COPYRIGHT NOTIFICATION

[0001] Unless defined Pursuant to 37 C.F.R. § 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application Number 61/283,148 (filed November 30, 2009). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.

STATEMENT CONCERNING GOVERNMENT SUPPORT

[0003] This invention was made with government support under Contract Nos. EY01 1254 and EY014174 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0004] The present invention generally relates to methods and compositions for treating ocular vascular disorders and ocular degenerative diseases.

BACKGROUND OF THE INVENTION

[0005] Retina vascular diseases such as age related macular degeneration (ARMD) and diabetic retinopathy (DR) are due to abnormal choroidal or retinal neovascularization respectively. They are the leading causes of visual loss in industrialized nations. Since the retina consists of well-defined layers of neuronal, glial, and vascular elements, relatively small disturbances such as those seen in vascular proliferation or edema can lead to significant loss of visual function. Inherited retinal degenerations, such as Retinitis Pigmentosa (RP), are also associated with vascular abnormalities, such as arteriolar narrowing and vascular atrophy. They affect as many as 1 in 3500 individuals and are characterized by progressive night blindness, visual field loss, optic nerve atrophy, arteriolar attenuation, and central loss of vision often progressing to complete blindness. While significant progress has been made in identifying factors that promote and inhibit angiogenesis, there are still no effective treatments to slow or reverse the progression of these retinal degenerative diseases.

[0006] Ischemic retinopathies are characterized by loss or dysfunction of the retinal vasculature which results in a reduction of blood flow and hypoxia. The retina responds to hypoxia by generating signals to grow new blood vessels, but these new vessels are usually fragile and disorganized. It is the growth of these abnormal new vessels that creates most of the threat to vision since they can leak, hemorrhage or lead to scarring that may end in retinal detachment. Current treatments for ischemic retinopathies seek to halt the growth of the pathological vessels but do not address the underlying ischemia that drives their growth. Furthermore, standard treatment for diabetic retinopathy, an ischemic retinopathy that affects millions, involves destruction of a portion of the retina with a laser in an attempt to stop new vessel growth and preserve central vision. Most recently, strategies have been employed to block the function of vascular endothelial growth factor (VEGF), a major promoter of vessel growth. In the short term, anti-VEGF therapy can improve vision, but it doesn't address the underlying ischemia and in fact may exacerbate this condition as it inhibits all vessel growth, including beneficial collaterals. There is also the serious concern of systemic exposure of these drugs in elderly and/or diabetic patients where new vessel growth may be required in ischemic brains, hearts or limbs.

[0007] There is a need in the art for better means for treating and preventing various ocular vascular diseases such as ischemic retinopathies. The present invention addresses this and other unfulfilled needs in the art.

SUMMARY OF THE INVENTION

[0008] In one aspect, the present invention provides methods for promoting normalization and repair of retinal vasculature in a subject. The methods entail administering to a subject suffering from an ocular vascular disorder a pharmaceutical composition comprising a therapeutically effective amount of a monocyte chemoattractant compound. Subjects suitable for treatment with the methods include one with an ocular vascular disorder such as ischemic retinopathy, diabetic retinopathy, retinopathy of prematurity, neo vascular glaucoma, central retinal vein occlusions, retina edema, macular degeneration or retinitis pigmentosa. Some methods of the invention are specifically directed to treating subjects afflicted with ischemic retinopathy.

[0009] In some of the methods, the employed chemoattractant compound is a chemokine. In some preferred embodiments, the chemoattractant compound is MCP-1, CX3CL1, CXCL13 or MIP-Ια. In some other embodiments of the invention, the employed monocyte chemoattractant compound is a peptide, a peptide mimetic, an antibody or a small organic compound. In still some other embodiments, the employed chemoattractant compound is an agent that specifically upregulates expression of a known monocyte chemoattractant compound such as MCP-1, CX3CL1, CXCL13 or MIP-Ια. For example, MCP-1 expression can be upregulated by agonists of the PPAR family of receptors. In some of the methods, the pharmaceutical composition is administered to the subject via intravitreal injection. In some preferred embodiments, the subject to be treated is a human.

[0010] In a related aspect, the invention provides methods for treating or preventing an ocular vascular disorder in a subject. The methods involve administering to a subject suffering from the ocular vascular disorder a pharmaceutical composition comprising an effective amount of a monocyte chemoattractant compound. The methods are suitable for therapeutic or prophylactic treatment of a subject that has or is at risk of developing an ocular vascular disorder such as ischemic retinopathy, diabetic retinopathy, retinopathy of prematurity, neovascular glaucoma, central retinal vein occlusions, retina edema, macular degeneration or retinitis pigmentosa. In some preferred embodiments, the ocular vascular disorder to be treated is ischemic retinopathy. Some of these methods employ a

chemoattractant compound that is a chemokine. For example, monocyte chemoattractant cytokines such as MCP-1, CXCL13 and MIP-Ια can be readily employed in the methods. Some other embodiments of the invention employ a monocyte chemoattractant compound that is a peptide, a peptide mimetic, an antibody or a small organic compound. The employed chemoattractant compound can also be an agent that specifically upregulates expression of a known monocyte chemoattractant compound such as MCP-1, e.g., an agonist of a receptor of the PPAR family of receptors. In some preferred embodiments, the pharmaceutical composition comprising the chemoattractant compound is administered to the subject via intravitreal injection.

[0011] The invention further provides a use of a monocyte chemoattractant compound in the manufacture of a medicament for treating or preventing an ocular vascular disorder (e.g., ischemic retinopathy) in a subject. In some of these embodiments, the employed monocyte chemoattractant compound is a chemokine such as MCP-1, CXCL13 or MlP-la. In some other embodiments, the employed chemoattractant compound is a peptide, a peptide mimetic, an antibody or a small organic compound. In still some other embodiments, the employed chemoattractant compound is an agent that specifically upregulates expression of a known monocyte chemoattractant compound such as MCP-1, e.g., an agonist of a receptor of the PPAR family of receptors. In some preferred embodiments, the medicament is for intravitreal injection. In some related embodiments, the invention provides a kit that contains a monocyte chemoattractant compound and an instruction for intravitreal injection of the compound to a subject suffering from an ocular vascular disorder.

[0012] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.

DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 shows that isolectin-stained retina whole mounts from the OIR model show massive increase in isolectin+ cells in right eyes the received MCP-1.

[0014] Figures 2A-2C show a significant increase of isolectin-labeled macrophages after injection of MCP-1 as measured by computer-aided quantification (top panel); representative images of quantified retinas (middle panel); and correlation between MCP-1 injection and enhanced vascular repair in the mouse OIR model.

[0015] Figure 3 shows in vivo observation of MCP-1 mediated cell recruitment. MCP-1 was injected intravitreally into the right eyes of adult Cx3CRl-GFP mice. Left eyes were untreated. Mice were imaged at various time points following injection on a Heidleberg Spectralis instrument. Evidence of GFP+ cell recruitment was seen in eyes treated with CCL2 (A, G), but not in control eyes (D, J). IR reflectance images are shown to indicate position of image on the retina (B,E, H, K). (C, F, I, L) Flat mount preparations were made from the same retinas imaged in vivo where a good correlation was shown between what can be observed in vivo and that which is observed on flat mount. [0016] Figure 4 shows that MCP-1 injection into the eye recruits Cell Tracer-labeled cells from the circulation into the retina after oxygen-induced injury.

[0017] Figure 5 shows time course of isolectin+ cell recruitment after MCP-1 injection in the OIR model.

[0018] Figure 6 shows characterization of MCP-1 recruited cells in the OIR retina. The vast majority of CD45+ cells are isolectin+, and all isolectin+ cells are CD1 lb+, indicating that recruited cells are largely macrophages.

[0019] Figure 7 shows that CXCL13 and MCP-1 recruit similar numbers of isolectin+ macrophages into the OIR retina (top panel); and that intra vitreal injection of these two chemokines is also correlated with similar degree of vascular repair in the OIR model (bottom panel).

[0020] Figure 8 shows that, like MCP-1, human fractalkine (aka CX3CL1) also effectively recruits cells into OIR retina after intravitreal administration.

DETAILED DESCRIPTION

I. Overview

[0021] The present invention relates to methods of using chemotactic compounds for monocytes and macrophages to treat or ameliorate symptoms of ocular vascular diseases or degenerative disorders. The invention is predicated in part on the discoveries by the present inventors that intraocular injection of the purified chemokines, MCP-1 (CCL2), CX ₃ CL1, CXCL13 or MIP-1 resulted in significantly enhanced repair of retinal damage in a mouse model of ischemic retinopathy. As detailed in the Examples below, it was discovered that intravitreal injection of chemotactic agents stimulates the influx of monocytes into the eye, which then differentiate into macrophages which exert the observed therapeutic activities. The inventors demonstrated that macrophages/microglia cells (i.e., isolectin positive cells) present in the retina after chemokine injection were in fact largely recruited from the circulation. In addition, it was found that the vast majority of CD45+ cells (i.e.,

hematopoietic cells) recruited to retina were positive for isolectin. The recruitment of macrophages/microglia by the chemokine injection was further evidenced by the fact that the isolectin positive cells were also positive for monocyte/macrophage marker CD1 lb but negative for lymphocyte marker CD3e or CD 19.

[0022] In accordance with these discoveries, the present invention provides methods for promoting normalization and repair of retinal vasculature in subjects with ocular vascular damage or neovascularization. The invention also provides methods of treating or ameliorating diseases or disorders related to or mediated by aberrant ocular vascularization. Related therapeutic compositions are also provided in the invention. The following sections provide more detailed guidance for practicing the invention.

II. Definitions

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1 ^st ed., 1992); Illustrated Dictionary of

Immunology, Cruse (Ed.), CRC Pr I Lie (2 ^nd ed., 2002); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3 ^rd ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1 ^st ed., 1999);

Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994);

Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4 ^th ed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0024] The term "agent" or "test agent" includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, peptide or mimetic, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms "agent", "substance", and "compound" are used interchangeably herein.

[0025] The term "analog" is used herein to refer to a molecule that structurally resembles a reference molecule (e.g., a known monocyte chemoattractant agent described herein) but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of analogs, to identify variants of known compounds having improved characteristics (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry.

[0026] Chemoattractant compounds for monocyte refer to any compounds that are capable of inducing chemotaxis of monocyte or macrophage. These include chemokines that are chemotactic for monocytes or macrophage, as well as other classes of compounds that can similarly promote or stimulate migration of the cells towards the compounds. Unless otherwise specified, these compounds also broadly encompass agents that directly or indirectly upregulate expression or cellular levels of a known monocyte chemoattractant compound (e.g., MCP-1), examples of which include PPAR receptor agonists. Unless otherwise noted, the term "chemoattractant compounds for monocyte" is used

interchangeably with the term "monocyte chemoattractant compounds" or "monocyte chemoattractant agents."

[0027] Glial cells, commonly called neuroglia or simply glia (Greek for "glue"), are non- neuronal cells that provide support and nutrition, maintain homeostasis, form myelin, and participate in signal transmission in the nervous system. In the human brain, there is roughly one glia for every neuron with a ratio of about two neurons for every three glia in the cerebral gray matter. Glial cells provide support and protection for neurons, the other main type of cell in the nervous system. Microglia refers to a type of glial cells which are the resident macrophages of the brain, and thus act as the first and main form of active immune defense in the central nervous system (CNS). Microglia constitute 20% of the total glial cell population within the brain. Microglia (and astrocytes) are distributed in large non- overlapping regions throughout the brain and spinal cord. Microglia are constantly moving and analyzing the CNS for damaged neurons, plaques, and infectious agents. Astrocytes (or astroglia) are characteristic star-shaped glial cells in the brain and spinal cord. They perform many functions, including biochemical support of endothelial cells which form the blood- brain barrier, the provision of nutrients to the nervous tissue, maintenance of extracellular ion balance, and a principal role in the repair and scarring process of the brain and spinal cord following traumatic injuries.

[0028] Chemokines are a family of small cytokines, or proteins secreted by cells. They are capable of inducing directed chemotaxis in nearby responsive cells (i.e., chemotactic cytokines). Proteins are classified as chemokines according to shared structural

characteristics such as small size (they are all approximately 8-10 kilodaltons in size), and the presence of four cysteine residues in conserved locations that are key to forming their 3- dimensional shape.

[0029] Neovascularization refers to the abnormal formation of new and fragile blood vessels, usually in response to ischemia following an occlusion (blockage) of an existing blood vessel. Neovascularization often occurs in the retina. It is widely considered to be highly undesirable as these fragile vessels are prone to hemorrhaging, thereby starving surrounding tissue of blood nutrients. This condition is traditionally treated by

photocoagulation which is the use of lasers to cauterise these new vessels.

Neovascularization differs from angiogenesis in that angiogenesis is mainly characterized by the protrusion and outgrowth of capillary buds and sprouts from pre-existing blood vessels. In ophthalmology, choroidal neovascularization is the formation of a microvasculature within the innermost layer of the choroid of the eye.

[0030] Revascularization or physiological revascularization refers to restoration or growth of existing retinal network of blood vessels.

[0031] Hematopoietic stem cells are stem cells that are capable of developing into various blood cell types e.g., B cells, T cells, granulocytes, platelets, and erythrocytes.

[0032] The cells that circulate in the bloodstream are generally divided into three types: white blood cells (leukocytes), red blood cells (erythrocytes), and platelets or thrombocytes. Leukocytes include granulocytes (polymorphonuclear leukocytes) and agranulocytes (mononuclear leucocytes). Granulocytes are leukocytes characterized by the presence of differently staining granules in their cytoplasm when viewed under light microscopy. There are three types of granulocytes: neutrophils, basophils, and eosinophils. Agranulocytes (mononuclear leucocytes) are leukocytes characterized by the apparent absence of granules in their cytoplasm. Although the name implies a lack of granules, these cells do contain non-specific azurophilic granules, which are lysosomes. Agranulocytes include

lymphocytes, monocytes, and macrophages.

[0033] Monocytes are produced by the bone marrow from haematopoietic stem cell precursors called monoblasts. Monocytes circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body. They constitute between three to eight percent of the leukocytes in the blood. In the tissues monocytes mature into different types of macrophages at different anatomical locations. Monocytes have two main functions in the immune system: (1) replenish resident macrophages and dendritic cells under normal states, and (2) in response to inflammation signals, monocytes can move quickly (aprox. 8-12 hours) to sites of infection in the tissues and divide/differentiate into macrophages and dendritic cells to elicit an immune response. Monocytes are usually identified in stained smears by their large bilobate nucleus.

[0034] Macrophages are white blood cells within tissues, produced by the division of monocytes. When a monocyte enters damaged tissue through the endothelium of a blood vessel (a process known as the leukocyte extravasation), it undergoes a series of changes to become a macrophage. Macrophages and monocytes are phagocytes, acting in both nonspecific defense (or innate immunity) as well as to help initiate specific defense mechanisms (or adaptive immunity) of vertebrate animals. Macrophages also play important roles in muscle regeneration and wound healing. They are also characterized by specific expressions of a number of surface proteins including CD14, CD1 lb, F4/80 (mice)/EMRl (human), Lysozyme M, MAC-l MAC-3 and CD68, which can be identified by flow cytometry or immunohistochemical staining.

[0035] As used herein, ocular vascular disorder or ocular neovascular disease refers to any pathological conditions characterized by altered or unregulated proliferation and invasion of new blood vessels into the structures of ocular tissues such as the retina or cornea. Examples of ocular neovascular diseases include ischemic retinopathy, diabetic retinopathy, retinopathy of prematurity, macular degeneration including age-related macular degeneration, retinitis pigmentosa, glaucoma, retinal degeneration, iris neovascularization, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, and diabetic retinal ischemia. Unless otherwise indicated, the term is used interchangeably with ocular degenerative disorder or ocular neovascular disease.

[0036] Other ocular vascular disorder or diseases associated with corneal

neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical bums, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal degeneration, mariginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis, Steven's Johnson disease, periphigoid radial keratotomy, and corneal graph rejection. [0037] Diseases associated with retinal/choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagets disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, retinitis pigmentosa, retina edema (including macular edema), Eales disease, Bechets disease, infections causing a retinitis or choroiditis, presumed ocular histoplasmosis, Bests disease, myopia, optic pits, Stargarts disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications. Other diseases include, but are not limited to, diseases associated with rubeosis (neovascularization of the angle) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy.

[0038] Retinopathy of prematurity (ROP) is a disease of the eye that affects prematurely born babies. It is thought to be caused by disorganized growth of retinal blood vessels which may result in scarring and retinal detachment. ROP can be mild and may resolve spontaneously, but may lead to blindness in serious cases. As such, all preterm babies are at risk for ROP, and very low birth weight is an additional risk factor. Both oxygen toxicity and relative hypoxia can contribute to the development of ROP.

[0039] Macular degeneration is a medical condition predominantly found in elderly adults in which the center of the inner lining of the eye, known as the macula area of the retina, suffers thinning, atrophy, and in some cases, bleeding. This can result in loss of central vision, which entails inability to see fine details, to read, or to recognize faces.

According to the American Academy of Ophthalmology, it is the leading cause of central vision loss (blindness) in the United States today for those over the age of fifty years.

Although some macular dystrophies that affect younger individuals are sometimes referred to as macular degeneration, the term generally refers to age-related macular degeneration (AMD or ARMD).

[0040] Age-related macular degeneration begins with characteristic yellow deposits in the macula (central area of the retina which provides detailed central vision, called fovea) called drusen between the retinal pigment epithelium and the underlying choroid. Most people with these early changes (referred to as age-related maculopathy) have good vision. People with drusen can go on to develop advanced AMD. The risk is considerably higher when the drusen are large and numerous and associated with disturbance in the pigmented cell layer under the macula. Large and soft drusen are related to elevated cholesterol deposits and may respond to cholesterol lowering agents or the Rheo Procedure.

[0041] Advanced AMD, which is responsible for profound vision loss, has two forms: dry and wet. Central geographic atrophy, the dry form of advanced AMD, results from atrophy to the retinal pigment epithelial layer below the retina, which causes vision loss through loss of photoreceptors (rods and cones) in the central part of the eye. While no treatment is available for this condition, vitamin supplements with high doses of

antioxidants, lutein and zeaxanthin, have, been demonstrated by the National Eye Institute and others to slow the progression of dry macular degeneration and in some patients, improve visual acuity.

[0042] Retinitis pigmentosa (RP) is a group of genetic eye conditions. In the progression of symptoms for RP, night blindness generally precedes tunnel vision by years or even decades. Many people with RP do not become legally blind until their 40s or 50s and retain some sight all their life. Others go completely blind from RP, in some cases as early as childhood. Progression of RP is different in each case. RP is a type of hereditary retinal dystrophy, a group of inherited disorders in which abnormalities of the photoreceptors (rods and cones) or the retinal pigment epithelium (RPE) of the retina lead to progressive visual loss. Affected individuals first experience defective dark adaptation or nyctalopia (night blindness), followed by reduction of the peripheral visual field (known as tunnel vision) and, sometimes, loss of central vision late in the course of the disease.

[0043] Macular edema occurs when fluid and protein deposits collect on or under the macula of the eye, a yellow central area of the retina, causing it to thicken and swell. The swelling may distort a person's central vision, as the macula is near the center of the retina at the back of the eyeball. This area holds tightly packed cones that provide sharp, clear central vision to enable a person to see form, color, and detail that is directly in the line of sight. Cystoid macular edema is a type of macular edema that includes cyst formation.

[0044] The terms "subject" and "patient" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as dogs, cats, sheeps, cows, pigs, rabbits, chickens, and etc.

Preferred subjects for practicing the therapeutic methods of the present invention are human. Subjects in need of treatment include patients already suffering from an ocular vascular disease or disorder as well as those prone to developing the disorder. [0045] As used herein, "treating" or "ameliorating" includes (i) preventing a pathologic condition (e.g., ischemic retinopathy) from occurring (e.g. prophylaxis); (ii) inhibiting the pathologic condition (e.g., ischemic retinopathy) or arresting its development; and (iii) relieving symptoms associated with the pathologic condition (e.g., ischemic retinopathy). Thus, "treatment" includes the administration of a monocyte chemoattractant compound or therapeutic composition to prevent or delay the onset of the symptoms, complications, or biochemical indicia of an ocular disease described herein, alleviating or ameliorating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. "Treatment" further refers to any indicia of success in the treatment or amelioration or prevention of the ocular disease, condition, or disorder described herein, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. Detailed procedures for the treatment or amelioration of an ocular disorder or symptoms thereof can be based on objective or subjective parameters, including the results of an examination by a physician.

III. Chemoattractant compounds for monocytes

[0046] Various chemotactic compounds can be employed in the practice of the present invention. In some embodiments, a chemokine is administered to the subject to recruit monocytes to the retina. Chemokines are a class of small proteins that are well known in the art. See, e.g., Fernandez and Lolis, Annu Rev Pharmacol Toxicol 42: 469-99; 2002; and Laing et al., Dev. Comp. Immunol. 28:443-60, 2004. There are four groups of molecules in the chemokine family, categorized by the spacing of their first two cysteine residues. They are CC chemokines, CXC chemokines, C chemokines and CX ₃ C chemokines.

[0047] The CC chemokine (or β-chemokine) proteins have two adjacent cysteines near their amino terminus. Beta chemokines induce cellular migration by binding to and activating CC chemokine receptors. CXC chemokines contain two N-terminal cysteines of CXC chemokines (or a-chemokines) which are separated by one amino acid, represented in this name with an "X". C chemokines (or γ chemokines) have only two cysteines; one N- terminal cysteine and one cysteine downstream. Two chemokines have been described for this subgroup and are XCL1 (lymphotactin-a) and XCL2 (lymphotactin-β). These chemokines attract T cell precursors to the thymus. The CX ₃ C chemokines have three amino acids between the two cysteines (also termed d-chemokines). An example of CX ₃ C chemokines is fractalkine (or CX3CL1). It is both secreted and tethered to the surface of the cell that expresses it, thereby serving as both a chemoattractant and as an adhesion molecule. In some preferred embodiments of the invention, a CX ₃ C chemokine such as CX3CL1 is used.

[0048] In some other preferred embodiments, the chemotactic compound employed in the invention is a member from a subgroup of the CC chemokines, CC chemokine ligands (CCL). There are at least 27 distinct members of this subgroup reported for mammals, called CCL1 to CCL28 (CCL10 being the same as CCL9). Chemokines of this subfamily usually contain four cysteines (C4-CC chemokines), but a small number of CC chemokines possess six cysteines (C6-CC chemokines). C6-CC chemokines include CCL1, CCL15, CCL21, CCL23 and CCL28. CC chemokines induce the migration of monocytes and other cell types such as NK cells and dendritic cells. Examples of CC chemokine include monocyte chemoattractant protein- 1 (MCP-1 or CCL2) which induces monocytes to leave the bloodstream and enter the surrounding tissue to become tissue macrophages. CCL 12 (SDF-1) specifically attracts eosinophils, monocytes and lymphocytes. CCL5 (or RANTES) attracts cells such as T cells, eosinophils and basophils that express the receptor CCR5.

[0049] Some other embodiments of the invention employ a chemokine that belong to the CXC chemokine group. There have been 17 different CXC chemokines described in mammals, that are subdivided into two categories, those with a specific amino acid sequence (or motif) of glutamic acid-leucine-arginine (or ELR for short) immediately before the first cysteine of the CXC motif (ELR-positive), and those without an ELR motif (ELR-negative). ELR-positive CXC chemokines specifically induce the migration of neutrophils, and interact with chemokine receptors CXCR1 and CXCR2. An example of an ELR-positive CXC chemokine is interleukin-8 (IL-8), which induces neutrophils to leave the bloodstream and enter into the surrounding tissue. Other CXC chemokines that lack the ELR motif, such as CXCL13, tend to be chemoattractant for lymphocytes.

[0050] Other than chemokines, other chemoattractants for monocytes can also be used in the practice of the present invention. These include any other compounds that are known in the art that are able to induce chemotaxis of monocytes or macrophage. For example, galectin-3 has been shown to be chemotactic for cultured human macrophages and alveolar macrophages (see, e.g., Sano et al., J. Immunol. 165:2156-64, 2000). In addition, leukotriene B4 (LTB4) has been reported to induce human monocytes and neutrophils to generate cellular superoxide and to undergo chemotactic migration (see, e.g., Kownatzki et al., Int. Arch. Allergy Appl. Immunol. 93:344-349, 1990; and Bomalaski et al., J. Leukoc. Biol. 47: 1-12, 1990). Further, Complement component 5a (C5a), a protein fragment released from complement component C5, is a well known chemoattractant for

polymorphonuclear leukocytes and monocytes (see, e.g., Emst et al., Infect. Immun. 46:81- 6, 1984; and Doherty et al., J. Clin. Invest. 86: 1065-75, 1990). Any of these chemotactic agents can be employed in the present invention.

[0051] Suitable monocyte chemotactic compounds that can be employed for practicing the present invention also include peptides, peptide mimetics and antibodies. For example, a number of peptides or peptide mimetics that mimic the functions of the above-described chemokines are known in the art. Specifically, U.S. Patent Publication No. 20060073114 disclosed several MCP-1 derived peptides that possess some of the chemotactic function of MCP-1. U.S. Patent No. 7,091,310 described chemokine analogs including agonists of MIP- la, MCP-1 , RANTES, IL-8 and CCL28. Similarly, U.S. Patent No. 7,368,425 disclosed peptide analogs and mimetic compounds of human chemokines that mimic the functions of SDF-1. U.S. Patent No. 7,378,098 disclosed CXC chemokine receptor 4 (CXCR4) agonist peptides, including SDF-1 polypeptides and SDF-1 polypeptide homologues. Any of these peptide agonists or chemokine analogs can be utilized by the skilled artisans in the practice of the methods of the present invention. Other than these peptide agonists, monocyte chemoattractant compounds that can be employed in the invention further include agonist antibodies of the chemokines described herein. Examples of such antibodies are known in the art. For example, U.S. Patent No. 6,084,075 described agonist antibodies that are capable of binding monocyte chemoattractant protein 1 (MCP-1) receptor CCR2. Additional antibodies can be readily produced with standard immunology techniques. Such antibodies can all be used in the practice of the presently disclosed methods. Additional monocyte chemoattractant agents of various chemical classes (e.g., peptides, peptide mimetics, proteins, or organic compounds) can be identified by screening test agents with methods routinely practiced in the art or described herein.

[0052] In some embodiments, small molecule organic compounds that mimic or agonize functions of the monocyte chemotactic compounds disclosed herein or that induce monocyte chemotactic migrations can be employed in the invention. Such chemoattractant agents for monocytes can be obtained via screening a library of small molecule compounds (e.g., organic compounds of less than about 500 or less than about 1 ,000 daltons) with appropriate functional assays. To screen for novel monocyte chemoattractant compounds, a library of test agents (e.g., small molecule compounds) can be randomly produced. Alternatively, they can also be analogs or variants of a known compound (e.g., MCP-1). Combinatorial libraries of chemical analogs of a known compound can be synthesized in a step-by-step fashion. Large combinatorial libraries of compounds can be constructed by the encoded synthetic libraries (ESL) method described in WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503 and WO 95/30642. Other methods for synthesizing analogs of various compounds are described in, e.g., by Overman, Organic Reactions, Volumes 1-62, Wiley- Interscience (2003); Broom et al., Fed Proc. 45: 2779-83, 1986; Ben-Menahem et al., Recent Prog. Horm. Res. 54:271-88, 1999; Schramm et al., Annu. Rev. Biochem. 67: 693-720, 1998; Bolin et al., Biopolymers 37: 57-66, 1995; Karten et al., Endocr. Rev. 7: 44-66, 1986; Ho et al., Tactics of Organic Synthesis, Wiley-Interscience; (1994); and Scheit et al., Nucleotide Analogs: Synthesis and Biological Function, John Wiley & Sons (1980).

[0053] Many functional assays can be employed to screen a library of test agents (e.g., small organic compounds) to identify novel monocyte chemoattractant compounds. For example, the test compounds can be examined for their ability to induce monocyte migration in a chemotaxis assay. Monocyte chemotaxis assay can be performed with commercially available systems, e.g., ChemoTx ^® Disposable Chemotaxis System from Neuro Probe, Inc. (Gaithersburg, MD). Detailed descriptions of materials and protocols for assaying monocyte chemotaxis are provided in, e.g., Matsushima et al., J. Exp. Med. 169: 1485, 1989; Rollins et al., Blood 78: 11 1, 1989; Dean et al., J. Immunol. Methods 14:65-72, 1997; and Entschladen et al., Exp. Cell Res. 307:418-426, 2005. When test agents used in the screening are analogs or variants of a known monocyte chemoattractant compounds, other functional assays can also be employed. For example, analogs or variants of a chemokine such as MCP-1 or SDF- 1 can be screened for their ability to compete with the chemokine for binding to the cognate receptor. Such assays are described in the art, e.g., U.S. Patent Nos. 6,084,075; 7,368,425; 7,091,310; and 7,378,098.

[0054] In some embodiments, the chemotactic compounds used in the invention are linked to or associated with other materials in order to increase the half-life of these compounds. For example, the monocyte chemoattractant compounds can be conjugated to biomaterials such as polyelthylene glycol (PEG) and hyaluronic acid. Alternatively, the half-life of a chemoattractant compound can be increased by encapsulation of the compound in liposomes. Methods for prolonging half-life of a therapeutic agent with these biomaterials are well known in the art. See, e.g., Nathan et al., Bioconjug. Chem. 4:54-62, 1993; Yadav et al., J. Drug Target 16:91-107, 2008; Langner et al., Pol. J. Pharmacol. 51 :21 1-22, 1999; Cantin et al., Am. J. Resp. Cell Mol. Biol. 27:659-665,2002; Sun et al., Cancer Res.

63:8377-8383, 2003; Yerushalmi et al., Arch. Biochem. Biophys. 313:267-73, 1994;

Coradini et al., Int. J. Cancer 81 :41 1-6, 1999.

[0055] Other than the monocyte chemoattractant compounds described above, the invention can also employ agonist agents that upregulate expression or cellular levels of these chemoattractant compounds. For example, the expression of MCP-1 (and some other monocyte chemoattractant compounds) can be induced through the PPAR family of receptors. Thus, PPAR agonist compounds can be used to recruit cells of monocyte lineage by upregulating MCP-1 or other monocyte chemoattractant molecules. Monocyte cells recruited by the upregulated monocyte chemoattractants will in turn mediate a therapeutic benefit under conditions of retinal disease. Examples of PPAR agonists suitable for the invention include troglitazone and ciglitazone (Panzer et al., Kidney Int'l. 62, 455-464, 2002), rosiglitazone (Shen et al., Arch. Ophthalmol. 126:793-799, 2008), 9-cis retinoic acid (Zhu et al., Arterioscler. Thromb. Vase. Biol.. 19:2105-2111, 1999), Mitomycin C (Chou et al, Invest Ophthalmol Vis Sci. 48:2009-16, 2007). These and other PPAR agonists known in the art can all be readily employed in the practice of the present invention.

IV. Treating ocular vascular diseases

[0056] The present invention provides methods of treating or preventing vascular disorders and neuronal degeneration in the retina of a mammal that suffers from an ocular disease. In accordance with the methods, monocyte chemoattractant compounds as described above can be administered to the retina of the mammal, preferably by intravitreal injection. The cells are administered in an amount sufficient to repair or reverse vascular and/or neuronal degeneration in the retina. Preferably, monocyte chemoattractant compounds are administered in a therapeutic composition that also contains a pharmaceutically acceptable carrier.

[0057] Not intended to be bound in theory, the administered monocyte chemoattractant compounds exert their therapeutic effect by recruiting monocytes and macrophages to the site of retinal vascular injury. At least some of the recruited cells can differentiate into cells with characteristics of microglia, the resident macrophage-like cell in the retina. Microglia cells (both endogenous and exogenous) are able to mediate vascular repair. There are many advantages afforded by the present invention. Unlike many other available treatments for retinal vascular injury, methods of the present invention can provide longer and enduring therapeutic effect. In addition, methods of the invention are intended to directly address the underlying pathology of ischemic retinal diseases, whereas other treatments only aim at relieving or ameliorating resulting symptoms. Further, many of the currently used drugs only inhibit the formation of pathologic vessels. In contrast, methods of the present invention are directed to promoting the normalization and repair of retinal vasculature. Therefore, with methods of the invention, vascular pathology and the subsequent complications such as hemorrhage, scarring and retinal detachment are less likely to occur. |0058] The subjects suitable for treatment with methods of the invention can be neonatal, juvenile or fully mature adults. In some embodiments, the subject to be treated with methods of the invention is one suffering from an ocular degenerative disease or ocular vascular disease, e.g., one at an early stage of the ocular disease. In some other

embodiments, the subject is one who is otherwise healthy but known to be predisposed to the development of an ocular degenerative disease (i.e., through genetic predisposition). In some embodiments, the subjects to be treated are neonatal subjects suffering from ocular disorders such as oxygen induced retinopathy or retinopathy of prematurity. In some preferred embodiments, the subjects are human.

[0059] Subjects suffering from various ocular vascular diseases or ocular degenerative disorders are suitable for treatment with the methods of the invention. These include ocular diseases such as retinal degenerative diseases, retinal vascular degenerative diseases, retina edema (including macular edema), ischemic retinopathies, vascular hemorrhages, vascular leakage, choroidopathies, retinal injuries and retinal defects involving an interruption in or degradation of the retinal vasculature. Specific examples of such diseases include age related macular degeneration (ARMD), diabetic retinopathy (DR), presumed ocular histoplasmosis (POHS), retinopathy of prematurity (ROP), sickle cell anemia, and retinitis pigmentosa, as well as retinal injuries.

V Pharmaceutical compositions and administration

[0060] The invention provides the use of a monocyte chemoattractant compound in the manufacture of a medicament for treating an ocular vascular disease or ocular degenerative disorder. Subjects in need of treatment or alleviation of such a condition can be

administered with a monocyte chemoattractant compound alone. However, the administration of a pharmaceutical composition that contains the monocyte chemoattractant or a pharmaceutically acceptable salt thereof is more preferred. Examples of monocyte chemoattractants that can be employed in the pharmaceutical compositions include the various compounds described herein, e.g., MCP-1. The invention also provides for a pharmaceutical combination, e.g. a kit. Such pharmaceutical combination can contain an active agent which is a monocyte chemoattractant disclosed herein, in free form or in a composition, one or more inactive agents or other components, as well as instructions for administration of the agents. In some embodiments, therapeutic kits of the invention can contain one or more doses of a monocyte chemoattractant (e.g., MCP-1 or CXCL13) present in a pharmaceutical composition described herein, a suitable device for intravitreal injection of the pharmaceutical composition, and an instruction detailing suitable subjects and protocols for carrying out the injection.

[0061] The pharmaceutical compositions that comprise a monocyte chemoattractant can be prepared in various forms. Suitable solid or liquid pharmaceutical preparation forms are, e.g., granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds. The pharmaceutical compositions of the invention can be prepared in accordance with the standard protocols well known in the art, e.g., Remington: The Science and Practice of Pharmacy, Gennaro (ed.), Lippincott Williams & Wilkins (20 ^th ed., 2003). The pharmaceutical compositions typically contain an effective amount of the monocyte chemoattractant that is sufficient to lessen or ameliorate symptoms of an ocular vascular disease or ocular degenerative disorder. In addition to the monocyte chemoattractants, the pharmaceutical compositions can also contain certain pharmaceutically acceptable carriers which enhance or stabilize the composition, or facilitate preparation of the composition. For example, the monocyte chemoattractant can be complexed with carrier proteins such as ovalbumin or serum albumin prior to their administration in order to enhance stability or pharmacological properties. The various forms of pharmaceutical compositions can also contain excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners and elixirs containing inert diluents commonly used in the art, such as purified water.

[0062] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered as well as by the particular method used to administer the composition. They should also be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral, sublingual, rectal, nasal, intravenous, or parenteral. For example, examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers for occlusive dressings can be used to increase skin permeability and enhance absorption. Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form.

[0063] For therapeutic or prophylactic applications, a pharmaceutical composition containing a monocyte chemoattractant can be administered locally or systemically in a therapeutically effective amount or dose. For example, they may be administered parenterally, enterically, by injection, rapid infusion, nasopharyngeal absorption, dermal absorption, and orally. However, in preferred embodiments, local administration of the composition is desired in order to achieve the intended therapeutic effect. In these embodiments, the compositions are typically administered to the subject in need of treatment via intravitreal injection. This can be performed in accordance with standard procedures known in the art. See, e.g., Ritter et al., J. Clin. Invest. 116:3266-76, 2006; Russelakis- Carneiro et al., Neuropathol. Appl. Neurobiol. 25: 196-206, 1999; and Wray et al., Arch. Neurol. 33:183-5, 1976.

[0064] A therapeutically effective amount means an amount that that is sufficient to reduce or inhibit the symptoms of the disorder or condition to be treated in a subject. In the practice of the present invention, the amount of the administered monocyte chemoattractant compound should be effective for repairing retinal damage of the eye, stabilizing retinal neo vasculature, maturing retinal neovasculature, and preventing or repairing vascular leakage and vascular hemorrhage. Such effective amount will vary from subject to subject depending on the ocular disorder afflicted by the subject, stage and severity of the disorder, the subject's general conditions (such as height, weight, age, and health), the particular compound administered, and other factors. For a given monocyte chemoattractant compound, one skilled in the art can easily identify the effective amount of the compound by using routinely practiced pharmaceutical methods. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the

pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders in human subjects (see Examples below). More often, a suitable therapeutic dose can be determined by clinical studies on mammalian species to determine maximum tolerable dose and on normal human subjects to determine safe dosage.

[0065] In general, except under certain circumstances when higher dosages may be required, the preferred dosage of a monocyte chemoattractant usually lies within the range of from about 0.001 to about 1000 mg, more usually from about 0.01 to about 500 mg per day. As a general rule, the quantity of a monocyte chemoattractant administered is the smallest dosage which effectively and reliably prevents or minimizes the conditions of the subjects. Also, the dosages to be administered and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively lower dosage may be administered at relatively infrequent intervals over a long period of time. Some subjects may continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively higher dosage at relatively short intervals may be required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of the ocular vascular disease. Thereafter, the subject can be administered a prophylactic regime. As is readily apparent, the above dosage ranges are intended to provide general guidance and support for the teachings herein, but are not intended to limit the scope of the invention. Additional guidance for preparation and administration of the pharmaceutical compositions of the invention has also been described in the art. See, e.g., Goodman & Gilman's The

Pharmacological Bases of Therapeutics , Hardman et al., eds., McGraw-Hill Professional (10 ^th ed., 2001); Remington: The Science and Practice of Pharmacy, Gennaro, ed.,

Lippincott Williams & Wilkins (20 ^th ed., 2003); and Pharmaceutical Dosage Forms and Drug Delivery Systems, Ansel et al. (eds.), Lippincott Williams & Wilkins (7 ^th ed., 1999).

EXAMPLES

[0066] The following examples are offered to illustrate, but not to limit the present invention.

Example 1. Injection of monocyte chemoattractants for repairing retinal damage

[0067] This Example describes treatment of ischemic retinopathy by recruiting monocytes into the eye with intravitreally injected monocyte chemoattractant compounds. Results from the study demonstrated that intraocular injection of the purified chemokines, MCP-1 (CCL2), CXCL13 or ΜΙΡ-1α results in significantly enhanced repair of retinal damage in a mouse model of ischemic retinopathy. These factors are potent chemoattractants for monocytes/macrophages. It was found that the observed repair effect is mediated by macrophages that are recruited into the retina in response to these chemotactic factors. Specifically, injection of 50ng/ml MCP-1 at postnatal day 12 (P12) resulted in a dramatic increase in the number of isolectin positive macrophages in the retina (Figure 1) when quantified one day later at PI 3 (Figures 2 A and 2B). In subsequent studies using a recruitment scoring method, a reproducible dose-response was observed when using MCP-1 doses ranging from 5ng to 250ng per injection. A second quantitative method showed that while a baseline number of cells (-200 cells/field) was present in retinas from vehicle- injected eyes, retinas from MCP-1 -injected eyes showed a greater than 6-fold increase in the number of isolectin positive cells (>1700 cells/field).

[0068] The recruited microglia progenitors promote vascular repair as evidenced by the improvement in the two major parameters of the oxygen-induced retinopathy (OIR) model, area of vascular obliteration and area of neovascularization. The functional consequence of the macrophage recruitment was shown when a single intravitreal injection of 0.5ng (50ng/ml) of MCP- 1 reduced retinal vascular obliteration by up to 50% and retinal neovascularization up to 76% (Figure 2C).

[0069] In addition to injection of MCP-1, virtually identical results were obtained after injection of another chemotactic agent, CXCL13, both in terms of the number of recruited macrophages and the repair effect mediated by the cells (Figure 7). It was also found that intravitreal injection human fractalkine (CX3CL1) into the OIR mice also effectively recruit cells into the retina at postnatal day 12 (Fig. 8). These results indicate that the observed effect is not specific to MCP-1. Instead, the effect is likely to be mediated by any agent that can recruit the appropriate cell type into the retina.

Example 2. Injection of monocyte chemoattractants leads to influx of monocytes

[0070] To demonstrate that the isolectin positive cells present in the retina after chemokine injection were in fact largely recruited from the circulation, we labeled circulating hematopoietic cells by intracardiac infusion of a dye (Cell Tracer). By analyzing OIR retinas from eyes injected with MCP-1, we were able to demonstrate the presence of significant numbers of labeled cell in the retina indicating that these cells were recruited from the circulation (Figure 4). The recruitment time course was studied in the OIR model by analyzing retinas at various times after injection of MCP-1 at PI 2. At 6 hrs after injection, we observed no difference between retinas that received MCP-1 and those with PBS. However, at 12 hrs we observed what appeared to be the early phase of recruitment in the MCP-1 treated eyes (Figure 5). Subsequent time points showed increasing number of isolectin+ cells, peaking at 72 hrs. In PBS-treated control eyes, we found a relatively low baseline number of isolectin+ cells. In summary, the data indicate that by 72 hrs post- injection of MCP-1, most of the recruited cells had distributed themselves uniformly throughout the retina and began to take on morphology similar to endogenous microglia, suggesting that this is the terminal differentiation state of these cells. These experiments demonstrated that intravitreal MCP-1 mediated rapid cell recruitment to the retina and that the recruited cells promptly adopt characteristics of microglia.

Example 3. CCL2 selectively recruits mveloid-derived progenitors of microglia

[0071] In an effort to better characterize the types of cells recruited into the retina after chemokine injection, we performed immunohistochemical analysis with a panel of antibodies against hematopoietic cells combined with isolectin labeling to identify macrophages/microglia. Using an antibody against CD45, which labels all hematopoietic cells, we found that the vast majority of CD45+ cells were also labeled positively for isolectin, identifying them as macrophages/microglia (Figure 6). This was further demonstrated by our observation that these isolectin positive cells were also positive for the monocyte/macrophage marker, CD1 lb+. Little or no labeling was observed for CD3e or CD19, which label lymphocytes. Specifically, it was found that the vast majority of cells (>90%) are positively labeled for CD1 lb, which is a marker for monocytes and

macrophages. In contrast, a relatively small number of cells (<10%) were labeled with CD3e, are marker present on T lymphocytes. These results demonstrate that intravitreal injection of chemokines such as MCP-1 and CXCL13 specifically recruit CD1 lb positive cells of the monocyte/macrophage lineage into the retina.

Example 4. In vivo imaging of MCP-1 mediated recruitment in adult animals

[0072] The studies described thus far were carried out in neonatal mice using dissected retinal flat mounts for analysis. The following describes the results of our evaluations of CCL2 activity in adult mice using an in vivo imaging approach. The CX3CR1-GFP+/- transgenic mouse has been described previously (REF) and been shown, in the heterozygous genotype, to be normal in all tested aspects (REFS). This mouse expresses GFP under the control of the promoter for CX3CR1, receptor for the chemokine CX3CL1 or fractalkine. CX3CR1 is expressed by microglia in the central nervous system and thus, in the retina, microglia express GFP, facilitating their visualization. We combined the use of this mouse with the scanning laser ophthalmoscope function of the Heidelberg Spectralis instrument which allowed visualization of microglia in the living animal. Intravitreal injection of CCL2 elicited a marked change in the pattern of detectable fluorescence compared to contralateral control eyes (Fig. 3). This pattern was similar to that seen during the early phase of recruitment in retinas from eyes treated with CCL2 where an increased number of cells was present near the major retinal vessels. Whole mount preparations of the same retinas demonstrated that the in vivo imaging approach provided a good representation of the recruitment activity in the retina (Fig. 3). These experiments demonstrated that MCP-1 (CCL2) mediates recruitment in the adult animal and that this process can be followed non- invasively.

Example 5. CCL2-mediated recruitment is not affected by diabetes

[0073] With reports of defective hematopoietic cell function in diabetes (REFS), we tested whether MCP-1 mediated cell recruitment would be influenced by hyperglycemia. For these experiments we used the streptozotocin induced diabetes model in mice. One week following administration of streptozotocin, we confirmed the presence of hyperglycemia and removed those mice with blood glucose levels below 250mg/dl. At two weeks, intravitreal injection of MCP-1 was performed and retinal flat mounts were prepared 24 hrs later. This experiment showed no detectable difference in the recruitment in diabetic animals compared to that observed in non-diabetic animals.

[0074] To show that MCP-1 mediated recruitment of microglia progenitors to the retina is not a species-specific phenomenon restricted to the mouse, we performed similar experiments in adult rats. When the dose of MCP-1 was scaled up to account for the larger eye of the rat, a similar degree of recruitment within the same 24hr time frame was observed like that seen in the mouse experiments.

[0075] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0076] All publications, GenBank sequences, ATCC deposits, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes as if each is individually so denoted.