflammatory agents, many have a target of action to block or ameliorate the actions of pro-inflammatory signals, such as histamine and cytokines. Although this provides some relief from the harmful effects of inflammation, it does not address the cause of the problem. Leukocytes and macrophages, which release pro-inflammatory factors into affected areas, are allowed access to the inflamed tissue following new blood vessel formation.

General

[0015] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation 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 or any two or more of the steps or features.

[0016] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

[0017] Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

[0018] The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[0019] The invention described herein may include one or more range of values (eg size, concentration etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. "foWβ] ^{■ '} -iWbύ^'gHoif this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law.

[0021] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

Summary of the Invention

[0022] According to the invention a method is disclosed for treating (eg controlling, ameliorating, or reducing), or preventing, in a patient, an inflammatory ailment. According to this method a patient is given an anti-vascular endothelial growth factor (VEGF) agent to treat or prevent the inflammatory ailment.

[0023] The invention also extends to a therapeutic method comprising the step of: providing to at least one inflammatory tissue in a patient a formulation comprising a therapeutically effective amount of an anti-VEGF agent, wherein the formulation ameliorates inflammation in the absence of angiogenesis.

[0024] Further, the invention extends to an ocular prophylaxis or treatment method comprising the step of: ocularly providing to a patient, having or at risk for developing an ocular inflammatory ailment, a formulation comprising an anti-VEGF agent under conditions sufficient to ameliorate a cause and/or effect of fluid leakage in the eye.

[0025] The invention also extends to the use of an anti-vascular endothelial growth factor (VEGF) agent in the manufacture of a medicament to treat or prevent the inflammatory ailment. It extends to the use of a therapeutically effective amount of an anti-VEGF agent in the manufacture of a medicament to treat at least one inflammatory tissue in a patient wherein the formulation ameliorates inflammation in the absence of angiogenesis. Further, it extends to the use of an anti-VEGF agent in the manufacture of a medicament for prophylaxis or treatment of a patient having or at risk for developing an ocular inflammatory ailment. 10020]. Aihti-VEΘF::' agents include but are not limited to bevacizumab (rhuMab VEGF, Avastin®, Genentech, South San Francisco CA), ranibizumab (rhuFAb V2, Lucentis®, Genentech), pegaptanib (such as pegaptanib sodium ie Macugen^®, Eyetech Pharmaceuticals, New York NY)₁ sunitinib maleate (Sutent®, Pfizer, Groton CT), TNP470, integrin av antagonists, 2-methoxyestradiol, paclitaxel, or P38 mitogen activated protein kinase inhibitors. Anti-VEGF siRNA (short double-stranded RNA to trigger RNA interference and thereby impair VEGF synthesis) may also be used as an anti-VEGF agent. In preferred embodiment of the invention the method is used to treat or prevent ocular inflammation. Ocular inflammation may be associated with underlying systemic disease or autoimmunity, or may occur as a direct result of ocular trauma or infectious agents (bacterial, viral, fungal, etc.).

[0027] In an embodiment, the anti-VEGF agent is administered to a patient either alone or with one or more agent(s) known to one skilled in the art under the classification of anti-inflammatory agents, to treat or prevent an inflammatory ailment. Anti-inflammatory agents include, but are not limited to, steroids, anti-prostaglandins, matrix metalloproteinase inhibitors, non-steroidal anti-inflammatory drugs (NSAIDS), macrolides, anti-proliferative agents, anti-cancer agents, etc. In one embodiment, the method is used to treat or prevent inflammation using an anti-VEGF agent such as bevacizumab alone.

[0028] In another embodiment, the method is employed to treat or prevent an inflammatory ailment using an anti-VEGF agent such as bevacizumab in combination with at least another anti-inflammatory agent.

[0029] According to the invention the method may be used to treat or prevent an inflammatory ailment at any stage, even early stage inflammation before occurrence of an angiogenic component. The method treats or prevents inflammation, and counteracts the action of angiogenic agents such as VEGF on the permeability of a vessel wall, thereby reducing or preventing the resulting tissue damage due to fluid leakage from the vessel (extravasation). The method is applicable to any tissue or site in the body, and to any cause of inflammation such as immune disease including autoimmune disease, viral and/or bacterial infection, trauma including surgical trauma, etc. fOΘ&G] '-^'' 'Fft ^••■arϊδtiVdr embodiment, sunitinib maleate (Sutent®) may be used to ameliorate (e.g., reduce, prevent, slow, etc.) age related macular degeneration (AMD), either alone or in combination with PDT or laser coagulation therapy (e.g., scatter threshold laser coagulation, etc.) (such therapies are described in U.S. Patent No. 6,942,655, which is expressly incorporated by reference herein in its entirety). Sunitinib maleate, alone or in combination with such therapies, is administered to improve vision, maintain vision, or reduce loss of visual acuity in a patient having or at risk for developing AMD. By reducing, slowing, or preventing its onset or progression, it thus reduces effects of AMD.

[0031] The invention also extends to the use of sunitinib maleate in the manufacture of a medicament to treat or prevent AMD, either alone or in combination with PDT or laser coagulation therapy.

[0032] Methods described herein may be used to control, reduce, or prevent cell or tissue damage resultant from an inflammatory ailment in or in close proximity to the brain or eye.

[0033] Other aspects and advantages of the invention will become apparent to those skilled in the art from a review of the ensuing description, which proceeds with reference to the following illustrative drawings.

Brief Description of the Drawings

[0034] Figure 1 is a schematic cross-sectional view of a mammalian eye 10 showing the anterior chamber 12, cornea 14, conjunctiva 16, iris 18, optic nerve 20, sclera 22, macula lutea 24, lens 26, retina 28 and choroid 30.

[0035] Figure 2 is an enlarged diagrammatic illustration of the circled area 2 in Figure 1 showing detailed retinal and choroids structures.

[0036] Figure 3 shows normalized areas of corneal neovascularization in bevacizumab-treated eye (n=10) and control eyes (n=6).

[0037] Figure 4 is a photograph of a representative bevacizumab-treated eye.

[0038] Figure 5 is a photograph of a representative control-treated eye. ^!'f003§} ^••'^'' Flgdre- "8^{; !!;:}ts a micrograph of a control eye histology (cornea with neovascularization and moderate inflammation).

[0040] Figure 7 is a micrograph of a treated eye histology (skip areas of vascularization alternating with clear stroma)

[0041] Figure 8 illustrates the effects of corticosteroids with or without bevacizumab on VEGF R1 expression on CECs. Unstimufated cells were not stimulated or treated. Untreated and all other treatment groups were stimulated with PMA and treated as designated on the x-axis one day after plating. Flow cytometric analysis was performed after 3 days in culture. Results are expressed as means ± SEM. Statistical significances are discussed in the text.

Detailed Description of the Invention

[0042] In its most general form, the invention concerns methods for treating or preventing, in a patient, an inflammatory ailment using at least an anti-vascular endothelial growth factor (VEGF) agent.

[0043] The invention also extends to a therapeutic method comprising the step of: providing to at least one inflammatory tissue in a patient a formulation comprising a therapeutically effective amount of an anti-VEGF agent, wherein the method ameliorates inflammation in the absence of angiogenesis. Thus-, inflammation and angiogenesis can occur independently and administration of anti-VEGF agents such as bevacizumab, either alone or to supplement known anti-inflammatory agents, ameliorates both inflammation without an angiogenic component (earlier stage inflammation), and inflammation that has progressed to an angiogenic component (later stage inflammation). Coexistence of inflammation and angiogenesis may lead to more severe, damaging, and persistent inflammation.

pfllf ^•'^' Furtϊi ef iffeFinvention extends to an ocular prophylaxis or treatment method comprising the step of: ocularly providing to a patient having or at risk for developing an ocular inflammatory ailment a formulation comprising an anti-VEGF agent under conditions sufficient to ameliorate a cause and/or effect of fluid leakage in the eye.

[0045] The invention also extends to the use of an anti-vascular endothelial growth factor (VEGF) agent in the manufacture of a medicament to treat or prevent the inflammatory ailment. It extends to the use of a therapeutically effective amount of an anti-VEGF agent in the manufacture of a medicament to treat at least one inflammatory tissue in a patient wherein the formulation ameliorates inflammation in the absence of angiogenesis. Further, it extends to the use of an anti-VEGF agent in the manufacture of a medicament for prophylaxis or treatment of a patient having or at risk for developing an ocular inflammatory ailment.

[0046] As used herein "inflammatory ailment" will be understood to include inflammatory states, responses and disorders. It includes inflammation, an antiinflammatory response, and/or effects of an anti-inflammatory response.

[0047] In one embodiment, the inventive method administers to a patient one or a combination of anti-VEGF agent(s) to treat or prevent an inflammatory ailment, and thus to control, reduce or prevent an inflammatory response or ameliorate the effects of an inflammatory response. In a preferred form of this embodiment, bevacizumab, ranibizumab or pegaptanib is used to enhance reabsorption of inflammatory exudates. Decreasing the level of exudates in the eye reduces the inflammatory process and the ensuing hyper-permeable state that occurs with allergies, infection, responses to ocular photodynamic therapy (PDT) and laser treatments, after ocular surgery or trauma, etc. In one embodiment, the anti-VEGF agent is administered to ameliorate an inflammatory process without an angiogenic component. Many inflammatory processes, such as early stage inflammation, are not associated with the formation of new blood vessels. Examples include, but are not limited to, inflammatory diseases of the central nervous system (brain and spinal cord) such as abscess, meningitis, encephalitis, vasculitis, and conditions resulting in cerebral edema; inflammatory diseases of the eye (uveitis, subsequently discussed), macular edema, and others known to one skilled in the art. '

embodiment, the anti-VEGF agent(s) is administered to ameliorate scarring and adhesions that are a part of the inflammatory process. Adhesions are bands of scar tissue that bind two internal body surfaces. They are an inflammatory response to tissue damage, and occur as a normal part of any healing process. As one example, adhesions frequently occur during the post-surgical healing process during which tissues have experienced mechanical trauma. However, adverse effects can occur when internal surfaces bind, and adhesions may persist even after the original trauma has healed. Surgery to repair adhesions itself results in recurrent or additional adhesions. The presence of adhesions may also complicate surgical procedures, for example, ocular conjunctival adhesions may complicate subsequent glaucoma surgery.

[0049] Adhesions can occur following any type of trauma or surgery, including but not limited to ocular surgery. Examples of ocular surgery that may result in adhesions include glaucoma filtration operations (i.e., iridencleisis and trephination, pressure control valves), extraocular muscle surgery, diathermy or scleral buckling surgery for retinal detachment, and vitreous surgery. Examples of ocular trauma include penetrating ocular injuries, intraocular foreign body, procedures such as PDT, scatter laser threshold coagulation, refractive surgery, and blunt trauma.

[0050] In yet another embodiment, anti-VEGF agents are used in the method to ameliorate disorders with both a vascular proliferative component and a scarring component. As one example, the invention may be used in patients with the ocular disease pterygia. In these patients, fibrovascular proliferation results in scarring of the conjunctiva. An elevated, superficial, external ocular mass, termed a pterygium, forms and extends onto the corneal surface. Patients may experience symptoms of inflammation (e.g., redness, swelling, itching, irritation) and blurred vision. The mass itself may become inflamed, resulting in redness and ocular irritation. Left untreated, pterygia can distort the corneal topography, obscure the optical center of the cornea, and result in altered vision.

[0051] The process whereby scar tissue forms (scarring) can occur without new blood vessels being formed (neovascularization). However, the neovascularization process always results in scarring because of the cell proliferation that occurs with the formation of new vessels also results in the proliferation of fibroblasts, glial cells, etc. ^:UIipSsύlt"fh*'!sβIr^;:liisue formation. The inventive method may be used to ameliorate the scarring process.

[0052] In a further embodiment, the anti-VEGF agent(s) is administered to treat or prevent and inflammatory ailment associated with uveal tissues (uveitis, an inflammation of tissues in the middle layer of the eye, mainly the iris (iritis) and the ciliary body). Ocular inflammation may be associated with underlying systemic disease or autoimmunity, or may occur as a direct result of ocular trauma or infectious agents (bacterial, viral, fungal, etc.). Inflammatory reactions in adjacent tissues, e.g., keratitis, can induce a secondary uveitis. There are both acute and chronic forms of uveitis. The chronic form is frequently associated with many systemic disorders and most likely occurs due to immunopathological mechanisms.

[0053] Uveitis presents with ocular pain, photophobia and hyperlacrimation, with decreased visual acuity ranging from mild blur to significant vision loss. Hallmark signs of anterior uveitis are cells and flare in the anterior chamber. If the anterior chamber reaction is significant, small gray to brown endothelial deposits known as keratic precipitates may arise, leading to endothelial cell dysfunction and corneal edema. There may be adhesions to the lens capsule (posterior synechia) or the peripheral cornea (anterior synechia). Granulomatous nodules may appear on the surface of the iris stroma. Intraocular pressure is initially reduced due to secretory hypotony of the ciliary body but, as the reaction persists, inflammatory by-products may accumulate in the trabeculum. If this debris builds significantly, and if the ciliary body resumes its normal secretory output, the pressure may rise sharply, resulting in a secondary uveitic glaucoma.

[0054] One skilled in the art will appreciate that scarring and adhesions in areas of the body other than the eye may be treated with the inventive method. Examples include adhesions associated with cardiac surgery (e.g., adhesions in the pericardial space), pulmonary surgery (e.g., in the peripleural space), abdominal surgery (e.g., appendectomy, gastric bypass surgery), gynaecological surgery (e.g., episiotomy, Caesarean section, hysterectomy), any type of laparoscopy or laparotomy surgery, reconstructive surgery (cosmetic or therapeutic), organ removal (partial or complete), etc. "[0OS*»3.^■■' fft ^••■•ari^'δtrøF" embodiment, the inventive method comprises the step of administering to a patient an anti-inflammatory agent simultaneously or concomitantly with an anti-VEGF agent such as bevacizumab to control, reduce, or prevent an inflammatory ailment. Other anti-VEGF agents such as Lucentis^®, Macugen^®, Sutent^®, geldanamycin, etc. may be included.

[0056] In a highly preferred embodiment, the method is used to treat or prevent inflammation using bevacizumab alone. Bevacizumab at a dose of 5 mg/0.1 ml has been found not to be toxic. In embodiments where bevacizumab or another anti- VEGF agent is administered as the sole agent to ameliorate inflammation, the dose of bevacizumab ranges between about 0.01 mg/0.1 ml to about 5 mg/0.1 ml. Accordingly the administered dose of bevacizumab is preferably less than about 5 mg/0.1 ml. In another embodiment, the administered dose of bevacizumab ranges from about 0.1 mg/ml to about 50 mg/ml. In another embodiment, the dose of bevacizumab administered systemically ranges from about 0.05 mg/ml to about 5 mg/ml. In one embodiment, the dose of bevacizumab administered intraocularly (e.g., intravitreally) is about 0.005 mg/ 0.1 ml to about 5 mg/ 0.1 ml. In one embodiment, the dose of bevacizumab administered topically to the eye is up to 5 mg/ml, and in another embodiment it may be higher. While these doses recite bevacizumab, one skilled in the art will appreciate that they may be used with other anti-VEGF agents, and that doses for a specific agent may be determined empirically, by patient disease severity, other patient variables, etc!

[0057] In an alternate highly preferred embodiment, the method is used to treat or prevent inflammation using ranibizumab or pegaptanib. The administered dose of ranibizumab (Lucentis^®), is either about 300 or about 500 microgram doses . Alternatively, if pegaptanib (e.g. Macugen^®) is used in the method it should be administered in a dose ranging from either about 0.3 mg to about 3.0 mg every four or six weeks.

[0058] The method may be used to treat or prevent an inflammatory ailment in any tissue including, but not limited to, eye (e.g., to ameliorate conjunctivitis (inflammation of the conjunctivae, the mucous membranes covering the sclera and inner eyelid), that may be associated with bacterial, viral, or Chlamydia infections, allergies, or susceptibility to irritants such as chemicals, smoke, etc.), lung (e.g., to ameliorate interstitial lung disease, inflammation of the interstitium (tissue between the air sacs in ^h'y^{! 8}IMg)T' Bon^:e"^"|[e~g., to ameliorate synovitis, inflammation of the synovium (the membranes lining joints) that may be associated with arthritis), brain (e.g., to ameliorate encephalitis (inflammation of brain tissue and/or membranes)), and muscle (e.g., to ameliorate myopathies (inflammation of muscles, such as muscles near a joint). The method may be used on patients at risk for developing inflammation. The method may be used on patients with inflammation and/or inflammatory processes from any cause, including but not limited to autoimmune diseases, diseases with an immune component, ischemic diseases, diabetes, age related macular degeneration, retinitis pigmentosa, infectious diseases, allergen-induced inflammation, other degenerative diseases, etc.

[0059] In an embodiment, the anti-VEGF agent is administered to a patient either alone or with one or more agent(s) known to one skilled in the art under the classification of anti-inflammatory agents, to treat or prevent an inflammatory ailment. Anti-inflammatory agents include, but are not limited to, steroids, anti-prostaglandins, matrix metalloproteinase inhibitors, non-steroidal anti-inflammatory drugs (NSAIDS), macrolides, anti-proliferative agents, anti-cancer agents, etc.

[0060] Examples of anti-inflammatory agents recognized by one skilled in the art include, but are not limited to, the following: a. colchicine; b._ a steroid such as triamcinolone (Aristocort®; Kenalog^®),. anecortave acetate (Retaane^®, Alcon), betamethasone (Celestone^®), budesonide cortisone, dexamethasone (Decadron-LA®; Decadron® phosphate; Maxidex^® and Tobradex^® (Alcon)), hydrocortisone methylprednisolone (Depo-Medrol^®, SoIu- Medrol^®), prednisolone (prednisolone acetate, e.g., Pred Forte^® (Aliergan), Econopred and Econopred Plus^® (Alcon), AK-Tate® (Akorn), Pred Mild^® (Aliergan), prednisone sodium phosphate (Inflamase Mild and lnflamase Forte^® (Ciba), Metreton^® (Schering), AK-Pred^® (Akorn)), fluorometholone (fluorometholone acetate (Fiarex^® (Alcon), Eflone^®), fluorometholone alcohol (FML^® and FML-Mild^®, (Aliergan), FluorOP^®), rimexolone (Vexol^®, Alcon), medrysone alcohol (HMS^®, Aliergan), lotoprednol etabonate (Lotemax^® and Alrex^®, Bausch & Lomb, and 11-desoxcortisol; c. an anti-prostaglandin such as indomethacin; ketorolac tromethamine ((±)-5- benzoyl-2,3-dihydro-1 H-pyrrolizine-1-carboxylic acid, a compound with 2-amino-

,3-propanediol (1 :1) (Acular^® Allegan), Ocufen^® (flurbiprofen sodium 0.03%), meclofenamate, fluorbiprofen, and the pyrrolo-pyrrole group of non-steroidal anti-inflammatory drugs; d. a macrolide such as sirolimus (rapamycin), pimecrolimus, tacrolimus (FK506), cyclosporine (Arrestase), everolimus 40-O-(2-hydroxymethylenrapamycin), ascomycin, erythromycin, azithromycin, clarithromycin, clindamycin, lincomycin, dirithromycin, josamycin, spiramycin, diacetyl-midecamycin, tylosin, roxithromycin, ABT-773, telithromycin, leucomycins, lincosamide, biolimus, ABT- 578 (methylrapamycin), and derivatives of rapamycin such as temsirolimus (CCI- 779, Wyeth) and AP23573 (Ariad); e. a non-steroidal anti-inflammatory drug such as derivatives of acetic acid (e.g. diclofenac and ketorolac (Toradol^®, Voltaren^®, Voltaren-XR^®, Cataflam^®)), salicylate (e.g., aspirin, Ecotrin^®), proprionic acid (e.g., ibuprofen (Advil^®, Motrin^®, Medipren^®, Nuprin^®)), acetaminophen (Tylenol^®), aniline (e.g., aminophenolacetaminophen, pyrazole (e.g., phenylbutazone), N-arylanthranilic acid (fenamates) (e.g., meclofenamate), indole (e.g., indomethacin (Indocin^®, Indocin-SR^®)), oxicam (e.g., piroxicam (Feldene^®)), pyrrol-pyrrole group (e.g., Acular^®), antiplatelet medications, choline magnesium salicylate (Trilisate^®), cox- 2 inhibitors (meloxicam (Mobic^®)), diflunisal (Dolobid^®), etodolac (Lodine^®), fenoprofen (Nalfon^®), flurbiprofen (Ansaid^®), ketoprofen (Orudis^®, Oruvail^®), meclofenamate (Meclomen^®), nabumetone (Relafen®),- naproxen (Naprosyn^®, Naprelan^®, Anaprox^®, Aleve^®), oxaprozin (Daypro^®), phenylbutazone (Butazolidine^®), salsalate (Disalcid^®, Salflex^®), tolmetin (Tolectin^®), valdecoxib (Bextra^®), sulindac (Clinoril^®), and flurbiprofin sodium (Ocufen^®); and f. an MMP inhibitor such as doxycycline, TIMP-1 , TIMP-2, TIMP-3, TIMP-4; MMP1 , MMP2, MMP3, Batimastat (BB-94), TAPI-2,10-phenanthroline, and marimastat.

[0061] The formulation may also contain anti-PDGF compound(s) such as imatinib mesylate (Gleevec^®), sunitinib malate (Sutent^®) which has anti-PDGF activity in addition to anti-VEGF activity, and/or anti-leukotriene(s) such as genleuton, montelukast, cinalukast, zafirlukast, pranlukast, zileuton, BAYX1005, LY171883, and MK-571 to account for the involvement of factors besides VEGF in neovascularization. The formulation may additionally contain other agents including, but not limited to, transforming growth factor β (TGFβ), interleukin-10 (IL-10), aspirin, a vitamin, and/or an antineoplastic agent. SfbSfeijl / S a liigiif ^;»preferred embodiment, the method is used to treat or prevent inflammation using an anti-VEGF agent such as bevacizumab and a steroid such as triamcinolone.

[0063] In another aspect of the invention, pharmaceutical formulations prepared according to the present invention may be prepared in combination with a glucocorticoid (e.g. prednisolone, prednisone), an oestrogen (e.g. oestrodiol), an androgen (e.g. testosterone) retinoic acid derivatives (e.g. 9-cis-retinoic acid, 13-trans- retinoic acid, all-trans retinoic acid), a vitamin D derivative (e.g. calcipotriol, calcipotriene), a non-steroidal anti-inflammatory agent, a vitamin D derivative, an anti- infective agent, a protein kinase C inhibitor, a MAP kinase inhibitor, an anti-apoptotic agent, a growth factor, a nutrient vitamin, an unsaturated fatty acid, and/or ocular anti- infective agents, for the treatment of the ophthalmic disorders set forth herein. In still other embodiments of the invention, a mixture of these agents may be used.

[0064] When the methods of the invention are employed to treat ocular inflammatory ailments the formulations may include ocular anti-infective agents such as penicillins (ampicillin, aziocillin, 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), miscellaneous agents such as aztreonam, bacitracin, ciprofloxacin, clindamycin, chloramphenicol, cotrimoxazole, fusidic acid, imipenem, metronidazole, teicoplanin, and vancomycin), antifungals (amphotericin B, clotrimazole, econazole, fluconazole, flucytosine, itraconazole, ketoconazole, miconazole, natamycin, oxiconazole, and terconazole), antivirals (acyclovir, ethyldeoxyuridine, foscamet, ganciclovir, idoxuridine, trifluridine, vidarabine, and (S)-1-(3-dydroxy-2-phospho- nyluethoxypropyl) cytosine (HPMPC)), antineoplastic agents (cell cycle (phase) nonspecific agents such as alkylating agents (chlorambucil, cyclophosphamide, mechlorethamine, melphalan, and busulfan), anthracycline antibiotics (doxorubicin, daunomycin, and dactinomycin), cisplatin, and nitrosoureas), antimetabolites such as antipyrimidines (cytarabine, fluorouracil and azacytidine), antifolates (methotrexate), antipurines (mercaptopurine and thioguanine), bleomycin, vinca alkaloids (vincrisine and vinblastine), podophylotoxins (etoposide (VP-16)), and nitrosoureas (carmustine, (BCNU)), immunosuppressant agents such as cyclosporin A and FK506, and anti-

factors (inhibitors), and inhibitors of proteolytic enzymes such as plasminogen activator inhibitors.

[0065] Where an anti-VEGF agent(s) is administered with an anti-infiammatory agent, an effective amount of the anti-inflammatory agent is administered to a patient at a standard dose known to one skilled in the art. As one example, prednisone is administered for a systemic dose in the range between about 5 mg to about 100 mg daily. As another example, Solu-medrol® is administered intravenously in a single dose of about 1 mg.

[0066] When the anti-VEGF agent employed in the method is formulated with an anti-prostaglandin the prostaglandin antagonist will be administered in a concentration sufficient to result in a prostaglandin inhibitory effect. As one example, anti- prostaglandins such as flurbiprofen may be administered at a concentration in the range of about 0.001 %^w/v to about 0.5%^w/v. As an example, OCUFEN® (flurbiprofen sodium 0.03% (Allergan), sodium (±)-2-(2-fluoro-4-biphenylyl)-propionate dihydrate) 0.03% may be administered at a concentration ranging from about 0.003% ^^/w to about 0.3% ^w/w. Anti-prostaglandins other than flurbiprofen may be included. The anti- prostaglandins may be administered at the doses and by the methods previously described, and include indomethacin, ketorolac, tromethamine 0.5% ((+)~5-benzoyl-2,3- dihydro-1 H-pyrrolizine-1-carboxylic acid, compound with 2-amino-2-(hydroxymethyl)- 1 ,3-propanediol (1 :1) (ACULAR^® Allegan, Irvine CA), meclofenamate, flurbiprofen, and compounds in the pyrrolo-pyrrole group of non-steroidal anti-inflammatory drugs (NSAIDs). For example, ACULAR® may be administered at a concentration ranging from about 0.003%^w/w to about 0.3%^w/w. In one embodiment, the concentration of ACULAR® is about 0.03% ^w/w

[0067] When the anti-VEGF agent employed in the method is formulated with a macrolide, the macrolide will be present in an amount sufficient to induce a therapeutic effect. For example it might be administered in a concentration ranging from about 20 μg/ml to about 200 μg/ml (about 0.002%w/v to about 0.02%w/v).

[0068] When the anti-VEGF agent employed in the method is formulated with a MMP inhibitor, the inhibitor will be present in an amount sufficient to inhibit MMPs and prevent pathogenic tissue destruction. For example, the concentration of doxycycline employed in this form of the invention will range from 0.01 μg/ml to about 30 mg/ml. iϋVJiolriϋ spelsiffclffyf ^!'iufoxycycline concentrations will range from about 0.05 mg/ml to about 1 mg/ml. Alternatively, doxycycline concentrations will range from about 0.05 mg/ml to about 10 mg/ml. Yet again doxycycline concentrations can range from about 1 mg/ml to about 20 mg/ml. These doses are substantially non-toxic to the patient.

[0069] According to the methods of the invention the formulations employed in the method may be administered by a variety of routes including enteral, parenteral, and ocular routes such as intravitreal injection, subconjunctival injection, retrobulbar injection, topical, intravenously, orally, ocularly, etc. One skilled in the art will appreciate that the route of administration may vary due to factors such as agent solubility, patient needs, dose required, etc. The active agent may be fast-acting, slow acting, or both. Further the formulation may be formulated for delayed and/or extended release to provide effects over a longer period of time. Preferably, methods of the invention are used to treat ocular inflammatory ailments and as such the formulations employed in the methods are administered by an ocular route, such as topical, subconjunctival, sub-Tenon, intraocular, etc. to the extent that the invention concerns the use of the above agents in the treatment of ocular inflammatory ailments doses for topical and sub-conjunctival administration of the above agents, as well as intravitreal dose and vitreous half-life may be found in Intravitreal Surgery Principles and Practice, Peyman G A and Shulman, J Eds., 2nd edition, 1994, Appleton-Longe, the relevant sections of which are expressly incorporated by reference herein.

[0070] The skilled reader will appreciate that the duration over which any of the formulations of the invention will dwell in a cellular environment will depend, inter alia, on such factors as the pharmacological properties of the compounds employed in the formulation, the concentration of the compound employed, the bioavailability of the compound, the inflammatory ailment to be treated, the mode of administration and the preferred longevity of the treatment. Where that balance is struck will often depend on the longevity of the effect required and the ailment being treated. Formulations prepared according to the invention will preferably have dwell times from hours to many months and possibly years, although the latter time period requires special delivery systems to attain such a duration. Some illustrative forms of such delivery systems are disclosed below. Most preferably the formulations described herein will have a dwell time of 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 formulation will have a dwell time of at I fϋaSI SS few^;M6n!HslsIch as, 1 month, 2 months, 3 months, with dwell times of greater than 4, 5, 6, 7 to 12 months being achievable.

[0071] The precise formulation used in the methods identified herein will vary according to a wide range of commercial and scientific criteria. That is the skilled reader will appreciate that the formulations used in the methods of the invention may contain other agents. For example the formulations may include a physiological saline solution as a carrier vehicle. The pH of the formulation may be maintained at a substantially neutral pH (for example, about 7.4, in the range of about 6.5 to about 7.4, etc.) with an appropriate buffer system as known to one skilled in the art (for example, acetate buffers, citrate buffers, phosphate buffers, borate buffers).

[0072] The formulation may additionally include at least a pharmaceutically acceptable additive (such as a diluent, carrier, adjunct, excipient or non-toxic, non- therapeutic, non-immunogenic stabilizers and the like). Preferably, the pharmaceutically acceptable additive should be ophthalmologically acceptable when used to treat ocular inflammatory ailments. More preferably the formulation will be compatible with the vitreous, and should not leave any vision impairing residue in the eye. Desirably, any pharmaceutically acceptable additive used in the formulation may preferably be suited to the delivery of the pharmaceutical formulation as an intravitreal depot injection.

[0073] Any diluent used in the preparation of the pharmaceutically acceptable formulation is preferably selected so as not to unduly affect the biological activity of the formulation. Examples of such diluents which are especially useful for formulation preparation are water, saline, organic or inorganic salt solutions, Ringer's solution, dextrose solution, and Hank's solution. For example, solutions may be prepared using a physiological saline solution as a vehicle. The pH of an ophthalmic solution may be maintained at a substantially neutral pH (for example, about 7.4, in the range of about 6.5 to about 7.4, etc.) with an appropriate buffer system as known to one skilled in the art (for example, acetate buffers, citrate buffers, phosphate buffers, borate buffers).

[0074] The formulations used in the invention may also contain pharmaceutically acceptable excipients known to one skilled in the art such as preservatives, stabilizers, surfactants, chelating agents, antioxidants such as vitamin C, etc. Preservatives include, but are not limited to, benzalkonium chloride, chlorobutanol, '

pfie'iny^'irife'rcuric acetate and phenylmercuric nitrate. A surfactant may be Tween 80. Other vehicles that may be used include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose, purified water, etc. Tonicity adjustors may be included, for example, sodium chloride, potassium chloride, mannitol, glycerin, etc. Antioxidants include, but are not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, butylated hydroxytόluene, etc.

[0075] In addition, the pharmaceutical formulation may include additives such as other buffers, diluents, carriers, adjuvants or excipients. Any pharmacologically acceptable buffer suitable for application to the eye may be used, e.g., tris or phosphate buffers. Other agents may be employed in the formulation for a variety of purposes. For example, buffering agents, preservatives, co-solvents, surfactants, oils, humectants, emollients, chelating agents, stabilizers or antioxidants may be employed. Water soluble preservatives which may be employed include, but are not limited to, benzalkonium chloride, chlorobutanol, thimerosal, sodium bisulfate, phenylmercuric acetate, phenylmercuric nitrate, ethyl alcohol, methylparaben, polyvinyl alcohol, benzyl alcohol and phenylethyl alcohol. A surfactant may be Tween 80. Other vehicles that may be used include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose, purified water, etc. Tonicity adjustors may be included, for example, sodium chloride, potassium chloride, mannitol, glycerin, etc. Antioxidants include, but are not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, butylated hydroxytoluene, etc. The indications, effective doses, formulations, contraindicatons, vendors etc, of the compounds in the formulations are available or are known to one skilled in the art.

[0076] These agents may be present in individual amounts of from about 0.001% to about 5% by weight and preferably about 0.01 % to about 2% by weight. Suitable water soluble buffering agents that may be employed are sodium carbonate, sodium borate, sodium phosphate, sodium acetate, sodium bicarbonate, etc., as approved by the US FDA for the desired route of administration. These agents may be present in amounts sufficient to maintain a pH of the system of between about 2 to about 9 and preferably about 4 to about 8. As such the buffering agent may be as much as about 5% on a weight to weight basis of the total formulation. Electrolytes such as, but not MiMi 4bf%6dϊiι# ^l!'ehloride and potassium chloride may also be included in the formulation.

[0077] The formulation employed in the methods of the invention may be administered as a slow release formulation, with a carrier formulation such as microspheres, microcapsules, liposomes, etc., as an intravenous solution or suspension, or in an intraocular injection, as known to one skilled in the art. A time-release drug delivery system may be administered intraocularly to result in sustained release of the agent over a period of time. The formulation may be in the form of a vehicle, such as a micro- or macro-capsule or matrix of biocompatible polymers such as polycaprolactone, polyglycolic acid, polylactic acid, polyanhydrides, polylactide-co- glycolides, polyamino acids, polyethylene oxide, acrylic terminated polyethylene oxide, polyamides, polyethylenes, φolyacrylonitriles, polyphosphazenes, poly(ortho esters), sucrose acetate isobutyrate (SAIB), and other polymers such as those disclosed in U.S. Patent Nos. 6,667,371 ; 6,613,355; 6,596,296; 6,413,536; 5,968,543; 4,079,038; 4,093,709; 4,131 ,648; 4,138,344; 4,180,646; 4,304,767; 4,946,931 , each of which is expressly incorporated by reference herein in its entirety, or lipids that may be formulated as microspheres or liposomes. A microscopic or macroscopic formulation may be administered through a needle, or may be implanted by suturing within the eye, for example, within the lens capsule. Delayed or extended release properties may be provided through various formulations of the vehicle (coated or uncoated microsphere, coated or Uncoated capsule, lipid or polymer components, unilamellar or multilamellar structure, and combinations of the above, etc.). The formulation and loading of microspheres, microcapsules, liposomes, etc. and their ocular implantation are standard techniques known by one skilled in the art, for example, the use a ganciclovir sustained-release implant to treat cytomegalovirus retinitis, disclosed in Vitreoretinal Surgical Techniques, Peyman et al., Eds. (Martin Dunitz, London 2001 , chapter 45); Handbook of Pharmaceutical Controlled Release Technology, Wise, Ed. (Marcel Dekker, New York 2000), the relevant sections of which are incorporated by reference herein in their entirety. For example, a sustained release intraocular implant may be inserted through the pars plana for implantation in the vitreous cavity. An intraocular injection may be into the vitreous (intravitreal), or under the conjunctiva (subconjunctival), or behind the eye (retrobulbar), or under the Capsule of Tenon (sub-Tenon), and may be in a depot form. Other intraocular routes of administration ϊirfc. lϊiij€icti^'έff'sϊfei ind forms are also contemplated and are within the scope of the invention.

[0078] In one embodiment, bevacizumab and/or other anti-VEGF agent(s) may be administered via a controlled release system (i.e., delayed release formulations and/or extended release formulations) such as polylactic or polyglycolic acid, silicone, hema, and/or polycaprolactone microspheres, microcapsules, microparticles, nanospheres, nanocapsules, nanoparticles, etc. A slow release system may release about 10 ng anti-VEGF agent/day to about 50 ng anti-VEGF agent/day for an extended period.

[0079] The route and form of administration of the formulations described herein will be by methods known to one skilled in the art, including as previously described. Administration may be by any suitable route, and where more than one formulation or agent is to be administered, administration may be by the same route or by different routes, including enteral, parenteral, and ocular routes such as intravitreal injection, subconjunctival injection, retrobulbar injection, topical, etc. As one example, the anti- VEGF agent (bevacizumab, sunitinib, etc.) may be topically administered to intact or compromised eyes, skin, mucous membranes, etc. to reduce scarring after trauma, surgery, radiation, burns, wounds, etc. As another example, it may be locally administered to a site in a surgical field to ameliorate inflammation (e.g., adhesions, scarring, effusions) of pleura, epicardium, etc. after thoracic, cardiac, abdominal, etc. surgery. As_. another example, it may be administered intrathecal^ (brain, spinal cord, etc.). As another example, it may be administered by inhalation, for example, to ameliorate inflammation in the respiratory tract (nose, trachea, bronchi, lungs, etc.). As another example, it may be instilled in a body cavity (ventricles, sinuses, bladder, etc.). As another example, the anti-VEGF agent may be administered systemically (e.g., a single dose/week for one month, then monthly re-evaluation of need) or topically (e.g., from about 10 ng/ml to about 100 ng/ml), or intraocularly (e.g., from about 7 ng/ml to about 20 μg/ml).

[0080] To the extent that the method is used to treat an ocular inflammatory ailment the formulation may be administered by topical, subconjunctival, and intraocular routes or ocular implants.

[0081] In one embodiment, the formulation is intraocularly injected, for example, into the vitreous. When administering the formulation by intravitreal injection, the active Sgelϋ sftol^'øf BI3JPicentratecl to minimise the volume for injection. For injection, a concentration less than about 20 mg/ml may be injected, and any amount may be effective depending upon the factors previously described. Preferably a dose of less than 7 mg/ml is administered, with doses of less than 6 mg/ml, 5 mg/ml, 4 mg/ml 3 mg/ml, 2 mg/ml and 1 mg/ml being more preferred. Sample concentrations include, but are not limited to, about 5 μg/ml to about 50 μg/ml; about 25 μg/ml to about 100 μg/ml; about 100 μg/ml to about 200 μg/ml; about 200 μg/ml to about 500 μg/ml; about 500 μg/ml to about 750 μg/ml; about 500 μg/ml up to i mg/ml; etc.

[0082] For example, in preparation for injection, topical alcaine was applied to the ocular surface, followed by 5% povidone iodine. A cotton-tipped applicator soaked in 4% lidocaine was then applied to the injection site, which was 4.0 mm posterior to the limbus in phakic eyes and 3.5 mm posterior to the limbus in pseudophakic eyes. A 27-gauge needle was used for injection at the superior pars plana. Indirect ophthalmoscopy can be used to confirm proper intravitreal placement of the suspension.

[0083] A suitable style of syringe is, for example, sold under the name of Uniject^™ manufactured by Becton Dickinson and Company. In this style of syringe, the material is expelled through the needle into the eye by pressure applied to the sides of a pliable reservoir supplying the needle, rather than by a plunger. As the name implies, the construction of the reservoir and needle forms a single unit._

[0084] Topical application of formulations of the invention may be as an in situ gellable aqueous formulation. Such a formulation comprises a gelling agent in a concentration effective to promote gelling upon contact with the eye or with lacrimal fluid in the exterior of 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., poloxamine); polycarbophil; and polysaccharides such as gellan, carrageenan (e.g., kappa-carrageenan and iota- carrageenan), chitosan and alginate gums.

[0085] The phrase "in situ gellable" as used herein embraces not only liquids of low viscosity that form gels upon contact with the eye or with lacrimal fluid in the exterior of the eye, but also more viscous liquids such as semi-fluid and thixotropic gels that exhibit substantially increased viscosity or gel stiffness upon administration to the eye. SMeiid/Oan^'' HKidlantageous to formulate a formulation of the invention as a gel, to minimize loss of the formulation immediately upon administration, as a result, for example, of lacrimation caused by reflex blinking. Although it is preferred that such a formulation exhibit further increase in viscosity or gel stiffness upon administration, this is not absolutely required if the initial gel is sufficiently resistant to dissipation by lacrimal drainage to provide the effective residence time specified herein.

[0086] To prepare a topical formulation for the treatment of ophthalmological disorders, a therapeutically effective amount of the formulation of the invention is placed in an ophthalmological vehicle as is known in the art. The amount of the therapeutic compound to be administered and the concentration of the compound in the topical formulations depend upon the diluent, delivery system or device selected, the clinical condition of the patient, the side effects and the stability of the compound in the formulation. Thus, the physician employs the appropriate preparation containing the appropriate concentration of the therapeutic compound and selects the amount of formulation administered, depending upon clinical experience with the patient in question or with similar patients.

[0087] Where the formulation contains two or more active agents, the active agents may be administered as a mixture, as an admixture, in the same formulation, in separate formulations, in extended release formulations, liposomes, microcapsules, or any of the previously described embodiments.

[0088] The formulation may be administered topically, or may be injected into the eye, or one active agent may be administered topically and the other agent(s) may be injected.

[0089] The method of the present invention may be performed alone, or in combination with one or more other therapies such as photodynamic therapy, laser treatment, or one or more biological or pharmaceutical treatments.

[0090] In another embodiment the invention resides in a method for reducing ocular irritation comprising the step of: administering to a patient a formulation as described above to a patient following corneal surgery (e.g., LASIK® surgery, photorefractive keratectomy (PRK), or other corneal procedures). ⁱ ΪJOUJPIIJ / \

$ with the method an effective amount of anti-VEGF agent; either as the sole active agent, or with one or more other non-anti-inflammatory agents as previously described, is administered to a patient.

[0092] In various embodiments, the formulations may contain other agents. The indications, effective doses, formulations, contraindications, vendors, etc. of these are available or are known to one skilled in the art. It will be appreciated that the agents include pharmaceutically acceptable salts and derivatives.

[0093] Administration of an anti-VEGF agent such as bevacizumab, and optionally other agents such as an anti-PDGF agent, another anti-VEGF agent, etc., may supplement or replace PDT and hence avoid the retinal damage frequently associated with PDT. PDT is frequently used to reduce or prevent damage from leaky vessels associated with age related macular degeneration and other diseases. A series of PDT treatments is often performed with a cumulative effect that, over time, results in retinal damage which in some cases may be severe. The present invention may obviate the need for PDT thus eliminating its associated damage.

[0094] Bevacizumab may be used to ameliorate (e.g., reduce, prevent, slow, etc.) corneal neovascularization. A subsequent example demonstrated the efficacy of bevacizumab on corneal neovascularization that was chemically induced. One skilled in the art, however, appreciates that the invention is not so limited and is applicable to amelioration of corneal neovascularization resulting from other etiologies. These include, but are not limited to, corneal transplant rejection, mechanical trauma, corneal ulcers caused by any mechanism including microorganisms, conjunctivitis sicca, use of contact lenses, presence of a foreign body, pemphigus, Sjorgen's disease, and other autoimmune diseases of the cornea and/or sclera.

[0095] In one embodiment, sunitinib maleate (Sutent®) may be used to ameliorate (e.g., reduce, prevent, slow, etc.) age related macular degeneration (AMD), either alone or in combination with PDT or laser coagulation therapy (e.g., scatter threshold laser coagulation, etc.) (such therapies are described in U.S. Patent No. 6,942,655, which is expressly incorporated by reference herein in its entirety). Sunitinib maleate, alone or in combination with such therapies, is administered to improve vision, maintain vision, or reduce loss of visual acuity in a patient having or at risk for ^•'^■'

slowing, or preventing its onset or progression, it thus reduces effects of AMD.

[0096] In one embodiment, a substantially non-toxic dose of sunitinib maleate is intraocularly administered. Because patients with early stage AMD may receive PDT, there may be cumulative inflammatory effects. Inflammation may result from an immune disease or reaction, including autoimmune diseases, or the presence of a foreign body or organism in the eye. It may be due to macular edema from any cause.

[0097] In one embodiment, sunitinib maleate is administered as the sole agent. It may be administered orally at a dose ranging between about 12.5 mg/day to about 50 mg/day. It may be administered topically at a dose ranging between about 10 ng/ml to about 100 ng/ml. It may be administered intraocularly at a dose between about 7 ng/ml to about 20 μg/ml. The agent may be formulated as a liquid, suspension (e.g., small particulates suspended in a liquid), etc. It will be appreciated that other formulations, including but not limited to emulsions, microspheres, liposomes, nanoparticles, nanospheres, etc. may also be delivered by the device. It may be administered by a controlled release system, as previously described, formulated as known by one skilled in the art, to release about 10 ng/day to about 50 ng/day over several years. The dose may be administered in any convenient volume (e.g. from about 0.1 ml to^about 0.5 ml).. One skilled in the art will appreciate that doses for a- specific patient may be determined empirically, by disease severity, the presence of other pathologies, other patient variables such as age and gender, etc.

[0098] The individual using the inventive method may be at risk for developing AMD, may present with one or more symptoms of AMD, and/or may be already undergoing therapy for AMD using other therapies, either singly or in combination. The method delays the onset or severity of the symptoms of AMD, improving visual acuity and/or preventing further vision loss, and/or reducing the need for retreatments. The combination of anti-VEGF agents with other anti-inflammatory agents results in collective or synergistic action to reduce or halt disease progression.

[0099] In one embodiment, and without being bound or limited by a specific theory, it is believed that the method achieves a synergistic effect when ocular phototherapy, for example, PDT, scatter threshold laser coagulation, other types of laser therapy,

conjunction with sunitinib maleate. The therapies damage the existing lesion of nascent vessels, and reduce the recurrence and slow the progression of additional new vessels. The therapies may be administered in any sequence, that is, sunitinib maleate may be administered before or after PDT, etc. or they may be administered essentially simultaneously, as discussed in more detail below. In one embodiment, sunitinib maleate may be administered prior to laser treatment. In this embodiment, sunitinib maleate treatment will decrease existing subretinal exudates, rendering subsequent laser treatment more effective. In this embodiment, sunitinib maleate treatment will reduce subsequent hyperpermeability that results because of the release of VEGF as a consequence of laser procedures.

[00100] AMD is a pathological, progressive age-related degeneration in the macula lutea 24 of the retina 28 (FIG. 1 is a schematic cross-sectional view of a mammalian eye 10 showing the anterior chamber 12, cornea 14, conjunctiva 16, iris 18, optic nerve 20, sclera 22, macula lutea 24, lens 26, retina 28 and choroid 30). AMD is the most common cause of legal blindness among individuals over the age of 60, with an incidence ranging from 11% to 18.5% in individuals over the age of 85. In the United States, AMD affects roughly 3.6 million individuals, with more than 200,000 new cases developing annually.

[00101] FIG. 2 is an enlarged diagrammatic illustration of the circled area 2 in FIG. 1 showing detailed retinal and choroidal structures. Between the retina 28 and the choroid 30 there is an outer segment of photoreceptor cells 32 including rods and cones, a subretinal space 34, and a layer of retinal pigment epithelium (RPE) 36. In a normal adult, retinal blood vessels 38, including capillaries, have walls or membranes 40 that contain no fenestrations or openings. In a normal adult, the large choroidal vessels 42 similarly have walls 44 that contain no fenestrations but the choriocapillaries 46 have walls that contain fenestrations 48. In an adult with AMD, there is either growth of new subretinal blood vessels whose walls or membranes are altered in that they also contain fenestrations, or the RPE cells are lost.

[00102] In exudative AMD, a lesion of subretinal neovascular tissue 52 develops in the choroid 30. The neovascular tissue 52 penetrates the RPE 36 and subretinal space 34, and extends into the area containing photoreceptor cells 32. The neovascular tissue 52 has membranes or walls 54 that are altered in having tertgΛatiαrt&/5©;.wMϋh permit fluid leakage into spaces surrounding photoreceptor cells 32, the subretinal space 34, and the RPE 36.

[00103] One type of AMD results in proliferation of new blood vessels in the subretinal area, typically the choroid. In the normal retina, both the large blood vessels and the capillaries have intact vessel walls. In the normal choroid, the large vessels have intact vessel walls, but the capillaries have fenestrations or openings in their walls. In patients with AMD, new blood vessels proliferate from the choriocapillaries through defects in Bruch's membrane beneath or on top of retinal pigment epithelium (RPE), and form vascular membranes. The resulting choroidal neovascularizations (new vessels in the choroid) occur in about 8-10% of all patients with AMD, and are also seen in patients with pathologic myopia and presumed ocular histoplasmosis syndrome, as well as other idiopathic conditions.

[00104] While the presence of the new vessels themselves is not problematic, any endogenous or exogenous fluid contained in these vessels (for example, blood, serous fluid, solubilized drug, etc.) will leak outside of the vessels and accumulate in the surrounding spaces. This accumulation of fluid can result in serous and hemorrhagic detachment of the RPE and neurosensory retina, and can lead to scarring in this area (fibrous deform scarring), resulting in decreased vision or even loss of vision. Thus, it is the fluid leakage from these new vessels in this type of AMD, called neovascular, exudative, or occult AMD that is the cause of the resulting visual, impairment. Therapies to prevent this form of AMD are directed to slowing or stopping the formation or proliferation of new vessels in the choroid 30. Therapies to treat neovascular AMD are directed to at least partially damaging or destroy existing neovascular tissue 52, and/or interfering with its function. In either case, leakage of fluid from the new vessels is decreased, and the concomitant scarring and loss of vision is likewise diminished or eliminated. Another type of AMD occurs less commonly and is due to dead RPE cells; this is termed atrophic AMD. In either type of AMD, without treatment, many of the affected individuals will become legally blind.

[00105] Patients with early stage AMD can be diagnosed in an examination by the presence of drusen, an accumulation of dead outer segments of photoreceptor cells, under the RPE. Hyaline excrescences that are located in Bruch's membrane (lamina basalis choroidea) also form. The presence of large, soft drusen in the eye indicates a pre-stage of exudative AMD, and places patients at higher-than-average risk for ^μieVfeF(3'ping|*π§c)V'a"Scii-l^'larizations, especially if one eye is already affected. To date, there are no known specific measures to prevent the occurrence of AMD. However, an anti-VEGF agent may have efficacy at an early stage of AMD (drusen), reducing or preventing its progression to full-fledged disease. Laser coagulation therapy results in drusen disappearance or reduction, but causes formation of scar tissue. The use of sunitinib maleate in the inventive method may preclude the need for such therapy and thus alleviate this problem. The use of sunitinib maleate in combination with PDT or

! laser coagulation may address this problem by ameliorating both AMD and resulting scar tissue.

[00106] For patients already diagnosed with AMD in one or both eyes, treatment involves targeting light (phototherapy) to the macular area to inhibit or impair the nascent defective blood vessels. For example, PDT uses a photosensitizing agent to locally and selectively destroy cells and/or tissues. The agent is administered into the vessels of a patient and transported to the retina. Immediately thereafter, or after an appropriate interval, the appropriate wavelength of light is directed to this specific area to activate the agent. Targeting low energy light to the area selectively activates the agent. The activated agent generates free radicals (e.g., singlet oxygen, hydroxyl radicals, other activated chemical species) that destabilize and destroy the new vessels in this area. For example, it damages the walls of the choriocapillaries and neovascular tissue, and leads to an initial vascular thrombus that may occlude the vessels. ^{. . .}

[00107] PDT is generally directed to the lesion, but may also be administered to a generally circular area surrounding the lesion, up to about five disk diameters from the lesion. In one embodiment, PDT is administered to the lesion and an area about three to about five disk diameters from the lesion. In another embodiment, PDT is administered to the lesion and an area about one-half to about one disk diameter from the lesion. In still another embodiment, PDT is administered to the lesion. The size of the applied laser treatments may be in the range of about 1 mm to about 9 mm.

[00108] Selection of a photosensitive agent depends on the site(s) of tissue distribution requiring treatment, the mechanisms of action of the agents themselves, and their specific optimal absorption wavelengths. For example, tin ethyl etiopurpurin (SnET2) is frequently used as a photosensitive agent. SnET2 has lower persistence and severity of skin photosensitivity, it absorbs at longer wavelengths yielding better #tls^UetpehefraMhrft^!!;h^'as a higher extinction coefficient resulting in increased potency and efficiency, ease of synthesis, and ability to be produced in a highly pure form. Protoporphyrin may be used as a photosensitizing agent. Protoporphyrin IX is a photoactive compound that is endogenously formed from 5-aminolevulinic acid (ALA) in the biosynthetic pathway of heme. ALA may be applied topically and is metabolized to protoporphyrin, the active photosensitizing agent. Laser irradiation is usually at a wavelength in the range of about 630 nm, or alternatively in the range of 670 nm. ALA may be administered orally in a bolus as an aqueous solution at a concentration of about 60 mg/kg body weight, or intravenously at a concentration of 30 mg/kg body weight. Other photosensitizing agents that may be used include, but are not limited to, benzoporphyrin derivative monoacid tube A (BPD-MA) and mono-l-aspartyl chlorine 6 (NPe6), with absorbance maxima in the range of about 660-690 nm, ATX- 106, and indocyanine green (ICG). Verteporfin, a synthetic, chlorin-like porphyrin, may be intravenously injected at a dose of about 1-2 mg/kg, and activated by light at 50 J/cm2 (absorbance peak of drug) from a non-thermal laser (for example, a diode laser) set at an intensity of 600 mW/cm² and a wavelength of 689 nm. Once activated, it generates singlet oxygen and other reactive oxygen radicals that selectively damage neovascular endothelial cells, and cause thrombus formation due to specific choroidal neovascular occlusion.

[00109] PDT has been reported to be of some benefit to patients having AMD. For example, one study (Arch. Ophthalmol. 17:1329-1345, 1999) evaluated PDT in four^" hundred and two eyes from patients diagnosed with AMD in at least one eye. Treatment outcome was assessed by comparing the patient's ability to accurately read a conventional vision chart (one having about five letters per line) pre-treatment and post-treatment. At twelve months post-PDT, 61 % of the eyes (246/402) lost fewer than 15 letters (that is, the patient lost less than about three lines on a standard visual chart), while 46% of the eyes (96/207) from patients undergoing treatment with a placebo lost fewer than 15 letters (p < 0.001 ). At twenty-four months post-PDT, the visual acuity and contrast sensitivity was sustained in patients receiving PDT. A significantly greater percentage of these patients (58%) lost fewer than 15 letters, compared to patients undergoing treatment with a placebo (38%). However, only 16% of the patients receiving PDT had improved vision, compared to 7% of the patients receiving a placebo. J[OMM]J /WiilS^' EtS ϋi: used to treat patients with AMD, it has some drawbacks. One problem with PDT is that its effects are transient; patients receiving PDT must be retreated about every three months. Furthermore, the patients require at least five retreatments within the first two years merely to stabilize their condition, and before any therapeutic effect occurs. Additionally, these cumulative treatments damage the retina, further reducing the patient's visual acuity.

[00111] Ranibizumab has been administered by intravitreal injection in combination with verteporfin PDT to determine its effect on choroidal neovascularization. The combination caused a greater reduction in angiographic leakage than PDT only, as reported by Husain et a!., Apr. 2005 Arch Opthamol. 123:309, which is expressly incorporated by reference herein in its entirety. As previously described, ranibizumab is a derivative of the full-length antibody bevacizumab (Fab fragment), and is further modified to increase its affinity for VEGF.

[00112] In one embodiment, the method prevents, alleviates, or delays the onset of AMD in a patient by administering PDT simultaneously or concomitantly with sunitinib maleate. The method also prevents or delays the progression of AMD, and reduces further loss of vision in a patient having AMD, by administering PDT simultaneously or concomitantly with sunitinib maleate. The combination of PDT with sunitinib maleate enhances alleviation of AMD and/or its symptoms or sequelae. One skilled in the art will appreciate that enhanced alleviation encompasses any reduction in the duration, severity, type, etc. of the underlying pathology and/or its symptoms, and is not limited to complete efficacy, although therapeutic efficacy is included. The method thus encompasses preventing or delaying the onset of AMD, and/or maintaining visual acuity and preventing further loss of vision in patients with AMD. The method may generate free radicals and other activated chemical species that destabilize and destroy the new vessels via PDT, and reduce the formation of new blood vessels via anti-VEGF action of sunitinib maleate.

[00113] In addition to AMD, other ocular conditions may be treated with the inventive method and device. These conditions include, but are not limited to, blepharitis, conjunctivitis, keratitis, episcleritis, scleritis, papillitis (optic neuritis), uveitis, and/or endophthalmitis. The inventive method and device also may be used to reduce postsurgical inflammation. ^^uv-w#j- ' iHs--.fep3oH:eiGi-that another anti-VEGF agent, bevacizumab, at a dose of 1 mg was administered as a single intravitreal injection to a patient with neovascular age- related macular degeneration. Rosenfeld et al. Ophthalmic Surg Lasers Imaging 2005; 36:331 , which is expressly incorporated by reference herein in its entirety. There was resolution of subretinal fluid after one week, with improved macular appearance maintained for at least four weeks, and no observed inflammation.

[00115] In one embodiment of the method, sunitinib maleate is administered without PDT or any other type of phototherapy. Thus sunitinib maleate alone, or in combination with another agent, provides therapy without phototreatment. For example, while the inventive method may be used in conjunction with other therapies such as thermal laser coagulation, it may also be used without PDT, laser treatment, etc. but may include the addition of anti-proliferative agents, steroids, etc. administered with the inventive device or separately (e.g., subconjunctival depot steroid therapy), as known to one skilled in the art.

[00116] In another embodiment of the method, both PDT and sunitinib maleate are administered, but their administration is not restricted to a particular sequence. In one embodiment, PDT is administered and, essentially simultaneously with or immediately thereafter, sunitinib maleate is administered. In another embodiment, PDT is administered and sunitinib maleate is administered in the same treatment session, within a time frame of a few minutes or within a few hours. In another embodiment, PDT is administered and sunitinib maleate is administered after an interval from about one day up to about 90 days. In another embodiment, sunitinib maleate is administered and, essentially simultaneously with or immediately thereafter, PDT is administered. In another embodiment, sunitinib maleate is administered and PDT is administered in the same treatment session, within a time frame of a few minutes or within a few hours. In another embodiment, sunitinib maleate is administered and PDT is administered after an interval from about one day up to about 90 days. For embodiments in which PDT and sunitinib maleate are not administered essentially simultaneously, either may be administered first.

[00117] In one embodiment, after administering the photosensitive agent (verteporfin, protoporphyrin, SnET2, NPe6, ATX-106, ICG, etc.), the patient is treated using a laser to administer low energy levels of light at a wavelength appropriate to activate the photosensitive agent. Treatment with sunitinib maleate is then initiated ^•-essentially'¹- -¹SMyWeOUsIy or concomitantly. Essentially simultaneous treatment includes administration of both sunitinib maleate and low energy light within the same treatment session. Concomitant treatment includes administration either immediately thereafter or within a few hours, within 24 hours, or after an interval from about one day to ninety days.

[00118] In another embodiment, the patient is treated with sunitinib maleate, and is thereafter treated with PDT. The photosensitive agent may be administered either before or after sunitinib maleate treatment, depending upon a variety of factors such as the specific photosensitive agent used, the specific treatment protocol, etc. PDT is then essentially simultaneously or concomitantly initiated, as previously described. Factors such as patient comfort, tolerance to treatment, and convenience may be considered in selecting the appropriate treatment regime.

[00119] The combination of PDT and sunitinib maleate may provide synergistic benefits. One benefit is that the combined therapies induce regression of neovascular tissue. Besides patients with AMD, patients with diabetes who are particularly prone to proliferative retinopathy, a frequent cause of blindness could benefit from this treatment. Another benefit is that the combined therapies do not produce additional neovascular tissue because of anti-angiogenic function. The combination of the two treatments thereby reduces the need for repetitive PDT treatments that damage the retina and further reduce the patient's visual acuity.

Non-limiting illustrations of Embodiments of the invention

[00120] Further features of the present invention are more fully described in the following description. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad description of the invention as set out above.

EXAMPLE 1

[00121] Sixteen Male Long Evans pigmented rats (200 g to 250 g) were administered general anaesthesia (94.7 mg/kg ketamine hydrochloride/xylazine i.p.) supplemented by topical anaesthesia (0.5% proparacaine hydrochloride). One cornea of each animal was cauterized by pressing an applicator stick (1.8 mm diameter) coated with 75% silver nitrate/25% potassium nitrate (Arzol Chemical Co., Keen NH) to the central Cornea¹ TOΓ røn-seconαs under the operating microscope. Excess silver nitrate was removed by rinsing the eyes with balanced salt solution (5 ml) and gentle blotting with tissue paper. To increase the reproducibility of the injuries, a single investigator cauterized all animals.

[00122] Animals were randomized to one of two groups: group 1 (n=10) received topical 4 mg/ml bevacizumab, and group 2 (n=6) received saline. Both treatments were topically administered two times per day for seven days, and began immediately after cauterization. Corneas from anesthetized animals were evaluated by slit-lamp biomicroscopy on the third and sixth day. Corneal photographs were taken with x25 magnification using a camera attached to the slit-lamp microscope (Topcon SL-7E, Tokyo Japan) on the seventh day. Neovascularization in each cornea was evaluated by an examiner blinded as to the treatment groups. For each eye, the extent of burn stimulus response was scored as follows: 0 (no blister, not raised above corneal surface), +1 (small blister, raised slightly above the surface), +2 (medium blister, raised moderately above the surface), +3 (large blister). Only corneas with a burn stimulus score of +2 or higher were included for the calculation of the mean burn stimulus and neovascularization scores in each group. All photographs were converted to high-resolution digital forms by scanner (Cano scan 9900F, Canon, Tokyo Japan). The corneal surface covered with neovascular vessels was measured on the photographs as the percentage of the total area of the cornea. Image analysis was performed on each cornea using an image processing and analysis software program (Image J 1.31v. - Wayne Rasband at the Research Services Branch, National Institute of Mental Health, Bethesda MD). The area of neovascularization was measured in terms of pixels and its ratio to the entire corneal area was determined as the percentage of corneal neovascularization. A drawing of corneal blood vessels was made by one investigator to compare with digital photos and to ensure that no vascular area was missed during calculation of percent area. After scoring the burn stimulus and the percentage of neovascularization for both groups, the animals were sacrificed on the seventh day.

[00123] Following sedation (previously described), enucleation was performed before the animals were euthanized. Immediately after enucleation, the globes were penetrated with a 27-gauge needle, 1.0 mm from the limbus at the 3 and 9 o'clock meridians which allowed the fixative to rapidly fill the eyes. The eyes were prepared fot^u"hiSto^'logriC^l ex'&ffii'fration using 10% formaldehyde. After fixation for twenty-four hours, the eyes were removed from the fixative. Corneas were dehydrated, sectioned, soaked in xylene and paraffin, embedded in paraffin, and cut at 1 μm for staining with hematoxylin and eosin (H&E) for light microscopy.

[00124] Light microscopic examination was performed on every microscopic section. Sections were examined by dividing the corneas into two halves through the centre of the lesion and were evaluated with regard to the intensity of new vessels, polymorphonuclear (PMN) leucocytes, edema, and fibroblastic activity.

[00125] The Mann-Whitney U test was used for comparisons. Statistical significance was defined as a probability (p) of less than 0.05 of the result being due to chance alone.

[00126] The burn stimulus score was +2 or higher in all eyes. The mean burn stimulus scores were not statistically different between the treatment and the placebo groups (P>0.05, Mann-Whitney U test).

[00127] FIG. 3 shows normalized areas of corneal neovascularization in bevacizumab-treated eyes (n=10) and control eyes (n=6). FIG. 4 is a photograph of a representative bevacizumab-treated eye, and FIG. 5 is a photograph of a representative control eye. The difference was statistically significant (p<0.02, Mann Whitney test). As seen in FIG. 3, bevacizumab-treated eyes had - less corneal neovascularization than control eyes. In bevacizumab-treated eyes, corneal neovascularization covered, on average, 38.2 ± 15.5% (mean ± standard deviation (SD) of the corneal surface. In control eyes, corneal neovascularization covered, on average, 63.5 ± 5.0% (mean ± SD) of the corneal surface (p<0.02, Mann-Whitney test). Topically administered bevacizumab at 4 mg/ml decreased corneal neovascularization by 40%.

[00128] Light microscopy evaluation of the histological preparations was consistent with the slit lamp evaluation. The bevacizumab treated group had less neovascularization and inflammation when compared to the control eyes (FIGS. 6 and 7). However they still showed peripheral vascularization of the cornea. S at U ¹BEXAMPLE-Z ffil

[00129] Animals are treated and prepared as in Example 1 , except that bevacizumab is administered by intravitreal injection using a 30 g needle. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 3

[00130] Animals are treated and prepared as in Example 1 , except that bevacizumab is administered by subconjunctival injection using a 30 g needle. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 4

[00131] Animals are treated and prepared as in Example 1 , except that bevacizumab is administered by subretinal injection using a 30 g needle. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 5

[00132] Animals are treated and prepared as in Example 1 , except that bevacizumab is administered by retrobulbar injection using a 30 g needle. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 6

[00133] Animals are treated and prepared as in Example 1, except that bevacizumab is replaced by ranibizumab. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 7

[00134] Animals are treated and prepared as in Example 2, except that bevacizumab is replaced by ranibizumab. Corneal neovascularization in treated eyes is reduced over untreated eyes.

[00135] Animals are treated and prepared as in Example 3, except that bevacizumab is replaced by ranibizumab. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 9

[00136] Animals are treated and prepared as in Example 4, except that bevacizumab is replaced by ranibizumab. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 10

[00137] Animals are treated and prepared as in Example 5, except that bevacizumab is replaced by ranibizumab. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 11

[00138] Animals are treated and prepared as in Example 1 , except that bevacizumab is replaced by pegaptanib. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 12

[00139] Animals are treated and prepared as in Example 2, except that bevacizumab is replaced by pegaptanib. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 13

[00140] Animals are treated and prepared as in Example 3, except that bevacizumab is replaced by pegaptanib. Corneal neovascularization in treated eyes is reduced over untreated eyes. EXAMPBE¹W

[00141] Animals are treated and prepared as in Example 4, except that bevacizumab is replaced by pegaptanib. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 15

[00142] Animals are treated and prepared as in Example 5, except that bevacizumab is replaced by pegaptanib. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 16

[00143] Animals are treated and prepared as in Example 1 , except that bevacizumab is replaced by sunitinib maleate. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 17

[00144] Animals are treated and prepared as in Example 2, except that bevacizumab is replaced by sunitinib maleate. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 18

[00145] Animals are treated and prepared as in Example 3, except that bevacizumab is replaced by sunitinib maleate. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 19

[00146] Animals are treated and prepared as in Example 4, except that bevacizumab is replaced by sunitinib maleate. Corneal neovascularization in treated eyes is reduced over untreated eyes. EXAiIPLE 20

[00147] Animals are treated and prepared as in Example 5, except that bevacizumab is replaced by sunitinib maleate. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 21

[00148] Animals are treated and prepared as in any of Example 1-20, except that the agent is administered using an intraocular device. Corneal neovascularization in treated eyes is reduced over untreated eyes.

EXAMPLE 22

[00149] This example describes in vitro effects of anti-inflammatory steroids and Avastin on inflammation.

[00150] In brief, VEGF R1 (flt-1 ) expression on the surface of choroidal endothelial cells (CECs) was assessed by flow cytometry in response to stimulation. The stimulants selected to induce inflammation were PMA and/or VEGF. The corticosteroids deoxycorticosterone acetate (DOCA), fludrocortisone acetate (FCA) and triamcinolone acetonide (TA) were used to treat stimulated cells at dosages shown previously to reduce VEGF R1 expression in response to PMA stimulation. The corticosteroids were used with or without 0.8 mg/ml Avastin (Spitzer efal., 2006).

[00151] Stimulation of the CECs with 1 μM PMA significantly increased VEGF R1 expression (p≤O.0001). Treatment of PMA stimulated CECs with DOCA (10 μM), FCA (10 μM) or TA (50 μM) statistically significantly inhibited VEGF R1 expression (reductions of 11.3%, 17.4% and 16.8% respectively: p-values = 0.0052, <0.0001 , <0.0001 respectively) (FlG. 8).

[00152] Addition of Avastin statistically significantly enhanced the reductions in VEGF R1 produced by DOCA (p=0.0016), FCA (p=0.0398) or TA (p≤0.0001) (FIG. 8). Treatment of PMA stimulated CECs with Avastin alone also significantly reduced VEGF R1 expression (9.3% reduction, p = 0.0198). Avastin plus DOCA (24.2% reduction) and Avastin plus FCA (25.5% reduction) produced a noticeable reduction in the individual treatments. However the magnitude of the reduction in VEGF R1 in HeδjiόήiSef^'td^ΦΑ-pltiS^vastin (39.9% reduction) was greater than the effects of either agent alone (FIG. 8).

[00153] When CECs were stimulated with VEGF, neither Avastin nor FCA alone significantly reduced VEGF R1 expression. However, when both Avastin and FCA were added to the cultures in combination there was a small but statistically significant reduction in VEGF R1 expression (p = 0.0108). This was the only significant reduction in VEGF R1 observed in response to VEGF stimulation (results not shown).

[00154] It can be concluded that bevacizumab (Avastin) significantly enhanced the effects of the corticosteroids tested. The effects of TA plus Avastin appeared to be synergistic, while the effects of DOCA plus Avastin and of FCA plus Avastin were not as pronounced. Such combinations may affect both the steroid dependent and independent pathways involved in inflammation and oedema.

[00155] The total abrogation of the effects of the glucocorticoid TA on VEGF R1 expression by PMA stimulated CECs by the mineralocorticoid receptor antagonist spironolactone, was initially surprising. However, spironolactone has been shown to display some antagonistic activity towards the glucocorticoid receptor (Williams et a/., 2006). The not quite statistically significant abrogation by spironolactone of the reduction in VEGF R1 by FCA indicates that FCA may be operating by both mineralocorticoid receptor dependent and independent pathways. However the strong lack of a significant difference . between FCA plus spironolactone treated PMA stimulated cells and PMA stimulated untreated cells support a mineralocorticoid receptor dependent mechanism for FCA.

[00156] The lack of a reduction in VEGF R1 expression following DOCA treatment of PMA stimulated CECs is indicative of the inter-experiment variability with this particular mineralocorticoid that has been observed throughout these series of experiments. The reduction in VEGF R1 seen with the addition of spironolactone may be due to significant mineralocorticoid receptor agonist activity that can be seen under certain experimental conditions and in a cell type specific manner with this agent (Williams et al., 2006; Massaad et al., 1997).

[00157] It should be understood that the embodiments of the present invention shown and described in the specification are only preferred embodiments of the

cΛam^i^, m~ inventive method may be used to treat cerebral edema associated with meningitis by intravenously administering bevacizumab. Therefore, various changes, modifications or alterations to these embodiments may be made or resorted to without departing from the spirit of the invention and the scope of the following claims.

[00158] Modifications of the above-described modes of carrying out the various embodiments of this invention will be apparent to those skilled in the art based on the above teachings related to the disclosed invention. The above embodiments of the invention are merely exemplary and should not be construed to be in any way limiting.

Claims

We ClaimT "

1. A method of treating or preventing, in a patient, an inflammatory ailment, the method comprising the step of: providing to the patient a formulation comprising a therapeutically effective amount of an anti-vascular endothelial growth factor (VEGF) agent.

2. A therapeutic method comprising the step of: providing to at least one inflammatory tissue in a patient a formulation comprising a therapeutically effective amount of an anti-VEGF agent, wherein the method ameliorates inflammation in the absence of angiogenesis.

3. An ocular prophylaxis or treatment method comprising the step of: ocularly providing to a patient having or at risk for developing an ocular inflammatory ailment a formulation comprising an anti-VEGF agent under conditions sufficient to ameliorate a cause and/or effect of fluid leakage in the eye.

4. A method according to any one of claims 1 to 3 wherein the anti-vascular endothelial growth factor agent is selected from the group comprising bevacizumab, ranibizumab, pegaptanib, anti-VEGF siRNA, TNP470, integrin av antagonists, 2-methoxyestradiol, paclitaxel, P38 mitogen activated protein kinase inhibitors, or sunitinib maleate.

5. The method of claim 2 wherein the inflammation in the absence of angiogenesis is from at least one of surgery, inflammatory diseases of the central nervous system, conditions resulting in cerebral edema, macular edema, or inflammatory diseases of the eye.

6. The method of claim 4 wherein the inflammation is a result of at least one of an immune disease, a microbial infection, trauma, ischemic diseases, diabetes, age related macular degeneration, retinitis pigmentosa, allergy, or a degenerative diseases.

7. The method of claim 4 wherein the patient has at least one of synovitis, uveitis, iritis, retinal vasculitis, optic nerve neuritis, papillitis, or diabetic retinopathy.

W

4 wherein the anti-VEGF agent ameliorates at least one of scars or adhesions.

9. The method of claim 4 wherein the anti-VEGF agent is administered is by a route selected from at least one of enteral, parenteral, ocular, topical, intrathecal, inhalation, or instillation.

10. The method of claim 4 wherein the dose of anti-VEGF agent is less than about 5 mg/0.1 ml.

11. The method of claim 4 wherein the dose of anti-VEGF agent ranges from 0.1 mg/ml to about 50 mg/ml.

12. The method of claim 4 wherein the anti-VEGF agent is administered systemically at a dose from about 0.05 mg/ml to about 5 mg/ml.

13. The method of claim 4 wherein the anti-VEGF agent is administered intraocularly at a dose from about 0.005 mg/ 0.1 ml to about 5 mg/ 0.1 ml.

14. The method of claim 4 wherein the anti-VEGF agent is administered topically to the eye at a dose up to about 5 mg/ml.

15. The method of claim 4 wherein the dose of the anti-VEGF agent ranges from about 0.01 mg/0.1 ml to about 5 mg/0.1 ml.

16. The method of claim 4 wherein the anti-VEGF agent is formulated in at least one of microspheres, nanospheres, microcapsules, or nanocapsules.

17. The method of claim 4 wherein the anti-VEGF agent is a controlled release formulation.

18. A method to ameliorate corneal neovascularization comprising ocularly administering an anti-vascular endothelial growth factor agent at a concentration ranging between about 0.01 mg/0.1 ml to about 5 mg/0.1 ml for a duration sufficient to ameliorate neovascularization.

19. The method of claim 18 wherein the agent is at least one of bevacizumab, ranibizumab, pegaptanib, sunitinib maleate, anti-VEGF siRNA, TNP470, ^'"Iri^'tdgrϊh "av antagonists, 2-methoxyestradiol, paclitaxel, or P38 mitogen activated protein kinase inhibitors.

20. The method of claim 18 wherein ocular administration is selected from topical, intraocular injection, or intraocular implantation.

21. A method to ameliorate corneal neovascularization comprising topically administering to an eye of a patient in need thereof a biocompatible formulation comprising bevacizumab at a concentration up to about 5 mg/0.1 ml for a duration sufficient to ameliorate neovascularization.

22. The method of claim 3 wherein the formulation also includes an antiinflammatory agent.

23. The method according to claim 22 wherein the anti-VEGF agent and the anti-inflammatory agent are present in an amount sufficient to reduce angiogenesis.

24. The method of claim 23 wherein the anti-VEGF agent is bevacizumab.

25. The method of claim 24 wherein the anti-inflammatory agent is a steroid

26. The method of claim 25 wherein the steroid is triamcinolone or a salt thereof.

27. The method of claim 22 further comprising providing another anti-VEGF compound, an anti-PDGF compound, an anti-leukothene, or combinations thereof.

28. The method of claim 24 wherein bevacizumab is at a dose up to about 5 mg/0.1 ml.

29. The method of claim 24 where bevacizumab is administered topically to an eye at a concentration up to about 5 mg/ml.

30. The method of claim 24 where bevacizumab is administered systemically at a concentration ranging from about 0.05 mg/ml to about 5 mg/ml.

31. The method of claim 24 where bevacizumab is administered intraocularly at a concentration ranging from about 0.05 mg/ml to about 5 mg/ml.

32."^""The^'lln1etRo^'criSf claim 24 wherein the anti-inflammatory agent is selected from at least one of colchicine, a steroid, a non-steroidal anti-inflammatory drug (NSAID), an anti-prostaglandin, an antihistamine, a matrix metalloproteinase inhibitor, or a macrolide.

33. The method of claim 24 ameliorating the inflammatory process in at least one of a brain, an eye, a joint, or a muscle.

34. A method for ameliorating an ocular inflammatory process in a patient comprising providing to the patient a therapeutic concentration of at least one anti-inflammatory agent and bevacizumab at a concentration sufficient to reduce ocular angiogenesis.

35. The method of claim 34 wherein bevacizumab is administered topically, systemically, or by intraocular injection.

36. A formulation comprising about 0.1 mg/ml to about 50 mg/ml bevacizumab and about 0.05 mg/ml to about 100 mg/ml of at least one anti-inflammatory agent in a biocompatible formulation.

37. The formulation of claim 36 further comprising at least one of an anti-PDGF compound, an anti-histamine, an anti-VEGF compound, or an anti- leukotriene.

38. The formulation of claim 36 wherein the anti-inflammatory agent is at least one of a steroid, a non-steroidal anti-inflammatory agent, colchicine, an antiprostaglandin, or a matrix metalloproteinase inhibitor.

39. An adjuvant to ocular steroid therapy comprising about 0.05 mg/ml to about 50 mg/ml bevacizumab and optionally one or a combination of an anti-PDGF compound and/or an anti-leukotriene in a biocompatible formulation for ocular administration.

40. An ocular prophylaxis or treatment method comprising ocularly providing to a patient having or at risk for developing an ocular disease of fluid leakage from new ocular blood vessels to a surrounding area, a formulation comprising sunitinib maleate under conditions sufficient to ameliorate a cause and/or effect of fluid leakage in the eye.

"41-M»Αfϊ ø&uiaFprbfiitøylaxis or treatment method comprising ocularly providing to a patient having or at risk for developing an ocular disease of fluid leakage from new ocular blood vessels to a surrounding area, a biocompatible formulation comprising sunitinib maleate under conditions sufficient to ameliorate a cause and/or effect of fluid leakage in the eye.

42. The method of claim 40 or claim 41 wherein sunitinib maleate is administered orally at a dose ranging between about 12.5 mg/day to about 50 mg/day.

43. The method of claim 40 or claim 41 wherein sunitinib maleate is administered topically at a dose ranging between about 10 ng/ml to about 100 ng/ml.

44. The method of claim 40 or claim 41 wherein sunitinib maleate is administered intraocularly at a dose between about 7 ng/ml to about 20 μg/ml.

45. The method of claim 40 or claim 41 wherein sunitinib maleate is controllably administered by an ocular device to release a dose of about 10 ng/day to about 50 ng/day into the eye.

46. The method of claim 40 or claim 41 additionally providing an effective amount of a photosensitive agent to the new ocular vessels and activating the agent in the vessels with a low energy light sufficient to damage the vessels.

47. The method of claim 40 or claim 41 wherein sunitinib maleate is provided prior to the photosensitive agent.

48. An ocular prophylaxis or treatment method comprising providing to a patient having or at risk for developing an ocular disease of fluid leakage from new ocular blood vessels to a surrounding area, a formulation comprising sunitinib maleate and a photosensitizing agent for photodynamic therapy.

- "THW-fhitHocf-dF-claim 48- further comprising performing ocular photodynamic therapy.

50. An ocular prophylaxis or treatment method comprising a. providing to a patient having or at risk for developing an ocular disease of fluid leakage from new ocular blood vessels to a surrounding area, a formulation comprising sunitinib maleate, and b. providing ocular laser coagulation therapy.

51. A method according to any one of claims 1 , 2, 3, 18, 21 , 34, 40, 41 , 48, or 50 substantially as herein described.

52. A formulation according to claim 36 substantially as herein described.

53. An adjuvant according claim 39 substantially as herein described.