WO2009051992A1

WO2009051992A1 - Compositions and methods for treating diseases involving ocular angiogenesis by inhibiting one or more selected receptor tyrosine kinases

Info

Publication number: WO2009051992A1
Application number: PCT/US2008/079134
Authority: WO
Inventors: Keith W. Ward
Original assignee: Bausch & Lomb Incorporated
Priority date: 2007-10-19
Filing date: 2008-10-08
Publication date: 2009-04-23

Abstract

Compositions and methods are provided for the treatment of diseases involving ocular angiogenesis. The compositions and methods provide treatments for ocular angiogenic diseases such as diabetic macula edema, age-related macular degeneration, and diabetic retinopathy, by inhibiting one or more selected receptor tyrosine kinases, such as by inhibiting recepteur d'origine nantais (RON) or by inhibiting selected combinations of receptor tyrosine kinases such as RON, c-Met, Tie-2 and/or one or more VEGF receptors.

Description

COMPOSITIONS AND METHODS FOR TREATING DISEASES INVOLVING

OCULAR ANGIOGENESIS BY INHIBITING ONE OR MORE SELECTED

RECEPTOR TYROSINE KINASES

FIELD OF THE INVENTION

The technical field involves the treatment of diseases involving ocular angiogenesis. The present invention relates to compositions and methods for the treatment of ocular angiogenic diseases by inhibiting one or more selected receptor tyrosine kinases, such as by inhibiting recepteur d'origine nantais (RON) or by inhibiting selected combinations of receptor tyrosine kinases such as RON, c-Met, Tie-2 and/or one or more VEGF receptors.

BACKGROUND OF THE INVENTION

The eyes of humans and other mammals include various types of ocular tissues, such as the aqueous humor, cornea, iris, ciliary body, vitreous humor, retina, choroid, and sclera. Various ocular tissues are vascularized to different extents, with some ocular tissues having little or no vascularization, which refers to the presence of blood vessels in the tissue. For example the cornea normally is not vascularized. Other ocular tissues, such as the retina, include some limited degree of vascularization, and the blood vessels supply various components to tissue and the eye. There are many diseases that cause inappropriate growth or formation of blood vessels in the eye. Some of these diseases tend to be localized in the eye, while others are or involve diseases that affect other organs as well.

One of the most common causes of blindness is inappropriate angiogenesis (growth or formation of blood vessels) in the eye, particularly in the retina, choroid or cornea. Inappropriate angiogenesis, which includes neovascularization and vascular permeability, are major causes for many ocular disorders including age-related macular degeneration (AMD), retinopathy of prematurity (ROP), sickle cell retinopathy, ischemic retinal vein occlusions and diabetic retinopathy, including proliferative diabetic retinopathy. Age-related macular degeneration and diabetic retinopathy are among the most common causes of severe, irreversible vision loss. In these and other diseases involving ocular angiogenesis, central vision loss is secondary to the development of new blood vessels from pre-existing vasculature, and alterations in vascular permeability properties.

Macular degeneration involves a gradual loss or impairment of eyesight due to cell and tissue degeneration of the yellow macular region in the center of the retina. Macular degeneration is often characterized as one of two types, non-exudative (dry form) or exudative (wet form). Although both types tend to impact both eyes and to grow progressively worse, each type may reflect different pathological processes. The wet form of age-related macular degeneration (AMD) is the most common form of choroidal neovascularization and a leading cause of blindness in the elderly. AMD affects millions of Americans over the age of 60, and is the leading cause of blindness among the elderly.

Choroidal neovascularization (CNV) refers to the sprouting of abnormal blood vessels that develop into a cluster under the macula. These new blood vessels tend to break, bleed, and leak fluid under the macula, causing it to lift up and pull away from its base. This causes damage to the photoreceptor cells which sense and receive light. Damage to the photoreceptor cells can result in a rapid and severe loss of central vision. There are several treatments currently used by clinicians for treating wet AMD, including the use of anti-angiogenic drugs, photodynamic therapy, macular translocation surgery, and thermal laser photocoagulation.

Photodynamic therapy (PDT) has been approved for AMD treatment by the U.S. Food and Drug Administration (FDA). PDT employs the intravenous administration of a photosensitive drug followed by the application of a low-dose, nonthermal (light only) laser to the affected area of the retina. The drug circulates throughout the body's blood vessels, and is particularly attracted to new blood vessels, including the abnormal vessels under the macula. The laser activates the drug at selected locations, which selectively seals off the leaking blood vessels without damaging the surrounding healthy retinal tissue. This feature allows PDT to be used directly beneath the center of the macula, unlike thermal (heat) laser photocoagulation, which can burn and destroy normal retinal tissue. Macular translocation is a surgical procedure that involves detaching the retina from its base, rotating it slightly, and replacing it in a different position, so that the macula sits on a new, healthy base.

Thermal laser photocoagulation is used by retinal surgeons to treat a number of eye conditions, including the exudative form of macular degeneration. A thermal (heat) laser is directed into the eye at abnormal blood vessels growing beneath the retina. The heat from the laser closes off the unwanted blood vessels, preventing further leakage and vision loss. Thermal laser photocoagulation does not restore lost vision, and it can also destroy surrounding healthy retinal tissue as it seals the leakage from abnormal blood vessel growth.

One anti-angiogenic drug approved for treating wet AMD is pegaptanib sodium (MACUGEN^®). This is a selective Vascular Endothelial Growth Factor (VEGF) antagonist which binds to VEGF. Pegaptanib sodium blocks VEGF binding to receptors and prevents the growth of abnormal new vessels and prevents leakage of fluid and blood in the retina. MACUGEN^® has been approved by the FDA for use in wet macular degeneration. The recommended dose of MACUGEN^® is 0.3 mg of intravitreous injection administered once every 6 weeks. Another anti-angiogenic drug is ranibizumab (LUCENTIS^®), which has also been approved by the FDA for treatment of wet AMD. LUCENTIS is administered once every four weeks.

Vascular endothelial growth factor (VEGF) is involved in angiogenesis (the growth of blood vessels from pre-existing vessels). As its name implies, VEGF activity has been found in connection with cells of the vascular endothelium, although it does have effects on other cells. VEGF has been shown in vitro to stimulate the proliferation and migration of endothelial cells. VEGF has been widely studied and discussed as having a key role in the regulation of normal and abnormal angiogenesis. VEGF actually refers to several isoforms that are splice variants encoded by the same gene. Various VEGF isoforms bind with high affinity to two different receptors, VEGFRl and VEGFR2 (KDR), which are selectively expressed on endothelial cells. VEGFRl and VEGFR2 are members of the Type III receptor tyrosine kinase (RTK III) family.

Protein kinases are enzymes that catalyze the addition of one or more phosphate groups to proteins, which is called phosphorylation. Protein kinases catalyze the addition of phosphate to polar groups of amino acids, particularly to tyrosine, serine and threonine residues of proteins. Many protein enzymes are switched "on" or activated by phosphorylation. Phosphorylation is involved in cell differentiation, cell proliferation, and many other aspects of cell life. Abnormal protein kinase activity has been related to many diseases, including various cancers.

Protein kinases can be categorized as receptor type or non-receptor type. Receptor-type protein kinases have an extracellular, a transmembrane, and an intracellular portion, while non-receptor type protein kinases are wholly intracellular. Protein kinases can also be categorized based on amino acids for which they catalyze phosphorylation. For example, tyrosine kinases are enzymes that transfer a phosphate group to a tyrosine residue in a protein, while serine/threonine kinases phosphorylate serine or threonine residues. Some kinases have mixed kinase activities and will phosphorylate serine, threonine and tyrosine.

Receptor tyrosine kinases (RTK) are transmembrane cell receptors that have tyrosine kinase activity. There are a large number of RTKs with diverse biological activity. In fact, about 20 different subfamilies of receptor-type tyrosine kinases have been identified. See Ibrahim et al., U.S. Patent App. Pub. 20070161651. One tyrosine kinase subfamily, designated the HER subfamily, is comprised of EGFR (HERl), HER2, HER3, and HER4. Ligands of this subfamily of receptors include epithelial growth factor, TGF-alpha, amphiregulin, HB-EGF, betacellulin and heregulin. Another subfamily of these receptor tyrosine kinases is the insulin subfamily, which includes rNS-R, IGF-IR, and IR-R. The PDGF subfamily includes the PDGF-alpha and beta receptors, CSFIR, c-Kit and FLK-II. Another family of receptor tyrosine kinases is the FLK family, which is comprised of the kinase insert domain receptor (KDR), fetal liver kinase-1 (FLK-I), fetal liver kinase-4 (FLK-4) and the fms-like tyrosine kinase-1 (flt-1).

There are also a large number of non-receptor types of tyrosine kinases. They can also be grouped in numerous subfamilies, such as Src, Frk, Btk, Csk, AbI, Zap70, Fes/Fps, Fak, Jak, Ack, and LIMK. Ibrahim et al., U.S. Patent App. Pub. 20070161651. Each of these subfamilies can be further sub-divided into varying receptors. For example, the Src subfamily is one of the largest and includes Src, Yes, Fyn, Lyn, Lck, BIk, Hck, Fgr, and Yrk. BRIEF SUMMARY OF THE INVENTION

The present invention contemplates that inhibiting an activity of one or more selected receptor tyrosine kinases, or a combination of selected receptor tyrosine kinases, would provide an effective and beneficial method for treatment (including prevention) of ocular angiogenesis.

As one aspect of the present invention, compositions are provided for the treatment of an ocular angiogenic disease, in which the compositions comprise an inhibitor of a selected receptor tyrosine kinase, for example an inhibitor of RON. In some embodiments of the present invention, the inhibitor is a selective inhibitor for RON, or the inhibitor is effective for inhibiting RON exclusively or predominantly. In other embodiments, the inhibitor is also effective for inhibiting the activity of one or more other receptor tyrosine kinases. In some embodiments, the composition includes a second receptor tyrosine kinase inhibitor, for example an inhibitor of the activity of one or more other receptor tyrosine kinases, such as c-Met, Tie-2 or one or more VEGF receptors.

As another aspect of the present invention, compositions are provided for the treatment of an ocular angiogenic disease, in which the compositions comprise an inhibitor of a selected receptor tyrosine kinase, for example an inhibitor of c-Met. In some embodiments of the present invention, the inhibitor is a selective inhibitor for c- Met, or the inhibitor is effective for inhibiting c-Met exclusively or predominantly. In other embodiments, the inhibitor is also effective for inhibiting the activity of one or more other receptor tyrosine kinases. In some embodiments, the composition includes a second receptor tyrosine kinase inhibitor, for example an inhibitor of the activity of one or more other receptor tyrosine kinases, such as RON, Tie-2 or one or more VEGF receptors.

The present invention relates to compositions useful in treating of ocular angiogenic diseases, and different types of compositions are provided for different routes of administration to a subject in need of treatment. Among the preferred embodiments of the present compositions are ophthalmic pharmaceutical compositions suitable for administration to the eye of a subject, such as injectable compositions for intraocular administration or local ocular administration. The ophthalmic pharmaceutical compositions can include an ophthalmically acceptable excipient. The ophthalmic pharmaceutical compositions can be suspensions or isotonic solutions formulated for ophthalmic administration.

As yet another aspect of the present invention, methods are provided for treatment of diseases involving ocular angiogenesis. The methods comprise administering to a subject having or at risk of ocular angiogenesis and/or in need of treatment of ocular angiogenesis, a receptor tyrosine kinase inhibitor such as an inhibitor of RON, or an inhibitor of c-MET, or an inhibitor of RON and/or c-MET and one or more other receptor tyrosine kinases. The methods comprise administering the inhibitor for a time and in an amount effective for prevention or treatment of ocular angiogenesis. The present methods can include administering the inhibitor in any fashion effective for the treatment of the ophthalmic infection, including for example, administering the inhibitor systemically or locally to the subject. hi some embodiments of the present methods, the method of treating ocular angiogenic disease comprises administering to a subject a receptor tyrosine kinase inhibitor that inhibits the activity of RON, wherein the inhibitor is administered for a time and in an amount effective to reduce a risk of ocular angiogenesis. hi one embodiment, the method comprises inhibiting the activity of c-MET, either with the same inhibitor that inhibits RON or by administering a second tyrosine kinase inhibitor, wherein the second receptor tyrosine kinase inhibitor is effective to inhibit the activity of c-Met. In another embodiment, the method comprises inhibiting the activity of one or more other receptor tyrosine kinases, for example Tie-2 or one or more of the VEGF receptors, either with the same inhibitor that inhibits RON or c-Met, or by administering a second, third or even more receptor tyrosine kinase inhibitors, wherein those receptor tyrosine kinase inhibitor(s) are effective to inhibit the activity of other receptor tyrosine kinases. In various embodiments, the methods can include administering a receptor tyrosine kinase inhibitor specifically inhibits RON; or inhibits both c-MET and RON; or inhibits RON and c-MET and at least one other receptor tyrosine kinase; or inhibits RON and c-MET and one or more VEGF receptors; or inhibits RON and c-MET and Tie-2; or inhibits c-MET and one or more VEGF receptors; or inhibits c-MET and Tie-2; or inhibits RON and one or more VEGF receptors; or inhibits RON and Tie-2. The present methods are particularly contemplated for treating (including preventative treatment) of ocular angiogenic diseases such as diabetic macula edema, age-related macular degeneration, and diabetic retinopathy. The methods may include administering to the subject as a co-therapy a compound selected from the group consisting of an additional receptor tyrosine kinase inhibitor, pegaptanib sodium, ranibizumab, and photodynamic therapy. The methods may also include additional steps such as diagnosing a subject at risk of an ocular angiogenic disease (such as diabetic macula edema, age-related macular degeneration, or diabetic retinopathy), and administering the inhibitor before the onset of ocular angiogenesis.

The present compositions and methods can also include an active agent in addition to the receptor tyrosine kinase inhibitor. For example, the present compositions can include an anti-VEGF agent in addition to an inhibitor of RON or an inhibitor of c- MET or both. The compositions can include an excipient such as salts, buffers, surfactants, polymers and acids or bases. In an ophthalmic pharmaceutical composition, an excipient is preferably selected from the group consisting of ophthalmically acceptable salts, ophthalmically acceptable buffers, ophthalmically acceptable surfactants, and ophthalmically acceptable polymers.

DETAILED DESCRIPTION OF THE INVENTION

Angiogenesis refers to the processes whereby new blood vessels are formed. Angiogenesis is sometimes referred to as neovascularization. Formation of new blood vessels is normal during embryogenesis and development, and angiogenesis also occurs in mature organisms as a part of healing. However, angiogenesis also occurs in various pathological conditions, including in ocular diseases such as diabetic retinopathy and macular degeneration. Angiogenesis can occur when endothelial cells that line blood vessels are induced to proliferate. Various growth factors and cytokines can induce endothelial cells to become active and begin to proliferate and migrate into unvascularized tissue, and to form new blood vessels. In addition to growth factors, angiogenesis depends on interaction of the endothelial cells with the extracellular matrix and with each other. The activation of endothelial cells by growth factors and the migration into and interaction with the extracellular matrix and with each other is dependent on cell surface receptors expressed by the endothelial cells. These cell surface receptors, which include growth factor receptors and integrins, interact specifically with particular molecules.

The present invention provides novel compositions and methods for treating or preventing diseases involving ocular angiogenesis by inhibiting one or more selected receptor tyrosine kinases. As described herein, ocular angiogenesis is prevented or treated by inhibiting certain receptor tyrosine kinases so as to reduce or mitigate ocular angiogenesis. The present invention relates to inhibiting particular or selected receptor tyrosine kinases, such as inhibiting the activity of RON. The present invention also relates to the use of one or more inhibitors that inhibit particular combinations of receptor tyrosine kinases, such as inhibiting the activity of both c-MET and RON, or inhibiting RON and c-MET and at least one other receptor tyrosine kinase. For example, the present invention contemplates that it is desirable to inhibit RON and c-MET and one or more VEGF receptors, or to inhibit RON and c-MET and Tie-2.

Several approaches have been suggested for the treatment of angiogenesis. Among the most commonly discussed approach is the inhibition of VEGF itself, and a number of compounds for inhibiting VEGF have been identified, including pegaptanib sodium and ranibizumab (discussed above) as well as bevacizumab (marketed as AVASTIN^®) which has been approved in the treatment of metastitic colorectal cancer and non-small cell lung cancer and has been explored as a treatment for proliferative diabetic retinopathy. See, for example, Spaide et al., Intravitreal bevacizumab (AVASTIN ) treatment of proliferative diabetic retinopathy complicated by vitreous hemorrhage, Retina 26(3):275-8 (2006).

However, the present inventors contemplate that inhibition of alternative signaling pathways in ocular angiogenesis, either as an alternative to or in addition to inhibiting VEGF would provide a beneficial and effective approach to the prevention and/or treatment of diseases involving ocular angiogenesis. By way of example, the present inventors contemplate that inhibiting c-MET in addition to VEGF would yield a synergistic reduction in angiogenic activity, and the combined inhibition of c-MET and VEGF is a promising approach to the prevention of treatment of ocular angiogenesis. In some embodiments of the present methods and compositions, an inhibitor of VEGF or its receptors is combined with an inhibitor of one or more receptor tyrosine kinases, such as an inhibitor of c-MET, RON or Tie-2. In other embodiments of the present methods and compositions, a compound is employed which inhibits (1) VEGF or one or more of its receptors and (2) c-MET or RON. By inhibiting alternative pathways, a greater likelihood of successful treatment or prevention of ocular angiogenesis is provided.

Moreover, the present inventors contemplate that some approaches for inhibiting angiogenesis may be particularly suited for use in treating or preventing ocular angiogenesis based on the expression of particular receptor tyrosine kinases in ocular tissues. By way of example, c-MET is highly expressed in corneal fibroblasts and epithelium. See Li et al., Three patterns of cytokine expression potentially involved in epithelial-fibroblast interactions of human ocular surface, Journal of Cellular Physiology 163(l):61-79 (1995). The present inventors contemplate that inhibition of c-MET can reduce the proliferation and/or migration of corneal fibroblasts and epithelial cells, c- MET is constitutively expressed and inducible in retinal pigment epithelium and in retinal endothelial cells. See He et al., Retinal pigment epithelial cells secrete and respond to hepatocyte growth factor, Biochem. & Biophysical Research Comm. 249(l):253-7 (1998); Cai et al., Mechanisms of hepatocyte growth factor-induced retinal endothelial cell migration and growth, Investigative Ophthalmology & Visual Science. 41(7): 1885-93 (2000). The present inventors contemplate that inhibition of c-MET can reduce the proliferation and/or migration of retinal pigment epithelium and in retinal endothelial cells.

Ocular angiogenic disease refers to any type of disease involving, characterized by or caused by inappropriate angiogenesis (including vascularization or neovascularization) in the eye or an ocular tissue. Ocular angiogenic disease refers to angiogenesis itself as well as to underlying diseases or conditions that cause or promote angiogenesis. For example, diseases involving ocular angiogenesis include diabetic macular edema, macular degeneration, diabetic retinopathy, retinal neovascularization, choroidal neovascularization, corneal neovascularization, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, and pterygium keratitis sicca. Diseases that cause inflammation or ischemia of the ocular tissues may also involve ocular angiogenesis. RON (recepteur d'origine nantais) is receptor tyrosine kinase that is expressed in epithelial cells. RON is a 180-kDa protein that is heterodimer of a 40-kDa alpha chain and a 150-kDa beta chain. Camp et al., RON a Tyrosine Kinase Receptor Involved in Tumor Progression and Metastasis, Annals of Surgical Oncology 12:273-281 (2005); Ronsin et al., A novel putative receptor protein tyrosine kinase of the met family, Oncogene 8(5): 1195-202 (1993). RON is initially created as a single-chain precursor, pro-RON, and is then cleaved into the two active chains. The alpha chain is completely extracellular, whereas the beta chain traverses the cell membrane and contains the intracellular tyrosine kinase and regulatory elements. Macrophage- stimulating protein (MSP) is the RON ligand whose binding to RON causes receptor activation. Angeloni et al., The Soluble Sema Domain of the RON Receptor Inhibits Macrophage-stimulating Protein-induced Receptor Activation, J. Biol. Chem. 279(5):3726-32 (2004); Wang et al., J. Biol. Chem. 272:16999-17004 (1997). Two binding sites for RON have been identified in MSP. MSP beta-chain contain a high- affinity binding site, and an alpha chain of MSP contains a second, low-affinity receptor binding site. Neither alpha nor beta chain of MSP alone induces RON tyrosine kinase phosphorylation and activation, which suggests that both binding sites of MSP are required for biological activity.

Inhibiting the activity of RON generally refers to inhibiting the kinase activity or the catalytic activity of the receptor to add a phosphate to tyrosine. However the present inventors contemplate that the inhibitors may inhibit other activities instead of or in addition to the direct inhibition of kinase activity, such as by inhibiting the activity of the receptor tyrosine kinase to dimerize or the activity of the receptor tyrosine kinase activity in binding to its ligands or substrates. Inhibiting RON includes any reduction, slowing, stopping or interfering with any activity of RON that directly or indirectly contributes to the tyrosine kinase activity of RON. Inhibiting RON also includes preventing, slowing, stopping, or reducing the binding of macrophage-stimulating protein to the extracellular domain of RON.

The c-MET (mesenchymal epithelial transition) receptor is a receptor tyrosine kinase that is encoded by the Met protooncogene. Chen et al., Co-Expression and Regulation of Met and Ron Proto-Oncogenes in Human Hepatocellular Carcinoma Tissues and Cell Lines, Hepatology 26(l):59-66 (1997). c-Met differs from RON with respect to the ligand that binds and activates the receptor. The c-Met receptor is activated by the ligand hepatocyte growth factor (HGF), which is also referred to as scatter factor (SF). See Chen et al.; Follenzi et al, Cross-talk between the proto- oncogenes Met and Ron, Oncogene 19(27):3041-49 (2000); and Comoglio et al., The HGF receptor family: unconventional signal transducers for invasive cell growth, Genes to Cells l(4):347-54 (1996). Chen et al., Follenzi et al., Comoglio et al. and other references also discuss similarities between c-Met and RON. c-MET and its ligand HGF are expressed in numerous tissues, especially epithelial and mesenchymal cells. See Bardelli et al. c-MET and HGF are required for normal mammalian development and are involved in cell migration, cell proliferation and survival, morphogenic differentiation, and organization of 3 -dimensional structures. c-MET-dependent tumor growth, invasion, and dissemination may be mediated by these cellular actions. See Vojkovsky et al., U.S. Patent App. Pub. 2005/0014755. In addition to its effects on epithelial cells, HGF/SF has been reported to be an angiogenic factor, and c-MET signaling in endothelial cells can induce many of the cellular responses necessary for angiogenesis (proliferation, motility, invasion).

Inhibiting the activity of c-MET generally refers to inhibiting the kinase activity or the catalytic activity of the receptor to add a phosphate to tyrosine. However, the present inventors contemplate that the inhibitors can inhibit other activities instead of or in addition to the direct inhibition of kinase activity, such as by inhibiting the activity of the receptor tyrosine kinase to dimerize or the activity of the receptor tyrosine kinase activity in binding to its ligands or substrates. Inhibiting c-MET includes any reduction, slowing, stopping or interfering with any activity of c-MET that directly or indirectly contributes to the tyrosine kinase activity of c-MET. Inhibiting c-MET also includes preventing, slowing, stopping, or reducing the binding of hepatocyte growth factor to the extracellular domain of c-MET.

Tie-2 is a receptor tyrosine kinase which is expressed primarily in endothelial cells and early hemopoietic cells. See Ibrahim et al., U.S. Patent App. Pub. 20070161651. Tie-2 has been shown to be involved in angiogenesis, in that it participates in endothelial cell migration, sprouting, survival and periendothelial cell recruitment during angiogenesis. When Tie-2 is bound by the ligands Angiopoietin-1 (Ang-1) or Ang-4, autophosphorylation by Tie-2 is induced and signaling based on the Tie-2 receptor is increased, and when Tie-2 is bound by the ligands Ang-2 and Ang-3, Tie-2 activity is downregulated. See Chen et al., U.S. Patent App. Pub. 20060293342. Ang-1 signaling through Tie-2 facilitates later stages of vascular development by modulating cell-cell, and cell-matrix interactions, resulting in the survival and stabilization of newly formed blood vessels. Compounds said to inhibit, regulate and/or modulate the Tie-2 receptor protein kinase are discussed in Ibrahim et al., U.S. Patent App. Pub. 20070161651 and Stieber et al., U.S. Patent App. Pub. 20030158199. Efficient sequence specific gene silencing is possible through the use of siRNA technology. The generation of small interfering RNAs for silencing the gene expression of the Tie-2 protein receptor is discussed in Khvorova et al., U.S. Patent App. Pub. 20070134697.

Inhibiting Receptor Tyrosine Kinases To Prevent or Treat Ocular Angiogenesis

The present invention relates to inhibiting particular or selected receptor tyrosine kinases, such as inhibiting the activity of RON, in methods or compositions for treating or preventing diseases involving ocular angiogenesis. In some embodiments, a method or a composition of the present invention includes an inhibitor that specifically inhibits RON, for example, without significantly inhibiting or without intentionally inhibiting other receptor tyrosine kinases. Alternatively, the present methods and compositions may include one or more inhibitors that inhibit particular combinations of receptor tyrosine kinases, such as inhibiting the activity of both c-MET and RON. In some embodiments of the present compositions and methods, RON and c-MET and at least one other receptor tyrosine kinase are inhibited. For example, the present compositions and methods can include one or more inhibitors that inhibit RON and c- MET and one or more VEGF receptors are inhibited. As another example, the present compositions and methods can include can include one or more inhibitors that inhibit RON and c-MET and Tie-2. As another example, the present compositions and methods can include can include one or more inhibitors that inhibit c-MET and one or more VEGF receptors. As another example, the present compositions and methods can include can include one or more inhibitors that inhibit c-MET and Tie-2. As yet another example, the present compositions and methods can include can include one or more inhibitors that inhibit RON and one or more VEGF receptors. As another example, the present compositions and methods can include can include one or more inhibitors that inhibit RON and Tie-2.

In various embodiments of the present invention, where two or more receptor tyrosine kinases are inhibited by a method or composition, it is envisioned that the receptor tyrosine kinases can be inhibited by a single compound that is effective as an inhibitor of those two or more receptor tyrosine kinases, and/or that the receptor tyrosine kinases may be inhibited by separate compounds that are each predominantly effective against a particular receptor tyrosine kinase. An example of a single compound said to be effective against more than one receptor tyrosine kinase is MGCD265 from MethylGene Inc. of Montreal, Quebec. MGCD265 is said to targets the c-MET, VEGFRl, VEGFR2, VEGFR3, Tie-2 and RON receptor tyrosine kinases. MethylGene press release, MethylGene Selects Multi-Targeted Kinase 'c-MET Clinical Candidate for Oncology, December 7, 2006. MGCD265 is said to be a clinical candidate potentially in tumor types in which c-MET has been shown to be overexpressed, such as non-small cell lung cancer (NSCLC), ovarian, head and neck, gastric, colorectal, breast, leukemias, multiple myeloma or hereditary papillary renal cell cancer.

Inhibiting the activity of a receptor tyrosine kinase generally refers to inhibiting the kinase activity or the catalytic activity of the receptor to add a phosphate to tyrosine. However, the present inventors contemplate that the inhibitors may inhibit other activities instead of or in addition to the direct inhibition of kinase activity, such as by inhibiting the activity of the receptor tyrosine kinase to dimerize or the activity of the receptor tyrosine kinase activity in binding to its ligands or substrates.

The receptor tyrosine kinases may be inhibited by one or more mechanisms of inhibition. For example, an inhibitor may act upon, such as by binding to, the receptor's extracellular domain so as to prohibit or prevent binding of the receptor tyrosine kinase to a ligand that will activate the receptor. As yet another example, an inhibitor may bind to the ligand for the receptor. Alternatively, the inhibitor may act upon, such as by binding to, the receptor's intracellular domain, particularly to a catalytic moiety that is active in phosphorylation. Alternatively, the inhibitor may act upon, such as by binding to, a ligand that would active the receptor if bound to the receptor, in that the inhibitor acts upon the ligand in such a way as to prevent binding to the receptor. Alternatively, the expression of the receptor tyrosine kinase may be inhibited. In general, unless indicated otherwise, the selected receptor tyrosine kinases can be inhibited by inhibiting any reaction that directly or indirectly relates to the receptor tyrosine kinase's expression, translation, insertion into the cell membrane, formation of a dimer, cleavage of a precursor of the receptor tyrosine kinase, signal transduction, binding with ligands, binding with substrates, or kinase activity.

Inhibitors of recepteur d'origine nantais (RON)

The present compositions and methods can include one or more inhibitors of recepteur d'origine nantais (RON). Several inhibitors that inhibit RON, such as by acting directly on RON or by inhibiting expression of RON, have been discussed. For example, Pereira et al., International Pub. WO 2005/120557 discusses antibodies specific for Macrophage-Stimulating Protein Receptor ("MSP-R" or "RON") for use in methods for treatment of tumors and other diseases in a mammal comprising administration of. O'Toole et al., Therapeutic implications of a human neutralizing antibody to the macrophage-stimulating protein receptor tyrosine kinase (RON), a c-MET family member, Cancer Research. 66(18):9162-70 (2006) discusses a human immunoglobulin Gl (IgGl) antibody that is said to bind with high affinity to RON. Compositions comprising antibodies and antibody fragments specific for RON, including human antibodies, that inhibit RON activation are discussed. Taira et al. U.S. Patent App. Pub. 20060247193 discusses a method for modulating expression of a target gene in a cell by introducing into the cell a polynucleotide that forms a duplex region with an mRNA transcribed from the target gene, wherein the duplex region comprises a mammalian mRNA target region. One of the target genes whose expression can be modulated is said to be the gene encoding the RON tyrosine kinase. Naito et al., U.S. Patent App. Pub. 20040116330 discusses a method of controlling the growth and differentiation of cancer, which comprises diagnosing or specifying a growth factor receptor expressed in cancer cells, and selectively inhibiting all or a part of the growth factor receptor. Among the growth factor receptor which are said to be targets for inhibiting are RON, Met, Sea, and Vascular endothelial growth factor receptor, along with many others. Angeloni et al. states that a soluble, secreted molecule representing the sema domain of RON (referred to as ron-sema) has a dominant negative effect on the ligand-induced receptor activation and is capable of inhibiting RON-dependent signaling pathways and cellular responses. It is suggested that RON-sema interferes with binding of MSP to RON either by sequestering MSP or by binding to the sema domain of RON.

The present inventors contemplate that additional inhibitors of RON can be developed, including compounds that include RON inhibiting moieties from the various inhibitors of RON described above. The present inventors further contemplate that additional inhibitors can be developed which are effective as inhibitors of other receptor tyrosine kinases in addition to RON. Inhibitors which are effective in inhibiting more than one of the selected receptor tyrosine kinases discussed herein are also desirable under some clinical conditions for treating an ocular angiogenic disease by addressing multiple and/or alternative pathways of inappropriate angiogenesis in the eye.

Inhibitors of c-MET

The present compositions and methods can include one or more inhibitors of c-MET. Several inhibitors that inhibit c-Met, such as by acting directly on c-Met or by inhibiting expression of c-Met, have been discussed. A variety of c-MET inhibitors have been described including, for example, tetracyclic compounds discussed in Cui et al., U.S. Patent App. Pub. 2005/0107391 and Vojkovsky et al., U.S. Patent App. Pub. 2005/0014755. Other inhibitors of c-Met are discussed in Barmen et al., U.S. Patent App. Pub. 20070179130 and Bannen et al., U.S. Patent App. Pub. 20070225307. Efforts to identify peptides capable of blocking both the kinase activity and the biological properties of the Met receptor are discussed in Bardelli et al., A peptide representing the carboxyl-terminal tail of the met receptor inhibits kinase activity and invasive growth, Journal of Biological Chemistry. 274(41):29274-81 (1999).

For example, antibodies can be prepared that specifically bind to various epitopes on c-Met and that inhibit the tyrosine kinase activity of c-Met. Morton et al., U.S. Patent App. Pub. 20040166544 discusses antibodies to c-Met for the treatment of cancers. It states that antagonist antibodies can be employed to block binding of HGF to c-Met or substantially inhibit c-Met activation. Michaud et al., U.S. Patent App. Pub. 20050054019 discusses antibodies including human antibodies and antigen-binding portions thereof that specifically bind to c-Met, preferably human c-Met, and that function to inhibit c-Met. The inhibitor of c-MET can be a compound that specifically binds the c-Met receptor or binds its ligand hepatocyte growth factor (HGF) in such a way as to prevent binding of HGF with the c-Met receptor. For example, an inhibitor of c-MET can be an antibody that binds to primate and human c-Met, and more preferably one that is a human antibody, and that inhibits the binding of HGF to c-Met or that inhibits autophosphorylation of tyrosine residues of c-Met.

Additional inhibitors of c-Met can be developed, including compounds that include c-Met inhibiting moieties from the various inhibitors of c-Met described above.

Inhibiting Other Receptor Tyrosine Kinases

In some embodiments of the present compositions and methods, in addition to inhibiting selected receptor tyrosine kinases such as RON, or selected combinations of receptor tyrosine kinases such as RON and c-MET and optionally others, other kinase receptors are also inhibited, such as a VEGF receptor or another receptor tyrosine kinase. The inhibitor may be the same compound that inhibits the activity of the selected receptor tyrosine kinase (such as RON or Tie-2 or c-MET or a VEGF receptor), or it may be a different compound that is included in the present compositions or methods. For example, Barmen et al. U.S. Patent App. Pub. 20070179130 discusses compounds for modulating protein kinase enzymatic activity which are said to inhibit, regulate and/or modulate the receptor kinases c-Met, KDR, and flt-3. Bannen et al., U.S. Patent App. Pub. 20070225307 discusses compounds for modulating protein kinase enzymatic activity which are said to inhibit, regulate, and/or modulate the kinase receptors c-Met, KDF, c-Kit, flt-3 and flt-4. In some embodiments of the present compositions and methods, an inhibitor is provided that inhibits the tyrosine kinase activity of EGFR (HERl), HER2, HER3, HER4, INS-R, IGF-IR, IR-R, the PDGF-alpha receptor, the PDGF-beta receptor, CSFIR, c-Kit, FLK-II, kinase insert domain receptor (KDR), fetal liver kinase- 1 (FLK-I), fetal liver kinase-4 (FLK-4) and the fms-like tyrosine kinase- 1 (fit- 1)- Inhibitors of other receptor tyrosine kinases may be included in the present methods and compositions. For example, inhibitors of the receptor tyrosine kinase Tie-2 are discussed in Bump et al., U.S. Patent App. Pub. 20030082622.

Receptor tyrosine kinase inhibitors suitable for use in the present compositions and methods may be a small molecule compound, a polypeptide, an antibody or antigen-binding fragment, a polysaccharide, a lipid, a nucleic acid or another type of compound, hi some embodiments, the inhibitor is selected from the group consisting of a small interfering RNA (siRNA), a microRNA, an antisense nucleic acid, a ribozyme, an expression vector encoding a transdominant negative mutant, an intracellular antibody, a peptide and a small molecule. For example, the inhibitor may be an siRNA, such as an siRNA selected from the group consisting of a double stranded oligonucleotide, a single stranded oligonucleotide, and a polynucleotide. hi some embodiments of the present compositions and methods, the inhibitor is an antisense nucleic acid, such as antisense RNA, that binds to mRNA that encodes an receptor tyrosine kinase such as RON or a VEGF receptor or Tie-2 or c-MET or to an activating ligand of one of those receptor tyrosine kinases. Antisense RNA is single- stranded RNA that is complementary to an RNA sequence. Antisense RNA may be introduced into a cell to inhibit translation of a complementary RNA sequence by base pairing to it. Antisense RNA has been widely discussed in the context of providing antisense RNA that is complementary to messenger RNA. By binding to mRNA, the antisense RNA physically obstructs the translation of that mRNA sequence into a protein.

It is also contemplated that antisense DNA can be used in conjunction with or in place of antisense RNA. Taira et al. U.S. Patent App. Publication No. 20060247193 discusses regulation of gene expression by DNA interference, where expression of a target gene in a cell is modulated by introducing into the cell a polynucleotide that forms a duplex region with an mRNA transcribed from the target gene.

In another embodiments, translation of mRNA encoding receptor tyrosine kinases such as RON or a VEGF receptor or Tie-2 or c-MET can be inhibited using an antisense nucleic acid sequence expressed by a plasmid vector. The antisense expressing vector is used to transfect a cell, thereby causing reduced expression of a receptor tyrosine kinase by the cell. hi some embodiments of the present compositions and methods, the inhibitor is an antibody or antigen-binding fragment that binds to an extracellular or intracellular domain of an receptor tyrosine kinase such as RON or a VEGF receptor or Tie-2 or c- MET. One aspect of the present invention contemplates intracellular delivery of an antibody or antigen-binding fragment that specifically binds to an extracellular domain of RON, Tie-2, c-MET, a VEGF receptor or another receptor tyrosine kinase inhibitor. Some expected advantages of trans-membrane antibodies include the specific target recognition characteristic of antibodies and antigen-binding fragments.

Methods of Treating a Disease Involving Ocular Angiogenesis

In some embodiments, the present methods of treatment comprise administering a composition which comprises an effective amount of at least one inhibitor of c-MET and/or RON. The composition may be an ophthalmic pharmaceutical composition.

Treatment of or treating ocular angiogenesis includes treating, alleviating, ameliorating, or reducing ocular angiogenesis or one or more symptoms of ocular angiogenesis (such as vision loss). In general, treatment of ocular angiogenesis also includes use of the present methods and compositions to prevent ocular angiogenesis, and preventing includes delaying or stopping the onset or the progression of ocular angiogenesis or one or more symptoms of ocular angiogenesis. Treatment includes stopping or slowing vision loss experienced by a subject. Treatment further includes arresting or suppressing the development of clinical symptoms; and/or relieving the disease or disorder (i.e., causing the regression of clinical symptoms). Prevention of or preventing ocular angiogenesis includes stopping or delaying ocular angiogenesis, including delaying the onset. Preventative uses of the present compositions include posttraumatic, post-surgical, and pre-surgical administration.

The present methods include embodiments in which one or more specific causes or risk factors for ocular angiogenesis are addressed with an inhibitor that is effective for inhibiting that cause or risk factor. For example, a method of preventing ocular angiogenesis can comprise administering an inhibitor of c-MET to a subject having diabetes before the onset of diabetic retinopathy. Alternatively or additionally, a method of preventing ocular angiogenesis can comprise administering an inhibitor of RON to a subject having diabetes before the onset of diabetic retinopathy.

After a subject reaches a certain age (such as 50, 55 or 60), it is desirable to take steps to diagnose an ocular angiogenic disease before the onset or progression of ocular angiogenesis or its symptoms (particularly vision loss or formation of new blood cells). Steps for diagnosis include periodic retinal examinations and fluorescein angiograms to monitor for the presence of subretinal fluid, blood, exudates, RPE detachment, cystic retinal changes, or the presence of grayish green subretinal neovascular membrane. When an ocular angiogenic disease is diagnosed, various embodiments of the present methods may be applied, with or without other co-therapies such as photodynamic therapy or photocoagulation.

In some embodiments, the present methods employ a composition comprising a receptor tyrosine kinase inhibitor that is administered topically in an effective amount to an eye that has elevated levels of one or more receptor tyrosine kinases, such as RON or c-MET. Accordingly, some embodiments of the present methods comprise the step of assessing the expression level of receptor tyrosine kinases, such as RON or c-MET. hi some embodiments, the present methods are used to administer a pharmaceutical composition comprising an inhibitor of c-MET or RON. An appropriate dosage, frequency and duration of administration, i.e., treatment regimen, to be used in any particular situation can be readily determined, and will depend, among other factors, on the particular active agent(s) present in the composition, on the particular disease being treated, on the age, weight and general physical condition of the subject, and on other medication being administered to the subject. It is preferred that response of the disease to treatment according to the present method be monitored and the treatment regimen be adjusted if necessary in light of the results of such monitoring.

The methods of treatment described herein may include the step of diagnosing an ocular angiogenic disease, or diagnosing the signs or symptoms of an ocular angiogenic disease. The methods may include (but do not have to include) the steps of diagnosing the overexpression of RON and/or c-MET in the ocular epithelial cells of a subject.

The methods can include identifying a subject having wet AMD, for example a subject in an early stage of wet AMD. It is advantageous to identify a subject having early stage wet AMD because the present inventors contemplate that the present methods will be especially beneficial in such subjects. The methods can include identifying a subject at risk of ocular angiogenic disease, for example, a subject having diabetes. In some embodiments, the subject to be treated by the present methods is preferably one of (i) a human subject diagnosed as suffering from diabetic macula edema, (ii) a human subject diagnosed as suffering from macular degeneration, (iii) a human subject diagnosed as suffering from diabetic retinopathy, and (iv) a human subject suffering from inappropriate angiogenesis of the cornea, the retina, the choroid or another ocular tissue.

In the present methods for treating an ocular angiogenic disease, an ophthalmic pharmaceutical composition as described herein in a therapeutically or prophylactically effective dose is administered to at least one eye of a subject in need thereof.

Administering The Inhibitor(s) of Receptor Tyrosine Kinase(s)

One or more inhibitors of selected receptor tyrosine kinases can be administered to a subject in any suitable manner. Methods of administration include but are not limited to ocular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically. Some methods of administration will result in the release of the composition or the inhibitor into the bloodstream, but preferably the method of administration will localize or preferentially localize the inhibitor at or near the eye, more preferably at or near the site of ocular angiogenesis. In the present methods, a composition for the treatment of an ocular angiogenesis disease can be administered systemically or locally. For example, a composition can be administered systemically by oral administration or by injection. As another example, a composition can be administered locally, such as by administration in eye drops, preferably as an isotonic solution. As other examples, a composition can be administered by local infusion during surgery, by topical application, e.g. in conjunction with a wound dressing after surgery, by a catheter, or by an implant.

In some embodiments of the present methods and compositions, the inhibitor is provided in a pharmaceutical composition, for example an ophthalmic pharmaceutical composition. The concentration of the inhibitor in such a pharmaceutical composition may be in the range from about 0.0001 to about 1000 mg/ml (or, alternatively, from about 0.001 to about 500 mg/ml, or from about 0.001 to about 300 mg/ml, or from about 0.001 to about 250 mg/ml, or from about 0.001 to about 100 mg/ml, or from about 0.001 to about 50 mg/ml, or from about 0.01 to about 300 mg/ml, or from about 0.01 to about 250 mg/ml, or from about 0.01 to about 100 mg/ml, or from about 0.1 to about 100 mg/ml, or from about 0.1 to about 50 mg/ml).

In some embodiments of the present methods, a subject in need of treatment of an ocular angiogenic disease is treated by multiple administrations of an inhibitor of one or more selected receptor tyrosine kinases. The present methods may comprise administering the inhibitor one or more times, such as by administering the inhibitor on an approximately regular basis. For example, an inhibitor may be administered to a subject on an approximately monthly, bi-quarterly, quarterly, semi-yearly, yearly, or other basis, for example, at a frequency of every 2-24 weeks, or every 1-12 months (including a frequency set by any number within those ranges). More particularly, the methods may comprise repeatedly administering an inhibitor of one or more selected receptor tyrosine kinases to a subject in need of treatment every four weeks, or every six weeks, every eight weeks, or every twelve weeks. Alternatively, a first dose of the inhibitor may be administered to the subject, and a second dose is administered one to four weeks after the first dose, and subsequent doses are administered every six weeks after the second dose. Alternatively, the present methods may comprise administering the inhibitor directly to an eye no more frequently than every four weeks, or between 0.5 and 12 times per year. The present methods can also comprise administering the inhibitor until one or more signs or symptoms of ocular angiogenesis are alleviated, ameliorated, reduced, or removed. Improvement of clinical symptoms are monitored by one or more methods known to the art, for example, indirect ophthalmoscopy, fundus photography, fluorescein angiopathy, electroretinography, external eye examination, slit lamp biomicroscopy, applanation tonometry, pachymetry, and autorefaction. Alternatively, the present methods can also comprise administering the inhibitor on a regular basis for a set period, such as for 1, 2, or more years, or for the lifetime of the subject.

hi some preferred embodiments of the present methods, the inhibitor of a selected receptor tyrosine kinase, such as c-MET or RON, is applied locally to the eye of a subject, such as by topical, intravitreal, subconjunctival, periocular, retrobulbar, juxtascleral, or intraocular administration. In some embodiments of the present invention, a composition comprising an inhibitor of selected receptor tyrosine kinase, such as c-MET or RON, is administered by injection to an eye that is suffering from or at risk of ocular angiogenic disease. Preferably, administration of the receptor tyrosine kinase inhibitor will be directly to the eye. Methods of administration directly to the eye include, for example, by subconjunctival injections or implants, intravitreal injections or implants, sub-Tenon's injections or implants, incorporation in surgical irrigating solutions, eye drops, and others.

For example, the compositions can be administered to the eye by injection, for example, by intraocular, subconjunctival, or intravitreal administration. Injectable compositions include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. The present methods may comprise injecting a composition comprising an inhibitor into the eye of a subject, for example, into the vitreous humor of the subject.

As another example, the compositions can be administered topically to the eye of a subject. Compositions suitable for topical administration are known to the art (see, for example, U.S. Patent Application 20050059639). In various embodiments, the present compositions can comprise a liquid comprising an active agent in solution, in suspension, or both. As used herein, liquid compositions include gels. The present methods may comprise administering a liquid composition comprising an inhibitor to the eye of a subject. Alternatively, the present composition can take form of an ointment, and the present methods may comprise administering an ointment comprising an inhibitor to the eye of a subject. When the composition is to be administered to the eye, the methods generally comprise administering a composition preferably have ophthalmically compatible pH and osmolality (discussed in more detail below).

Preferably, the composition is administered to the eye of a warm-blooded subject, preferably a mammalian subject. Suitable mammalian subjects include domestic mammals, farm and exotic mammals, and humans. The present methods can be useful, for example, in treatment of ocular angiogenic diseases of dogs, cats, horses, cattle, sheep and pigs. The present methods are particularly contemplated for use where the subject is human. A composition comprising an inhibitor of a selected receptor tyrosine kinase, such as c-MET or RON, can be administered directly to the front of an eye. For example, eye drops may be used alone or with independent, or in the contact lenses. The pH of the composition should be adjusted so that there is no irritation of the eye, which in turn would lead to possibly infection by other organisms, and possibly to damage to the eye. In some embodiments, the pH of the composition is in the range from about 4 to about 11. Alternatively, the pH of the composition is in the range from about 5 to about 9, from about 6 to about 9, or from about 6.5 to about 8. In another aspect, the composition comprises a buffer having a pH in one of said pH ranges. In another aspect, the composition has a pH of about 7. Alternatively, the composition has a pH in a range from about 7 to about 7.5. In still another aspect, the composition has a pH of about 7.4.

Other embodiments of the present compositions and methods include the use of a contact lens as a vehicle for administration. An inhibitor of a selected receptor tyrosine kinase may be applied to a lens before the lens is placed in the eye. Contact lenses may be contacted or treated with an inhibitor contained in an aqueous solution, for example, by storing or soaking the contact lens in the solution or by spraying the lens with the solution for sufficient time to wet the surfaces thereof. The treated lens can be placed directly in the eye or, alternately, the lens can be first rinsed before being placed in the eye. Drops of an ophthalmic formulation comprising the inhibitor can be placed on the lens surface and the treated lens placed in the eye, or the formulation may be directly applied to the eye in the form of eye-drops while the contact lens is being worn. The specific lens care regimen used may depend on the other compounds or ingredients present in the solution, as will be appreciated by those skilled in the art.

Alternatively, various implants or drug delivery devices can be used to administer the inhibitor of a selected receptor tyrosine kinase. For example, the inhibitor may be administered by implanting an intraocular implant in the eye of a subject in need of treatment, wherein the intraocular implant comprises and is adapted to release an inhibitor of a selected receptor tyrosine kinase. Such implants may be biodegradable and/or biocompatible implants, or may be non biodegradable implants. The implants may be permeable or impermeable to the inhibitor, and may be inserted into a chamber of the eye, such as the anterior or posterior chambers or may be implanted in the sclera, transchoroidal space, or an avascularized region exterior to the vitreous. In a preferred embodiment, the implant may be positioned over an avascular region, such as on the sclera, so as to allow for transcleral diffusion of the inhibitor to the desired site of treatment, such as the intraocular space and macula of the eye. Furthermore, the site of transcleral diffusion is preferably in proximity to the macula. Examples of various drug delivery devices are found in the following patents: U.S. Patent App. Pub. 20020086051 (Viscasillas); U.S. Patent App. Pub. 2002/0106395 Al (Brubaker); U.S. Patent App. Pub. 2002/0110591 Al (Brubaker et al.); U.S. Patent App. Pub. 20020110592A1 (Brubaker et al.); U.S. Patent App. Pub. 20020110635 (Brubaker et al.); U.S. Pat. No. 5,378,475 (Smith et al.); U.S. Pat. No. 5,773,019 (Ashton et al.); U.S. Pat. No. 5,902,598 (Chen et al.); U.S. Pat. No. 6,001,386 (Ashton et al); U.S. Pat. No. 6,217,895 (Guo et al.); U.S. Pat. No. 6,375,972 (Guo et al.).

The present compositions can be provided in an ophthalmic depot formulation comprising an inhibitor, such as for subconjunctival administration. For example, microparticles comprising an inhibitor can be embedded in a biocompatible pharmaceutically acceptable polymer or a lipid encapsulating agent. The depot formulations may be adapted to release all or substantially all the active material over an extended period of time. The polymer or lipid matrix, if present, may be adapted to degrade sufficiently to be transported from the site of administration after release of all or substantially all the active agent. The depot formulation can be a liquid formulation, comprising a pharmaceutical acceptable polymer and a dissolved or dispersed active agent, upon injection, the polymer forms a depot at the injections site, e.g. by gelifying or precipitating. The composition can comprise a solid article that can be inserted in a suitable location in the eye, such as between the eye and eyelid or in the conjuctival sac, where the article releases the active agent. Solid articles suitable for implantation in the eye in such fashion generally comprise polymers and can be bioerodible or non- bioerodible.

In other embodiments of the present methods, the receptor tyrosine kinase inhibitor is applied systemically (oral, subcutaneous, intramuscular, intravenous) as a treatment for an ocular angiogenic disease.

In some embodiments of the present methods, it may be desirable to administer the inhibitor or a pharmaceutically acceptable salt thereof. The inhibitor can be provided as or a pharmaceutically acceptable derivative, salt, solvate, hydrate, prodrug, enantiomer or racemic mixture thereof, of one or more compounds that includes an inhibitor of a selected receptor tyrosine kinase. hi some embodiments of the present methods and compositions, an inhibitor of one or more selected receptor tyrosine kinases is used in the manufacture of a medicament useful for the treatment of ocular angiogenic diseases.

Ophthalmic Pharmaceutical Compositions

The present compositions include, but are not limited to, ophthalmic pharmaceutical compositions intended for treating ocular angiogenic disease. Ophthalmic pharmaceutical compositions are suitable for administration to the eye of a subject, and they generally include one or more ophthalmically acceptable excipients.

A composition, compound or excipient is ophthalmically acceptable when it has no long-term adverse effect on the eye being treated or its function, or on the general health of the subject being treated. There may be short-term adverse effects such as minor irritation or a stinging sensation, but such short-term adverse effects are not uncommon with topical ophthalmic administration of active agents. The existence of such short-term adverse effects is not inconsistent with a composition, compound or excipient being ophthalmically acceptable. However, preferred compositions, compounds and excipients are those that do not cause a substantial short-term adverse effect.

Factors that affect the bioavailability of ocular drugs include pH, salt form of the active agent, osmolality/tonicity and viscosity. The ophthalmic pharmaceutical composition may be an aqueous solution or a suspension. For example, some embodiments of the present composition may be a suspension comprising mannitol, carbomer, tyloxapol, edetate sodium, sodium chloride, HCl or NaOH to adjust the pH, and purified water. Other embodiments of the present composition may be an injectable composition may be an aqueous solution comprising sodium chloride, one or more sodium or potassium phosphates, and HCl or NaOH to adjust the pH, and water for injection. Preferred compositions for ophthalmic administration include injectable compositions suitable for intraocular, subconjunctival, or intravitreal administration. Injectable compositions include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. An injectable composition may comprise the inhibitor and a carrier. A variety of aqueous carriers can be used, for example, water, buffered water, saline, phosphate buffered saline (PBS), balanced salt solution (BSS), Ringers lactate solution, and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol, vegetable oils, gelatin, hydrogenated naphalenes, and injectable organic esters, such as ethyl oleate, and mixtures thereof.

Ophthalmic pharmaceutical compositions may also contain one or more excipients or other substances, such as preservatives, antioxidants, pH adjusting agents, buffering agents, salts, co-solvents, diluents, carriers, adjuvants, oils, humectants, emollients, stabilizers, emulsifying agents, and/or dispersing agents. Other agents may be employed in the compositions for a variety of purposes. By way of example, injectable compositions may contain various exicipients or other substances, such as preservatives, antioxidants, pH adjusting agents, buffering agents, salts, emulsifying agents, and/or dispersing agents. For example, an injectable composition may be a sterile, preservative-free solution comprising an inhibitor of one or more selected receptor tyrosine kinases, one or more sodium or potassium phosphates, and HCl or NaOH to adjust the pH, and water for injection.

Injectable compositions may include one or more polymers. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene- polyoxypropylene copolymers may be used to control the release of the inhibitor of selected receptor tyrosine kinases. Injectable compositions may also include liposomes which encapsulate the inhibitor.

Other compositions for ophthalmic administration include those which prolong the time during which the inhibitor will remain on the surface of eye. These include gels, ointments, solid inserts, soft contact lens, and collagen shields. Ophthalmic gels (for example, pilocarpine 4% gel) release active agents by diffusion following erosion of soluble polymers. Suitable polymers include cellulosic ethers, polyvinyl alcohol, carbopol, polyacrylamide, polymethylvinyl ether-maleic anhydride, poloxamer 407, and puronic acid. Ointments usually contain a mineral oil and a petrolatum base. Solid inserts, such as OCUSERT^® PILO-20 and PILO-40 release the active agent by steady-state diffusion, in which the active agent is released at a relatively constant rate to the eye.

The present compositions may be an aqueous solution and may be in the form of eye drops. By means of a suitable dispenser, a desired dosage of the active agent can be metered by administration of a known number of drops into the eye, and most preferably by one drop. Where ophthalmic administration of the inhibitor is contemplated, it is desirable to be able to administer an effective amount of the inhibitor in a relatively small volume, without irritating the eye.

The present compositions can optionally include one or more preservatives, preferably a water-soluble preservative. Examples of water-soluble preservatives include sodium bisulfite, sodium bisulfate, sodium thiosulfate, benzalkonium chloride, chlorobutanol, thimerosal, ethyl alcohol, methylparaben, polyvinyl alcohol, benzyl alcohol, and phenylethyl alcohol. These agents may be present in individual amounts of from about 0.001 to about 5% by weight (preferably, about 0.01% to about 2% by weight).

The present compositions can optionally include an ophthalmically acceptable antioxidant. Preferred antioxidants included in the composition include, but are not limited to: sodium bisulfite, sodium thiosulfate, acetyl cysteine, cysteine, thioglycerol, sodium sulfite, acetone sodium bisulfite, dithioerythreitol, dithiothreitol, thiourea, and erythorbic acid. Alternatively, the antioxidant included in the composition can be selected from the group consisting of sodium bisulfite, sodium thiosulfate, acetyl cysteine, cysteine, thioglycerol. Alternatively, the antioxidant may be sodium bisulfite.

The present compositions optionally further include at least one ophthalmically acceptable salt in an amount sufficient to bring osmolality of the composition into an ophthalmically acceptable range. In some cases, the salts can also be antioxidants, such as those described above. Salts suitable for use in adjusting osmolality include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, acetate, bicarbonate, sulfate, thiosulfate or bisulfite anions; preferred salts include sodium acetate, sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate, with sodium chloride being especially preferred. Other solutes suitable for adjustment of osmolality include sugar (such as dextrose, lactose, xylitol, and mannitol) and glycerine.

The present compositions can also include an ophthalmically acceptable pH adjusting agent, such as an acid, base and/or buffer. For example, the compositions can include an acid such as acetic, boric, citric, lactic, phosphoric, sulfuric, and hydrochloric acids; a base such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylarainomethane, triethanolamine; and/or a buffer such as citrate/dextrose, sodium bicarbonate and ammonium chloride or an amino acid. Such an acid, base and/or buffer can be included in an amount sufficient to adjust pH of the composition to an ophthalmically acceptable range.

Buffers, acids and/or bases can be included for buffering or adjusting the pH of the composition, and they may also be used for adjusting the tonicity (osmolality) of the ophthalmic pharmaceutical compositions. Alternatively, additional tonicity adjusting agents may be included in the compositions. Preferably, the pH of the present ophthalmic pharmaceutical compositions maintained within the range of about 5 to about 8, more preferably about 6 to about 8, most preferably about 6.5 to about 7.8. The tonicity agents may be employed in an amount effective to adjust the osmotic value of the final composition to a desired value, typically from about 250 to about 350 mθsmols/kg in order to approximate the osmotic pressure of normal lachrymal fluids which is equivalent to a 0.9 percent solution of sodium chloride. Some embodiments of the ophthalmic pharmaceutical composition comprise a pH adjusting agent and/or an agent for adjusting osmolality in amounts whereby the solution is substantially isotonic and has a physiologically acceptable pH. Preferably, the ophthalmic pharmaceutical composition is an isotonic solution.

The present compositions may comprise a sequestering agent (or chelating agent). Examples of preferred sequestering agents include ethylenediaminetetraacetic acid (EDTA) and its salts, with the disodium salt (sodium edetate) being preferred.

Clear aqueous solutions and suspensions are often preferred by subjects as being more comfortable, especially for daytime administration. The present ophthalmic pharmaceutical compositions can be in the form of an ointment. However, they are preferably in the form of an aqueous solution or suspension, more preferably in the form of a clear aqueous solution.

The present ophthalmic pharmaceutical compositions are preferably formulated to account for the high rate of drug loss that often occurs when a drug is administered to the eye. A relatively small volume of fluid can be accommodated in the exterior of the eye, including the conjunctival sac, and under normal conditions lacrimal fluid fills most of the available volume. Lacrimal fluid tends to wash away drugs administered to the eye. By increasing the viscosity of the ophthalmic pharmaceutical composition, the rate of drug loss can be reduced and residence time of the drug in of the eye can be increased. One effect of the loss of an ophthalmic composition from a treated eye is a reduced concentration of the inhibitor in the lacrimal fluid and hence in the infected eye. Suspensions, gels, and ointments are often used as ophthalmic formulations for this reason.

The present ophthalmic pharmaceutical compositions preferably further include at least one ophthalmically acceptable excipient that reduces the rate of removal of the inhibitor from the eye by lacrimation, such that the inhibitor has an effective residence time in the eye of about 2 to about 24 hours. Lacrimation is the production of tear fluid, and can remove matter from the eyes both by external wash-out and by lacrimal drainage into the nasopharyngeal cavity via the nasolacrimal ducts. For sustained anti-infective action, the concentration in the lacrimal fluid and in the target eye tissue, for example, the conjunctiva or the cornea, must remain at or above an effective concentration for the inhibitor. In some embodiments, the ophthalmic pharmaceutical compositions is viscous or mucoadhesive, or both viscous or mucoadhesive. By way of example, the compositions can contain carboxymethylcellulose, a viscosity enhancer and promoter of mucoadhesion. Other compounds that may be used to enhance the viscosity of the composition include, but are not limited to: monomeric polyols, such as, glycerol, propylene glycol, ethylene glycol; polymeric polyols, such as, polyethylene glycol; various polymers of the cellulose family, such as hydroxypropylmethyl cellulose ("HPMC"), carboxymethyl cellulose ("CMC") sodium, hydroxyethylcellulose ("HEC"), hydroxypropyl cellulose ("HPC"); polysaccharides, such as hyaluronic acid and its salts, chondroitin sulfate and its salts, dextrans, such as, dextran 70; water soluble proteins, such as gelatin; vinyl polymers, such as, polyvinyl alcohol, polyvinylpyrrolidone, povidone; carbomers (acrylic acid polymer), such as carbomer 934P, carbomer 941, carbomer 940, or carbomer 974P; and acrylic acid polymers. Other excipients which may be used in the present compositions include an ophthalmically acceptable mucoadhesive polymer, such as, poly(methylmethacrylate), polyacrylamide, polycarbophil, polyethylene oxide, acrylic acid/butyl acrylate copolymer, and sodium alginate. In general, a desired viscosity can be in the range from about 1 to about 400 centipoise ("cps") (or mPa.s).

Other ophthalmically acceptable excipients can be included in the present ophthalmic pharmaceutical compositions to increase retention of the inhibitor in an eye. For example, an ophthalmically acceptable viscosity enhancer can be included. Alternatively, a thermosetting polymer that forms a gel at a human body temperature can be included. Upon placement of such a liquid composition in an eye, a gel will form, thereby retarding loss of the inhibitor from the eye by lacrimal drainage.

In some embodiments, the ophthalmic pharmaceutical composition is an in situ gellable aqueous composition, such as an in situ gellable aqueous solution. An in situ gellable composition comprises a gelling agent in a concentration effective to promote gelling upon contact with the eye or with lacrimal fluid in the exterior of the eye. Suitable gelling agents include thermosetting polymers such as terra-substituted ethylene diamine block copolymers of ethylene oxide and propylene oxide (e.g., poloxamine 1307); polycarbophil; and polysaccharides such as gellan, carrageenan (e.g., kappa-carrageenan and iota-carrageenan), chitosan and alginate gums.

Optionally, the present compositions can include one or more ophthalmically acceptable surfactants, preferably nonionic surfactants. For example, the present compositions can comprise one or more non-ionic surfactants, such as polysorbates (such as polysorbate 80 (polyoxyethylene sorbitan monooleate), polysorbate 60 (polyoxyethylene sorbitan monostearate), polysorbate 20 (polyoxyethylene sorbitan monolaurate), commonly known by their trade names of Tween® 80, Tween® 60, Tween® 20), poloxamers (synthetic block polymers of ethylene oxide and propylene oxide, such as those commonly known by their trade names of Pluronic®; e.g., Pluronic® F 127 or Pluronic® F 108) ), or poloxamines (synthetic block polymers of ethylene oxide and propylene oxide attached to ethylene diamine, such as those commonly known by their trade names of Tetronic®; e.g., Tetronic® 1508 or Tetronic® 908, etc., other nonionic surfactants such as Brij®, Myrj®, and long chain fatty alcohols (i.e., oleyl alcohol, stearyl alcohol, myristyl alcohol, docosohexanoyl alcohol, etc.) with carbon chains having about 12 or more carbon atoms (e.g., such as from about 12 to about 24 carbon atoms). Such compounds are delineated in Martindale, 34^th ed., pp 141 1-1416 (Martindale, "The Complete Drug Reference," S. C. Sweetman (Ed.), Pharmaceutical Press, London, 2005) and in Remington, "The Science and Practice of Pharmacy," 21^st Ed., p. 291 and the contents of chapter 22, Lippincott Williams & Wilkins, New York, 2006); the contents of these sections are incorporated herein by reference. The concentration of a non-ionic surfactant, when present, in the present compositions can be in the range from about 0.001 to about 5 weight percent (or alternatively, from about 0.01 to about 4, or from about 0.01 to about 2, or from about 0.01 to about 1, or from about 0.01 to about 0.5 weight percent).

An ophthalmic pharmaceutical composition can also include glycerin to increase viscosity of the composition and for adjustment of osmolality and/or a cyclodextrin as a solubility agent.

Alternatively, in some embodiments of the present compositions and methods, one or more ophthalmic lubricating agents can optionally be included in the composition to promote lacrimation. Such agents include polyvinyl alcohol, methylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, and others. It will be understood that promotion of lacrimation is beneficial only in certain situations where lacrimation is naturally deficient, to restore a normal degree of secretion of lacrimal fluid. Where excessive lacrimation occurs, residence time of the inhibitor in the eye can be reduced.

Additional Active Agents

In some embodiments of the present methods, the present compositions are used in co-therapy, co-administration, or coformulated with at least one active agent other than a receptor tyrosine kinase inhibitor. In some embodiments, the present compositions further comprise a therapeutically and/or prophylactically effective amount of at least one other active agent. The other active agent(s) can cooperate with the inhibitor in the composition in treating an ophthalmic infection, or can be used to treat a related or unrelated condition simultaneously affecting the eye. Other active agents for use in the present compositions and methods may include additional anti-angiogenic agents, corticosteroids, corticosteroid mimetics, antiinflammatory agents, anti-infective agents, local anesthetic agents, destructive therapy agents, and antiandrogens.

Corticosteroids that may be used include betamethasone dipropionate, fluocinolone acetonide, betamethasone valerate, triamcinolone acetonide, clobetasol propionate, desoximetasone, diflorasone diacetate, amcinonide, flurandrenolide, hydrocortisone valerate, hydrocortisone butyrate, and desonide.

Local anesthetics include tetracaine, tetracaine hydrochloride, lidocaine, lidocaine hydrochloride, dyclonine, dyclonine hydrochloride, dimethisoquin hydrochloride, dibucaine, dibucaine hydrochloride, butambenpicrate, and pramoxine hydrochloride. A preferred concentration for local anesthetics is about 0.025% to 5% by weight of the total composition. Anesthetics such as benzocaine may also be used at a preferred concentration of about 2% to 25% by weight.

The present compositions also may include additional therapeutic drugs such as agents for treating glaucoma and anti-inflammatory drugs. Examples of anti- glaucoma drugs include but are not limited to timolol-base, betaxalol, athenolol, levobanolol, epinenephrin, dipivalyl, oxonolol, acetazilumide-base and methazalomide. Examples of anti-inflammatory drugs include but are not limited to non-steroids such as piroxicam, indomethacin, naproxen, phenyylbutazone, ibuprofen and diclofenac.

Any active agent having utility in an ophthalmic application can be used in co-therapy, co-administration or coformulation with the present compositions as described above. A method of treating a disease involving ocular angiogenic by co- therapy comprises administering an receptor tyrosine kinase inhibitor and an additional active agent to a subject at the same time or at different times of administration, in a single composition or in separate compositions, by the same route of administration or by different routes of administration. A method of treating a disease involving ocular angiogenic by co-administration comprises administering an receptor tyrosine kinase inhibitor and an additional active agent to a subject at the same time and by the same route of administration, but may be in a single composition or in separate compositions. A method of treating a disease involving ocular angiogenic by co-formulation comprises administering an receptor tyrosine kinase inhibitor and an additional active agent to a subject in a single composition.

Other examples of active agents for use with the present methods and compositions include without limitation: antibiotics; antimycotics, antivirals and other anti-infectives; acetylcholine blocking agents; adrenergic agonists, beta-adrenergic blocking agents and other antiglaucoma agents; antihypertensives; antihistamines; anticataract agents; and topical and regional anesthetics. Examples of specific drugs for use as an additional active agent include acebutolol, aceclidine, acetylsalicylic acid (aspirin), acetylsulfisoxazole, alclofenac, alprenolol, amfenac, amiloride, aminocaproic acid, p-aminoclonidine, aminozolamide, anisindione, apafant, atenolol, bacitracin, benoxaprofen, benoxinate, benzofenac, bepafant, betamethasone, betaxolol, bethanechol, bimatoprost, brimonidine, bromfenac, bromhexine, bucloxic acid, bupivacaine, butibufen, carbachol, carprofen, celecoxib, cephalexin, chloramphenicol, chlordiazepoxide, chlorprocaine, chlorpropamide, chlortetracycline, cicloprofen, cinmetacin, ciprofloxacin, clidanac, clindamycin, clonidine, clonixin, clopirac, cromolyn, cyclopentolate, cyproheptadine, demecarium, dexamethasone, dibucaine, diclofenac, diflusinal, dipivefrin, dorzolamide, enoxacin, epinephrine, erythromycin, eserine, estradiol, ethacrynic acid, etidocaine, etodolac, fenbufen, fenclofenac, fenclorac, fenoprofen, fentiazac, flufenamic acid, flufenisal, flunoxaprofen, fluoroquinolone, fluorometholone, flurbiprofen and esters thereof, fluticasone propionate, furaprofen, furobufen, furofenac, furosemide, gancyclovir, gentamicin, gramicidin, hexylcaine, homatropine, hydrocortisone, ibufenac, ibuprofen and esters thereof, idoxuridine, indomethacin, indoprofen, interferons, isobutyhmethylxanthine, isofluorophate, isoproterenol, isoxepac, ketoprofen, ketorolac, labetolol, ketorolac, latanoprost, levo- bunolol, lidocaine, lonazolac, loteprednol, meclofenamate, medrysone, mefenamic acid, mepivacaine, metaproterenol, methanamine, methylprednisolone, metiazinic, metoprolol, metronidazole, minopafant, miroprofen, MK-663, modipafant, nabumetome, nadolol, namoxyrate, naphazoline, naproxen and esters thereof, neomycin, nepafenac, nitroglycerin, norepinephrine, norfloxacin, nupafant, olfloxacin, olopatadine, oxaprozin, oxepinac, oxyphenbutazone, oxyprenolol, oxytetracycline, parecoxib, penicillins, perfloxacin, phenacetin, phenazopyridine, pheniramine, phenylbutazone, phenylephrine, phenylpropanolamine, phospholine, pilocarpine, pindolol, pirazolac, piroxicam, pirprofen, polymyxin, polymyxin B, prednisolone, prilocaine, probenecid, procaine, proparacaine, protizinic acid, rimexolone, rofecoxib, salbutamol, scopolamine, sotalol, sulfacetamide, sulfanilic acid, sulindac, suprofen, tenoxicam, terbutaline, tetracaine, tetracycline, theophyllamine, timolol, tobramycin, tolmetin, travoprost, triamcinolone, trimethoprim, trospectomycin, valdecoxib, vancomycin, vidarabine, vitamin A, warfarin, zomepirac, and pharmaceutically acceptable salts thereof.

Additional Tyrosine Kinase Inhibitors

It is particularly contemplated that a second tyrosine kinase inhibitor agent can be administered in co-therapy, co-administration, or coformulation with or in the present compositions and methods. For example, if the inhibitor is effective against c- MET or RON, but does not inhibit VEGF, it may be desired to also administer an inhibitor of VEGF or another tyrosine kinase. Such co-therapy, co-administration, and coformulation are embodiments of the present compositions and methods. Various receptor tyrosine kinases and inhibitors are discussed above.

Co-therapies for Diseases Involving Ocular Angiogenesis

The present methods and compositions can include the use of one of the inhibitors of selected receptor tyrosine kinases as a co-therapy. Preferably, an inhibitor of a selected receptor tyrosine kinase or of a selected combination of receptor tyrosine kinases can be administered in co-therapy with an additional receptor tyrosine kinase inhibitor, pegaptanib sodium, ranibizumab, and photodynamic therapy. Other co- therapies for the treatment of retinal and choroidal neovascularization include destruction of new vessels using photocoagulation or cryotherapy.

Photodynamic therapy (PDT) is discussed in Strong et al. U.S. Patent No. 7,060,695. Methods are discussed for treating conditions of the eye characterized by ocular neovascularization. Patients are given and initial photodynamic therapy (PDT) treatment to destroy the neovasculature, and then are re-evaluated at least twice during the following 6 months, and retreated as necessary. Preferably, three retreatments are provided. Methods of Screening for an Inhibitor of One or More Selected Receptor Tyrosine

Kinases

The present invention also relates to methods of screening a compound as an inhibitor of one or more selected receptor tyrosine kinases, for example as an inhibitor of RON, or as an inhibitor of c-MET, or an inhibitor of both RON and c-MET and optionally other receptor tyrosine kinases. The methods comprise providing a test compound and determining the effect of the test compound on the activity of a kinase, a mixture comprising a receptor tyrosine kinase and c-MET and/or RON under conditions suitable for expression of c-MET and/or RON. The test compound can be a member of a library of compounds. Methods of screening compounds for inhibition of c-MET and/or RON are known, and the present inventors contemplate that such methods can be adapted for methods of screening a compound as an inhibitor of c-MET and/or RON.

Example 1

An ophthalmic pharmaceutical composition comprises an inhibitor of one or more selected receptor tyrosine kinases, sodium chloride, sodium phosphate monohydrate, dibasic sodium phosphate heptahydrate, and HCl or NaOH to adjust the pH, and water for injection. The composition has a pH in the range of 6 to 7.5 and an osmolality of 250 to 350 mθsm/kg. The composition is a sterile, clear solution. The composition is an injectable composition suitable for intraocular administration, such as by injection in the vitreous of a subject.

Example 2

A method of treating ocular angiogenic disease comprises administering an inhibitor of RON to an eye of a subject suffering from or at risk of ocular angiogenesis, for example, subject having diabetes mellitus. The inhibitor is administered directly to an eye no more frequently than every four weeks, or between 0.5 and 12 times per year. After several months, one or more signs or symptoms of ocular angiogenesis are alleviated, ameliorated, reduced, or removed.

Example 3

A method of treating ocular angiogenic disease comprises administering a composition comprising an inhibitor of c-Met, RON, Tie-2 and one or more VEGF receptors by injection into an eye of a subject suffering from or at risk of ocular angiogenesis, for example, subject having diabetes mellitus. The inhibitor is administered directly to an eye no more frequently than every four weeks. After several months, one or more signs or symptoms of ocular angiogenesis are alleviated, ameliorated, reduced, or removed.

Example 4

A subject in need of treatment for age-related macular degeneration is treated with an intravitreal injection of an ophthalmic pharmaceutical composition comprising an inhibitor of RON. The purpose of this treatment is to reduce or prevent the development of neovascularization, macular disease, and retinal damage.

In the present specification, use of the singular includes the plural except where specifically indicated. The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.

Wherever an open-ended term is used to describe a feature or element of the invention, it is specifically contemplated that a closed-ended term can be used in place of the open-ended term without departing from the spirit and scope of the invention. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention. In any set of alternatives, one alternative should not be presumed to be an equivalent of another alternative unless it is indicated otherwise. Where alternatives are equivalent for one purpose, they may not be equivalent for other purposes.

Pn the present specification, any of the functions recited herein may be performed by one or more compositions, compounds, or excipients for performing such functions. With respect to the methods described in the specification, it is intended that the specification also provides a description of the products of those methods. With respect to the compositions and combinations described in the specification, it is intended that the specification also provides a description of the components, parts, portions, of such compositions and combinations. With respect to any list of particular materials, the present inventors contemplate that the specification provides a description of a smaller list of such materials which excludes some of the listed materials. Any materials listed together should not be presumed to be interchangeable for each other.

All of the references cited herein, including patents, patent applications, and publications, are hereby incorporated in their entireties by reference.

While particular elements, embodiments and applications of the present invention have been shown and described, it will be understood, of course, that the invention is not limited thereto since modifications can be made by those skilled in the art without departing from the scope of the present disclosure, particularly in light of the foregoing teachings.

Although the dependent claims have single dependencies in accordance with U.S. patent practice, each of the features in any of the dependent claims can be combined with each of the features of other dependent claims or the main claim.

Claims

CLAIMS We claim:

1. An ophthalmic pharmaceutical composition for treating an ocular angiogenic disease, the composition comprising a receptor tyrosine kinase inhibitor that inhibits the activity of recepteur d'origine nantais (RON), and an ophthalmically acceptable excipient, wherein said receptor tyrosine kinase inhibitor is present in an amount sufficient for said treating.

2. The ophthalmic pharmaceutical composition of claim 1, wherein the receptor tyrosine kinase inhibitor also inhibits the activity of c-MET.

3. The ophthalmic pharmaceutical composition of claim 2, wherein the receptor tyrosine kinase inhibitor also inhibits the activity of one or more other receptor tyrosine kinases.

4. The ophthalmic pharmaceutical composition of claim 1 wherein the receptor tyrosine kinase inhibitor specifically inhibits RON; or inhibits both c-MET and RON; or inhibits RON and c-MET and at least one other receptor tyrosine kinase; or inhibits RON and c-MET and one or more VEGF receptors; or inhibits RON and c-MET and Tie-2; or inhibits c-MET and one or more VEGF receptors; or inhibits c-MET and Tie-2; or inhibits RON and one or more VEGF receptors; or inhibits RON and Tie-2.

5. The ophthalmic pharmaceutical composition of claim 1 wherein the composition comprises a second receptor tyrosine kinase inhibitor, and the second receptor tyrosine kinase inhibitor is an inhibitor of c-MET.

6. The ophthalmic pharmaceutical composition of claim 1 wherein the composition comprises a second receptor tyrosine kinase inhibitor, and the second receptor tyrosine kinase inhibitor is an inhibitor of the activity of one or more other receptor tyrosine kinases.

7. The ophthalmic pharmaceutical composition of claim 1 wherein the composition comprises a second receptor tyrosine kinase inhibitor, and the second receptor tyrosine kinase inhibitor is an inhibitor of one or more VEGF receptors or Tie-2.

8. The ophthalmic pharmaceutical composition of claim 1 wherein the inhibitor is a compound that specifically binds to an extracellular domain of the receptor tyrosine kinase.

9. The ophthalmic pharmaceutical composition of claim 1 wherein the inhibitor is a compound that specifically binds to a catalytic moieity of the receptor tyrosine kinase.

10. The ophthalmic pharmaceutical composition of claim 1 wherein the ophthalmic pharmaceutical composition is an isotonic solution.

11. The ophthalmic pharmaceutical composition of claim 1 wherein the ophthalmic pharmaceutical composition is a suspension.

12. The ophthalmic pharmaceutical composition of claim 1 wherein the ophthalmic pharmaceutical composition is an injectable composition.

13. An ophthalmic pharmaceutical composition for treating an ocular angiogenic disease, the composition comprising a receptor tyrosine kinase inhibitor that inhibits the activity of c-Met, and an ophthalmically acceptable excipient, wherein the composition is an injectable composition and said receptor tyrosine kinase inhibitor is present in an amount effective for said treating.

14. A method of treating ocular angiogenic disease comprising administering to a subject a receptor tyrosine kinase inhibitor that inhibits the activity of RON, wherein the inhibitor is administered for a time and in an amount effective to reduce a risk of ocular angiogenesis.

15. The method of claim 14, wherein the receptor tyrosine kinase inhibitor also inhibits the activity of c-MET.

16. The method of claim 14, wherein the receptor tyrosine kinase inhibitor also inhibits the activity of one or more other receptor tyrosine kinases.

17. The method of claim 14, wherein the receptor tyrosine kinase inhibitor specifically inhibits RON; or inhibits both c-MET and RON; or inhibits RON and c- MET and at least one other receptor tyrosine kinase; or inhibits RON and c-MET and one or more VEGF receptors; or inhibits RON and c-MET and Tie-2; or inhibits c-MET and one or more VEGF receptors; or inhibits c-MET and Tie-2; or inhibits RON and one or more VEGF receptors; or inhibits RON and Tie-2.

18. The method of claim 14, wherein the ocular angiogenic disease is diabetic macula edema.

19. The method of claim 14, wherein the ocular angiogenic disease is age- related macular degeneration.

20. The method of claim 14, wherein the ocular angiogenic disease is diabetic retinopathy.

21. The method of claim 14, further comprising administering to the subject as a co-therapy a compound selected from the group consisting of an additional receptor tyrosine kinase inhibitor, pegaptanib sodium, ranibizumab, and photodynamic therapy.

22. The method of claim 14, wherein the method comprises diagnosis a subject at risk of diabetic macula edema, age-related macular degeneration, or diabetic retinopathy, and administering the inhibitor before the onset of ocular angiogenesis.

23. The method of claim 14, wherein the inhibitor is administered by intravitreous injection.