WO2018182527A1 - Method for treating inflammatory complications in eye diseases - Google Patents

Abstract

The invention relates to methods of using inhibitors of CSF-1 R for reducing microglial activation in the eye as well as treating an inflammation associated with an eye disease, such as age-related macular degeneration (AMD), retinitis pigmentosa and diabetic retinopathy. The methods can further comprise the administration of anti-VEGF antibody. In preferred embodiments, the CSF-1 R inhibitor is Pexidartinib (PLX3397).

Description

METHOD FOR TREATING INFLAMMATORY COMPLICATIONS IN

EYE DISEASES

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62/479,217, filed March 30, 2017, the entire contents of which are incorporated by reference herein for all purposes.

BACKGROUND OF THE INVENTION

[0002] Microglia are tissue macrophages of the central nervous system and eye. Within the eye, microglia reside in the inner retina and continually cycle to the outer retina. Resident microglia in the eye play physiological roles in tissue repair and maintenance. However, under pathological conditions in the eye, microglia activation (cytokine release, tissue damage, reactive oxygen production, recruitment of blood monocytes to retina) and localization to the outer retina is a common feature, particularly in Age-related Macular Degeneration (AMD) (dry and wet forms with the latter having choroidal neovascularization), Retinitis Pigmentosa, and Diabetic Retinopathy (reviewed in Jin N et al. 2016). The current standard for care for treating AMD (wet CNV form only) is anti-VEGF antibody therapy. Anti-VEGF antibody therapy removes leaky, newly formed blood vessels derived from the choroid capillary system. Anti-VEGF therapy involves lifetime, monthly intravitreal needle injections of anti-VEGF antibody. While currently an essential treatment for wet AMD, intravitreal drug injection poses risks of infection (endophthalmitis), intraocular inflammation, rhegmatogenous retinal detachment, intraocular pressure elevation, ocular hemorrhage, and overtreatment to patients, and is inconvenient (Falavarjani KG et al. 2013). In addition, anti-VEGF therapy does not address the underlying microgliosis. There are currently no treatments for dry form of AMD (a.k.a. geographic atrophy), Retinitis Pigmentosa or Diabetic Retinopathy.

[0003] Colony Stimulating Factor Receptor 1 (CSFR1) is a cell surface tyrosine kinase receptor found on the surface of microglia and monocytes that is the receptor for Macrophage Colony Stimulating Factor (M-CSF) and IL34 (Metcalf 1985). CSFRl is essential for survival and differentiation of microglia. Inhibition of CSFRl results in reversible depletion of microglia from brain and reduction of peripheral monocytes from blood (Elmore et al. 2014). This disclosure relates to inhibiting the CSFRl to treat diseases of the eye.

BRIEF SUMMARY OF THE INVENTION

[0004] The instant disclosure provides methods of treating eye diseases with inhibitors of CSFRl . In some embodiments, the eye diseases are associated with microgliosis and/or inflammation of the eye tissue. In some embodiments, the eye tissue is the retina.

[0005] In some embodiments, a method for reducing microglial activation in the eye is described, the method comprising administering to a subject in need thereof an effective amount of a CSFRl inhibitor. In some embodiments, the CSFRl inhibitor is a small molecule or antibody (or antibody fragment). In some embodiments, the CSFRl inhibitor is a small molecule or antibody selected from those shown in Table 1. In one embodiment, the CSFRl inhibitor is Pexidartinib (PLX3397). In some embodiments, the CSFRl inhibitor is a humanized or chimeric antibody. In some embodiments, provided is the use of an effective amount of a CSFRl inhibitor in a method for reducing microglial activation in the eye. In some

embodiments, the use of an effective amount of a CSFRl inhibitor in the manufacture and/or preparation of a medicament for reducing microglial activation in the eye is provided.

[0006] In some embodiments, the treatment comprises contacting retinal tissue of the eye with the CSFRl inhibitor. Contacting the retinal tissue can be achieved by administering an inhibitor of CSFRl (e.g., a small molecule drug) that is capable of crossing the blood-eye barrier to enter the eye tissue. In some embodiments, the retinal tissue is contacted with the CSFRl inhibitor by injecting the inhibitor into the eye.

[0007] Also provided is a method for treating inflammation associated with an eye disease, the method comprising administering to a subject in need thereof an effective amount of a CSFRl inhibitor. In some embodiments, the use of an effective amount of a CSFRl inhibitor in a method for treating inflammation associated with an eye disease is provided. In some embodiments, the use of an effective amount of a CSFRl inhibitor in the manufacture and/or preparation of a medicament for treating inflammation associated with an eye disease is provided. In some embodiments, the eye disease is selected from age-related macular degeneration (AMD), retinitis pigmentosa, or diabetic retinopathy.

[0008] In some embodiments, the methods described herein further comprise administering an effective amount of an anti-VEGF therapy to the subject. In some embodiments, an effective amount of an anti-VEGF antibody is administered to the subject. In some embodiments, use of an effective amount of a CSFRl inhibitor in combination with an effective amount of an anti- VEGF therapy or anti-VEGF antibody in a method for an eye disease is provided. In another embodiment, a medicament comprising an effective amount of a CSFRl inhibitor and an effective amount of an anti-VEGF therapy or anti-VEGF antibody is provided. In one embodiment, use of an effective amount of a CSFRl inhibitor and an effective amount of an anti-VEGF therapy or anti-VEGF antibody in the manufacture or preparation of a medicament for treating eye diseases is provided. In some embodiments, the eye disease is selected from age- related macular degeneration (AMD), retinitis pigmentosa, or diabetic retinopathy.

[0009] In some embodiments, the disclosure provides for the use of an effective amount of a CSFRl inhibitor in a method for treating eye diseases. In some embodiments, the use of an effective amount of CSFRl inhibitor in the manufacture and/or preparation of a medicament for treating eye diseases is provided. In some embodiments, the eye disease is associated with microgliosis and/or inflammation of the eye tissue. In some embodiments, the eye disease is selected from age-related macular degeneration (AMD), retinitis pigmentosa, or diabetic retinopathy.

[00010] In some embodiments, the disclosure provides a medicament comprising a CSFRl inhibitor for the treatment of eye diseases. In some embodiments, the eye disease is associated with microgliosis and/or inflammation of the eye tissue. In some embodiments, the eye disease is selected from age-related macular degeneration (AMD), retinitis pigmentosa, or diabetic retinopathy.

[00011] In another aspect, described herein is a composition comprising an effective amount of a CSFRl inhibitor for use in a method for reducing microglial activation in the eye. In some embodiments, the method comprises administering the composition to a subject in need thereof. In some embodiments, the CSFRl inhibitor is a small molecule or antibody. In some

embodiments, the composition comprises a small molecule CSFRl inhibitor selected from those shown in Table 1. In one embodiment, the composition comprises Pexidartinib (PLX3397). In some embodiments, the CSFRl inhibitor is an antibody selected from those shown in Table 1. In some embodiments, the CSFRl inhibitor is a humanized or chimeric antibody. In some embodiments, the use comprises contacting retinal tissue of the eye with an effective amount of the CSFRl inhibitor. In some embodiments, the composition for use in a method for reducing microglial activation in the eye further comprises an effective amount of an anti-VEGF antibody.

DEFINITIONS

[00012] The term "comprising" in the claims can be substituted with "consisting essentially of and "consisting of. The terms are terms of art, having the meaning understood by one of skill in the art. The term "comprising" is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The term "consisting of excludes any element, step, or ingredient not specified in the claim. The term "consisting essentially of limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention.

[00013] It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural references unless the content clearly dictates otherwise.

[00014] The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +/- 20% or +/- 10%, +/- 5%, +/- 1%, or +/- 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[00015] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. It is understood that any methods and materials similar or equivalent to those described herein can be used in the practice of the methods described herein. [00016] "Vertebrate," "mammal," "subject," "mammalian subject," or "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, cows, horses, goats, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as mice, sheep, dogs, cows, avian species, ducks, geese, pigs, chickens, amphibians, and reptiles.

[00017] "Treating" or "treatment" refers generally to either (i) the prevention of a disease or pathological condition, e.g., prophylaxis, or (ii) the reduction or elimination of symptoms of a disease of interest, e.g., therapy. Treating a subject with the compositions described herein can prevent or reduce microglial activation and/or inflammation of the eye, and eye diseases associated therewith. Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

[00018] "Preventing" or "prevention" refers to prophylactic administration with compositions described herein.

[00019] "Therapeutically-effective amount" or "an amount effective to reduce or eliminate microglial activation and/or inflammation of the eye" or "an effective amount" refers to an amount of a composition described herein that is sufficient to prevent inflammation of the eye or to alleviate (e.g., mitigate, decrease, reduce) at least one of the symptoms associated with microglial activation and/or inflammation of the eye. It is not necessary that the administration of the composition eliminate the symptoms of the condition, as long as the benefits of administration of the composition outweigh the detriments. Likewise, the terms "treat" and "treating" in reference to the diseases or pathological conditions described herein, as used herein, are not intended to mean that the subject is necessarily cured of the pathological condition or that all clinical signs thereof are eliminated, only that some alleviation or improvement in the condition of the subject is effected by administration of the composition.

[00020] The term "small organic molecule" generally refers to an organic molecule having a molecular weight of less than about 900, 800, 700, 600, or 500 daltons, or a molecular weight between 100 and 900 daltons (e.g., 100, 200, 300, 400, 500, 600, 700, 800, or 900 daltons, or any value in between 100 and 900 daltons). [00021] As used herein, the term "antibody" refers to any immunoglobulin or intact molecule as well as to fragments thereof that bind to a specific epitope. Such antibodies include, but are not limited to polyclonal, monoclonal, chimeric, humanized, single chain, Fab, Fab', F(ab)' fragments and/or F(v) portions of the whole antibody and variants thereof. All isotypes are encompassed by this term, including IgA, IgD, IgE, IgG, and IgM.

[00022] As used herein, the term "antibody fragment" refers specifically to an incomplete or isolated portion of the full sequence of the antibody which retains the antigen binding function of the parent antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[00023] An intact "antibody" comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or V.sub.H) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH.sub. l, CH.sub.2 and CH.sub.3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or

V.sub.L) and a light chain constant region. The light chain constant region is comprised of one domain, C.sub.L. The V.sub.H and V.sub.L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term antibody includes antigen- binding portions of an intact antibody that retain capacity to bind. Examples of binding include (i) a Fab fragment, a monovalent fragment consisting of the V.sub.L, V.sub.H, C.sub.L and CHI domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the V.sub.L and V.sub.H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., 1989), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).

[00024] As used herein, the term "single chain antibodies" or "single chain Fv (scFv)" refers to an antibody fusion molecule of the two domains of the Fv fragment, V.sub.L and V.sub.H. Although the two domains of the Fv fragment, V.sub.L and V.sub.H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V.sub.L and V.sub.H regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al., 1988; Huston et al., 1988). Such single chain antibodies are included by reference to the term "antibody" fragments and can be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.

[00025] As used herein, the term "human sequence antibody" includes antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences. The human sequence antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo). Such antibodies can be generated in non-human transgenic animals, e.g., as described in PCT App. Pub. Nos. WO 01/14424 and WO 00/37504. However, the term "human sequence antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., humanized antibodies).

[00026] Also, recombinant immunoglobulins can be produced. See, Cabilly, U.S. Pat. No. 4,816,567, incorporated herein by reference in its entirety and for all purposes; and Queen et al., 1989.

[00027] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions (if present) derived from human germline immunoglobulin sequences. In one aspect, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[00028] As used herein, the term "humanized antibody," refers to at least one antibody molecule in which the amino acid sequence in the non-antigen binding regions and/or the antigen-binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.

[00029] In addition, techniques developed for the production of "chimeric antibodies" (see Morrison, et al., 1984, incorporated herein by reference in its entirety) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. For example, the genes from a mouse antibody molecule specific for an autoinducer can be spliced together with genes from a human antibody molecule of appropriate biological activity. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.

[00030] In addition, techniques have been developed for the production of humanized antibodies (see, e.g., U.S. Pat. No. 5,585,089 and U.S. Pat. No. 5,225,539, which are

incorporated herein by reference in their entirety). An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, referred to as complementarity determining regions (CDRs). Briefly, humanized antibodies are antibody molecules from non-human species having one or more CDRs from the non-human species and a framework region from a human immunoglobulin molecule.

[00031] Alternatively, techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies against an immunogenic conjugate of the present disclosure. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Fab and F(ab')2 portions of antibody molecules can be prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See e.g., U.S. Pat. No. 4,342,566. Fab' antibody molecule portions are also well-known and are produced from F(ab')2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.

BRIEF DESCRIPTION OF THE DRAWINGS

[00032] Figs. 1A and IB. Elimination of retinal microglia and restoration of retinal anatomy from PLX3397 treatment in Mfsd2a KO mice. Fig. 1A. Immunolocalization of the microglia/macrophage marker lba- 1 and apical membrane of retinal pigment epithelium (RPE) marker Ezrin (asterisk) was performed on eye sections from 6 week old mice, lba- 1 positive cells appeared rounder and hypertrophic in Mfsd2a KO on chow diet as compared to wild-type on chow diet, indicative of an activated state (dotted arrows). Moreover, lba-1 positive cells, which normally reside in the inner eye, were found in the sub-retinal space (solid arrows). Scale bar = 50μπι. WT, n=2 ; KO, n=2. In mice treated with PLX3397, microglia population is depleted, as seen from the absence of lba-1 positivity. Scale bar = 50μπι. WT, n=3 ; KO, n=3. Fig. IB. Immunolocalization of Ezrin (black arrow head with white outline) was performed on eye sections from 6 week old mice as done in panel A. A fluorescence marker (asterisk) was used to visualise photoreceptor outer segments (OS). In chow-fed wild-type, the RPE apical membrane interacts with the OS, but gaps are seen in the sub-retinal space (solid arrows) in chow-fed Mfsd2a KO. Scale bar = 20μπι. WT, n=2 ; KO, n=2. In PLX3397-treated wild-type, the RPE apical membrane interacts with the OS, similar to chow-fed wild-type. In PLX3397-treated Mfsd2a KO however, gaps are no longer seen in the sub-retinal space (dotted arrows), as the RPE apical membrane interacts with the OS. Scale bar = 20μπι. WT, n=3 ; KO, n=3. INL: inner nuclear layer, ONL: outer nuclear layer, PR: photoreceptor, OS: outer segments, RPE: retinal pigment epithelium.

[00033] Figs. 2A and 2B. Agents targeting human and mouse CSF-1R pathways. Fig. 2A. CSF-1 and CSF-1R targeting agents currently in clinical development for cancer therapeutics. The two different ligands of CSF-1R, CSF-1 and IL-34 respectively, bind to overlapping yet distinct epitopes within the ligand-binding domain. RG7155 is a ligand- dependent and ligand-independent dimerization inhibitor, while all other antibodies targeting the CSF-1R (FPA008 (see US2011/0274683), IMC-CS4 (see US2011/030148), and AMG820 (seeUS2008/073611) bind to epitopes in the ligand binding domain (D1-D3). MCS110 (US2006/029186) binds to the ligand CSF- 1. Small molecules targeting the tyrosine kinase domain (ARRY-382, JNJ-40346527, PLX3397 and PLX7486 do not exclusively target CSF-IR but also e.g., c-Kit, a receptor important in the development of myeloid cells making it difficult to dissect effects solely mediated by the CSF-IR pathway. Fig. 2B. Agents targeting the mouse CSF-l/CSF-lR for preclinical evaluation. Antibodies targeting the mouse CSF-IR, M279, AFS98 and 2G2, bind within the ligand-binding domain, while 5A1 targets the ligand CSF-1 . 2G2 is the only murine chimeric antibody (with the complementary determining region still hamster-derived), that allows long-term treatment without development of anti-drug antibodies (C. Ries and S. Noyes, unpublished data). Small molecules targeting the tyrosine kinase domain of CSF-IR used in mouse cancer models include ΚΪ20227, BLZ945, CYC11645, GW2580 and PLX3397 (see Ries et al 2015).

[00034] Figs. 3A and 3B. Elimination of retinal microglia after 3 weeks of PLX3397 treatment by oral gavage, but repopulates retina 1 week following drug cessation. Fig. 3A. Images of retinas obtained from Cx3crl^GFP/+ mice. Microglia is depleted following 3 weeks of PLX3397 treatment by oral gavage as seen from the absence of GFP positive cells, but repopulates 1 week following drug cessation. Control mice received only LPC18:0 by oral gavage. Control, n=6; 3 weeks PLX3397, n=8; 3 weeks PLX3397 + 1 week off, n=9. Scale bar = 0.5mm. Fig. 3B. Close-up images of retinas obtained from Cx3crl-GFP mice shown in panel A. Repopulating microglia have a ramified morphology typical of non -inflammatory microglia. Control, n=6; 3 weeks PLX3397, n=8; 3 weeks PLX3397 + 1 week off, n=9. Scale bar = 50μΜ.

[00035] Figs. 4A and 4B. PLX3397 treatment reduced microglia population at peak disease stage, reduced photoreceptor loss and preserved outer limiting membrane and Bruch's membrane. Cx3crl^GFP/+, RdlO/RdlO mice were treated with either LPC18:0 (control) or PLX3397 by daily intraperitoneal injections from postnatal day 13 to 24 and eyes were harvested for histology on postnatal day 25. Fig. 4 A. Ramified microglia is seen in the inner eye (solid arrows) while activated microglia is seen in the outer nuclear layer and sub-retinal space (dashed arrows) in control mice. PLX3397 treatment reduced microglia population in the eye as seen from the absence of GFP positive cells. Control, n=3; PLX3397, n=4. Scale bar = ΙΟΟμΜ. Fig. 4B. H&E images of eyes of LPC18:0 (control) or PLX3397 treated mice. Starting treatment at onset of peak photoreceptor loss reduced photoreceptor segments loss and preserved the outer limiting membrane and Bruch's membrane. Control, n=3; PLX3397, n=4. Scale bar = 20μΜ.

[00036] Figs. 5A and 5B. PLX3397 treatment reduced microglia population after peak photoreceptor loss, and preserved outer limiting membrane and Bruch's membrane.

Cx3crl^or7+, RdlO/RdlO mice were fed with normal chow or PLX3397 chow at 3 weeks of age for 3 weeks. Fig. 5A. Ramified microglia is seen in the inner eye (solid arrows) while activated microglia is seen in the outer nuclear layer and sub-retinal space (dashed arrows) in control mice. PLX3397 treatment reduced microglia population in the eye as seen from the significant reduction of GFP positive cells. Control, n=3; PLX3397, n=5. Scale bar = 50μΜ. Fig. 5B. H&E images of eyes of mice fed with normal chow or PLX3397 chow at 3 weeks of age for 3 weeks. Starting treatment after peak photoreceptor death still preserved the outer limiting membrane and Bruch's membrane. Control, n=3; PLX3397, n=5. Scale bar = 20μΜ.

[00037] Fig. 6A. Gene microarray analysis from eye cups of 6 weeks old WT and Mfsd2a KO mice indicates PLX3397 treatment reduced activated microglia in both wild-type and Mfsd2a KO mice. WT and Mfsd2a KO were fed with normal chow or PLX3397 chow at 3 weeks of age for 3 weeks before eyecups were harvested for microarray. Upregulation of microglia/interferon pathway in eyes of Mfsd2a KO mice was reported in Wong et. al., 2016. This is illustrated by an MA plot, where M stands for "log ratio" and A stands for "mean average", which is used to visualize intensity-dependent ratio of raw gene microarray data. Red dots represent individual targets in this pathway over the background of expressed genes.

PLX3397 treatment repressed microglia/interferon pathway in both wild-type and Mfsd2a KO mice. WT Chow, n=6; KO Chow, n=6; WT PLX3397, n=6; KO PLX3397, n=6.

DETAILED DESCRIPTION OF THE INVENTION

[00038] The present disclosure is related to methods for reducing microglial activation in the eye and methods for reducing or treating inflammation in the eye, for example, inflammation associated with an eye disease. Examples of eye diseases associated with microglial activation and/or inflammation of the eye include age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy. The methods described herein comprise contacting eye tissue with an effective amount of a Colony Stimulating Factor 1 (CSFR1) inhibitor. In some embodiments, the eye tissue is retinal tissue. In some embodiments, the methods comprise administering to a subject in need thereof an effective amount of a CSFRl inhibitor. The inventors have unexpectedly found that contacting eye tissue with CSFRl inhibitors results in a significant reduction in the numbers of microglia in the retina and restoration of the retinal anatomy in eye diseases associated with microglia activation.

METHODS OF TREATMENT

[00039] Described herein are methods of treating inflammation associated with an eye disease and methods for reducing microglial activation in the eye. The methods comprise administering an effective amount of a CSFRl inhibitor to a subject in need thereof. In some embodiments, the CSFRl inhibitor is a small molecule. In some embodiments, the CSFRl inhibitor is an antibody or fragment thereof. In some embodiments, provided herein is the use of an effective amount of a CSFRl inhibitor in a method for treating inflammation associated with an eye disease described herein, or a method of treating an eye disease described herein. In some embodiments, provided herein is the use of an effective amount of a CSFRl inhibitor in the manufacture and/or preparation of a medicament for treating an eye disease described herein, or inflammation associated with an eye disease described herein.

DISEASES

[00040] The methods described herein are useful for treating diseases or symptoms of diseases associated with microglial activation in the eye, and/or diseases associated with inflammation of the eye. In some embodiments, the disease condition results in microglial activation in the retina, which can disrupt the anatomy of the retina. Microglia activation can result in cytokine release, tissue damage, reactive oxygen species production, and recruitment of blood monocytes to the retina. Microglia can also migrate to the outer retina under certain pathological conditions. In some embodiments, the methods result in a reduction in the number of activated microglia in the eye, or a reduction in the total number of microglia in the eye or retina. In some

embodiments, the methods described herein result in a reduction in the number of monocytes and/or macrophages that infiltrate the retinal tissue.

[00041] Diseases or conditions that can be treated by the methods described herein also include diseases associated with degeneration of the retina. In some embodiments, the disease or condition is selected from age-related macular degeneration (AMD), including dry and wet forms (including the wet form with choroidal neovascularization), retinitis pigmentosa, diabetic retinopathy, or glaucoma.

CSFRl INHIBITORS

[00042] The CSFRl inhibitors described herein can be small organic molecules. The term "small organic molecule" generally refers to an organic molecule having a molecular weight of less than about 900, 800, 700, 600, or 500 daltons, or a molecular weight between 100 and 900 daltons (e.g., 100, 200, 300, 400, 500, 600, 700, 800, or 900 daltons, or any value in between 100 and 900 daltons). Small organic molecule inhibitors will generally be selected to have specific binding to CSFRl, have oral bioavailability, and be able to cross the blood-brain and blood-eye barrier. In some embodiments, the small organic molecule has the properties specified by "Lipinski's rule of five" or the "rule of five" (described in Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (March 2001). "Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings". Adv. Drug Deliv. Rev. 46 (1-3): 3-26; and Lipinski CA (December 2004). "Lead- and drug-like compounds: the rule- of-five revolution". Drug Discovery Today: Technologies. 1 (4): 337-341 ; which are incorporated by reference herein) and summarized below:

[00043] No more than 5 hydrogen bond donors (the total number of nitrogen-hydrogen and oxygen-hydrogen bonds)

[00044] No more than 10 hydrogen bond acceptors (all nitrogen or oxygen atoms) [00045] A molecular mass less than 500 daltons

[00046] An octanol-water partition coefficient log P not greater than 5.

[00047] In some embodiments, the CSFRl inhibitors described herein are antibodies or fragments thereof. The structure of antibodies is well known in the art. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric or humanized antibody.

[00048] Examples of representative CSFRl inhibitors are shown in Table 1 below: [00049] Table 1.

Novartis CSFR1 Small molecule Advanced Phase 1 Likely Oral yes

Synovitis [00050] In some embodiments, the CSFRl inhibitor is a nucleic acid that inhibits expression of CSFRl in microglia. In some embodiments, the nucleic acid is a micro RNA (miRNA) or small interfering RNA (siRNA) that inhibits expression of CSFRl in microglia. In some

embodiments, the nucleic acid is administered by injection into the eye, for example, intravitreal injection.

PHARMACEUTICAL COMPOSITIONS

[00051] In some embodiments, the CSFRl inhibitors described herein are administered as part of a pharmaceutical composition. The pharmaceutical composition can contain a

pharmaceutically acceptable carrier or adjuvant. In some embodiments, the carrier is

pharmaceutically acceptable for use in humans. The carrier or adjuvant should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, ammo acid copolymers and inactive virus particles.

[00052] Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonate and benzoates.

[00053] Pharmaceutically acceptable carriers in therapeutic compositions can additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, can be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient.

[00054] The compositions of the presently disclosed subject matter can further comprise a carrier to facilitate composition preparation and administration. Any suitable delivery vehicle or carrier can be used, including but not limited to a microcapsule, for example a microsphere or a nanosphere (Manome et al. 1994; Saltzman & Fung et al. 1997), a glycosaminoglycan (U.S. Pat. No. 6, 106,866), a fatty acid (U.S. Pat. No. 5,994,392), a fatty emulsion (U.S. Pat. No.

5,651,991), a lipid or lipid derivative (U.S. Pat. No. 5,786,387), collagen (U.S. Pat. No. 5,922,356), a polysaccharide or derivative thereof (U.S. Pat. No. 5,688,931), a nanosuspension (U.S. Pat. No. 5,858,410), a polymeric micelle or conjugate (Goldman et al. 1997 and U.S. Pat. Nos. 4,551,482, 5,714, 166, 5,510, 103, 5,490,840, and 5,855,900), and a polysome (U.S. Pat. No. 5,922,545).

[00055] Antibody sequences can be coupled to active agents or carriers using methods known in the art, including but not limited to carbodiimide conjugation, esterification, sodium periodate oxidation followed by reductive alkylation, and glutaraldehyde crosslinking (Goldman et al. 1997; Cheng 1996; Neri et al. 1997; Nabel 1997; Park et al. 1997; Pasqualini et al. 1997;

Bauminger & Wilchek 1980; U.S. Pat. No. 6,071,890; and European Patent No. 0 439 095).

[00056] A therapeutic composition described herein comprises in some embodiments a pharmaceutical composition that includes a pharmaceutically acceptable carrier. Suitable formulations include aqueous and non-aqueous sterile injection solutions which can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics and solutes which render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze -dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example water for injections, immediately prior to use. Some exemplary ingredients are SDS in the range of in some embodiments 0.1 to 10 mg/ml, in some embodiments about 2.0 mg/ml; and/or mannitol or another sugar in the range of in some embodiments 10 to 100 mg/ml, in some embodiments about 30 mg/ml; and/or phosphate -buffered saline (PBS). Any other agents conventional in the art having regard to the type of formulation in question can be used. In some embodiments, the carrier is pharmaceutically acceptable. In some embodiments, the carrier is pharmaceutically acceptable for use in humans.

[00057] Pharmaceutical compositions of the present disclosure can have a pH between 5.5 and 8.5, preferably between 6 and 8, and more preferably about 7. The pH can be maintained by the use of a buffer. The composition can be sterile and/or pyrogen free. The composition can be isotonic with respect to humans. Pharmaceutical compositions of the presently disclosed subject matter can be supplied in hermetically-sealed containers. [00058] Pharmaceutical compositions can include an effective amount of one or more antibodies as described herein. In some embodiments, a pharmaceutical composition can comprise an amount that is sufficient to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic effect. Therapeutic effects also include reduction in physical symptoms. The precise effective amount for any particular subject will depend upon their size and health, the nature and extent of the condition, and therapeutics or combination of therapeutics selected for administration. The effective amount for a given situation is determined by routine experimentation as practiced by one of ordinary skill in the art.

TREATMENT REGIMENS: PHARMACOKINETICS

[00059] The pharmaceutical compositions described herein can be administered in a variety of unit dosage forms depending upon the method of administration. Dosages for typical

pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisory in nature and are adjusted depending on the particular therapeutic context or patient tolerance. The amount of CSFRl inhibitor described herein adequate to accomplish this is defined as a "therapeutically effective dose." The dosage schedule and amounts effective for this use, i.e., the "dosing regimen," will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, pharmaceutical formulation and concentration of active agent, and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration. The dosage regimen must also take into consideration the pharmacokinetics, i.e., the pharmaceutical composition's rate of absorption, bioavailability, metabolism, clearance, and the like. See, e.g., Remington's Pharmaceutical Sciences, 1976; Egleton, 1997; Langer, 1990, which are incorporated by reference herein.

[00060] In some embodiments, the pharmaceutical compositions described herein are administered in therapeutically effective amounts for periods of time effective to treat a disorder of the eye described herein. One of ordinary skill in the art can determine the effective amount of small molecule compound drugs. Examples of dosage amounts for a mammal include from about 0.5 to about 200 mg of active compound per kilogram of body weight, about 0.5 to about 150 mg/kg, about 0.5 to 100 mg/kg, about 0.5 to about 75 mg/kg, about 0.5 to about 50 mg/kg, about 0.01 to about 50 mg/kg, about 0.05 to about 25 mg/kg, about 0.1 to about 25 mg/kg, about 0.5 to about 25 mg/kg, about 1 to about 20 mg/kg, about 1 to about 10 mg of active compound per kg of body weight, or any range therein. In some embodiments, the active dose is about 20 mg/kg, about 10 mg/kg, about 5 mg/kg, about 2.5 mg/kg, about 1.0 mg/kg, or about 0.5 mg of active compound per kg of body weight. The compositions described herein can be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day, or once every 2 days, 3 days, 4 days, 5 days, 6 days, weekly, or monthly. The compositions described herein can also be administered for various treatment cycles, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 treatment cycles. In some embodiments, the small molecule compound is administered to a subject at a dose of up to 1 g/day.

[00061] A therapeutically effective amount of a composition comprising an anti-CSFRl antibody, contains about 0.05 to 1500 μg protein, about 10 to 1000 μg protein, about 30 to 500 μg and about 40 to 300 μg, or any integer between these values. For example, antibodies described herein can be administered to a subject at a dose of about 0.1 μg to about 200 mg, e.g., from about 0.1 μg to about 5 μg, from about 5 μg to about 10 μg, from about 10 μg to about 25 μg, from about 25 μg to about 50 μg, from about 50 μg to about 100 μg, from about 100 μg to about 500 μg, from about 500 μg to about 1 mg, or from about 1 mg to about 2 mg. It is understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific antibody employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

[00062] Routes of administration include, but are not limited to, oral, topical, subcutaneous, intramuscular, intravenous, intradermal, transdermal and subdermal. In some embodiments, the CSFR1 inhibitors described herein are administered by intravitreal injection, e.g., of a therapeutic compound that does not readily cross the blood-brain or blood-eye barrier. In some embodiments, the CSFR1 inhibitor is an anti-CSFRl antibody described herein and is administered via intravitreal injection. Depending on the route of administration, the volume per dose is preferably about 0.001 to 10 ml, more preferably about 0.01 to 5 ml, and most preferably about 0.1 to 3 ml. Compositions can be administered in a single dose treatment or in multiple dose treatments on a schedule and over a time period appropriate to the age, weight and condition of the subject, the particular antibody formulation used, and the route of administration.

[00063] Further, doses of drugs administered to animals can be converted to equivalent doses for humans based on the body surface area (BSA) (represented in mg/m ) normalization method (see, e.g., Reagan-Shaw, S. et al. 2007; and U.S. Department of Health and Human Services 2005; which are incorporated by reference herein). For example, the human equivalent dose (HED) based on BSA can be calculated by the following formula I:

0 33

[00064] I. HED = animal dose in mg/kg x (animal weight in kg/human weight in kg)

[00065] Alternatively, the HED can be determined by the following formula II:

[00066] II. HED (mg/kg) = animal dose (mg/kg) x (animal K_m/human K_m)

[00067] The Km factor is determined based on the following Table (see Guidance for Industry, Id.):

[00068] Table 2: Conversion of Animal Doses to Human Equivalent Doses Based on Body Surface Area

Assumes 60 kg human.

[00069] Thus, a 5 mg/kg dose in mice is equivalent to a 0.4 mg/kg dose in a 60 kg human. A 0.4 mg/ml dose in a 60 kg human is equivalent to a dose of 14.8 mg/m .

KITS

[00070] Also provided are kits comprising the CSFRl inhibitors described herein which can be used, for instance, for therapeutic applications described above. The article of manufacture comprises a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition which includes an active agent that is effective for therapeutic applications, such as described above. The active agent in the composition can comprise the CSFRl inhibitors described herein. The label on the container indicates that the composition is used for a particular therapy or non-therapeutic application, and can also indicate directions for either in vivo or in vitro use, such as those described above.

ANTI-VEGF THERAPY

[00071] Abnormal overproduction of vascular endothelial growth factor (VEGF) in the eye is associated with vision loss in AMD. In the so-called "wet" or "neovascular" form of AMD, new blood vessels grow into or under the retina, and these vessels depend on VEGF for their growth and proliferation. These vessels are fragile and leak fluids, and their proliferation and associated fluid buildup lead to macular damage and increasing loss of vision in the central visual field. Anti-VEGF antibody therapy is currently used to treat wet AMD. Anti-VEGF antibodies are administered by injection into the eye up to every four weeks. Examples of anti-VEGF antibodies that are effective at treating AMD include Lucentis® (ranibizumab), Avastin® (bevacizumab), antibody fragments that bind to the VEGF protein, and Eylea® (aflibercept). Thus, in some embodiments, the methods described herein can be combined with anti-VEGF therapies for treating eye disorders associated with activated microglia and/or inflammation.

EXAMPLES

EXAMPLE 1.

[00072] This example demonstrates that the CSFR1 inhibitor PLX3397 eliminates retinal microglia in a mouse model of retinal microgliosis.

[00073] Our laboratory has demonstrated that deficiency in the lysolipid transporter Mfsd2a resulted in severe microgliosis in the retina and pathological changes in retinal anatomy (e.g. collapsed microvilli of retinal pigment epithelium, and disorganized outer rod segments of photoreceptor cells) (Wong B et al. 2016). Based on these findings, we tested if treating Mfsd2a KO mice with PLX3397 resulted in reduced microgliosis. Wild-type and Mfsd2a KO mice were weaned at the age of 4 weeks onto a diet containing PLX3397 for a total of 3 weeks of treatment (see Materials and Methods below). Microglia were effectively eliminated in both wild-type and Mfsd2a KO mice (Figure 1A). Moreover, the collapsed microvili seen in Mfsd2a KO mice as visualized by the apical microvilli marker ezrin is restored to an elongated morphology similar to wild-type mice. This is the first evidence that indicates that PLX3397 can eliminate retinal microglia and have beneficial effects on restoring retinal pigment epithelial anatomy in Mfsd2a KO mice, a model for microgliosis.

MATERIALS AND METHODS

[00074] Microglia depletion with PLX3397:

[00075] This protocol is based on Elmore M.R., et al. 2014.

[00076] Drug: Pexidartinib (PLX3397), 99% Cat#: 206178 from MedKoo

[00077] Dose: 290mg/kg of chow, mice treated upon weaning, treated for 21 days

[00078] Chow diet: Global 18% Protein Rodent Diet from Harlan, Envigo

[00079] Gender: Male and Female

[00080] Age: 6 weeks old (Treatment started at 3 weeks of age)

[00081] Antibodies: The following antibodies were used in these studies: Ezrin (Cat#: ab4069, Abeam), lba-1 (Cat#: 019-19741, Wako).

[00082] Animals:

[00083] Wild-type mice used in the experiments were C57BL6/NTac. Mfsd2a Knockout (KO) mice were generated as described previously (Berger et al, 2012). Pups were weaned at 3 weeks of age onto chow diet (Global 18% Protein Rodent Diet from Harlan, Envigo) or PLX3397- coated chow diet, and maintained on the same diet for 3 weeks. Both male and female mice were used in all experiments. Mice were anaesthetized with a combination of ketamine (20mg/kg body weight) and xylazine (2mg/kg body weight) for terminal experiment. Experimental protocols were approved by SingHealth Institutional Animal Care and Use Committee.

[00084] Preparation of PLX3387 diet:

[00085] 290 mg/kg PLX3397 (Pexidartinib, 99%, MedKoo, Cat#: 206178) were incorporated into chow diet. 128.3 mg of PLX3397 powder, dissolved in 500 ml 100% ethanol, was sprayed directly onto 420 g of chow diet (Global 18% Protein Rodent Diet from Harlan, Envigo) that was spread out on aluminum foil, and the ethanol was allowed to evaporate in a biosafety hood. Food pellets were allowed to dry overnight in the biosafety hood. [00086] Histological Studies:

[00087] For immunofluorescence staining, 6 week old wild-type and Mfsd2a KO mice were deeply anaesthetized and eyes were enucleated. Eyes were transferred to a small petri dish containing 4% paraformaldehyde (PFA), where an incision was made at the cornea with a sharp blade before cuts were made along the ora serrata. Cornea, lens and optic nerve were removed, leaving behind eye cups. Eye cups were transferred to a 2 ml tube containing 4% PFA and allowed to fix at room temperature (RT) for 1-2 hours. Eye cups were rinsed briefly in PBS, and dehydrated in 10% sucrose for 1 hour at RT, 20% sucrose for 2-3 hours at RT and eventually in 30% sucrose at 4 °C overnight. Eyes were embedded in 1: 1 of 30% sucrose:Tissue-Tek O.C.T. Compound (Sakura, USA), and allowed to freeze slowly on dry ice. 12 μπι cryosections were prepared using a cryostat (CM 1520, Leica) from frozen blocks and processed for

immunocytochemistry procedure using antibodies against Ezrin (1:50), and lba-1 (1 : 100) overnight at 4 degrees and incubated for 1 min in 4', 6-diaminodino-2-phenylindole (DAPI, 1: 1000) before washing and mounting. Images were acquired on Zeiss LSM 710 upright fluorescence microscope (Carl Zeiss, Singapore).

[00088] As shown in Fig. 1, treatment of Mfsd2a KO mice with PLX3387 eliminated microglia from the retina and restored the retinal anatomy. Therefore, this Example demonstrates that treating an animal model of an eye disease with CSFRl inhibitors reduced symptoms associated with microgliosis.

EXAMPLE 2.

MATERIALS AND METHODS:

Mice models

[00089] Mice models purchased from The Jackson's Laboratory, USA:

1. Cx3crl^GFP/Cx3crl^GFP mice: B6.129P-Cx3crltmlLitt/J (Stock No: 005582)

2. RdlO/RdlO mice: B6.CXB 1-Pde6brdl0/J (Stock No: 004297) - Retinitis pigmentosa model.

[00090] Cx3crl^GFP/Cx3crl^GFP mice were first crossed to RdlO/RdlO mice to obtain

Cx3crl^or7+, Rdl0/+ mice. Cx3crl^or7+, Rdl0/+ mice were then intercrossed to obtain

Cx3crl^or7 Cx3crl , RdlO/RdlO mice. Finally, RdlO/RdlO mice were then crossed with Cx3crr 7 Cx3crl , RdlO/RdlO mice to generate Cx3crr 7+, RdlO/RdlO for use in these experiments.

PLX3397 treatment by oral gavage in Cx3crl^GFP/+ mice

[00091] Based on a dose of lg a day for humans (ClinicalTrials.gov Identifier: NCT02371369, NCTO 1349049), an equivalent dose was calculated for mice based on the formula described by Nair and Jacob (Nair and Jacob, 2016). With a Human Equivalent Dose (HED) of 16.67mg/kg, to calculate Animal Equivalent Dose (AED), multiply HED with 12.3 (K_m ratio, provided on Table 1 in Nair and Jacob, 2016) for an AED of 205mg/kg (AED = HED x K_m ratio). To prepare PLX3397 (Pexidartinib, 99%, MedKoo, Cat#: 206178) for oral gavages, the drug was first dissolved in chloroform. 4μΜ lysophosphotidylcholme 18:0 (LPC18:0) in chloroform was added (10 times above critical micelle concentration) to improve the solubility of PLX3397 in water. Aliquots of the PLX3397-LPC18:0 mix were dried down under nitrogen and stored at -20°C till use. Just before PLX3397 treatment in mice, sterile water was added to the dried PLX3397- LPC18:0 mix, and sonicated at 60V in an ice bath with alternating intervals of 30 seconds on and 10 seconds off for 30 minutes. Treatment conditions were as follows: Cx3crl^GFP/+ mice received a daily gavage of 205mg/kg of PLX3397 for 3 weeks, while control Cx3crl^GFP/+ mice received 4μΜ LPC18:0 alone for 3 weeks before eyes were harvested for histology. To test if microglia is able to repopulate following drug cessation, Cx3crl^GFP/+ mice received a daily gavage of 205mg/kg of PLX3397 for 3 weeks before eyes were harvested a week later for histology.

PLX3397 treatment by intraperitoneal injections from postnatal day 13 to 24

[00092] PLX3397 and LPC18:0 for intraperitoneal injections were prepared as above for oral gavages. Cx3crl^or7+, RdlO/RdlO mice were treated with either 4μΜ LPC18:0 or 205mg/kg PLX3397 by daily intraperitoneal injections from postnatal day 13 to 24 and eyes were harvested for histology on postnatal day 25.

PLX3397 treatment by ad libitum feeding in wild-type or Mfsd2a KO mice

[00093] To prepare PLX3387 chow diet, 290 mg/kg PLX3397 (Pexidartinib, 99%, MedKoo, Cat#: 206178) was incorporated into chow diet. 128.3 mg of PLX3397 powder was dissolved in 500 ml 100% ethanol, poured over 420 g of chow diet (Global 18% Protein Rodent Diet from Harlan, Envigo) that was spread out on aluminum foil. Chow pellets were allowed to soak in PLX3397 and the ethanol was allowed to evaporate in a biosafety hood. Chow pellets were allowed to dry overnight in the biosafety hood. 3 -week old wild-type or Mfsd2a KO mice were fed PLX3397 chow or control chow for 3 weeks before eyes were harvested for gene microarray analysis.

Tissue preparation for microarray and pathway analysis

[00094] To prepare tissues for gene microarray, control or PLX3397 treated wild-type and Mfsd2a KO mice were deeply anesthetized, and eyes were enucleated. Eyes were transferred to a small Petri dish containing cold PBS, where an incision was made at the cornea with a sharp blade before cuts were made along the ora serrata. Cornea, lens and optic nerve were removed, leaving behind eye cups. Excess PBS was drained with Kimwipes and tissues were flash-frozen immediately in liquid nitrogen. Eye cups were ground with a cell pellet homogenizer (VWR) on ice in TRIzol (Roche Applied Science). RNA was extracted and purified with RNeasy mini kit (Qiagen) and quantified by Nanodrop. Equal amounts of RNA from six eye cups were pooled for each genotype or treatment group, and RNA integrity was verified using Bioanalyzer (Agilent Technologies). Microarray profiling was done on pooled samples with an RNA integrity cutoff of 7.0 using Mouse 430 2.0 arrays (Affymetrix). Filtered gene list (genes with >50 expression in KO or WT and fold-change >1.25 or <0.75) were used to identify significantly altered canonical pathways and predicted activation or inhibition of upstream inhibitors of Mfsd2a and how that changes with PLX3397 treatment. MA plots, which are used to visualize intensity-dependent ratio of raw gene microarray data, were used to represent the data.

Histological studies

[00095] For images in Figs. 3A and 3B, Fig. 4A and Fig. 5A, control or PLX3397 treated Cx3crlGFP/+, RdlO/RdlO mice were deeply anaesthetized and eyes were enucleated. Eyes were transferred to a small petri dish containing 4% paraformaldehyde (PFA), where an incision was made at the cornea with a sharp blade before cuts were made along the ora serrata. Cornea, lens and optic nerve were removed, leaving behind eye cups. Eye cups were transferred to a 2 ml tube containing 4% PFA and allowed to fix at room temperature for 1-2 hours. Eye cups were rinsed briefly in PBS, and dehydrated in 10% sucrose for 1 hour at room temperature, 20% sucrose for 2-3 hours at RT and eventually in 30% sucrose at 4 °C overnight. Eyes were embedded in 1: 1 of 30% sucrose:Tissue-Tek O.C.T. Compound (Sakura, USA), and allowed to freeze slowly on dry ice. 12-μπι cryosections were prepared using a cryostat (CM1520, Leica) from frozen blocks and where applicable, incubated for 1 min in Hoechst (1 : 1000) before washing and mounting. Images were acquired on Zeiss LSM 710 upright fluorescence microscope (Carl Zeiss, Singapore).

[00096] For hematoxylin and eosin (H&E) staining, control or PLX3397 treated P25

Cx3crl^or7+, RdlO/RdlO mice, were deeply anesthetized as before. Eyes were enucleated and placed immediately in Perfix solution (4% paraformaldehyde, 20% isopropyl alcohol, 2% trichloroacetic acid, 2% zinc chloride) at room temperature for 48 h. Whole eyes were dehydrated and processed using increasing concentrations of ethanol and xylene and embedded in paraffin. 5-μπι sections were prepared using a microtome (RM2255, Leica), and H&E staining was performed. Images were taken on a DMi8 fluorescent microscope (Leica).

[00097] References:

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Kitazawa M, Matusow B, Nguyen H, West BL, Green KN (2014). Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron. 2014 Apr 16;82(2):380-97.

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[00098] 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. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method for reducing microglial activation in the eye, comprising administering to a subject in need thereof an effective amount of a CSFRl inhibitor.

2. The method of claim 1, wherein the CSFRl inhibitor is a small molecule or antibody.

3. The method of claim 2, wherein the CSFRl inhibitor is a small molecule selected from those shown in Table 1.

4. The method of claim 3, wherein the CSFRl inhibitor is Pexidartinib

(PLX3397).

5. The method of claim 2, wherein the CSFRl inhibitor is an antibody selected from those shown in Table 1.

6. The method of claim 2, wherein the CSFRl inhibitor is a humanized or chimeric antibody.

7. The method of claim 1, wherein the administering comprises contacting retinal tissue of the eye with the inhibitor.

8. A method for treating inflammation associated with an eye disease, comprising administering to a subject in need thereof an effective amount of a CSFRl inhibitor.

9. The method of claim 8, wherein the eye disease is selected from age- related macular degeneration (AMD), retinitis pigmentosa, or diabetic retinopathy.

10. The method of claims 1-9, further comprising administering an effective amount of an anti-VEGF antibody to the subject.

11. A composition comprising an effective amount of a CSFRl inhibitor for use in a method for reducing microglial activation in the eye, the method comprising administering the composition to a subject in need thereof.

12. The composition of claim 11, wherein the CSFRl inhibitor is a small molecule or antibody.

13. The composition of claim 12, wherein the CSFRl inhibitor is a small molecule selected from those shown in Table 1.

14. The composition of claim 12, wherein the CSFRl inhibitor is Pexidartinib

(PLX3397).

15. The composition of claim 12, wherein the CSFRl inhibitor is an antibody selected from those shown in Table 1.

16. The composition of claim 15, wherein the CSFRl inhibitor is a humanized or chimeric antibody.

17. The composition of claim 11, wherein the method comprises contacting retinal tissue of the eye with an effective amount of the CSFRl inhibitor.

18. The composition of claim 11, further comprising an effective amount of an anti-VEGF antibody for use in a method for reducing microglial activation in the eye.

19. Use of an effective amount of a CSFRl inhibitor in the manufacture of a medicament for treating inflammation associated with an eye disease.

20. The use of claim 18, wherein the eye disease is selected from age-related macular degeneration (AMD), retinitis pigmentosa, or diabetic retinopathy.