WO2023019218A1 - Modified fibroblast growth factors for treating fibrotic diseases - Google Patents

Abstract

Described herein are methods of preventing, treating or ameliorating a disease, disorder or a condition associated with fibrotic diseases, comprising administering to a subject having a fibrotic disease a pharmaceutically effective amount of composition comprising modified fibroblast growth factor (FGF) polypeptides.

Description

MODIFIED FIBROBLAST GROWTH FACTORS FOR TREATING FIBROTIC

DISEASES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/232,481, filed August 12, 2021, which is incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] This application incorporates by reference a Sequence Listing submitted with this application as a text file entitled “45341-711.601. xml” created on August 11, 2022 and having a size of 315 ,392 bytes.

BACKGROUND

[0003] FGFs are large polypeptides widely expressed in developing and adult tissues (Baird et al., Cancer Cells, 3 :239-243, 1991) and play roles in multiple physiological functions (McKeehan etal., Prog. Nucleic Acid Res. Mol. Biol. 59:135-176, 1998; Burgess, W. H. etal., Annu Rev. Biochem. 58:575-606 (1989). The FGF family includes at least twenty -two members (Reuss etal., Cell Tissue Res. 313 :139-157 (2003)).

SUMMARY

[0004] Provided herein in one embodiment is a method of treating glaucoma, or preventing or delaying the onset or progression of glaucoma, the method comprising: administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide, wherein the modified FGF-1 polypeptide comprises a sequence comprising one or more mutations relative to the wild-type FGF-1 sequence of SEQ ID NO: 1. In some embodiments, the modified FGF-1 comprises at least two mutations with respectto the wild type FGF-1 of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide comprises an N-terminal methionine upstream of the first residue of SEQ ID NO: 1 . In some embodiments, the modified FGF-1 comprises an amino acid sequence of that is at least about 80% identical to the sequence of SEQ ID NO: 2. In some embodiments, the glaucoma is open-angle glaucoma. In some embodiments, the glaucoma is angle-closure glaucoma. In some embodiments, the subject has an elevated intra ocular pressure (IOP), increased aqueous humor production, impaired outflow/deflow of aqueous humor, or any combinations thereof. In some embodiments, the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide is administered to the subject at an early stage of glaucoma. In some embodiments, the pharmaceutical composition comprising a therapeutically effective amount of the modifiedFGF-1 polypeptide is administered to the subject prior to scleral canal wall expansion and/or posterior deformation of the lamina cribrosa. In some embodiments, the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF- 1 polypeptide is administered to the subject at an advanced stage of the glaucoma, wherein the advanced stage of the glaucoma is characterized by loss of retinal ganglion cells (RGC) in the subject. In some embodiments, the pharmaceutical composition comprising a therapeutically effective amount of the modifiedFGF-1 polypeptide is administered to the subject at a stage prior to trabeculectomy. In some embodiments, administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide prevents or delays the onset or progression of glaucoma. In some embodiments, administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF- 1 polypeptide prevents or decreases the need for surgical intervention. In some embodiments, administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide reduces at least one clinical symptom associated with glaucoma. In some embodiments, administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide results in at least one of: reduced IOP, reduced aqueous humor production, increased outflow/deflow of aqueous humor, or any combinations thereof. In some embodiments, administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide reduces or eliminates epithelial-mesenchymal like transition of trabecular meshwork (TM) cells. In some embodiments, administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide reduces macrophage infiltration in ocular tissue as observed by scanning electron microscopy. In some embodiments, administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide reduces at least one of: eye pain, headache, nausea, poor vision, optic nerve damage, alteration in corneal thickness, narrowed drainage angle and loss of vision associated with glaucoma. In some embodiments, the pharmaceutical composition is formulated for ophthalmic administration. In some embodiments, the pharmaceutical composition comprises a formulation for ocular delivery. In some embodiments, the formulation for ocular delivery comprises a humectant at a concentration of at least about 5%. In some embodiments, the humectant is selected from the group consisting of : Glycerin, Maltitol, Propylene Glycol, Sorbitol, and Triacetin. In some embodiments, the formulation comprises at least about 0.1% (w/v) of a surfactant. In some embodiments, the surfactant is polysorbate 80 or polysorbate 20. In some embodiments, the pharmaceutical composition comprises: about lOmM citrate or Histidine, about 5% sorbitol, about 0.1% polysorbate 80, and has a pH of about 5.8. In some embodiments, the pharmaceutical composition comprises phosphate buffered saline, about 0.1% polysorbate 80, and has a pH of about 7.4. In some embodiments, the pharmaceutical composition is administered at a dose comprising about 10 ng to about 1000 ng of the modified FGF-1 polypeptide per eye per administration, wherein the administration is intracameral. In some embodiments, the pharmaceutical composition is administered at a dosage comprising the modified FGF-1 polypeptide at a concentration of about 0.1 to 10 pg/ml, wherein the administration is topical. In some embodiments, the pharmaceutical composition is administered at a dose comprising about 0.3 mg/kgto about 10 mg/kg of the modified FGF-1 polypeptide. In some embodiments, the pharmaceutical composition is administered at a dose comprising about 0.3 pg/eye to about 3 pg/eye of the modified FGF-1 polypeptide, per administration. In some embodiments, the pharmaceutical composition is administered one to five times a day. In some embodiments, the pharmaceutical composition is administered about two times a day. In some embodiments, the pharmaceutical composition is administered about three times a day. In some embodiments, the pharmaceutical composition is administered for at least five consecutive days. In some embodiments, the pharmaceutical composition is administered for at least seven consecutive days. In some embodiments, the pharmaceutical composition is administered at least for 15 days, 21 days, 24 days, 28 days, 30 days. In some embodiments, the pharmaceutical composition is administered via intracameral or intravitreal injection every 7 to 30 days. In some embodiments, administering the pharmaceutical composition for about 7 days reduces the IOP in the eye by at least about 1 .12% .

[0005] Provided herein in one embodiment is a method of treating an ocular disease, disorder or a condition associated with a fibrotic or profibrotic induction in a tissue, or a condition associated with a risk of a fibrotic induction in a tissue, the method comprising: administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide, wherein the modified FGF-1 polypeptide comprises a sequence comprising an N-terminal methionine and a sequence comprising one or more mutations relative to the wild-type FGF-1 sequence of SEQ ID NO: 1 . In some embodiments, the disease, disorder or condition is Geographic atrophy (GA). In some embodiments, administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide reduces at least one symptom associated with GA, wherein the at least one symptom is selected from poor vision, loss of melanin from the retina, GA lesions, central RPE loss, choriocapillary thinning and choroidal enhancement, andRPE cell migration. In some embodiments, the RPE cell migration occurs from a native location of the RPE cells in the outer retinal layer to ectopic locations in the inner retinal layers. In some embodiments, the disease, disorder or condition is drusen. In some embodiments, the disease, disorder or condition is Stevens-Johnson Syndrome (SJS). In some embodiments, the disease, disorder or condition is Graves’ orbitopathy. In some embodiments, the disease, disorder or condition is pingueculae or pterygia. In some embodiments, the disease, disorder or condition is Limbal epithelial stem cell deficiency (LSCD). In some embodiments, the disease, disorder or condition is chronic cicatrizing conjunctivitis. In some embodiments, the pharmaceutical composition comprises a formulation for ocular delivery. In some embodiments, the formulation comprises a humectant at a concentration of at least about 5% concentration. In some embodiments, humectant is selected from the group consisting of: Glycerin, Maltitol, Propylene Glycol, Sorbitol, and Triacetin. In some embodiments, the formulation comprises at least about 0.1% of a surfactant. In some embodiments, the surfactant comprises polysorbate 80 or polysorbate 20. In some embodiments, the pharmaceutical composition comprises: about lOmM citrate or Histidine, about 5% sorbitol, about 0.1% polysorbate 80, and has a pH of about 5.8. In some embodiments, the pharmaceutical composition comprises: wherein the pharmaceutical composition comprises phosphate buffered saline, about 0.1% polysorbate 80, and has a pH of about 7.4.

[0006] Provided herein in one embodiment is a method of treating a disease, disorder or a condition resulting from a fibrotic or profibrotic induction in a tissue, the method comprising: administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide, wherein the modified FGF-1 polypeptide comprises a sequence comprising the mutations Cysl6Ser, Ala66Cys, and Cysl 17Val relative to the wild-type FGF-1 sequence of SEQ ID NO: 1, and wherein the disease, disorder, or a condition is not an ocular disease, disorder, or condition. In some embodiments, the disease, disorder or condition is a fibrotic disease, disorder or condition of the lung. In some embodiments, the disease, disorder or condition is idiopathic pulmonary fibrosis. In some embodiments, the disease, disorder or condition is a fibrotic disease, disorder or condition of the cardiac tissue. In some embodiments, the disease, disorder or condition is myocardial infarction. In some embodiments, the disease, disorder or condition is a fibrotic disease, disorder or condition of the renal tissue. In some embodiments, the disease is selected from anti-glomerular basement membrane glomerulonephritis, lupus nephritis, antigen-induced immune complex glomerulonephritis, renal allograft injury, ischemia reperfusion injury, and nephropathy. In some embodiments, the disease, disorder or condition results from pneumonia, rheumatoid arthritis, mixed connective tissue disease, systemic erythematosus lupus, scleroderma, dermatomyositis, polymyositis, systemic sclerosis or sarcoidosis. In some embodiments, the disease is associated with an elevated expression of TGF-b eta, TNF-alpha, IFN-gamma, IL-6, alpha-SMA, vimentin, fibronectin, ZEB-1 or ZEB-2, Slug, Snail or Twist, and administering the pharmaceutical composition reduces the elevated expression levels of any one or more of TGF-beta, TNF-alpha, IFN-gamma, IL-6, alpha-SMA, vimentin, fibronectin, ZEB-1 or ZEB-2, Slug, Snail and Twist. In some embodiments, administering the pharmaceutical composition reduces alpha-smooth muscle actin (a-SMA) upregulation in TM cells and/or RPE cells.

[0007] Provided herein in one embodiment is a method of treating a disease, disorder or a condition associated with activation of myeloid cells, the method comprising: administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide, wherein the modified FGF-1 polypeptide comprises a sequence comprising the mutations Cysl6Ser, Ala66Cys, and Cysl 17 Vai relative to the wildtype FGF-1 sequence of SEQ ID NO: 1, and wherein the disease, disorder, or a condition is not an ocular disease, disorder, or condition. In some embodiments, the myeloid cell is an antigen presenting cell, a macrophage, a dendritic cell, or a neutrophil. In some embodiments, the myeloid cell is macrophage. In some embodiments, the disease disorder or condition is characterized by macrophage infiltration in an affected tissue of the subject. In some embodiments, administering the pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide alters at least one gene expression signature associated with macrophage infiltration in the affected tissue of the subject. In some embodiments, the myeloid cell is an M2 polarized macrophage. In some embodiments, administering the pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide increases the expression of a M2 polarized macrophage specific biological marker in an affected tissue of the subject. In some embodiments, the M2 macrophage specific biological marker is CD 163, and administering the pharmaceutical composition reduces the number of CD 163+ cells in the ocular tissue. In some embodiments, the disease disorder or condition is characterized by macrophage to myofibroblast transformation. In some embodiments, administering the pharmaceutical composition reduces at least one gene expression signature associated with macrophage to myofibroblast transformation. In some embodiments, the disease disorder or condition is characterized by increased inflammatory gene expression. In some embodiments, the disease disorder or condition is characterized by increased expression ofLy6C, Ibal, and/or Argl, and administering the pharmaceutical composition reduces the expression of one or more of Ly6C, Ibal, and/or Argl . In some embodiments, the disease disorder or condition is characterized by increased expression of TGF- beta, TNF-alpha, IFN-gamma, IL-6, alpha-SMA, vimentin, fibronectin, ZEB-1 or ZEB-2, Slug, Snail or Twist. In some embodiments, the disease disorder or condition is characterized by increased expression of genes related to vasculogenesis or fibrogenesis. In some embodiments, the disease, disorder or condition is characterized by increased VEGF, PDGF, peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), matrix metalloproteinases, a disintegrin and metalloprotease (ADAM), caspases, or components of the renin-angiotensin- aldosterone system (ANG II). In some embodiments, administering the pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide reduces the gene expression of one or more of VEGF, PDGF, peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), matrix metalloproteinases, a disintegrin and metalloprotease (ADAM), caspases, or components of the renin-angiotensin-aldosterone system (ANGII). In some embodiments, the disease, disorder or condition is characterized by altered gene expression of a cytokine or a chemokine gene. In some embodiments, the cytokine or chemokine is IL4, IL-13, IL-21, TGF-pi, MCP-1, or MIP-ip. In some embodiments, administering the pharmaceutical composition reverses the alteration of gene expression of one or more of IL4, IL-13, IL-21, TGF-pi, MCP-1 and MIP-ip induced by the disease, disorder or condition. In some embodiments, the gene expression is detected by RT-PCR of a biological sample from the subject. In some embodiments, the biological sample is obtained from a tissue or cells from the subject that is affected by the disease, disorder or condition. In some embodiments, the biological sample is obtained from peripheral blood. In some embodiments, the disease, disorder or condition is associated with aberrant myeloid cell activation and is further characterized by activation of an epithelial or endothelial cell. In some embodiments, the modified FGF-1 comprises a modified human

FGF-1 . In some embodiments, the modified FGF-1 comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 2. In some embodiments, the subject is human.

[0008] Also provided herein in one embodiment is a kit comprising (a) a pharmaceutical composition comprising a modified FGF-1 polypeptide, and (b) a container; wherein the modified FGF-1 polypeptide comprises a sequence comprising the mutations Cysl6Ser, Ala66Cys, and Cysl 17Val relative to the wild -type FGF-1 sequence of SEQ ID NO: 1, and wherein the disease, disorder, or a condition is not an ocular disease, di sorder, or condition. In some embodiments, the container is an eye dropper or a vial. In some embodiments, the container is a glass container or a non-glass (e.g., resin) container. In some embodiments, the container comprises a polyolefin resin, a polyester resin, a polysulfone resin, a polyamide resin, a polycarbonate resin, a polyphenylene ether resin, a polyvinyl chloride resin, a styrene resin, or combinations thereof. In some embodiments, the container comprises a polyolefin resin. In some embodiments, the container consists essentially of a polyolefin resin. In some embodiments, the container is made of a polyolefin resin. In some embodiments, the polyolefin resin is a cyclic polyolefin resin. In some embodiments, the container comprises a resin and an UV light absorbing agent or an UV light scattering agent. In some embodiments, the container is compatible with high and low pH conditions. In some embodiments, the container comprises a low protein and peptide adsorption. In some embodiments, the container is a Crystal Zenith vial or Crystal Zenith syringe. In some embodiments, the container is a Crystal Zenith vial.

INCORPORATION BY REFERENCE

[0009] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entireties to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF DRAWINGS

[0010] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

[0011] FIG. 1 shows data illustrating changes in intraocular pression (IOP) of one eye of New Zealand White (NZW) rabbits 7-day after administering different doses of TTHX1114.

TTHX1 114 was administered three times daily for 7 days. At the end of the 7^th day, IOP was measured using rebound tonometry.

DETAILED DESCRIPTION

[0012] The regulation of events leading to fibrosis typically involves at least two major events. One is the proliferation and differentiation of fibrocytes. Fibrocytes are a distinct population of fibroblast-like cells derived from peripheral blood monocytes that normally enter sites of tissue injury to promote angiogenesis and wound healing. Fibrocytes differentiate from CD 14+ peripheral blood monocytes, and may differentiate from other PBMC cells. The presence of SAP, IL-12, Laminin-1, anti-FcyR antibodies, crosslinked IgG and/or aggregated IgG may inhibit or at least partially delay this process. The second major event is the formation and maintenance of fibrotic tissue. Fibrotic tissue may be formed and maintained by the recruitment and proliferation of fibroblast cells, the formation of new extracellular matrix, and the growth of new vascular tissue. In pathologic fibrosis, such as following chronic inflammation, injury, or idiopathic fibrosis, it is this excess fibrotic tissue that can lead to tissue damage and destruction. [0013] A second source of cells that contribute to fibrotic diseases is the epithelial (or endothelial) to mesenchymal transition (EMT) of the endogenous myeloid cells or of other cells within a tissue including keratocytes, keratinocytes, and other cells. EMT is fibrotic induction as cells that have undergone EMT become myofibroblasts and contribute to the fibrotic tissue. [0014] In some embodiments the methods of this disclosure comprise administering a composition comprising a modified FGF-1 polypeptide that is suitable for the inhibition or delay of fibrocyte differentiation and/or of EMT to a target location, that is suitable for the inhibition or antagonizing of pro fibrotic factors. The present disclosure thus provides methods for treating fibrotic and fibroproliferative disorders, e.g., by administering a fibrocyte suppressor. The methods provide for treatment of fibrotic diseases, including those affecting the lung, liver, heart, kidney and eye.

[0015] Examples of fibroproliferative diseases include but are not limited to: glaucoma; glomerulonephritis (GN); diabetic nephropathy; renal interstitial fibrosis; renal fibrosis resulting from complications of drug exposure; HIV-associated nephropathy; transplant necropathy; liver cirrhosis due to all etiologies; disorders of the biliary tree; hepatic dysfunction attributable to infections; pulmonary fibrosis; adult respiratory distress syndrome (ARDS); chronic obstructive pulmonary disease (COPD); idiopathic pulmonary fibrosis (IPF); acute lung injury (ALI); pulmonary fibrosis due to infectious or toxic agents; congestive heart failure; dilated cardiomyopathy; myocarditis; vascular stenosis; progressive systemic sclerosis; polymyositis; scleroderma; Grave's disease; dermatomyositis; fascists; Raynaud's syndrome, rheumatoid arthritis; proliferative vitreoretinopathy; fibrosis associated with ocular surgery, and excessive or hypertrophic scar or keloid formation in the dermis occurring during wound healing resulting from trauma or surgical wounds. Still other exemplary fibrotic disorders that can be treated with the methods of this disclosure, comprising administering a modified FGF polypeptide, or a pharmaceutical composition comprising the same, are described in further detail below. The etiology may be due to any acute or chronic insult including toxic, metabolic, genetic and infectious agents.

[0016] As used herein the terms “fibroproliferative disorder” and “fibrotic disorder” refer to conditions involving fibrosis in one or more tissues. As used herein the term “fibrosis” refers to the formation of fibrous tissue as a reparative or reactive process, rather than as a norm al constituent of an organ or tissue. Fibrosis is characterized by fibroblast accumulation and collagen deposition in excess of normal deposition in any particular tissue. As used herein the term “fibrosis” is used synonymously with “fibroblast accumulation and collagen deposition”. Fibroblasts are connective tissue cells, which are dispersed in connective tissue throughoutthe body. Fibroblasts secrete a nonrigid extracellular matrix containing type I and/or type III collagen. In response to an injury to a tissue, nearby fibroblasts migrate into the wound, proliferate, and produce large amounts of collagenous extracellular matrix. Collagen is a fibrous protein rich in glycine and proline that is a major component of the extracellular matrix and connective tissue, cartilage, and bone. Collagen molecules are triple-stranded helical structures called a-chains, which are wound around each other in a ropelike helix. Collagen exists in several forms or types; of these, type I, the most common, is found in skin, tendon, and b one; and type III is found in skin, bloodvessels, and internal organs. Fibrotic disorders include, but are not limited to, systemic and local scleroderma, keloids and hypertrophic scars, atherosclerosis, restenosis, pulmonary inflammation and fibrosis, idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis, sarcoidosis, fibrosis as a result of chronic hepatitis B or C infection, kidney disease, heart disease resulting from scar tissue, and eye diseases such as glaucoma, and retinal and vitreal retinopathy. Additional fibrotic diseases include, but are not limited to, fibrosis resulting from chemotherapeutic drugs, radiation-induced fibrosis, and injuries and burns.

[0017] Described herein, in some embodiments, are methods of preventing, treating or ameliorating a disease, disorder or a condition associated with a fibrotic or profibrotic induction in a tissue, or a condition associated with a risk of a fibrotic induction in a tissue, comprising administering to a subject having a fibrotic disease by administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising modified fibroblast growth factor (FGF) polypeptides (e.g., a modified FGF-1 polypeptide comprising one or more mutations relative to a wild-type human FGF-1).

[0018] In some embodiments, a fibrotic or profibrotic induction in a tissue is a condition that develops into progressive fibrotic diseases induced by the action of a component cause. In some embodiments, the component cause is a cause that leads to a fibrotic condition, for example, a persistent inflammatory condition, scarring, and so on. In some embodiments, for a given disease, a fibrotic or profibrotic induction appears at an early stage of the disease or at a later stage or as a sequela of the disease itself. In some embodiments, an exemplary component cause comprises inflammatory exacerbation. In some embodiments, the fibrotic or profibrotic induction as described herein comprises a pre-fibrotic condition. In some embodiments, the fibrotic or profibrotic induction as described herein includes a risk of developing a pro-fibrotic or fibrotic condition.

[0019] In some embodiments, the fibrotic diseaseis characterized by progressive scarring. Progressive scarring (fibrosis) is a pathological feature of many chronic inflammatory diseases and is an important cause of morbidity and mortality worldwide. In some embodiments, fibrosis is characterized by the accumulation of excess extracellular matrix components (e.g., collagen, fibronectin) that forms fibrous connective tissue in and around an inflamed or damaged tissue. Fibrosis may cause overgrowth, hardening, and/or scarring that disrupts the architecture of the underlying organ or tissue. While controlled tissue remodeling and scarring is part of the normal wound healing process promoted by trans differentiation of fibroblasts into myofibroblasts, excessive and persistent scarring due to severe or repetitive injury or dysregulated wound healing (e.g. , persistence of myofibroblasts) can eventually result in permanent scarring, organ dysfunction and failure, and even death. These processes separately or together are involved in many diseases and conditions. These diseases or conditions may be systemic or may be relatively localized in all main tissue and organ systems (e.g., lung, liver, kidney, heart, skin or eye).

[0020] Homeostasis of the eye, as in tissues elsewhere in the body, depends on the presence of normal vasculature, extracellular matrix (ECM), and various cell types. If homeostasis is disturbed by infection, inflammation, or metabolic disease, visual function becomes impaired. The end result of these conditions is often fibrosis. In the central nervous system (CNS), of which the retina is a part, such wound-healing responses and associated fibrosis are mediated by glial cells, which perform functions in the CNS similar to those performed by fibroblasts in the rest of the body. Therefore, gliosis is frequently used to describe the glial cell-mediated woundhealingresponse observed in the CNS, much as fibrosis (which is fibroblast mediated) is used to describe similar processes in non-CNS tissues. In the skin, fibrosis can lead to a cosmetic blemish in the form of a scar; in the eye this can have disastrous consequences for vision — mechanically disrupting the visual axis or sufficiently disturbing the tissue microenvironment such that proper cellular functioning is no longer possible. For example, fibrosis of the cornea can occur after a viral infection, leading to corneal opacification and thereby loss of vision. In the posterior segment of the eye, uncontrolled retinal vascular proliferation, as a result of diabetes-associated retinal hypoxia, can lead to fibrosis and traction retinal detachment, a dreaded complication of advanced diabetic retinopathy (DR). Fibrosis in the retina, for example by RPE cells undergoing EMT, can result in photoreceptor degeneration and loss of vision. Collectively, these conditions of fibrosis in the eye lead to vision loss in millions of individuals worldwide.

[0021] In some embodiments, the disclosure provides a method of treating glaucoma, or preventing or delaying the onset or progression of glaucoma by administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide. Glaucoma is a common degenerative disease affecting the retinal ganglion cells (RGC) and the optic nerve axons, with progressive and chronic course that affected approximately 60.5 million people worldwide in 2010. This number is expected to rise approximately to 76.0 million by 2020 and 112.0 million by 204. Glaucoma is the second most common eye disease after cataracts. Glaucoma encompasses a number of age-related degenerative eye diseases, the most common form being open-angle glaucoma (OAG), with other types including angle-closure glaucoma and uveitic glaucoma. Uveitis is inflammation of the middle layer of the eye, the uvea, which functions to provide the vascular support required for phototransduction. It is known as the “silent thief of vision” because diagnosis may be difficult due to the lack of symptoms in early stages of disease and in many cases its symptoms are usually felt in the late stages of the disease when the visual field and vision are seriously impaired and a severe neuronal damage may have already occurred. Yet, it is the most common cause of vision loss internationally. Glaucoma is a neurodegenerative disorder of the optic nerve, most typically associated with high intraocular pressure (IOP) resulting from a block of aqueous humor outflow from the anterior chamber of the eye. It is a form of optic neuropathy, characterized by damage of the optic disc, the place where the optic nerve and blood vessels enter the retina. The retina is a thin layer on the rear eye part that collects light and it consists of neuronal and non-neuronal parts. One of five types of retinal neurons are retinal ganglion cells (RGCs). These are unique retinal cells, which can produce action potentials that are transmitted to the brain through the optic nerve, thus enabling vision. Of all retinal cells, only ganglion cells, in particular their axons, are affected by glaucomatous changes. This sensitivity is because: a part of ganglion cell axons, between the retina and the lamina cribrosa, is not myelinated, and ganglion cell axons are very long and susceptible to numerous disorders. The lamina cribrosa can be posteriorly displaced and thicker and the scleral canal can be enlarged atBruch’s membrane and at the anterior laminar insertion in the early -glaucoma eyes compared with the contralateral normal eyes. Although glaucomatous havoc affects the retinal ganglion cell stroma, photoreceptors, the lateral geniculate body, and the visual cortex, the main reason for vision loss is the impairment of retinal ganglion cell (RGC) axons within the lamina cribrosa of the optic nerve head. The change of the optic disc, known as “cupping of the opt known as “cupping of the optic disc ” is its vertical elongation and is accompanied by the loss of the neuroretinal rim, which can be visualized on the superior and inferior poles of the optic disc. Most frequently, glaucomatous changes of the optic disc occur as a result of elevated intraocular pressure (IOP). It has been proventhat lowering IOP in ocular hypertension delays or even stops changes in optic nerve axons. In both open-angle and closed-angle glaucoma, inflammatory cytokines are elevated in the aqueous humor. However, the biggest threat to vision from this increased IOP is the death of retinal ganglion cells (RGCs), whose axons exit the retina through the optic disc where they are bundled together to form the optic nerve. As a result of RGC death, glaucoma patients experience loss of vision, beginning with their peripheral vision. Within the high-IOP normal eyes, the lamina cribrosa can be posteriorly displaced compared with that in the low-IOP normal eyes. Within the high-IOP early -glaucoma eyes, the lamina cribrosa can be posteriorly displaced and thicker and the scleral canal enlarged, compared with both low-IOP normal eyes and high-IOP normal eyes. Since functional and anatomic changes due to glaucoma are often irreversible, early detection still remains an important strategy to prevent loss of vision. This goal has been achieved so far evaluating optic nerve structure and function through retinographies and perimetries. New techniques are emerging to complement the use of these consolidated procedures, including analysis of nerve fibers and detection of apoptosis of in vivo ganglion cells. In the advanced stage of glaucoma, the retinas can be almost completely depleted of ganglion cells, which have degenerated and therefore could not be stained. The few remaining ganglion cells, which can be considered to be resistant to glaucoma, show drastic morphologic alterations, such as abnormal axonal beading. In some cases, the cell bodies are normal in size but have irregular silhouettes or swellings, and there are fewer dendritic bifurcations. Once RGCs die in glaucoma, they are not replaced. Unlike peripheral nerve cells in other parts of the body, RGCs are part of the body’s central nervous system, which does not regenerate once damaged.

[0022] In some embodiments, the disclosure provides methods of treating ocular hypertension (OHT), or preventing or delaying the progression of OHT to glaucoma by administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide. Ocular hypertension is when the pressure inside the eye (intraocular pressure or IOP) is higher than normal. With ocular hypertension, the eye does not drain fluid properly. This causes eye pressure to build up. Higher than normal eye pressure can cause glaucoma and the high eye pressure damages the optic nerve, causing vision loss. Ocular hypertension is not the same as glaucoma. The Ocular Hypertension Treatment Study (OHTS) identified baseline risk factors for the conversion from OHT to glaucoma. Strong evidence supports that a higher baseline IOP, thinner central cornea, older age, and increased vertical cup-to-disc ratio are significant independent risk factors for glaucomatous progression. With ocular hypertension, the optic nerve looks normal and there are no signs of vision loss. However, people with ocular hypertension are at increased risk for glaucoma and are considered “glaucoma suspects.”

[0023] Provided herein in some embodiments is a method of treating a disease, disorder or a condition associated with a fibrotic or profibrotic induction in a tissue, or a condition associated with a risk of a fibrotic induction in a tissue by administering a modified fibroblast growth factors (e.g., FGF-1) polypeptides, or pharmaceutical composition or medicaments that include such modified peptides. In some embodiments, the fibrotic induction is caused by EMT. In some embodiments, the method comprises treating ocular fibrotic diseases, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating glaucoma, by administering modified FGF (e.g., FGF-1) described herein. In some embodiments, the method comprises treating glaucoma at a stage prior to trabeculectomy, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating Grave’ s orbitopathy (GO), by administering modified FGF (e.g. , FGF-1) polypeptides described herein. In some embodiments, the method comprises treating Geographic atrophy (GA), by administering modified FGF-1 polypeptides described herein. In some embodiments, the method comprises treating pingueculae and pterygia (fibrovascular growths on the surface of the cornea), by administering modified FGF (e.g, FGF-1) polypeptides described herein. In some embodiments, the method comprises treating limbal epithelial stem cell deficiency (LSCD), by administering modified FGF-1 polypeptides described herein. In some embodiments, the method comprises treating chronic cicatrizing conjunctivitis, by administering modified FGF (e.g, FGF-1) polypeptides described herein. In some embodiments, the method comprisestreating a number of ocular surface diseases accompanied by corneal inflammation and defects in wound healing, such as keratoconjunctivitis sicca, recurrent corneal erosion, persistent epithelial defects, Stevens- Johnson Syndrome, or post-refractive surgery keratitis, by administering modified FGF (e.g., FGF-1) polypeptides described herein.

[0024] Also provided herein is a method of treating non-ocular fibrotic diseases, by administering modifiedFGF-1 polypeptides described herein. In some embodiments, the method comprises treating idiopathic pulmonary fibrosis, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating activation of myeloid cells, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprisestreating myocardial injury and infarction, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating Stevens-Johnson Syndrome (SJS), by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating Sarcoidosis, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating fibrotic kidney disease, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating cardiac fibrosis, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating liver fibrosis, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating intestinal fibrotic strictures associated with Crohn’s disease, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating a fibrotic disease, disorder or condition of the lung, by administering modified FGF-1 polypeptides described herein. In some embodiments, the method comprises treating idiopathic pulmonary fibrosis, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating a fibrotic disease, disorder or condition of the cardiac tissue, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating myocardial infarction, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating a fibrotic disease, disorder or condition of the renal tissue, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating anti- glomerular basement membrane glomerulonephritis, lupus nephritis, antigen -induced immune complex glomerulonephritis, renal allograft injury, ischemia reperfusion injury, and nephropathy, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating the fibrotic disease, disorder or condition results from pneumonia, rheumatoid arthritis, mixed connective tissue disease, systemic erythematosus lupus, scleroderma, dermatomyositis, polymyositis or sarcoidosis, by administering modified FGF (e.g., FGF-1) polypeptides described herein. In some embodiments, the method comprises treating the disease, disorder or condition is associated with activation of myeloid cells, wherein the myeloid cell is an antigen presenting cell, a macrophage, a dendritic cell, or a neutrophil, by administering modified FGF (e.g., FGF-1) polypeptides described herein.

Certain Terminology

[0025] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0026] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood to which the claimed subject matter belongs. In the event that there is a plurality of definitions for terms herein, those in this section prevail. All patents, patent applications, publications and published nucleotide and amino acid sequences (e.g. , sequences available in GenBank or other databases) referred to herein are incorporated by reference. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0027] As used herein, the term “Percent (%) amino acid sequence identity” with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alignment for purposes of determining percent amino acid sequence identity can for example be achieved using publicly available sequence comparison computer program ALIGN- 2. The source code for the ALIGN-2 sequence comparison computer program is available with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program can be compiled for use on a UNIX operating system, such as a digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0028] Definition of standard chemistry terms may be found in reference works, including but not limited to, Carey and Sundberg “ADVANCED ORGANIC CHEMISTRY 4™ED.” Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology.

[0029] Unless specific definitions are provided, the nomenclature employedin connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those recognized in the field. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Reactions and purification techniques can be performed e.g., using kits of manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures canbe generally performed of conventional methods and as describedin various general and more specific references that are cited and discussed throughout the present specification.

[0030] It is to be understood that the methods and compositions described herein are not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods, compounds, compositions described herein.

[0031] The terms “treat,” “treating” or “treatment” include alleviating, abating or ameliorating a disease, disorder or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease, disorder, or condition, e.g., arresting the development of the disease, disorder or condition, relieving the disease, disorder or condition, causing regression of the disease, disorder or condition, relieving a condition caused by the disease, disorder or condition, or stopping the symptoms of the disease, disorder or condition. The terms “treat,” “treating” or “treatment”, include, but are not limited to, prophylactic and/or therapeutic treatments.

[0032] The term “acceptable” or “pharmaceutically acceptable”, with respect to a formulation, composition or ingredient, refers to having no persistent detrimental effect on the general health of the subject being treated or does not abrogate the biological activity or properties of the modified FGF described herein, and is relatively nontoxic.

[0033] The term “amelioration” of the symptoms of a particular disease, disorder or condition by administration of a particular modified FGF or pharmaceutical composition refers to any lessening of severity, delay in onset, slowing of progression, or shortening of duration, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the modified FGF or pharmaceutical composition.

[0034] The term “combination” or “pharmaceutical combination” as used herein, means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that one active ingredient (e.g., a modified FGF) and a co-agent are both administered to a patient simultaneously in the form of a single entity or dosage. The term “nonfixed combination” means that one active ingredient (e.g., a modified FGF) and a co-agent are administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific intervening time limits, wherein such administration provides effective levels of the two agents in the body of the patient. The latter also applies to cocktail therapy, e.g. , the administration of three or more active ingredients.

[0035] The term “pharmaceutical composition” as used herein refers to one or more modified FGF-1 polypeptides with one or more other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the modified FGF-1 polypeptides to an organism. Multiple techniques of administering a modifiedFGF-1 polypeptide exist in the art including, but not limited to: topical, ophthalmic, intraocular, periocular, intravenous, oral, aerosol, parenteral, and administration.

[0036] The term “carrier,” as used herein, refers to relatively nontoxic chemical compounds or agents that facilitate the incorporation of an agent of interest (e.g., a modified FGF) into cells or tissues.

[0037] The term “diluent” refers to chemical compoundsthat are used to dilute the agent of interest (e.g., a modified FGF) prior to delivery. Diluents can also be used to stabilize agents because they can provide a more stable environment. Salts dissolved in buffered solutions (which also can provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution.

[0038] The terms “co-administration” or the like, are meant to encompass administration of the selected agents (e.g., a modified FGF or composition thereof and a co-agent) to a single patient, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different time.

[0039] The terms “effective amount” or “therapeutically effective amount,” refer to a sufficient amount of a modified FGF-1 polypeptide, agent, combination or pharmaceutical composition described herein administered which will relieve to some extent one or more of the symptoms of the disease, disorder or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic usesis the amount of the modified FGF, agent, combination or pharmaceutical composition required to provide a desired pharmacologic effect, therapeutic improvement, or clinically significant decrease in disease symptoms without undue adverse side effects. An appropriate “effective amount” in any individual case may be determined using techniques, such as a dose escalation study. The term “therapeutically effective amount” includes, for example, a prophylactically effective amount. It is understood that “an effect amount” can vary from subject to subject due to variation in metabolism of the modifiedFGF, combination, or pharmaceutical composition, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. By way of example only, therapeutically effective amounts may be determined by routine experimentation, including but not limited to a dose escalation clinical trial.

[0040] The term “prophylactically effective amount,” refers that amount of a modified FGF, compound, agent, combination or pharmaceutical composition described herein applied to a patient which will relieve to some extent one or more of the symptoms of a disease, condition or disorder being treated. In such prophylactic applications, such amounts may depend on the patient's state of health, weight, and the like. It is considered well within the skill of the art for one to determine such prophylactically effective amounts by routine experimentation, including, but not limited to, a dose escalation clinical trial.

[0041] The term “subject” or “patient” as used herein, refers to an animal, which is the object of treatment, ob servation or experiment. By way of example only, a subject may be, but is not limited to, a mammal including, but not limited to, a human.

[0042] The terms “enhance” or “enhancing” means to increase or prolong either in potency or duration a desired effect. By way of example, “enhancing” the effect of therapeutic agents singly or in combination refers to the ability to increase or prolong, either in potency, duration and/or magnitude, the effect of the agents on the treatment of a disease, disorder or condition. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.

[0043] The term “modulate,” means to interact with a target (e.g., a FGF receptor) either directly or indirectly so as to alter the activity of the target, including, by way of example only, to enhance the activity of the target, to inhibit or antagonize the activity of the target, to limit the activity of the target, or to extend the activity of the target. In some embodiments, modified FGF-1 polypeptides and pharmaceutical compositions described herein can modulate the activity of one or more respective targets (e.g., one or more FGF receptors). In some embodiments, the modified FGF-1 polypeptides described herein modulate (e.g., increase) the activity of one or more FGF receptors on a cell (e.g., a corneal endothelial cell), resulting, e.g., in cell migration and/or cell proliferation.

[0044] As used herein, the term “target” or refers to a biological molecule (e.g., a target protein or protein complex), such as an FGF receptor, or a portion of a biological molecule capable of being bound by a selective binding agent (e.g., a modifiedFGF) or pharmaceutical composition described herein. As used herein, the term “non-targef ’ refers to a biological molecule or a portion of a biological molecule that is not selectively bound by a selective binding agent or pharmaceutical composition described herein.

[0045] The term “target activity” or “cell response” refers to a biological activity capable of being modulated by a modified FGF or any cellular response that results from the binding of a modified FGF to a FGF receptor. Certain exemplary target activities and cell responses include, but are not limited to, binding affinity, signal transduction, gene expression, cell migration, cell proliferation, cell differentiation, and amelioration of one or more symptoms associated with an ocular disease, disorder or condition.

Modified FGF-1 polypeptides for methods of treating fibrotic diseases

[0046] FGFs stimulate a family seven FGF receptor isoforms, and each FGF stimulates a different pattern of receptors to achieve its specific effect. See, e.g., Omitz etal. (1996) The Journal of biological chemistry, 1996, 271(25):15292-7; Zhang e tai. (2006) The Journal of biological chemistry, 2006, 281(23): 15694-700). In some embodiments, modifiedFGF-1 polypeptide is preferable because it binds to and stimulates all seven FGF receptor isoforms. See Ornitz et al. (1996) The Journal of biological chemistry, 1996, 271(25): 15292-7.

[0047] Certain embodiments disclosed herein relate to methods comprising administering a modified FGF (e.g., FGF-1) polypeptide or a pharmaceutical composition (e.g., an ophthalmic formulation) comprising a modified FGF (e.g., FGF-1 polypeptide) described hrein. In some embodiments, the methods comprise treating fibrotic diseases by administering a modified FGF (e.g., FGF-1) polypeptide described hrein or a pharmaceutical composition (e.g. , an ophthalmic formulation) comprising the modified FGF (e.g., FGF-1) polypeptide described hrein. A modified FGF-polypeptide, as used herein, refers to a recombinant FGF that includes a substitution or mutation of one or more different amino acid residues and/or one or more deletions of one or more amino acid residues and/or one or more additions of one or more amino acid residues of a wild-type FGF-polypeptide. In some embodiments, a modified FGF- polypeptide comprises a modified FGF-1 polypeptide a substitution or mutation of one or more different amino acid residues and/or one or more deletions of one or more amino acid residues and/or one or more additions of one or more amino acid residues relative to the amino acid sequence of SEQ ID NO: 1 .

[0048] Provided herein, in some embodiments, are methods comprising administering a modified FGF-1 polypeptide comprising the sequence set forth as SEQ ID NO: 1, with one or more mutations, wherein the modified polypeptide further comprises a methionine residue upstream to the first residue of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide comprising the N-terminal methionine (N-Met) residue is a mature form of the polypeptide. In some instances, the modified FGF-1 polypeptide, according to the first embodiment, comprises one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide is expressed in a host cell with a methionine residue upstream to the first residue of SEQ ID NO: 1 . In some embodiments, the modified FGF-1 polypeptide is not subject to N-terminal processing for removal of the N-Met residue during maturation. Thus, in some embodiments, the mature form of a modifiedFGF-1 comprises an N-Met residue and one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1 . An exemplary modified FGF-1 sequence, comprising an N-Met residue, is disclosed herein as SEQ ID NO: 2.

[0049] In some embodiments described herein, where the modified FGF-1 polypeptide is expressed with an N-terminal methionine (N-Met) residue, the polypeptide is subsequently purified without a step requiring proteolytic cleavage for removal of an N-terminal peptide sequence (e.g., a leader sequence). Accordingly, in some embodiments, the present disclosure provides a method comprising administering a modified FGF-1 polypeptide that is prepared by a rapid purification method, without involving a proteolytic cleavage step for removal of anN- terminal peptide. In some instances, this is particularly advantageous for production of the modified FGF-1 polypeptides per good manufacturing practice (GMP) guidelines. The advantages include the lack of a cleavage step, including eliminating the need for subsequent purification of the cleaved product and removal of the reagents used for cleavage. The further advantage of this is an increase in yield due to decreased handling and the alleviation of the need to test for residual cleavage reagents and contaminants introduced for the cleavage and subsequent separation of cleaved from uncleaved material. In some embodiments, the modified FGF-1 polypeptides described herein, can have increased stability (e.g. thermostability), reduced number of buried free thiols, and/or increased effective heparan sulfate proteoglycan (HSPG) affinity.

[0050] Several other advantages are associated with the use of the modified FGF-1 polypeptides in the methods described herein. For example, the modified FGF-1 polypeptides described herein can be administered without heparin in its pharmaceutical composition or formulation (e.g., an ophthalmic formulation), avoiding potential safety issues related to the biologic origin of heparin and its complex preparation process. In addition, avoidance of heparin allows the use of higher doses of the modified FGF-1 polypeptides without complications resulting from local heparin-induced adverse events or preexisting anti-heparin antibodies or immune reactions. Furthermore, in the absence of heparin, immediate binding of the modified FGF to tissue is maximized and systemic distribution is significantly reduced. The modified FGF-1 polypeptides described herein also have the advantage of having enhanced local sequestration and reduced redistribution kinetics, thus increasing the elimination half-life and mean residence time (MRT) at the site of delivery, and allowing for a reduced dosing frequency. This can be the result of modified FGF-1 polypeptides described herein that have increased stability (e.g. thermostability), reduced number of buried free thiols, and/or increased effective heparan sulfate proteoglycan (HSPG) affinity.

[0051] The FGF-1 polypeptides of the present disclosure comprise, in various embodiments, modifications at the N-terminus of the polypeptide, such as an addition, a truncation, or a combination of additions and truncations. In some embodiments, the modification is the addition of a single N-terminal methionine residue. In some embodiments, the modification is the addition of an extension peptide. In some embodiments, the modification is a truncation of one or more of the first five residues of a FGF-1 polypeptide. In some embodiments, the FGF-1 polypeptides comprise a sequence as set forth in SEQ ID NO: 1, with one or more mutations, in addition to the N-terminal modification. Several examples of the modified FGF-1 polypeptides for use in the methods disclosed herein comprise an N-terminal methionine (N-Met) residue in a mature form of the polypeptide. The retention of biological activity when amino acids are added to the N-terminus of a protein is unpredictable. Some proteins are tolerant of this and some are not, and the retention of biological activity and the potential for changes in stability are only determined empirically. In some embodiments, the methods of this disclosure comprise administering a modified FGF-1 polypeptide comprising an N-terminal Met residue that retain biological activity and stability.

[0052] In some embodiments, the methods of this disclosure comprise administering a modified FGF-1 as described herein, comprising an N-Met residue in its mature form, that has at least similar biological activity as a version without the N-Met residue. N-terminal methionine removal, or excision, is a co-translational process that occurs as soon as a polypeptide emerges from the ribosome. The removal of the N-terminal methionine involves the substrate specificities of a cleavage enzyme, methionine aminopeptidase (metAP), which recognizes a methionine residue which is followed by an amino acid residue with a small side chain, such as alanine, glycine, proline, serine, threonine, or valine. Due to this substrate sequence specificity, the modified FGF-1 of the first embodiment, which comprises an N-Met residue followed by phenylalanine, see position 1 of SEQ IDNO: 1, is not processed by metAP. Thus, by expressing the modified FGF-1 with a methionine residue directly upstream of SEQ ID NO: 1, a mature modified FGF-1, comprising methionine as its N-terminal residue, canbe obtained. In some embodiments, the modified FGF-1 according to the first embodiment is not expressed with an N-terminal peptide and therefore is not subject to proteolytic cleavage for removal of the same, during sub sequent purification. [0053] Provided herein, in some embodiments, is a method comprising administering a modified FGF-1 polypeptide comprising the sequence set forth as SEQ ID NO: 1, with one or more mutations, wherein the modified polypeptide further comprises a methionine residue upstream to the first residue of SEQ ID NO: 1, and one or more amino acids of the peptide set forth as SEQ ID NO: 3. A peptide comprising one or more residues of SEQ ID NO: 3 is herein referred to as an “extension peptide.” Thus, the modified FGF-1 according to the second embodiment comprises the sequence set forth as SEQ ID NO: 1, with one or more mutations, a methionine residue upstream to the first residue of SEQ ID NO: 1 , and an extension peptide positioned between the methionine residue and the first residue of SEQ ID NO: 1 . In some embodiments, the modified FGF-1 polypeptide comprising the N-terminal methionine and an extension peptide, positioned between the methionine residue and the first residue of SEQ ID NO: 1, is a mature form of the polypeptide. In some embodiments, the modified FGF-1 polypeptide comprises one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1, which polypeptide is expressed in a host cell with a methionine residue upstream to the first residue of SEQ ID NO: 1, and further an extension peptide positioned between the methionine residue and the first residue of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide is expressed with an extension peptide comprising five residues of SEQ ID NO: 3, positioned between the methionine residue and the first residue of SEQ ID NO: 1 . In some embodiments, the modified FGF-1 polypeptide is expressed with four residues of SEQ ID NO: 3, positioned between the methionine residue and the first residue of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide is expressed with three residues of SEQ ID NO: 3, positioned between the methionine residue and the first residue of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide is expressed with two residues of SEQ ID NO: 3, positioned between the methionine residue and the first residue of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide is expressed with one residue of SEQ ID NO: 3, positioned between the methionine residue and the first residue of SEQ ID NO: 1. Exemplary sequences of the extension peptide include SEQ ID NOS: 4-8.

[0054] In some instances, the methods of this disclosure comprise administering a modified FGF-1 polypeptide comprising an extension peptide and an N-terminal methionine residue, is not subject to N-terminal processing for removal of the methionine residue, whereas in some instances the methionine is excised by a cleavage enzyme. Typically, the cleavage enzyme is methionine aminopeptidase (metAP). Thus, in some examples, the mature form of the modified FGF-1 polypeptide comprises an N-Met residue followed by an extension peptide as described herein. Exemplary sequences of mature forms of modified FGF-1 polypeptides comprising an N-terminal methionine, and one or more residues of the extension peptide, positioned between the methionine residue and the first residue of SEQ ID NO:1, are set forth as SEQ ID NOS: 9- 13, wherein the sequences further comprise one or more mutations at amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. Additional exemplar sequences of mature modified FGF-1 polypeptides comprising an N-terminal methionine, and an extension peptide are set forth as SEQ ID NOS: 14-18. In some other examples, the mature form of the modified FGF-1 polypeptide does not comprise an N-Met residue but includes only an extension peptide. Exemplary sequences of mature forms of modified FGF-1 polypeptides comprising an extension peptide, positioned upstream to the first residue of SEQ ID NO:1 are set forth as SEQ ID NOS: 19-23 , wherein the sequences further comprise one or more mutations at amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. Additional exemplar sequences of mature modified FGF-1 polypeptides comprising one or more residues of the extension peptide are set forth as SEQ ID NOS: 24-28. In some embodiments, the methionine residue is cleaved by metAP when the extension peptide starts with an alanine (as in SEQ ID NO: 4) or with a threonine (as in SEQ ID NO: 5). In those instances, the mature FGF-1 polypeptide does not comprise an N-terminal methionine residue, e.g., SEQ ID NOS: 19, 21, 24, and 26.

[0055] Provided herein, in some embodiments, is a method of this disclosure comprising administering a modified FGF-1 polypeptide comprisingthe sequence setforth as SEQ ID NO: 1 , with one or more mutations, wherein the modified polypeptide further comprises an extension peptide positioned upstream to the first residue of SEQ ID NO: 1 . In some embodiments, the modified FGF-1 polypeptide comprising an extension peptide is a mature form of the polypeptide. In some embodiments, the modified FGF-1 polypeptide comprising one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1, which polypeptide is expressed in a host cell with one or more amino acid residues of the extension peptide positioned upstream to the first residue of SEQ ID NO: 1 . Exemplary sequences of the modified FGF-1 polypeptides comprising an extension peptide, expressed without an N-terminal methionine residue, are setforth as SEQ ID NOS: 19-23, wherein the sequences further comprise one or more mutations at amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1 . Additional exemplar sequences of mature modified FGF-1 polypeptides comprising one or more residues of the extension peptide, and expressed without an N-terminal methionine residue, are set forth as SEQ ID NOS: 24-28.

[0056] Provided herein, in some embodiments, is a method of this disclosure comprising administering a modified FGF-1 polypeptide comprisingthe sequence set forth as SEQ ID NO: 1 , with one or more mutations, wherein the modified polypeptide further comprises a truncation of one or more of the first five residues of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide comprising the truncation of one or more of the first five residues of SEQ ID NO: 1 is the mature form of the polypeptide. In some embodiments, the modified FGF-1 polypeptide comprises one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1, wherein one or more of the first five residues of SEQ ID NO: 1 is deleted. In some cases, the modified FGF-1 polypeptide comprising truncations is expressed with an N-terminal methionine residue. For instance, the modified FGF-1 polypeptide, in some embodiments, comprises a sequence wherein the N-Met residue is followed by the second residue, asparagine, of SEQ ID NO: l . In some cases, the modified FGF-1 polypeptide comprises an N-Met residue followed by the third residue, leucine, of SEQ ID NO: 1. In some cases, the modifiedFGF-1 polypeptide comprises an N-Met residue followed by the fourth residue, proline, of SEQ ID NO: 1. In some cases, the modifiedFGF-1 polypeptide comprises an N-Met residue followed by the fifth residue, proline, of SEQ ID NO:1 . An extension peptide canbe positioned in between the N-Met residue and the first, second, third, fourth, or fifth residue of SEQ ID NO: 1 . Examples of a mature form of the modified FGF-1 polypeptide wherein an N-Met residue is followed by the second, third, fourth, or fifth residue of SEQ ID NO: 1 are shown in SEQ ID NOS: 37-40, wherein the sequences further comprise one or more mutations at amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1 . Additional examples of modified FGF-1 polypeptides comprising truncations and an N-Met residue, are provided in SEQ ID NOS: 41- 44.

[0057] The present disclosure also relates to a method of administering a modifiedFGF-1 polypeptide comprising one or more mutations of SEQ ID NO: 1 , wherein the polypeptide is expressed with an N-Met residue followed by an extension peptide, and the extension peptide is followed by truncation of one or more of the first five residues of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide comprises one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1, wherein the polypeptide is expressed with an N-Met residue followed by an extension peptide, and the extension peptide is followed by truncation of one or more of the first five residues of SEQ ID NO: 1. Examples of such sequences expressed with an N-Met residue followed by an extension peptide, which extension peptide is followed by truncation of one or more of the first five residues of SEQ ID NO: 1 are disclosed as SEQ ID NOs: 45-68, wherein the sequences further comprise one or more mutations at amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: l . In some examples, the N- terminal methionine is cleaved off by N-terminal processing and accordingly the mature form of the modified FGF-1 polypeptide comprises only one or more residues of the leader fragment followed by truncation of one or more of the first five residues of SEQ ID NO: 1 , as exemplified in SEQ ID NOS: 69-92, wherein the exemplary sequences further comprise one or more mutations at amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. Additional examples of sequences without N-Met residue but including an extension peptide and truncations of N-terminal residues, are provided in SEQ ID NO: 93, 94, and 96-117.

[0058] In some examples, the N-Met residue is retained in the mature modified FGF-1 polypeptide sequence, and accordingly the mature forms comprise sequences as exemplified in SEQ ID NO: 45-68, further comprising one or more mutations at amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1 . Additional examples of sequences comprising an N-Met residue, an extension peptide and truncations of N-terminal residues, are provided in SEQ ID NO: 118-141 and 207.

[0059] The truncated versions of the modified FGF-1 polypeptides comprising one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1, are expressed without an N- terminal methionine residue, and further without an extension peptide. In some examples, mature modified FGF-1 polypeptides comprise a sequence as set forth in SEQ ID NOS: 29-32, wherein the sequences further comprise one or more mutations at amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1 . In some examples, the modified FGF-1 polypeptides comprise a sequence selected from the group consisting of SEQ ID NOS: 33-36. [0060] In instances where the modified FGF-1 polypeptide, or its truncated version, comprising one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1, is expressed with an N-terminal methionine followed by an extension peptide, the methionine residue is either retained or cleaved off of the N-terminus during maturation of the polypeptide after expression. In some examples, where the modified FGF-1 polypeptide is expressed with an alanine next to the N-Met residue, e.g., SEQ ID NO: 14, the methionine is cleaved, to yield a mature FGF-1 polypeptide that doesnot comprise an N-Met residue, e.g., SEQ ID NO: 19. In some examples, where the modified FGF-1 polypeptide is expressed with a threonine next to the N-Met residue, e.g., SEQ ID NO: 16, the methionine is cleaved, to yield a mature FGF-1 polypeptide that does not comprise an N-Met residue, e.g., SEQ ID NO: 20. In some examples, where the modified FGF-1 polypeptide is expressed with a glutamic acid next to the N-Met residue, e.g, SEQ ID NO: 17, the methionine is not cleaved, to yield a mature FGF-1 that comprise an N-terminal methionine and has the same sequence as the expressed form.

[0061] Provided herein, in some embodiments, is a method comprising administering a modified FGF-1 polypeptide comprising the sequence setforth as SEQ ID NO: 1, comprising a mutation at position 67. In some embodiments, the modifiedFGF-1 polypeptide comprisesa mutation at position 67 of SEQ ID NO: 1, one or more further mutations at positions 12, 16, 66, 117, and 134, and is expressed with an N-Met residue. The internal methionine at position 67 can be replaced, for example, with an alanine residue. In absence of the internal methionine at position 67, the N-terminal methionine of the modified FGF-1 polypeptide can be cleaved, postexpression; using cyanogen bromide (CNBr), an agent that specifically cleaves the amide bond after methionine residues. In some cases, the modified FGF-1 polypeptides are expressed with an extension peptide. In some other cases, modified FGF-1 polypeptides are expressed in a form comprising truncations of one or more of the first five residues of SEQ ID NO: 1 , as exemplified in SEQ ID NOS: 142-149, wherein the sequences further comprise one or more mutations at amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. In yet other examples, the modified FGF-1 polypeptides are expressed in a form comprising an extension peptide and truncations of one or more of the first five residues of SEQ ID NO : 1 , as exemplified in SEQ ID NOS: 151-175. Additional examples of the modified FGF-1 polypeptides, in their mature forms, are set forth in SEQ ID NOS: 174-204 Among the modified FGF-1 polypeptides expressed in a form that comprises an internal methionine mutation, in cases where the polypeptide is expressed with an N-terminal methionine followed by an alanine or a threonine residue from the extension peptide, e.g., SEQ ID NO: 175 and SEQ ID NO: 177, respectively, the N-terminal methionine can be cleaved off during maturation of the polypeptide either by metAP or using CNBr.

[0062] Provided herein, in some embodiments, is a method comprising administering a modified FGF-1 polypeptide comprising the sequence set forth as SEQ ID NO: 205, for use in a method as described herein. Provided herein, in some embodiments, is a method comprising administering a modified FGF-1 polypeptide comprising the sequence set forth as SEQ ID NO: 206, for use in a method as described herein.

[0063] The present disclosure further relates to methods comprising administering modified FGF-1 polypeptides comprising any combination of deletion, insertion, and substitution of SEQ ID NO: 1 . Amino acid substitutions may be introduced into a modified FGF-1 polypeptide and the products screened for a desired activity, e.g., retained/improved effectivity in treating fibrotic diseases. Amino acid substitutions may also be introduced into a modified FGF-1 polypeptide and the products screened for a desired physicochemical property, e.g., less prone to aggregation, improved solubility, prolonged half-life, ease of formulating as an ophthalmic pharmaceutical, enhanced stability, improved shelf-life. Both conservative and non-conservative amino acid substitutions are contemplated.

[0064] In some instances, the modified FGF-1 polypeptide, as in any of the above embodiments, is expressed in a form that comprises at least 136 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form that comprises 137 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form that comprises 138 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form that comprises 139 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form that comprises 140 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form that comprises 141 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form that comprises 142 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form that comprises 143 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form that comprises 144 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form that comprises 145 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form that comprises 146 amino acids.

[0065] The modified FGF-1 polypeptide, as in any of the above embodiments, comprises at least 136 amino acids in the mature form. In some examples, the modified FGF-1 polypeptide comprises 137 amino acids in the mature form. In some examples, the modified FGF-1 polypeptide comprises 138 amino acids in the mature form. In some examples, the modified FGF-1 polypeptide comprises 139 amino acids in the mature form. In some examples, the modified FGF-1 polypeptide comprises 140 amino acids in the mature form. In some examples, the modified FGF-1 polypeptide comprises 141 amino acids in the mature form. In some examples, the modified FGF-1 polypeptide comprises 142 amino acids in the mature form. In some examples, the modified FGF-1 polypeptide comprises 143 amino acids in the mature form. In some examples, the modified FGF-1 polypeptide comprises 144 amino acids in the mature form. In some examples, the modified FGF-1 polypeptide comprises 145 amino acids in the mature form. In some examples, the modified FGF-1 polypeptide comprises 146 amino acids in the mature form.

[0066] In some embodiments, methods of this disclosure comprises administering a modified FGF-1 polypeptide comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 , provided that said polypeptide comprises an N-Met residue in the mature form of the polypeptide. In some embodiments, the sequence of the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences selected from SEQ ID NO: 9-13, provided that said polypeptide comprises the N-Met residue in its mature form, and the polypeptide comprises one or more mutations at amino acid positions corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: l . In some embodiments, the sequence of the modifiedFGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences selected from SEQ ID NO: 14-18, provided that said polypeptide comprises the N-Met residue in its mature form. In some embodiments, the sequence ofthe modifiedFGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences selected from SEQ ID NO: 19 -23, provided that said polypeptide does not comprise the N-Met residue in its mature form, and the polypeptide comprises one or more mutations at amino acid positions corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1 . In some embodiments, the sequence of the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences selected from SEQ ID NO: 24-28, provided that said polypeptide does not comprise an N-Met residue in its mature form. In some embodiments, the sequence of the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences selected from SEQ ID NO: 19-23, provided that said polypeptide does not comprise an N-Met residue in its mature form, and the polypeptide comprises one or more mutations at amino acid positions corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. In some embodiments, the sequence of the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences selected from SEQ ID NO: 37-40, provided that said polypeptide comprises an N- Met residue in its mature form, and the polypeptide comprises one or more mutations at amino acid positions corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. In some embodiments, the sequence of the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any ofthe sequences selected from SEQ ID NO: 41 -44, provided that said polypeptide comprises an N-Met residue in its mature form. In some embodiments, the sequence of the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any ofthe sequences selected from SEQ ID NO: 45-68, provided that said polypeptide comprises one or more mutations at amino acid positions corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1, and said polypeptide does not comprise an N-Met residue in its mature form. In some embodiments, the sequence of the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences selected from SEQ ID NO: 69-92, comprises one or more mutations at amino acid positions corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1, and said polypeptide comprises an N-Met residue in its mature form. In some embodiments, the sequence of the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any ofthe sequences selected from SEQ ID NO: 93-117, provided that said polypeptide does not comprise an N-Met residue in its mature form. In some embodiments, the sequence of the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences selected from SEQ ID NO: 118-141 and 207, provided that said polypeptide comprises an N-Met residue in its mature form. In some embodiments, the sequence of the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences selected from SEQ ID NOS: 29-32, provided that said polypeptide comprises one or more mutations at amino acid positions corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. In some embodiments, the sequence of the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences selected from SEQ ID NOS: 33-36. In some embodiments, the sequence of the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences selected from SEQ ID NOS: 142-204. In some embodiments, the modifiedFGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 mutated at position 12 with, for example, the mutation Lysl2Val, and wherein said modified FGF-1 polypeptide comprises an N-terminal methionine in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence with a mutation at position 12 of SEQ ID NO: 1, for example the mutation Lysl2Val, and with truncation of one or more of the first five residue of SEQ ID NO: 1, wherein said modified FGF-1 polypeptide comprises an N-Met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence with a mutation at position 12 of SEQ ID NO: 1 , for example the mutation Lysl2Val, with an extension peptide, and with truncation of one or more of the first five residue of SEQ ID NO: 1, wherein said modified FGF-1 polypeptide comprises an N- met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence with mutations at position 12 of SEQ ID NO: 1, for example the mutationLysl2Val, wherein the polypeptide further comprises a mutation of the methionine at position 67 of SEQ ID NO: 1 , and is expressed with a methionine at the N-terminus, which methionine is cleaved off of the polypeptide in its mature form.

[0067] In some embodiments, the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 mutated at position 16 with, for example, the mutation Cysl6Ser, and wherein said modified FGF-1 polypeptide comprises an N- met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence with a mutation at position 16 of SEQ ID NO: 1 , for example the mutation Cysl 6 Ser, and with truncation of one or more of the first five residue of SEQ ID NO: 1 , wherein said modified FGF-1 polypeptide comprises an N- met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence with a mutation at position 16 of SEQ ID NO: 16, for example the mutation Cysl6Ser, with an extension peptide, andwith truncation of one or more of the first five residue of SEQ ID NO: 1, wherein said modified FGF- 1 polypeptide comprises an N- met residue. In some embodiments, the modified FGF-1 polypeptide comprises a sequence with mutations at position 16 of SEQ ID NO: 1, for example the mutation Cysl6Ser, wherein the polypeptide further comprises a mutation of the methionine at position 67 of SEQ ID NO: 1, and is expressed with a methionine at the N-terminus, which methionine is cleaved off of the polypeptide in its mature form.

[0068] In some embodiments, the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 mutated at position 66 with, for example, the mutation Ala66Cys, and wherein said modifiedFGF-1 polypeptide comprisesan N-terminal methionine in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence with a mutation at position 66 of SEQ ID NO: 1, for example the mutation Ala66Cy s, and with truncation of one or more of the first five residue of SEQ ID NO: 1, wherein said modified FGF-1 polypeptide comprises an N- met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence with a mutation at position 66 of SEQ ID NO: 1 , for example the mutation Ala66Cys, with an extension peptide, and with truncation of one or more of the first five residue of SEQ ID NO: 1, wherein said modifiedFGF-1 polypeptideis expressed with an N-Met residue. In some embodiments, the modified FGF-1 polypeptide comprises a sequence with mutations at position 66 of SEQ ID NO: 1, for example the mutation Ala66Cys, wherein the polypeptide further comprises a mutation of the methionine at position 67 of SEQ ID NO: 1 , and is expressed with a methionine at the N-terminus, which methionine is cleaved off of the polypeptide in its mature form.

[0069] In some embodiments, the modifiedFGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 mutated at position 117 with, for example, the mutation Cysll7Val, and wherein said modified FGF-1 polypeptide comprises an N- met residue in its mature form. In some embodiments, the modifiedFGF-1 polypeptide comprises a sequence with a mutation at position 117 of SEQ ID NO: 1, for example the mutation Cysl 17 Vai, and with truncation of one or more of the first five residue of SEQ ID NO: 1, wherein said modifiedFGF-1 polypeptide comprises anN- met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence with a mutation at position 117 of SEQ ID NO: 1, for example the mutation Cysl 17Val, with an extension peptide, and with truncation of one or more of the first five residue of SEQ ID NO: 1 , wherein said modified FGF-1 polypeptide comprises an N- met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence with mutations at position 117 of SEQ ID NO: 1 , for example the mutation Cysl 17Val, wherein the polypeptide further comprises a mutation of the methionine at position 67 of SEQ ID NO: 1, and is expressed with a methionine at the N-terminus, which methionine is cleaved off of the polypeptide in its mature form.

[0070] In some embodiments, the modifiedFGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 mutated at position 134 with, for example, the mutation Pro 134 Vai, and wherein said modified FGF-1 polypeptide comprisesan N-terminal methionine in its mature form. In some embodiments, the modifiedFGF-1 polypeptide comprises a sequence with a mutation at position 134 of SEQ ID NO: 1 , for example the mutation Prol 34Val, and with truncation of one or more of the first five residue of SEQ ID NO: 1, wherein said modifiedFGF-1 polypeptide comprises an N- met residue in its mature form. In some embodiments, the modifiedFGF-1 polypeptide comprises a sequence with a mutation at position 134 of SEQ ID NO: 1, for example the mutation Pro 134Val, with an extension peptide, and with truncation of one or more of the first five residue of SEQ ID NO: 1 , wherein said modified FGF-1 polypeptide comprises an N- met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence with mutations at position 134 of SEQ ID NO: 1, for example the mutation Prol 34Val, wherein the polypeptide further comprises a mutation of the methionine at position 67 of SEQ ID NO: 1 , and is expressed with a methionine at the N-terminus, which methionine is cleaved off of the polypeptide in its mature form.

[0071] In some embodiments, the modifiedFGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 mutated atpositions 16,66, and 117 of SEQ ID NO: 1, with, for example, the mutation Cysl6Ser, Ala66Cys, and Cysll7Val, and wherein said modified FGF-1 polypeptide comprises an N- met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence with mutations at positions 16, 66, and 117 of SEQ ID NO: 1, with, for example, the mutation Cysl6Ser, Ala66Cys, and Cysl 17Val, and with truncation of one or more of the first five residue of SEQ ID NO: 1, wherein said modifiedFGF-1 polypeptide comprises an N- met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence with mutations atpositions 16, 66, and 117 of SEQ ID NO: 1, with, for example, the mutation Cysl6Ser, Ala66Cys, and Cysl 17Val, with an extension peptide, and with truncation of one or more of the first five residue of SEQ ID NO: 1, wherein said modified FGF-1 polypeptide comprises an N- met residue. In some embodiments, the modifiedFGF-1 polypeptide comprises a sequence with mutations at positions 16, 66, and 117 of SEQ ID NO: 1 , with, for example, the mutation Cysl6Ser, Ala66Cys, and Cysl 17 Vai, wherein the polypeptide further comprises a mutation of the methionine at position 67 of SEQ ID NO: 1, and is expressed with a methionine at the N-terminus, which methionine is cleaved off of the polypeptide in its mature form.

[0072] In some embodiments, the sequence of the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 2, 94, 96-204, and 207. In some embodiments, the sequence of the modified FGF-1 polypeptide comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 205 or206.

[0073] In some embodiments, the modifiedFGF-1 polypeptide is thermostable. As used herein, a thermostable FGF (e.g., a thermostable FGF-1) refers to an FGF having a modified amino acid sequence relative to SEQ ID NO: 1 that is also more stable than the polypeptide of SEQ ID NO: 1 under the same conditions. Examples of mutations capable of conferring thermostability to FGF (e.g., FGF-1) and methods for assessing thermostability are described, for example, in U.S. Patent Nos. 7,790,682; 7,595,296; 7,696,171; 7,776,825; 7,659,379;

8,119,776; 8,153,770; 8,153,771; and 8,461,111; U.S. Patent Application Publication Nos. 2011/0224404 and2013/0130983; and in Xia e/aZ. PloSone. (2012) 7(ll):e48210. In some embodiments, positions 12 and/or 134 are mutated in FGF-1 to generate a modified FGF-1 that is thermostable. An FGF-1 formulation may be considered “stable” for a duration of time at a certain temperature, which is understood as the formulation in which the FGF-1 is present in its original purity and form for the designated period of time at the designated temperature. In some embodiments, the FGF-1 may be considered as remainingin its original purity and form, if there is less than 5%, less than 2% or less than 1% degradation or change in its monomeric form. Such a change may be detectable by any of the analytic procedures discussed herein, for example, chromatographic procedures, ELISA, SDS-PAGE and western blot.

[0074] In some embodiments, the modified FGF-1 polypeptide includes one or more modifications that reduce the number of reactive thiols (e.g. , free cysteines). Examples such modifications in FGF-1 are described, for example, in U.S. PatentNos. 7,790,682; 7,595,296; 7,696,171; 7,776,825; 7,659,379; 8, 119,776; 8,153,770; 8,153,771 ; and 8,461,111 ; U.S. Patent Application Publication Nos. 2011/0224404 and 2013/0130983; and in Xia etal. PloS one. (2012) 7(1 l):e48210. In some embodiments, positions 83 and/or 117 are mutated in SEQ ID NO: 1 to generate a modified FGF-1 that reduces the number of reactive thiols. In some embodiments, the modified FGF includes one or more modifications that enable formation of an internal disulfide linkage. In some embodiments, position 66 is mutated in SEQ ID NO: 1 to generate a modified FGF-1 that comprises an internal disulfide linkage.

[0075] In some embodiments, the modifiedFGF-1 polypeptides described herein canbe administered without exogenous heparin in the formulation for stability, they can be formulated and applied without heparin and thus are more able to bind to the tissue heparans. Such modified FGF-1 polypeptides have a high affinity fortissue heparans that are exposed in a surgical, traumatic or dystrophic conditions and disease-states and so bind to diseased tissue on application. In addition, the modifiedFGF-1 polypeptides beingmore thermally stable are suitable for formulation and storage at room temperature. The stability of the modifiedFGF-1 polypeptides also makes them suitable for administration in both solution (e.g. , immediate release) and sustained-release formulations.

[0076] In some embodiments, the modifiedFGF-1 polypeptide is SEQ ID NO: 1 thathas been modified at one or more of positions 12, 16, 66, 1 17, and 134. In some embodiments, the modified FGF is SEQ ID NO: 1 that has been modified at positions 16, 66, and 117. The amino acid positions can be substituted with, e.g., Ser, Cys, Vai, or other amino acids to create disulfide linkages between modified amino acids and wild-type amino acids. In some embodiments, the modified FGF comprises the amino acid sequence of SEQ ID NO: 2, also referred to as N-Met THXl 114. In some embodiments, the modifiedFGF-1 polypeptide comprises one or more mutations selected from the group consisting of: Lysl2Val, Prol34Val, Ala66Cys, Cysl 17Val, andProl34Val. In some embodiments, the modified FGF-1 polypeptide comprises the sequence of SEQ ID NO: 2.

[0077] In some embodiments, the modifiedFGF-1 polypeptides or compositions described herein may be prepared as prodrugs. A “prodrug” refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. The modified FGF-1 polypeptides described herein may be labeled isotopically (e.g., with a radioisotope) or by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, photoactivatable or chemiluminescent labels. The present discloser further relates to modified FGF polypeptides comprising N-terminal modification(s), wherein the modified FGF polypeptide can be any member of the FGF family, including FGF-1 (SEQ ID NO: 1), FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, and FGF-23, and FGF-24. In some embodiments, the synthesis of modified FGF-1 polypeptides as described herein is accomplished using means described in the art, using the methods described herein, or by a combination thereof. In some embodiments, the sequence of the modified FGF comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 mutated at one or more positions 16, 66, and 117 with, for example, the mutations Cysl6Ser, Ala66Cys, and Cysl l7Val. In some embodiments, the modified FGF comprises the wild-type human FGF-1 sequence with a mutation at positions 16, 66 and 117, for example the mutations Cysl6Ser, Ala66Cys, and Cysl 17Val.

Recombinant Techniques for Preparation of Modified FGF-1 Polypeptides

[0078] A variety of host-expression vector systems maybe utilized to produce the modified FGF-1 polypeptides provided herein for use in methods of this disclosure. Such host-expression systems represent vehicles by which the modified FGF-1 polypeptides may be produced and subsequently purified, but also represent cells that may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the modified gene product//? situ. Examples of host-expression systems include but are not limited to, bacteria, insect, plant, mammalian, including human host systems, such as, but not limited to, insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing nucleotide sequences coding for the modified FGF-1 polypeptides; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing coding sequences for the modified FGF-1 polypeptides; or mammalian cell systems, including human cell systems, e.g., HT1080, COS, CHO, BHK, 293, 3T3, harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells, e.g., metallothionein promoter, or from mammalian viruses, e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter, or from yeast-derived plasmids e.g., pSH19 and pSH15, or from bacteriophages such as lambda phase and derivatives thereof. Examples of bacterial expression systems include but are not limited to Escherichia coli-derived plasmids (e.g., pBR322, pBR325, pUC12, pUC13, and pET-3); Bacillus subtilis-derived plasmids (e.g. , PUB 110, pTP5, andpC194). In some embodiments the bacterial expression system comprises a pMKet vector. In some embodiments, a method comprising use of the pMKet bacterial expression vector to express modified FGF-1 polypeptide improves yield of the modified FGF-1 by about 5 fold to about 60-fold, compared to a method comprising subcloning a sequence encodingthe modifiedFGF-1 into a pET vector. In some embodiments, the method comprising the subcloning of modified FGF-1 in a pET vector results in an yield of about 0.5 g- about0.7 g/ 100 L following a fermentation run. In some embodiments, the method comprising the subcloning of modified FGF-1 in a pMKet vector results in an yield of about 20 g- about 40 g / 100 L following a fermentation run, for example 37 g/ 100 L.

[0079] In some embodiments, a host cell strain is chosen such that it modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications and processing of protein products may be important for the function of the protein. Different host cells have specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells, including human host cells, include but are not limited to HT1080, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.

[0080] For long-term, high-yield production of recombinant peptides, stable expression is desired. For example, cell lines that stably express the recombinant modified FGF-1 polypeptides maybe engineered. In some embodiments, rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements, e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, and the like, and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines. In some examples, this method may advantageously be used to engineer cell lines that express the modified FGF-1 polypeptide product. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the biological of the gene product.

[0081] In some embodiments, bacterial cells are used for expressingthe recombinant FGF protein. In some embodiments the bacterial cell is an Escherichia coli cell (E. coll). In some embodiments, the A. coli strain is selected fromBLA21 Al, KI 2 HMS174, and W3110. For long-term, high-yield production of recombinant peptides, stable expression is desired. For example, cell lines that stably express the recombinant modified FGF-1 polypeptides may be engineered. In some embodiments, rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements, e.g. , promoter, enhancer, sequences, transcription terminators, poly adenylation sites, and the like, and a selectable marker. In some embodiments, the recombinant nucleic acid comprising a sequence encoding the FGF-1 polypeptide optimized for maximizing codon usage by the strain or cell in which it is expressed. In some embodiments, the recombinant nucleic acid comprising a sequence encoding the FGF-1 polypeptide is operably linked to a promoter, 3 ’ UTR regulatory sequence, for example a poly A sequence or a sequence that stabilizes the transcript, and helps in translation. In some embodiments the plasmid vector comprises a selection marker, such as an antibiotic resistance gene, such as kanamycin.

[0082] In some embodiments the promoter for bacterial expression is a T7 promoter. In some embodiments the promoter for bacterial expression is a Tac promoter. In some embodiments, the plasmid comprises a pBR322 of sequence. In some embodiments, the bacterial expression vector is modified from a commercially available vector backbone, such as pBR322, pBR325, pUC12, pUC13, and pET-T3, or pET-T7 vectors.

[0083] In some embodiments, a periplasmic expression of the protein is intended. The recombinant protein may accumulate in inclusion bodies. In some embodiments, the cytoplasmic expression of the protein is intended. In some embodiments, the cytoplasmic expression of the protein is intended, wherein the protein is an insoluble protein. In some embodiments, the recombinant polypeptide may be desired for extracellular release. In some embodiments, the recombinant nucleic acid encoding the polypeptide may comprise a suitable leader sequence, such as an ompA leader sequence. In some embodiments, the recombinant nucleic acid encoding the modified FGF-1 polypeptide does not comprise a leader sequence, such as an omp A leader sequence. In some embodiments, during manufacture, the modified FGF-1 polypeptide, in certain step, is directed to the periplasmic space. The periplasmic space comprises inclusion bodies, where the polypeptide is likely to accumulate. Inclusion bodies may then be harvested after cell fractionation to recover the polypeptide. In some embodiments, the recombinant nucleic acid does not contain a leader sequence. In some embodiments the modified FGF-1 is directed for cytoplasmic expression in the cell.

[0084] In some embodiments, the recombinant nucleic acid construct comprises one or more modification for increasing yield of the modified FGF-1 polypeptide from the cell. In some embodiments, the one or more modifications comprise sequence optimization for increased expression of the modified FGF-1 polypeptide in the cell. In one embodiment, the one or more modifications comprise modifications in the plasmid. In one embodiment, the one or more modifications comprise selecting a suitable promoter for increasing yield of the modified FGF-1 polypeptide from the cell.

[0085] In further embodiments, one or more modifications are considered towards developing the host cell for expressing the polypeptide. In some embodiments, one or more modifications may be considered resulting in adjusting the adequate nutrient media for maximizing cell proliferation. In one embodiment, the adequate nutrient media comprises a carbon source. In one embodiment, the carbon source is glucose or glycerol.

[0086] In one embodiment, one or more modifications are made in the plasmid to increase the copy number and expression efficiency of the plasmid in the host cell. In one embodiment, one or more modifications are considered for maximizing the yield of the modified FGF-1 polypeptide from the cell, wherein the one or more modifications maybe selected from: i. modification within the recombinant nucleic acid encoding the mutant FGF-1 polypeptide; ii. modification within the recombinant nucleic acid comprising one or more regulatory elements related to the recombinant nucleic acid encoding the mutant FGF-1 polypeptide, selected from a promoter, an enhancer, a 5’- untranslated region, a 3 ’ -untranslated region, a poly A tail, a transcript stabilizing element; iii. modification of the plasmid comprising the recombinant nucleic acid; iv. modification of the cell strain or selection of a cell strain for maximizing cell proliferation; v. modification of the cell growth media; and vi. modifications in the processes of recovering the modified FGF-1 polypeptide from the cell.

[0087] In some embodiments, the bacterial cells are electroporated or chemically transformed with a plasmid comprising a recombinant nucleic acid comprising a sequence encoding the FGF- 1 polypeptide. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. In some embodiments, one or more carbon sources are used for maximizing the bacterial cell growth within a period of time for expansion of the expressed FGF polypeptide for increased production. In some embodiments the carbon source for the bacterial cell may be glucose. In some embodiments the carbon source for the bacterial cell may be glycerol.

[0088] The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines. In some examples, this method may advantageously be used to engineer cell lines that express the modified FGF-1 polypeptide product. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the biological of the gene product.

[0089] The method of production described herein can be easily scaled up to form large scale productions of bacterial cultures expressingthe modified FGF-1. In some embodiments, the method can be scaled to produce FGF-1 in IL bacterial cultures, or in lOL bacterial cultures, or in 100L bacterial cultures, or in 500L bacterial cultures. In some embodiments, the method is scalable to produce 1 g of modified FGF-1 polypeptide per batch. In some embodiments, the method is scalable to produce 10 g of modifiedFGF-1 polypeptide per batch. In some embodiments, the method is scalable to produce 100 g of modified FGF-1 polypeptide per batch. In some embodiments, the methodis scalable to produce 1 Kg of modified FGF-1 polypeptide per batch. In some embodiments, the method is scalable to produce 10 Kg of modified FGF-1 polypeptide per batch. In some embodiments, the method is scalable to produce 100 Kg of modified FGF-1 polypeptide per batch.

[0090] In general, a seed culture is formed from transformed bacterial cells. The inoculated seed flasks may be incubated at about 235 RPM and 37 °C. Following a 10-14 h incubation, a sample of the seed culture from each of the flasks may be tested for purity (microscopic observation of a wet mount with no contaminating organisms observed), pH, optical density at 600 nm (OD₆₀o), and sterility hold. The seed cultures are desired to exhibit optimal growth by demonstrating an OD₆oo > 1.0 and no contaminating organisms. Six of the seed flasks from each production run may be selected for scale-up. Selection criteria may include a growth time of 12 ± 2 h, an OD₆oo > 1 .0, and having six flasks. To create the fermentor inoculum, the contents of the six flasks may be pooled into a 10 L bag (sterile single-use bioprocess container) in a BSC for a total seed culture volume of approximately six liters.

[0091] One or more 150 L fermentors may be prepared for the fermentation of culturedE. coli expressing mFGF-1 with production medium (comprising nutrients, for example, soytone 12 g/L, yeast extract 24 g/L, glycerol 15.1 g/L, potassium phosphate dibasic 12.5 g/L, potassium phosphate monobasic 3.8 g/L, and P2000 antifoam 0. 1 mL/L). The production medium is sterilized in situ. The sterile medium is then supplemented with a 5 + 0.1 L sterile solution that contained magnesium sulfate heptahydrate 0.4 g/L, kanamycin 0.050 g/L. The one or more than one fermentors may be inoculated with six liters of pooled seed culture at the appropriate time. Fermentation cultures are then monitored every 60 ± 30 min and the samples were processed for pH, purity (microscopic observation of a wet mount), and OD₆₀o. Dissolved oxygen may be maintained by controlling agitation and air flow rates. The pH may be maintained within the desired range by making appropriate aseptic additions of phosphoric acid and/or ammonium hydroxide.

[0092] In some embodiments, the expression of mFGF-1 induced with addition of 0.2-0.4 g/L isopropyl-P-D-l- thio-galactopyranoside (IPTG) and 5.0 g/L L-Arabinose when the culture reaches an OD₆oo of ~ 4.5 at 3 hours post-inoculation. After induction, the fermentations may be continued for an additional three hours. Prior to centrifugation samples may be taken from each fermentor for analysis by SDS- PAGE and culture purity. The fermentations may be evaluated intermittently and may be grown for another 1 -20 hours. In some embodiments, the final culture reaches an optical density of 50-100, 50-200, 50-250, 70-260 or about 100, 200 or 250.

[0093] In some embodiments, the harvesting of the lots may be performed by transferring the fermentation broth to the centrifuge via a peristaltic pump and tubing (at 0.5 - 0.8 liters per minute) and centrifuged at 20,000 x g while coolingusing a water-circulation jacket. The mass of the harvested cell paste maybe measured, collected, divided into four containers, and placed in a < -70°C freezer.

[0094] Cell Lysis: Frozen cells comprising the modifiedFGF-1 may be thawed at a suitable time, and resuspended in a suitable buffer, for example, the buffer may comprise Tris and EDTA. In an exemplary embodiment, the cells may be thawed in TES buffer (50 mM Tris, 20 mM EDTA, 100 mMNaCl, pH7.4) containing 1 mMDTE at a ratio of 1 :5 (w/v), i.e. 1 gram of cell paste in 5 mL of buffer. The suspension may be chilled to below 16 °C before running through a high-pressure homogenizer. OD₆₀o is monitored after each pass until no significant decrease. An equal volume of TES + 5% Triton X-100 may then be mixed into the ruptured-cell suspension. The ruptured cell suspension may be used for recovery of the expressed FGF-1 proteins. The expressed protein may be collected from the ruptured cell by passing the lysate through a specific capture method, such as affinity column, that specifically binds the FGF-1 protein, and is later eluted. However, for maximizing the FGF-1 recovery and yield, the protein may be directed for expression in the IBs, and the protein may be collected from the inclusion bodies. In an exemplary method, the mixture may be centrifuged at 15,900 x g for 60 min at 4 °C to collect the mFGF-1 -containing inclusion bodies. Following lysis, the overexpressed proteins may be recovered from inclusion bodies (IB) from E. coli paste by centrifugation. [0095] Recovery from inclusion bodies: In an exemplary embodiment, for the recovery of FGF-1, the cell paste may be thawed at 2 - 8 °C, resuspended in a suitable buffer. The buffer may comprise Tris and EDTA. For example, the cell paste may be thawed in 4.5 L of TES buffer, pH 7.4 (50 mM Tris, 100 mMNaCl, 20 mMEDTA) and the cells may then be lysed by pressure homogenization. Five homogenization passes (approximately 8,000 psi) may be performed to achieve maximum cell lysis. An equal volume of TES buffer and 5 %Triton X- 100, pH 7.4 may then be added to the lysate to obtain a 2.5% Triton concentration.

[0096] The mixture may be divided into 6-20 centrifuge bottles, as per convenience, which are then incubated for at least 30 min at approximately 15-20 °C with shaking at 225 RPM. The bottles may be centrifuged for 60 min at 15,900 x g and 4°C. The supernatant was discarded as waste. Using a tissue homogenizer (Model Omni GLH850), the recovered inclusion bodies were individually resuspended in TE Buffer (~1 L, 50 mM Tris, 20 mM EDTA, pH 7.4) with 2.5% (w/v) Triton X-100, and the bottles were incubated for at least 30 min at 15 -20 °C with shaking at 225 RPM. After incubation, the bottles were centrifuged for 45 min at 15,900 x g and 4 °C. The inclusion body washing process suspension, incubation, and centrifugation was performed a total of three times. The recovered inclusion bodies may be stored overnight at 2 °C - 8 °C. The inclusion bodies were washed with TE buffer without Triton (~ 1 L, 50 mM Tris, 20 mMEDTA, pH 7.4) and the bottles were incubated for at least 15 min at 15-20°C with shaking at 225 RPM. In some embodiments, the IBs may be washed in a buffer comprising polysorbate 20 or polysorbate 80. After incubation, the bottles were centrifuged for 30 min at 15,900x g and 4 °C. Washing without Triton, incubation, and centrifugation may be performed a total of five times. Samples may be removed and submitted for total protein and SDS-PAGE Coomassie Stain/densitometry analysis at this stage. In some embodiments, a total of 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1,000 g of inclusion bodies may be recovered. The centrifuge bottles containing the washed inclusion bodies for each lot may be stored at< -70 °C.

[0097] Solubilization of Washed Inclusion Bodies: Solubilization of the washed inclusion bodies for each lot may be performed using a solubilization buffer. A solubilization buffer may comprise chaotropic components, such as urea, or guanidine salts. The washed inclusion bodies may be removed from storage and thawed at 2-8 °C (15 h - 19 h). Inclusion bodies may be centrifuged at 15,900 x G, 4°C for 60 min, and after removing the liquid phase the net weight of the pellets was determined. The inclusion bodies may then be solubilized in buffer. An exemplary solubilization buffer may comprise 4-8M Guanidine. An exemplary solubilization buffer may comprise 6M Guanidine. An exemplary solubilization buffer may comprise 4-6M Urea. Additionally, such a buffer may comprise lOOmMTris, 2mMEDTA, (pH 8.0). The buffer may be mixed at 10 mL per g ratio at 2-8 °C using a tissue homogenizer (e.g., Model Omni GLH850) at 10,000 RPM until the solution was visually homogenous. Guanidine is a chaotropic agent that results on protein denaturation. Dithioerythritol (DTE) may be used to a final concentration of 10 mg/mLto the solubilized inclusion bodies at 2 -8 °C to reduce disulfide bonds to thiols. This reaction may continue for 2-6 hours. The solubilized inclusion bodies may then be centrifuged for 40 min at 15,900 x g and 4 °C. The complete process including centrifugation may be less than five hours. The supernatant may be collected into 2 L PETG bottles and stored at 2-8 °C (25-50 min) while the protein concentration was tested. Based on the protein results, the solubilized inclusion bodies maybe diluted to a target concentration of 2.0 ± 0.5 mg/mL with dilution buffer (6M Guanidine, 100 mM Tris, 2 mMEDTA, pH 8.0). DTE may be added to a target concentration of lOmg/mL and mixed. The solubilized inclusion bodies may be stored at < -70 °C. The IBs may be solubilized in 6 M guanidine hydrochloride in 100 mM Tris, 2 mM EDTA, pH 8.0 at a ratio of 10 mL buffer per gram IB. DTE may be added (10 mg/ml) and after 3 -5 h mixing (initially with tissue homogenizer, Polytron PT 3100, followed by magnetic stir bar), the mixture may be centrifuged (15,900 x g > 40 min). The supernatant may be filtered through a 0.45 pm filter.

[0098] Refolding of denatured protein : Guanidine-solubilized IBs (2 ± 0.5 mg/mL) may be added into cold refolding buffer. The refolding buffer may comprise L-arginine. (e.g., 0.5 ML- Arginine, 100 mM Tris, 2mM EDTA, pH 9.5). In some embodiments the refolding buffer may contain oxidized glutathione. In some embodiments, the refolding buffer may contain reduced glutathione. The solubilized mFGF-1 may be added slowly, e.g., dropwise into the vortex of the refold solution and mixing continued at 2-8 °C for 2 h. An equal volume of 3 M ammonium sulfate may be added to the refolding solution and stirred at 2-8 °C for 1 h.

[0099] The recovered protein may be detected by SDS-PAGE. Total protein content may be measured by one or more methods known in the art. For example, total protein content may be measured by Coomassie stain of proteins resolved in SDS-PAGE. The recovered protein maybe further purified using HPLC, for example size exclusion chromatography (SEC)- HPLC.

[00100] The biological activity of the protein may be assessedby cell proliferation assay in vitro. For this purpose, endothelial cell lines may be used. In some embodiments, fibroblasts may be used for in vitro proliferation assay. [00101] In some embodiments one or more modifications are made to improve the recovery yield of the modified FGF-1 from the cell. The one or more improvements comprise improvements of the plasmid vector; improvement in choice of a suitable bacterial strain; improvement in the growth media; improvement in induction time with IPTG; improvement in incubation time for maximal growth of the bacteria; and optimizing the temperature for growth of the bacteria. In some embodiments, the one or more modifications lead to at least 2 fold, at least 3 fold, at least 4 fold, to at least 6 fold, at least 7 fold, at least 8 fold, to at least 9 fold, at least 10 fold, atleast 12 fold, atleast 15 fold, to atleast20 fold, atleast25 fold, atleast 30 fold or at least 50 fold increase in the yield of modified FGF-1.

Disulfide Bond Formation in Modified FGF-1 Polypeptides

[00102] In some embodiments, the modified FGF-1 polypeptide of the present disclosure comprises the following mutations in SEQ ID NO: 1- Cysl6Ser, Ala66Cys, and Cysll7Val, wherein the polypeptide includes an internal disulfide bond between the cysteine residues at positions 66 and 83. For many recombinant proteins, the formation of correct disulfide bondsis vital for attaining their biologically active three-dimensional conformation. The formation of erroneous disulfide bonds can lead to protein misfolding and aggregation into inclusion bodies. In E. coli, cysteine oxidation typically takes places in the periplasm, where disulfide bonds are formed in disulfide exchange reactions catalyzed by a myriad of enzymes, mainly from the Dsb family (Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advancesand challenges. Frontiers in Microbiology, 5, 172). By contrast, disulfide bond formation in the cytoplasm is rare. This situation affects the production of recombinant proteins with disulfide bonds that are produced in the cytoplasm, such as a modified FGF-1 polypeptide comprising an internal disulfide linkage between Cys66 and Cys83. Accordingly, in some examples, an engineered E. coli strain that possess an oxidative cytoplasmic environment that favors disulfide bond formation is selected as a host cell for expression of the modifiedFGF-1 polypeptides (Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology, 5, 172). Examples of such strains include but are not limited to Origami (Novagen), which has a trxB- gor- genotype in the K-12 background, and SHuffle® T7 Express strain (NEB), which has a trxB-gor-genotype in a BL21 (DE3) background and constitutively expresses a chromosomal copy ofthe disulfide bond isomerase DsbC. Ithas been shown that DsbC promotes the correction of mis-oxidized proteins into their correct form and is also a chaperone that can assist in the folding of proteins that do not require disulfide bonds. Without being bound by a particular theory, it is contemplated that due to the action of DsbC, less target protein, such as the modified FGF-1 polypeptide comprising an internal disulfide linkage between Cys66 and Cys 83, aggregates into inclusion bodies. Thus, in certain embodiments, the present disclosure identifies an improved method for cytoplasmic production of a modified FGF-1 polypeptide comprising internal disulfide linkage between Cysl 6 and Cys83.

[00103] In some embodiments where the modified FGF-1 polypeptide is expressed with an N- Met residue, the polypeptide is subsequently purified without a step requiring proteolytic cleavage for removal of an N-terminal peptide. Accordingly, in some embodiments, the present disclosure provides a method of rapid purification of the modified FGF-1 polypeptides described herein, without involving a proteolytic cleavage step for removal of an N-terminal peptide. This is particularly advantageous for production of the modifiedFGF-1 polypeptides per good manufacturing practice (GMP) guidelines. The advantages include the lack of a cleavage step, including eliminating the need for subsequent purification of the cleaved product and removal of the reagents used for cleavage. The further advantage of this is an increase in yield due to decreased handling and the alleviation of the need to test for residual cleavage reagents and contaminants introduced for the cleavage and subsequent separation of cleaved from uncleaved material.

Methods of Use

[00104] Provided herein, in one embodiment, is a method of treating a disorder or condition in a mammal comprising administering to the mammal a modified FGF-1 polypeptide as described in the above embodiments, wherein the disease, disorder, or condition is associated with fibrosis, fibrotic or profibrotic induction.

[00105] Also provided herein, in another embodiment, are uses of the modified FGF-1 polypeptides as disclosed herein, in the manufacture of a medicament for treating a disorder or condition (e.g., an ocular disease) in mammal in need of.

[00106] Also provided herein, in yet another embodiment, the modified FGF-1 polypeptides as disclosed herein for use in treating a disorder or condition (e.g., an ocular disease) in mammal in need of.

[00107] Also provided herein, in yet another embodiment, are pharmaceutical compositions comprising the modifiedFGF-1 polypeptides as disclosed herein for use in treating a disorder or condition (e.g., an ocular disease) in mammal in need of.

[00108] In some instances, the modifiedFGF-1 polypeptide for use in the methods described herein comprises a sequence selected from SEQ ID NOs: 2, 9-94, 96-204, and 207. Provided herein, in one embodiment, is a method of treating a glaucoma in a mammal comprising administeringto the mammal a modified FGF-1 polypeptide comprising a sequence as set forth in SEQ ID NOs: 2, 9-94, 96-204, 205, 206, or 207.

[00109] In some embodiments, the disease, disorder or condition comprising an ocular disease, disorder or condition. In some embodiments, the disease, disorder or condition to be treated is a disease, disorder, or condition of the retina, nerve fiber layer, ocular tissue or macrophage. Diseases, disorders, or conditions of the retina, nerve fib er layer, ocular tissue or macrophage include, but are not limited to, , open-angle glaucoma, angle-closure glaucoma, normal tension glaucoma, grave’s orbitopathy, geographic atrophy (GA), pinguecula and pterygia, corneal fibrosis, and limbal epithelial stem cell deficiency (LSCD).

[00110] In some embodiments, the ocular disease, disorder or condition to be treated is tissue stiffness and fibrosis in the trabecular meshwork (TM) caused by long term exposure to steroids, which is thought to mimic the effects of aging on the TM in patients with primary open angle glaucoma.

[00111] In some embodiments, the disclosure provides methods of treating ocular hypertension (OHT), or preventing or delaying the progression of OHT to glaucoma by administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide. Ocular hypertension can cause glaucoma and the high eye pressure damages the optic nerve, causing vision loss. Ocular hypertension is not the same as glaucoma. Strong evidence supports that a higher baseline IOP, thinner central cornea, older age, and increased vertical cup-to-disc ratio are significant independent risk factors for glaucomatous progression. With ocular hypertension, the optic nerve looks normal and there are no signs of vision loss. However, people with ocular hypertension are at increased risk for glaucoma. In some embodiments, a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide of this disclosure is used to reduce ocular pressure.

[00112] A pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide of this disclosure, in some embodiments, is used to reduce elevated intraocular pressure (IOP). In some embodiments, the ocular disease, disorder or condition to be treated comprises impairment of outflow pathway that is responsible for maintaining intraocular pressure. The outflow pathway is responsible for elevated IOP in glaucoma and managing elevated IOP in patients is a unique therapeutic tool for glaucoma. In some cases of people with primary open-angle glaucoma (POAG), the most common type of glaucoma, develop ocular hypertension (OHT, /.< ., elevated IOP) after long term exposure to steroids. It is likely because such exposure increases extracellular matrix (ECM) material, cell contractility, and stiffness in an already dysfunctional conventional outflow pathway. The conventional outflow pathway drains the majority of the aqueous humor, and its hydrodynamic resistance is the primary determinant and homeostatic regulator of IOP. Thus, pro-fibrotic changes in this outflow pathway frequently lead to significantly elevated lOPs. It has been shown that reducing IOP can slow down the deterioration of the visual field, and thus prevent the development and progression of glaucoma. In some embodiments, administering a method of this disclosure comprises administering modified FGF-1 polypeptide for 7 days and reduces the IOP in the eye by at least about 1% to about 50%, about 2% to about 40%, about 3% to about 30%, about 4% to about 20%, about 5% to about 15%, or about 6% to about 10%, such as, for about 1 .12%. [00113] In some embodiments, a method of this disclosure comprises administering a modified FGF-1 polypeptide as described herein or a pharmaceutical composition comprising the same to a subject who is at risk of developing glaucoma to prevent damage to the retina and the optic nerve, and prevent the death of retinal ganglion cells (RGCs), whose axons exit the retina through the optic disc where they are bundled together to form the optic nerve. As a result of RGC death, glaucoma patients experience loss of vision, beginning with their peripheral vision. In some embodiments, a modified FGF-1 polypeptide of this disclosure is used to increase fluid drainage from the eye, lower the amount of fluid made by the eye, suppress inflammation in the anterior or posterior chambers, or modulate an inflammatory response.

[00114] In some embodiments, a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide of this disclosure is used to treatthe subject at an early-onset stage of glaucoma. The lamina cribrosa can be posteriorly displaced and thicker and the scleral canal can be enlarged at Bruch’s membrane and at the anterior laminar insertion in the early -glaucoma eyes compared with the contralateral normal eyes (plastic deformation). Within the high-IOP normal eyes, the lamina cribrosa can be posteriorly displaced compared with that in the low-IOP normal eyes. Within the high-IOP early -glaucoma eyes, the lamina cribrosa can be posteriorly displaced and thicker and the scleral canal enlarged, compared with both low-IOP normal eyes and high-IOP normal eyes (hypercompliant deformation). In some embodiments, a modified FGF-1 polypeptide of this disclosure is used to treatthe subject prior to scleral canal wall expansion and/or posterior deformation of the lamina cribrosa. The retina is a thin tissue in the back of the eye containing different kinds of nerve cells. There are over a million retinal ganglion cells (RGCs) in the human retina, and they allow people to see as they send the image to the brain. In the advanced stage of glaucoma, the retinas can be almost completely depleted of ganglion cells, which have degenerated and therefore could not be stained. The few remaining ganglion cells can be considered to be resistant to glaucoma. They show drastic morphologic alterations, such as abnormal axonal beading, the cell bodies are normal in size but have irregular silhouettes or swellings, and there are fewer dendritic bifurcations. Once RGCs die in glaucoma, they are not replaced. Unlike peripheral nerve cells in other parts of the body, RGCs are part of the body’s central nervous system, which does not regenerate once damaged.

[00115] In some embodiments, a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide of this disclosure is used to treatthe subject at an advanced stage of glaucoma, which is characterized by loss of retinal ganglion cells (RGC) in the subject. A trabeculectomy is a surgical procedure to treat glaucoma that creates a new pathway for fluid inside the eye to be drained. In some embodiments, a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide of this disclosure is used to treat the subject at a stage prior to trabeculectomy. In some embodiments, a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide of this disclosure is used to prevent or delay the progression of glaucoma. In some embodiments, a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide of this disclosure is used to prevent or decrease the need for surgical intervention.

[00116] In some embodiments, a modified FGF-1 polypeptide of this disclosure or a pharmaceutical composition comprising the same is administered to treat an increase in the stiffness of the trabecular meshwork (TM) and the lymphatic endothelial cells that line Schlemm’s canal. These features have been linked to changes in the biomechanics of the constituent cells, and the activation of pro-fibrogenic pathways. In open angle glaucoma, stiffness of the TM is also associated with increased deposition of matricellular proteins including tenascin-C and thrombospondin, both associated with wound-repair and fibrosis. The alterations to the matrix microenvironment in glaucoma are accompanied by changes to the cytoskeleton of TM stromal cells that contribute to a fibrotic outcome. The increased stiffness of Schlemm’s canal in glaucoma is also linked to increased presence of actin stress fibers as well as assembly of vinculin-rich focal adhesions in its endothelial cells. The stiffening of the TMis a feature shared among most types of glaucoma and is linked to the induction of both TGF- p/SMAD and Rho/Rock signaling pathways, the increased production of matricellular proteins, and fibrosis. Resident stromal cells, fibrocytes (mesenchymal cells that form from monocyte precursors), and other mesenchymal cell populations are all potential myofibroblast progenitors in the TM, and contributors to fibrosis.

[00117] In some embodiments, administering a modified FGF-1 polypeptide as described herein or a pharmaceutical composition comprising the same, reduces or eliminates epithelial- mesenchymal like transition of trabecular meshwork (TM) cells. In some embodiments, administering a pharmaceutical composition as disclosed herein, comprising a modified FGF-1 polypeptide reduces macrophage infiltration in ocular tissue as observed by scanning electron microscopy. In some embodiments, administering a modified FGF-1 polypeptide as described herein or a pharmaceutical composition comprising the same, reduces at least one clinical symptom associated with glaucoma, wherein the at least one clinical symptom is selected from eye pain, headache, nausea, poor vision, optic nerve damage, alteration in corneal thickness, narrowed drainage angle and loss of vision. In some embodiments, administering a modified FGF-1 polypeptide as described herein or a pharmaceutical composition comprising the same, reduces an elevated expression of at least one biomarker associated with glaucoma, wherein the at least one biomarker is selected from TGF-beta, TNF-alpha, IFN-gamma, IL-6, alpha-SMA, vimentin, fibronectin, ZEB- 1 or ZEB-2, Slug, Snail or Twist.

[00118] In some embodiments, a modified FGF-1 polypeptide of this disclosure or a pharmaceutical composition comprising the same is administered to treat post-cataract surgery fibrosis. Cataract surgery removes the differentiated lens fiber cells that comprise the bulk of the lens, leaving behind the matrix capsule surrounding the lens and the closely associated wounded lens epithelial cells amongst which the resident immune cells reside. An intraocular lens is implanted within this capsular bag that replaces the function of focusing images on the retina. While generally a highly successful procedure, a large number of post-cataract surgery patients develop a fibrotic vision-impairing condition called Posterior Capsule Opacification (PCO) along the fiber cell-denuded region of posterior lens capsule. At the cellular and molecular level, PCO is linked to the appearance of Collagen Lproducing, aSMA+ myofibroblasts, of which one well-characterized source is the epithelial -mesenchymal transition (EMT) of lens epithelial cells. The resident immune cells of the lens also have been identified as a progenitor source of aSMA+ myofibroblasts post-cataract surgery. These immune cells are highly susceptible to being signaled to transition to a myofibroblast phenotype, an outcome promotedby factors in their microenvironment. In addition, the identification of a population of resident APCs in the lens is consistent with their recruitment of neutrophils and monocytes to the post-cataract surgery lens. These immune cell types have been demonstrated to convert to aSMA+ myofibroblasts that are associated with the development of fibrosis

[00119] In some embodiments, the ocular disease, disorder or condition to be treated is uveitis which is a multifactorial inflammatory disease that has destructive effects in both anterior and posterior segments of the eye. It is characterized by inflammation within the uvea, the middle layer of the eye that contains most of its vasculature. The uvea is located between the sclera and the retina, and includes the iris, the ciliary body, and the choroid. While uveitis is linked to diseases, injuries and/or infections of the eye, it can also be a component of autoimmune or inflammatory disorders that occur in other parts of the body, such as sarcoidosis. The most common type of uveitis occurs in the front of the eye, anterior uveitis, in the region between the cornea and lens. Other forms of uveitis are intermediate (occurring primarily in the vitreous), posterior (retina and choroid) and panuveitis. When untreated, uveitis can lead to scarring, swelling or detachment of the retina, optic nerve damage, glaucoma, and lens cataract, all of which are associated with vision loss. While this inflammatory disease has been linked to cataract formation, the exact mechanism is not yet known. In contrast, there is a significant literature showing post-cataract surgery complications in patients with uveitis, particularly if inflammation is not controlled prior to surgery. The complications of cataract surgery in patients with uveitis include PCO, glaucoma, macular edema, and retinal detachments, evidence that many regions of the eye are impacted by ocular inflammation after cataract surgery.

[00120] In some embodiments, the ocular disease, disorder or condition to be treated is drusen which are small yellow deposits of fatty proteins (lipids) that accumulate in Bruch’s membrane (BrM) under the retina. The retina is a thin layer of tissue that lines the back of the inside of the eye, near the optic nerve. The optic nerve connects the eye to the brain. The retina contains light-sensing cells that are essential for vision. Drusen are like tiny pebbles of debris that build up overtime. Malfunction of choroidal macrophages may lead to the development of basal laminar deposits and drusen. Drusen consists of cholesterol-rich lipids and various oxidized proteins, which can induce inflammasome activation, and complement activation leading to the death of RPE and photoreceptors.

[00121] In some embodiments, the ocular disease, disorder or condition to be treated is Graves’ orbitopathy (GO) wherein GO is an autoimmune inflammatory disease affecting the orbit. Orbital fibroblasts are a key component in GO pathogenesis, which includes inflammation, adipogenesis, hyaluronic acid (HA) secretion, and fibrosis. Macrophages are thought to participate in the immunological stage of GO.

[00122] In some embodiment, administering the pharmaceutical composition disclosed herein may reduce acute, unilateral, usually painless visual loss that evolves over several hours to days. [00123] In some embodiments, the ocular disease, disorder or condition to be treated is Geographic atrophy (GA), wherein GA is a major cause of blindness. GA is also characterized by EMT of the retinal pigment epithelial cells (RPE) and RPE fibrosis. In some embodiments, administering the pharmaceutical composition disclosed herein reduces at least one symptom associated with GA, wherein the at least one symptom is selected from poor vision, loss of melanin from the retina, GA lesions, central RPE loss, choriocapillary thinning and choroidal enhancement, and RPE cell migration. The RPE cell migration occurs from a native location of the RPE cells in the outer retinal layer to ectopic locations in the inner retinal layers. [00124] In some embodiments, the ocular disease, disorder or condition to be treated is pinguecula and pterygia wherein fibrovascular growths occur on the surface of the cornea. A statistically significant positive correlation between MT (metallothionein) expression and lymphocyte subsets, macrophages and Langerhans' cells were found in pterygium.

[00125] In some embodiments, the ocular disease, disorder or condition to be treated is limbal epithelial stem cell deficiency (LSCD). The limbal epithelial stem cells (LESC) are important for epithelial cell renewal and closure of wound defects. LSCD is the most severe form of corneal surface diseases. A dysfunction or depletion of LESC in combination with destruction of their stem cell niche may result in a limbal stem cell deficiency (LSCD). It can be primary as a result of inherited eye disease, but more commonly it is the result of acquired conditions such as chemical or thermal bum injuries, systemic autoimmune disease, contact lens keratopathy, recurrent ocular surgeries, or Stevens-Johnson Syndrome (SJS).

[00126] In some embodiments, the ocular disease, disorder or condition to be treated is a number of ocular surface diseases accompanied by comeal inflammation such as keratoconjunctivitis sicca, recurrent comeal erosion, or post-refractive surgery keratitis. In some embodiments, the ocular disease, disorder or condition to be treated is chronic cicatrizing conjunctivitis wherein it is a condition where the conjunctival surface becomes fibrotic usually associated with inflammation and or Stevens-Johnson syndrome.

[00127] In additional embodiments are described methods of treating non-ocular fibrotic diseases by administering a modified FGF-1 polypeptide according to the present disclosure, or a pharmaceutical composition comprising the same. Exemplary fibrotic diseases that can be treated with the disclosed modified FGF-1 polypeptides include but are not limited to idiopathic pulmonary fibrosis, a fibrotic disease, disorder or condition of the cardiac tissue, myocardial injury or infarction, a fibrotic disease, disorder or condition of the renal tissue, kidney diseases, Stevens-Johnson Syndrome (SJS), Sarcoidosis, cardiac fibrosis, liver fibrosis and intestinal fibrotic strictures associated with Crohn’s disease.

[00128] In some embodiments, the non-ocular disease, disorder or condition to be treated is Idiopathic pulmonary fibrosis (IPF). IPF is a pathological consequence resulted from altered wound healing in response to persistent lung injury. M2 macrophages are designated to resolve wound healing processes or terminate inflammatory responses in the lung. FGF-1 has been shown to have a role in the fibrosis response in animal models of IPF including the bleomycin model.

[00129] In some embodiments, the non-ocular disease, disorder or condition to be treated is myocardial injury / infarction. Tissue repair occurs following acute myocardial infarction (MI) wherein different populations of IL-4 and/or IL- 13 -activated inflammatory monocytes and resident tissue macrophages play unique roles for in the resolution of inflammation, tissue repair, and fibrosis.

[00130] In some embodiments, the non-ocular disease, disorder or condition to be treated is a kidney disease. In some embodiments, the disease is selected from anti-glomerular basement membrane glomerulonephritis, lupus nephritis, antigen -induced immune complex glomerulonephritis, renal allograft injury, ischemia reperfusion injury, and nephropathy. Renal fibrosis is a direct consequence of the kidney's limited capacity to regenerate after injury. Renal scarring results in a progressive loss of renal function, ultimately leading to end-stage renal failure and a requirement for dialysis or kidney transplantation. Kidney disease consists of a diverse range of etiologies, including immunological, mechanical, metabolic and toxic insults amongst others. These variously affect the three functional compartments of the kidney; the vasculature, glomerulus and tubulointerstitium. It is these compartments that are collectively responsible for the delivery of blood, plasma filtration and modification of the glomerular filtrate respectively. Regardless of etiology, all patients with chronic kidney disease show a decline in renal function with time. The process is irreversible, inevitably leading to end-stage renal failure, a condition that requires life-long dialysis or renal transplantation. End-stage kidney disease manifests itself as fibrotic lesions affecting each compartment; glomerulosclerosis, vascular sclerosis and tubulointerstitial fibrosis. Even though matrix synthesis is of course part of the normal repair process that occurs after injury, excessive synthesis of extracellular matrix is itself destructive, further exacerbating injury in a vicious cycle.

[00131] In some embodiments, the fibrotic disease, disorder or condition results from pneumonia, rheumatoid arthritis, mixed connective tissue disease, systemic erythematosus lupus, scleroderma, dermatomyositis, polymyositis or sarcoidosis. In some embodiments, the disease, disorder or condition is associated with activation of myeloid cells, wherein the myeloid cell is an antigen presenting cell, a macrophage, a dendritic cell, or a neutrophil. In some embodiments, the myeloid cell is macrophage. In some embodiments, the disease disorder or condition is characterized by macrophage infiltration in an affected tissue of the subject. In some embodiments, administering a pharmaceutical composition comprising a modified FGF-1 polypeptide as disclosed herein reduces at least one gene expression signature associated with macrophage infiltration in the affected tissue of the subject. In some embodiments, the myeloid cell is an M2 polarized macrophage. In some embodiments, administering the pharmaceutical composition disclosed herein increases the expression of a M2 polarized macrophage specific biological marker in an affected tissue of the subject. In some embodiments, the M2 macrophage specific biological marker is CD 163, and administering the pharmaceutical composition reduces the number of CD 163+ cells in the ocular tissue. In some embodiments, the disease disorder or condition is characterized by macrophage to myofibroblast transformation. In some embodiments, administering the pharmaceutical composition disclosed herein reduces at least one gene expression signature associated with macrophage to myofibroblast transformation. In some embodiments, the disease disorder or condition is characterized by increased inflammatory gene expression. In some embodiments, the disease disorder or condition is characterized by increased expression ofLy6C, Ibal, and/or Argl, and administering the pharmaceutical composition reduces the expression of one or more of Ly 6C, Ibal, and/or Argl . In some embodiments, the disease disorder or condition is characterized by increased expression of TGF- beta, TNF-alpha, IFN-gamma, IL-6, alpha-SMA, vimentin, fibronectin, ZEB-1 or ZEB-2, Slug, Snail or Twist. In some embodiments, the disease disorder or condition is characterized by increased expression of genes related to vasculogenesis or fibrogenesis. In some embodiments, the disease, disorder or condition is characterized by increased VEGF, PDGF, peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), matrix metalloproteinases, caspases, or components of the renin-angiotensin-aldosterone system (ANG II). In some embodiments, administering the pharmaceutical composition disclosed herein reduces the gene expression of one or more of VEGF, PDGF, peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), matrix metalloproteinases, caspases, or components of the renin-angiotensin-aldosterone system (ANG II).

[00132] In some embodiments, the disease, disorder or condition is characterized by altered gene expression of a cytokine or a chemokine gene. In some embodiments, the cytokine or chemokine is IL4, IL-13, IL-21, TGF- H, MCP-1, or MLP-1 ?. In some embodiments, administering the pharmaceutical composition disclosed herein reverses the alteration of gene expression of one or more of IL4, IL-13, IL-21, TGF- H, MCP-1 and MIP-1/? by the disease, disorder or condition. In some embodiments, the gene expression is detected by RT-PCR of a biological sample from the subject. In some embodiments, the biological sample is obtained from a tissue or cells from the subject that is affected by the disease, disorder or condition. In some embodiments, the biological sample is obtained from peripheral blood. In some embodiments, the disease, disorder or condition associated with aberrant myeloid cell activation is further characterized by activation of an epithelial or endothelial cell. In some embodiments, the disease, disorder or condition associated with aberrant myeloid cell activation is further characterized by activation of a mesenchymal cell. In some embodiments, administering the pharmaceutical composition disclosed herein reduces the activation of the epithelial or endothelial or mesenchymal cell. [00133] A modified FGF-1 polypeptide of this disclosure, in some embodiments, is used to treat Stevens-Johnson Syndrome (SJS) wherein patients experience fibrosis of the ocular surface driven by the inflammatory process. SJS is a rare but serious disorder that affects the skin, mucous membrane, genitals and eyes. The mucous membrane is the soft layer of tissue that lines the digestive system from the mouth to the anus, as well as the genital tract (reproductive organs) and eyeballs. Stevens-Johnson syndrome is usually caused by an unpredictable adverse reaction to certain medications. It can also sometimes be caused by an infection.

[00134] In some embodiments, the non-ocular disease, disorder or condition to be treated is sarcoidosis wherein sarcoidosis is a chronic inflammatory condition associated with fibrosis. It is also associated with interstitial pulmonary fibrosis. It is a disease characterized by the growth of tiny collections of inflammatory cells (granulomas) in any part of your body; most commonly the lungs and lymph nodes. But it can also affect the eyes, skin, heart and other organs.

[00135] In some embodiments, the non-ocular disease, disorder or condition to be treated is cardiac fibrosis which is a process of pathological extracellular matrix (ECM) remodeling, leading to abnormalities in matrix composition and quality, as well as an impaired heart muscle function. The exact composition of the ECM varies from tissue to tissue. The major component of the adult human cardiac muscle is fibrilliar collagen type I (approximately 85%) and collagen type III (approximately 11%). Collagen synthesis and turnover is mainly regulatedby cardiac fibroblasts. In the heart muscle, collagen fibers are highly important for the transmission of contractile forces. Cardiac fibrosis is a scarring event in the cardiac muscle that is characterized by an increased collagen type I deposition as well as cardiac fibroblast activation and differentiation into myofibroblasts. These pathological changes lead to an increased matrix stiffness and lead to abnormalities in cardiac function. In further embodiments, the modified FGF-1 polypeptides described herein can be used to induce cardiac repair. With this approach, residing cells in the heart can be targeted and cell processes like survival, migration, proliferation and differentiation can be impacted. In further embodiments, the modified FGF-1 polypeptides described herein can be used to reduce the scar size.

[00136] In some embodiments, the non-ocular disease, disorder or condition to be treated is liver fibrosis. In most types of chronic liver diseases, extracellular matrix proteins including collagen are accumulated. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires liver transplantation. Liver fibrosis is associated with major alterations in both the quantity and composition of ECM.

[00137] In some embodiments, the non-ocular disease, disorder or condition to be treated is intestinal fibrotic strictures associated with Crohn’s disease. Crohn's disease (CD) is a disease with chronic inflammation of unknown etiology involving any part of the gastrointestinal tract. In further embodiments, the modified FGF-1 polypeptides described herein can be used to decrease, delay or prevent strictures.

[00138] In some embodiments, a therapeutic composition comprising a modified FGF-1 polypeptide as described herein is administered upon detection of at least one cellular or molecular biomarker in a biological sample of the subject, that is associated with a disease or condition. For example, a biomarker associated with a disease or condition described anywhere in this section can be, but not limited to elevated levels of VEGF, PDGF, peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), caspases, or and components of the renin-angiotensin-aldosterone system (ANG II), compared to a control sample, wherein the control sample is a corresponding sample obtained from one or more subjects who do not have the disease or the condition e.g., a healthy subject). In some embodiments, at least one biological marker associated with the fibrogenesis is the level of myofibroblasts in the tissue of the diseased subject, compared to a healthy subject, (e.g., a subject who do not have any detectable disease or condition). In some embodiments, the at least one biological marker is a marker associated with an activated myeloid cell function. In some embodiments, the activated myeloid cell function is recruitment of activated myeloid cells to the diseased tissue. In some embodiments, the activated myeloid cell function is expression of a activated myeloid cell protein or a peptide. In some embodiments, the activated myeloid cell function is identified by detection of M2 macrophage recruitment to the tissue of the subject with the disease or condition. In some embodiments, the activated myeloid cell function is identified by detection of elevated M2 macrophages in the tissue, compared to that of a healthy subject or sample. In some embodiments, the activated myeloid cell function is release of a cytokine or a chemokine associated with fibrogenesis.

[00139] In some embodiments, administering a pharmaceutical composition comprising a modified FGF-1 polypeptide as described herein reduces the expression of a Ml polarized macrophage specific biological marker and increases the expression of a M2 polarized macrophage specific biological marker in an affected tissue of the subject. In some embodiments, the M2 macrophage specific biological marker is CD 163, and administering the pharmaceutical composition disclosed herein reduces the number of CD 163+ cells in the ocular tissue. In some embodiments, the disease disorder or condition is characterized by macrophage to myofibroblast transformation.

[00140] In some embodiments, administering a pharmaceutical composition comprising a modified FGF-1 polypeptide as described herein reduces at least one gene expression signature associated with macrophage to myofibroblast transformation. In some embodiments, the disease disorder or condition is characterized by increased inflammatory gene expression. In some embodiments, the disease disorder or condition is characterized by increased CCL18, collagen or PDGF expression and administering the pharmaceutical composition disclosed herein reduces the expression of CCL18, collagen or PDGF.

[00141] In some embodiments, the disease, disorder or condition is characterized by increased expression of genes related to vasculogenesis or fibrogenesis.

[00142] In some embodiments, the disease, disorder or condition is characterized by increased VEGF, PDGF, peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), matrix metalloproteinases, caspases, or components of the renin-angiotensin-aldosterone system (ANG II).

[00143] In some embodiments, administering the pharmaceutical composition disclosed herein reduces the gene expression of one or more of VEGF, PDGF, peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), matrix metalloproteinases, caspases, or components of the renin-angiotensin-aldosterone system (ANGII).

[00144] In some embodiments, the disease, disorder or condition is characterized by altered gene expression of a cytokine or a chemokine gene.

[00145] In some embodiments, the cytokine or chemokine is IL4, IL-13, IL-21, TGF- H, MCP- 1, orMIP-1 ?.

[00146] In some embodiments, administering the pharmaceutical composition disclosed herein reverse the alteration of gene expression of one ormore of IL4, IL-13, IL-21, TGF- H, MCP-1 and MIP- I// by the disease, disorder or condition.

[00147] In some embodiments, the gene expression is detected by RT-PCR of a biological sample from the subject.

[00148] In some embodiments, the biological sample is obtained from a tissue or cells from the subject that is affected by the disease, disorder or condition.

[00149] In some embodiments, the biological sample is obtained from peripheral blood.

[00150] In some embodiments, the disease, disorder or condition associated with aberrant myeloid cell activation is further characterized by activation of an epithelial or endothelial cell.

[00151] In some embodiments, the disease, disorder or condition associated with aberrant myeloid cell activation is further characterized by activation of a mesenchymal cell.

[00152] In some embodiments, administering the pharmaceutical composition disclosed herein reduces the activation of the epithelial or endothelial or mesenchymal cell.

Pharmaceutical Compositions., Methods of Administration, and Dosing

[00153] Pharmaceutical compositions comprising a modified FGF-polypeptide as described herein, for use in methods of this disclosure, in some embodiments, is formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Additional details about suitable excipients for pharmaceutical compositions described herein may be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, JohnE., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkinsl999), herein incorporated by referencefor such disclosure.

[00154] A pharmaceutical composition, as used herein, in some instances, refers to a mixture of a modified FGF polypeptides with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients, and, optionally, other therapeutic and/or prophylactic ingredients. The pharmaceutical composition facilitates administration of the modified FGF to an organism. In practicingthe methods of treatment or use provided herein, therapeutically effective amounts of modified FGF- 1 polypeptides described herein are administered in a pharmaceutical composition to a mammal having an ocular disease, disorder, or condition to be treated. In some embodiments, the mammal is a human. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. A pharmaceutically acceptable or suitable composition includes an ophthalmologically suitable or acceptable composition.

[00155] A pharmaceutical composition (e.g., for delivery by injection or for application as an eye drop) in some embodiments is in the form of a liquid or solid. A liquid pharmaceutical composition includes, for example, one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils that may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents; antioxidants; chelating agents; buffers and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is commonly used as an excipient, and an injectable pharmaceutical composition or a composition that is delivered ocularly (for example, as an eye drop) is preferably sterile.

[00156] A modified FGF-polypeptide or pharmaceutical composition described herein canbe delivered to a subjectby any suitable means, including, for example, topically, intraocularly, intracam erally, orally, parenterally, intravenously, intraperitoneally, intranasally (or other delivery methods to the mucous membranes, for example, of the nose, throat, and bronchial tubes), or by local administration to the eye, or by anintraocular or periocular device. Modes of local administration can include, for example, topical application, eye drops, intraocular injection or periocular injection. Periocular injection typically involves injection of the compound under the conjunctiva or into the Tennon's space (beneath the fibrous tissue overlying the eye). Intraocular injection typically involves injection of the modified FGF or pharmaceutical composition into the vitreous. In certain embodiments, the administration is non-invasive, such as by topical application or eye drops. In some embodiments, the administration is via a combination of topical and intracameral method.

[00157] A modified FGF or pharmaceutical composition described herein in some embodiments is formulated for administration using pharmaceutically acceptable (suitable) carriers or vehicles as well as techniques routinely used in the art. A pharmaceutically acceptable or suitable carrier includes an ophthalmologically suitable or acceptable carrier. A carrier is selected according to the solubility of the particular modified FGF. Suitable ophthalmological compositions and formulations include those that are administrable locally to the eye, such as by eye drops, injection or the like. In the case of eye drops, the formulation can also optionally include, for example, ophthalmologically compatible agents such as isotonizing agents such as sodium chloride, concentrated glycerin, and the like; buffering agents such as sodium phosphate, sodium acetate, and the like; surfactants such as polyoxyethylene sorbitan mono-oleate (also referred to as Polysorbate 80), polyoxyl stearate40, polyoxyethylene hydrogenated castor oil, and the like; stabilization agents such as sodium citrate, sodium edentate, and the like; preservatives such as benzalkonium chloride, parabens, and the like; and other ingredients. Preservatives can be employed, for example, at a level of from about 0.001 to about 1.0% weigh t/volume. The pH of the formulation is usually within the range acceptable to ophthalmologic formulations, such as within the range of about pH 4 to 8.

[00158] For injection, the modified FGF or pharmaceutical composition in some embodiments is provided in an injection grade saline solution, in the form of an injectable liposome solution, slow-release polymer system or the like. Intraocular and periocular injections are known to those skilled in the art and are described in numerous publications including, for example, Spaeth, Ed., Ophthalmic Surgery: Principles of Practice, W. B. Sanders Co., Philadelphia, Pa., 85-87, 1990.

[00159] In some embodiments, the modified FGF polypeptide or pharmaceutical composition (e.g. , an ophthalmic formulation) is administered via microneedles into the cornea (Jiang etal. (2007). Invest Ophthalmol Vis Sci 48(9): 4038-4043). A microneedle array is coated with the modified FGF or pharmaceutical composition and pressed against the cornea such that the microneedles penetrate into the corneal stroma but do not penetrate the entire cornea. It is then removed, and the modified FGF or pharmaceutical composition is left behind in the corneal stroma. This modified FGF or pharmaceutical composition can stimulate the corneal cells to proliferate and migrate, and suppressesthe scarring response thatthe stromal cells normally have.

[00160] In some embodiments, the composition is formulated for intraocular delivery. Intraocular delivery comprises intravitreal delivery, corneal injections, intracameral delivery. In some embodiment the composition is formulated for intracameral delivery. In some embodiments the composition is formulated for intravitreal delivery. The formulation is an injectable liquid, may comprise a very small volume, and the density of the injectable liquid formulation maybe adjusted such its release in the targeted space doesnot incur injury to the tissue. In some embodiments, the volume for intracameral delivery is less than about 20 microliters, less than about 10 microliters, less than about 5 microliters, less than about2.5 microliters, or about 1 microliter. Provided herein is an injectable formulation for intraocular delivery, comprising: a modified FGF-1 polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, or having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 1, and comprising atleast 1, 2, 3, 4 or 5 single amino acid mutations; and L-methionine. The modified FGF-1 polypeptide in the formulation in some embodiments is present at greater than about 95% pure and the polypeptide is in monomeric form in the formulation. In some embodiments, the polypeptide further comprises an extension peptide positioned between the N- terminal methionine residue and the first residue of SEQ ID NO: 1 . In some embodiments, the injectable formulation comprises a modified FGF-1 comprising an amino acid sequence set forth in any one of the following sequences, SEQ ID NOs: 2, 205, 206, 207, 3-8, 14-18, 24-28, 93, 94, 96-117, 118-141, 146-149, and 174-204, or a sequence that is at least 90% identical to the sequences, or is a fragment thereof.

[00161] In some embodiments, an injectable formulation is hereby provided, the formulation comprises a required dose and concentration of the modified FGF-1, and an excipient, comprising one or more of sodium chloride; ammonium sulfate; monobasic potassium phosphate; dibasic sodium phosphate dihydrate; ethylenediaminetetraacetic; andL-Methionine. In some embodiments, the injectable formulation comprises: a modified FGF-1 polypeptide; at least about 50 mM dibasic sodium phosphate dihydrate; at least about 100 mM sodium chloride; at least about 10 mM ammonium sulfate; at least about 0.1 mM ethylenediaminetetraacetic acid (EDTA); atleast about 5 mML-Methionine; and at least about 0.01% polysorbate 80 (w/v). The injectable formulation comprising the modified FGF-1 polypeptide comprises one or more mutations selected from the group consisting of: Cysl6Ser, Ala66Cys, and Cysll7Val, Lysl2Val, Cysl6Ser, Ala66Cys, Cysl 17Val, and Prol34Val, and wherein the modified FGF-1 polypeptide further comprises at least one residue of the peptide ALTEK. In some embodiments, the modified FGF-1 polypeptide comprises one or more mutations comprisingthe following mutations of SEQ ID NO: 1 : Cysl6Ser, Ala66Cys, and Cysll7Val, wherein the modified FGF-1 polypeptide comprises a methionine residue positioned upstream to the first residue of SEQ ID NO: 1 , and at least one residue of the peptide ALTEK located between the N-terminal methionine and position 1 of SEQ ID NO: l . In some embodiments, the modified FGF-1 polypeptide comprises one or more mutations comprising the following mutations of SEQ ID NO: 1 : Cysl6Ser, Ala66Cys, and Cysl 17Val, wherein the modifiedFGF-1 polypeptide comprises a methionine residue positioned upstream to the first residue of SEQ ID NO: 1 . In some embodiments, the modified FGF-1 polypeptide comprises one or more mutations comprisingthe following mutations of SEQ ID NO: 1 : Cysl6Ser, Ala66Cys, and Cysl 17Val. [00162] In some embodiments, the formulation or the pharmaceutically suitable excipient therein comprises human serum albumin (HSA) and/or polysorbate 80. In some embodiments, the formulation comprises L-Methionine. In some embodiments, the L-Methionine is present at a concentration between 1 mM to 20 mM in the formulation. In some embodiments, the L- Methionine is present at a concentration between 2 mM to 10 mM in the formulation. In some embodiments, the L-Methionine is present at a concentration between 1 mM to 10 mM in the formulation. In some embodiments, the L-Methionine is present at a concentration between 2.5 mM to 15 mM in the formulation. In some embodiments, the L-Methionine is present at a concentration of about 5 mM in the formulation. For delivery of a composition comprising at least one of the modifiedFGF-1 polypeptides described herein via a mucosal route, which includes delivery to the nasal passages, throat, and airways, the composition may be delivered in the form of an aerosol. The compound in some embodiments is in a liquid or powder form for intramucosal delivery. For example, the composition may be delivered via a pressurized aerosol container with a suitable propellant, such as a hydrocarbon propellant (e.g., propane, butane, isobutene). The composition in some embodiments is delivered via a non-pressurized delivery system such as a nebulizer or atomizer.

[00163] For delivery of a composition comprising at least one of the modified FGF-1 polypeptides described herein via a mucosal route, which includes delivery to the nasal passages, throat, and airways, the composition may be delivered in the form of an aerosol. The compound in some embodiments is in a liquid or powder form for intramucosal delivery. For example, the composition may be delivered via a pressurized aerosol container with a suitable propellant, such as a hydrocarbon propellant (e.g., propane, butane, isobutene). The composition may be delivered via a non-pressurized delivery system such as a nebulizer or atomizer.

[00164] Suitable oral dosage forms include, for example, tablets, pills, sachets, or capsules of hard or soft gelatin, methylcellulose or of another suitable material easily dissolved in the digestive tract. Suitable nontoxic solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. See, e.g., Remington: The Science and Practice of Pharmacy (Gennaro, 21^st Ed. MackPub. Co., Easton, PA (2005)). [00165] The modified FGF-1 polypeptides or pharmaceutical compositions described herein may be formulated for sustained or slow-release. Such compositions may generally be prepared using well known technology and administered by, for example, periocular, intraocular, rectal, oral or subcutaneous implantation, or by implantation at the desired target site, or by topical application. Sustained-release formulations may contain an agent dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Excipients for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. The amount of active compound contained within a sustained-release formulation depends upon the site of implantation, the rate and expected duration of release, and the nature of the condition to be treated or prevented.

[00166] In some embodiments, methods of this disclosure comprise administering a composition comprising a modified FGF-1 polypeptide, citrate or histidine, sorbitol, and polysorbate. The concentrations, in some embodiments, are about 10 mM citrate or Histidine, about 5% sorbitol, about 0.1% polysorbate 80, and the formulation has a pH of about 5.8. In some embodiments, the concentration of citrate or Histidine comprises 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, 10 mM, 10.5 mM, 11 mM, 11.5 mM, 12 mM, 12.5 mM, and 13 mM, for example from about 1 mMto about 50 mM, from about 5 mMto about 40 mM, from about 10 mM to about 30 mM, from about 15 mM to about 20 mM, from about 7 mM to about 12 mM, and from about 10 mMto about 15 mM. In some embodiments, the pH of the formulation is about4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, or 7.0, for example from about4.0 to about 8.0, from about4.5 to about 7.2, from about 5 to about 7.0, from about 5.5 to about7.2, and from about4.8to about6.5. In some embodiments, the concentration of sorbitol comprises 3.8%, 4.0%, 4.2%, 4.4%, 4.6%, 4.8%, 5.0%, 5.2%, 5.4%, 5.6%, 5.8%, 6.0%, and 6.2%, for example from about 3.6% to about 6.5%, from about 4.0% to about 6.0%, from about 4.5% to about 6.5%, from about 5.0% to about6.2%, and from about 5.5% to about 6.5%. In some embodiments, the concentration of polysorbate 80 comprises 0.02%, 0.04%, 0.06%, 0.08%, 0.10%, 0.12%, 0.14%, 0.16%, 0.18%, 0.2%, and 0.22%, for example from about 0.02% to about 0.2%, from about 0.03% to about 0.9%, from about 0.04% to about 0.18%, from about 0.06% to about 0.15%, and from about 0.08% to about 0.14%.

[00167] In some embodiments, methods of this disclosure comprise administering a composition comprising a modified FGF-1 polypeptide, sodium chloride, ammonium sulfate, and di-sodium hydrogen phosphate. The concentrations, in some embodiments, are 3 mg/mL of the modified FGF-1 polypeptide, about 800 mM of sodium chloride, about 320 mM of ammonium sulfate, about 20 mM of di-sodium hydrogen phosphate, and has a pH of about 7.4. In some embodiments, the concentration of the modified FGF-1 comprises 0.5 mg/mL, 1 mg/mL, 1.5 mg/mL, 2 mg/mL, 2.5 mg/mL, 3 mg/mL, 3.5 mg/mL, 4 mg/mL, 4.5 mg/mL, and 5 mg/mL, for example from 0.5 mg/mL to 5 mg/mL, from 1 mg/mL to 4.5 mg/mL, from 1.5 mg/mL to 4 mg/mL, from 2 mg/mL to 3.5 mg/mL, and from 2.5 mg/mL to 3 mg/mL. In some embodiments, the concentration of sodium chloride comprises about 500 mM, about 550 mM, about 600 mM, about 650 mM, about 700 mM, about 750 mM, about 800 mM, about 850 mM, about 900 mM, and about 950 mM, for example from about 500mMto about 950 mM, from about 550 mMto about 900 mM, from about 600 mMto about 850 mM, from about 650 mM to about 800 mM, and from about 700 mMto about 750 mM. The concentration of ammonium sulfate comprises about 260 mM, about 280 mM, about 300 mM, about 320 mM, about 340 mM, about 360 mM, about 380 mM, and about 400 mM, for example from about 260 mMto about400 mM, from about280 mMto about380 mM, fromabout 300 mMto about360mM, and from about 320 mM to about 340 mM. The concentration of di-sodium hydrogen phosphate comprises about 10 mM, about 15 mM, about20 mM, about25 mM, about 30 mM, about35 mM, about40 mM, about45 mM, and about 50 mM, for example from about 10 mMto about 50 mM, from about 15 mMto about 45 mM, from about20mMto about40 mM, and from about 25 mMto about 35 mM. In some embodiments, the pH of the formulation is about 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, and 8.6 for example from about 5.6 to about 8.6, from about 5.8 to about 8.4, from about 6 to about 8.2, from about 6.2 to about 8.0, from about 6.4 to about 7.8, from about 6.6 to about 7.6, from about 6.8 to about 7.4, and from about 7.0 to about 7.2.

[00168] In some embodiments, methods of this disclosure comprise administering a composition comprising a modified FGF-1 polypeptide at a dose of about 10 ng- 1000 ng per eye per administration, wherein the administration is intracam eral. In some embodiments, a modified FGF-1 polypeptide or pharmaceutical compositions described herein is administered at a concentration of about 0.1 to 10 pg/ml, wherein the administration is topical. In some embodiments, a modified FGF-1 polypeptide or pharmaceutical compositions described herein is administered at a dose of about 0.3 mg/kgto about 10 mg/kg. In some embodiments, a modified FGF-1 polypeptide or pharmaceutical compositions described herein is administered at a dose of about 0.3 pg/eye to about 3 pg/eye per administration.

[00169] In some embodiments, methods of this disclosure comprise administering a composition comprising a modified FGF-1 polypeptide about one to five times a day. In some embodiments, a modified FGF-1 polypeptide or pharmaceutical compositions described herein is administered about two times a day. In some embodiments, a modified FGF-1 polypeptide or pharmaceutical composition described herein is administered about three times a day. In some embodiments, a modified FGF-1 polypeptide or pharmaceutical composition described herein is administered for at least five consecutive days. In some embodiments, a modified FGF-1 polypeptide or pharmaceutical composition described herein is administered for at least seven consecutive days. In some embodiments, a modified FGF-1 polypeptide or pharmaceutical compositions described herein is administered at least for 15 days, 21 days, 24 days, 28 days, 30 days. In some embodiments, a modified FGF-1 polypeptide or pharmaceutical compositions described herein is administered via intracameral or intravitreal injection every 7 to 30 days.

[00170] Systemic drug absorption of a drug or composition administered via an ocular route is known to those skilled in the art (see, e.g., Lee et al., Ini. J. Pharm. 233 : 1-18 (2002)). In one embodiment, a compound described herein is delivered by a topical ocular delivery method (see, e.g., Curr. Drug Metab. 4:213-22 (2003)). The composition may be in the form of an eye drop, salve, or ointment or the like, such as, aqueous eye drops, aqueous ophthalmic suspensions, nonaqueous eye drops, and non-aqueous ophthalmic suspensions, gels, ophthalmic ointments, etc. For preparing a gel, for example, carboxyvinyl polymer, methyl cellulose, sodium alginate, hydroxypropyl cellulose, ethylene maleic anhydride polymer and the like can be used.

[00171] In another embodiment, the modified FGF solution or pharmaceutical composition (e.g., an ophthalmic formulation) contains hyaluronic acid, carboxymethyl cellulose, or other polysaccharides that provide increased ocular tolerability, viscosity and osmolality to produce a comfortable ocular solution.

[00172] The dose of the modified FGF or pharmaceutical composition comprising at least one of the modified FGF-1 polypeptides described herein may differ, depending upon the patient's (e.g., human) condition, that is, stage of the ocular disease, disorder, or condition, general health status, age, and other factors that a person skilled in the medical art will use to determine dose. When the composition is used as eye drops, for example, one to several drops per unit dose, preferably 1 or 2 drops (about 50 pl per 1 drop), may be applied about 1 to about 6 times daily. [00173] Pharmaceutical compositions may be administered in a manner appropriate to the disease, disorder, or condition to be treated (or prevented) as determined by persons skilled in the medical arts. An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease, disorder, or condition, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provides the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (e.g., an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free, or a lessening of symptom severity). For prophylactic use, a dose should be sufficient to prevent, delay the onset of, or diminish the severity of an ocular disease, disorder, or condition. Optimal dosesmay generally be determined using experimental models and/or clinical trials. The optimal dose may depend upon the body mass, weight, or blood volume of the patient.

[00174] In various embodiments, a modified FGF-1 polypeptide of the present disclosure may be administered as a daily dose over a period of time to a subject. The doses of the modified FGF-1 polypeptides or pharmaceutical compositions can be suitably selected depending on the clinical status, condition and age of the subject, dosage form and the like. In some embodiments, a modified FGF-1 polypeptide of the present disclosure may be administered chronically or long-term. In some embodiments, a modified FGF-1 polypeptide of the present disclosure may be administered for a period of days, weeks, months, years or continued therapy over the lifetime of a subject. In some embodiments, a modified FGF-1 polypeptide of the present disclosure may be administered for a period of about 7 days, 15 days, about 21 days, about 30 days, about 3 months, about 6 months, about 12 months, about 18 months, about2 years, about 5 years, about 7 years, about 10 years, about 15 years, about 20 years, about 25 years, about 30 years, about 35 years, or about 40 years. In some embodiments, a treatment regime may be determined for an individual subject dependent on various factors. In some embodiments, the treatment regimen is about 2 weeks for an acute exposure and several months to a year for a longterm exposure. In some embodiments, the treatment regimen is chronic. [00175] At least one modified FGF described herein can be administered to human or other nonhuman vertebrates, for use in a method as provided herein. In certain embodiments, the modified FGF is substantially pure, in that it contains less than about 5% or less than about 1%, or less than about 0.1%, of other organic molecules, such as contaminating intermediates or byproducts that are created, for example, in one or more of the steps of a synthesis method. In other embodiments, a combination of one or more modified FGF-1 polypeptides described herein can be administered. [00176] The compositions described herein can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, the compositions are administered to a patient already suffering from a disease or condition (e.g. , a fibrotic disease, disorder, or condition), in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition. Amounts effective for this use will depend on the severity and course of the disease or condition, previous therapy, the patient's health status, weight, and response to the drugs, and the judgment of the treating physician. In prophylactic applications, compositions described herein are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition. Such an amount is defined to be a "prophylactically effective amount or dose. " In this use, the precise amounts also depend on the patient's state of health, weight, and the like. In the case wherein the patient’s condition does not improve, upon the doctor’s discretion the administration of the compositions may be administered chronically, that is, for an extended period of time, including throughout the duration of the patient’ s life in order to ameliorate or otherwise control or limit the symptoms of the patient’ s disease or condition. In the case wherein the patient’s status does improve, upon the doctor’ s discretion the administration of the compositions may be given continuously; alternatively, the dose of drug being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained. Patients can, however, require intermittent treatment on a long-term basis upon any recurrence of symptoms. The desired dose may conveniently be presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

[00177] The pharmaceutical compositions described herein may be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more modified FGF-1 polypeptides. The unit dosage may be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension or solution compositions can be packaged in single-dose non- reclosable containers. Alternatively, multiple-dose reclosable containers can be used, in which case it is typical to include a preservative in the composition. By way of example only, formulations for parenteral injection may be presented in unit dosage form, which include, but are not limited to ampoules, or in multi-dose containers, with an added preservative. [00178] Toxicity and therapeutic efficacy of such therapeutic regimens can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD₅₀ and ED₅₀. Compounds exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies canbe used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with minimal toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

Combination Treatments

[00179] The modified FGF-1 polypeptides and pharmaceutical compositions may also be used in combination with other therapeutic agents that are selected for their therapeutic value for the condition to be treated. The modified FGF-1 polypeptides and pharmaceutical compositions may also be used in combination with other therapeutic agents that are selected for their therapeutic value for treating the fibrotic diseases. Such agents do not have to be administered in the same pharmaceutical composition, and may, because of different physical and chemical characteristics, have to be administered by different routes. The determination of the mode of administration and the advisability of administration, where possible, in the same pharmaceutical composition, is well within the knowledge of the clinician. The initial administration can be made according to established protocols recognized in the field, and then, based upon the observed effects, the dosage, modes of administration and times of administration can be modified by the clinician.

[00180] The particular choice of these optional additional agents used will depend upon the diagnosis of the attending physicians and their judgment of the condition of the patient and the appropriate treatment protocol. The agents may be administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially, depending upon the nature of the disease, disorder, or condition, the condition of the patient, and the actual choice of agents used. The determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during a treatment protocol, is well within the knowledge of the physician after evaluation of the disease being treated and the condition of the patient.

[00181] The pharmaceutical agents which make up the combination therapy disclosed herein may be a combined dosage form or in separate dosage forms intended for substantially simultaneous administration. The pharmaceutical agents that make up the combination therapy may also be administered sequentially, with either therapeutic compound being administered by a regimen calling for two-step administration. The two-step administration regimen may call for sequential administration of the active agents or spaced -apart administration of the separate active agents. The time period between the multiple administration steps may range from, a few minutes to several hours, depending upon the properties of each pharmaceutical agent, such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the pharmaceutical agent. Circadian variation of the target molecule concentration may also determine the optimal dose interval.

[00182] Therapeutically-effective dosages can vary when the drugs are usedin treatment combinations. Methods for experimentally determining therapeutically-effective dosages of drugs and other agents for use in combination treatment regimens are described in the literature. For example, the use of metronomic dosing, i.e., providing more frequent, lower doses in order to minimize toxic side effects, has been described extensively in the literature. Combination treatment further includes periodic treatments that start and stop at various times to assist with the clinical management of the patient.

[00183] In some embodiments, the therapeutic agent comprises a profibrotic factor antagonist that targets a profibrotic factor. As used herein the term “profibrotic factors” refers to cytokines, growth factors or chemokines which have been observed to promote the accumulation of fibroblasts and deposition of collagen in various tissues. A number of cytokines and growth factors have been reported to be involved in regulating tissue remodeling and fibrosis. These include the “profibrotic cytokines” such as transforming growth factor beta (TGF-P), interleukin-4 (IL- 4), interleukin-5 (IL-5), and interleukin- 13 (IL-13), which have been shown to stimulate collagen synthesis and fibrosis in fibrotic tissues (Letterio etal. Ann Rev. Immunol. 16, 137- 161 (1998), Fertin etal., Cell Mol. Biol. 37, 823-829 (1991), Doucet etal., J. Clin. Invest. 101 , 2129-2139 (1998). Interleukin-9 (IL-9) has been shown to induce airway fibrosis in the lungs of mice (Zhu et al., J. Clin. Invest. 103, 779-788(1999)). In addition to TGF-P, other cytokines or growth factors which have been reported to increase fibrosis in the fibrotic disorder idiopathic pulmonary fibrosis (IPF) include granulocyte/macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-a), interleukin-1 beta (IL- ip), and connective tissue growth factor (CTGF) (Kelly et al. Curr Pharmaceutical Pes 9: 39- 49 (2003)). Cytokines and growth factors reported to be involved in promoting pulmonary fibrosis occurring in scleroderma include TGF-P, interleukin-1 beta (IL-I P), interleukin-6 (IL-6), oncostatinM (OSM), platelet derived growth factor (PDGF), the type 2 cytokines IL- 4 and IL-13, IL-9, monocyte chemotactic protein 1 (CCL2/MCP-1), and pulmonary and activation-regulated chemokine (CCL1 8/P ARC) (Atamas etal., Cyto Growth Fact Rev 14: 537-550 (2003)). [00184] Profibrotic factors that may be targeted with profibrotic factor antagonists as part of the combination therapy with a modified FGF polypeptide of this disclosure, include without limitation, a growth factor type P (TGF-P, including TGF- [31-5), VEGF, EGF, PDGF, IGF, RANTES, members of the interleukin family (e.g., IL-I, IL- 4, IL-5, IL-6, IL-8 and IL-13), tumor necrosis factor type alpha(TNF-a), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), monocyte chemoattractant protein type 1 (MCP-I), macrophage inflammatory protein (e.g., MIP- la, MIP-2), connective tissue growth factor (CTGF), endothelin-1, angiotensin-II, leptin, chemokines (e.g., CCL2, CCL12, CXCL12, CXCR4, CCR3, CCR5, CCR7, SLC/CCL21), integrals (e.g., ctl[31, a2pi avPo, avP3), tissue inhibitors of matrix metalloproteinases (e.g., TIMP-I, TIMP-2) and other factors known to promote or be related to the formation, growth, or maintenance of fibrotic tissue.

[00185] In some embodiments, the modifiedFGF-1 polypeptide is incorporated into formulations that contain other active ingredients such as steroids, antibiotics, antiinflammatories, cytokines such as IL-1 or analogs of IL-1, or antagonists of cytokines such as inhib itors of IL- 17.

[00186] Other exemplary cytokines include, but are not limited to, interleukins (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-la, IL-ip, and IL-1 RA), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), oncostatin M, erythropoietin, leukemia inhibitory factor (LIF), interferons, B7.1 (also known as CD80), B7.2 (also known as B70, CD86), TNF family members (TNF-a, TNF-P, LT-P, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, Trail), and migration inhibitory factor MIF.

[00187] In some embodiments, combinations or pharmaceutical compositions described herein are administered in immunosuppressive therapy to reduce, inhibit, or prevent activity of the immune system. Immunosuppressive therapy is clinically used to: prevent the rejection of transplanted organs and tissues; treatment of autoimmune diseases or diseases that are most likely of autoimmune origin; and treatment of some other non-autoimmune inflammatory diseases.

[00188] In some embodiments, the modifiedFGF-1 polypeptides and pharmaceutical compositions described herein are administered with one or more anti-inflammatory agent including, but not limited to, non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids (glucocorticoids). [00189] NSAIDs include, but are not limited to: aspirin, salicylic acid, gentisic acid, choline magnesium salicylate, choline salicylate, choline magnesium salicylate, choline salicylate, magnesium salicylate, sodium salicylate, diflunisal, carprofen, f enoprofen, f enoprofen calcium, fluorobiprofen, ibuprofen, ketoprofen, nabutone, ketolorac, ketorolac tromethamine, naproxen, oxaprozin, diclofenac, etodolac, indomethacin, sulindac, tolmetin, meclofenamate, meclofenamate sodium, mefenamic acid, piroxicam, meloxicam, and COX-2 specific inhibitors (such as, but not limited to, celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib, lumiracoxib, CS-502, JTE-522, L-745,337 and NS398).

[00190] Corticosteroids, include, but are not limited to: betamethasone, prednisone, alclometasone, aldosterone, amcinonide, beclometasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortisone, cortivazol, deflazacort, deoxycorticosterone, desonide, desoximetasone, desoxy cortone, dexamethasone, diflorasone, diflucortolone, difluprednate, fluclorolone, fludrocortisone, fludroxycortide, flumetasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fluocortolone, fluoromethoIone, fluperolone, fluprednidene, fluticasone, formocortal, halcinonide, halometasone, hydrocortisone/cortisol, hydrocortisone aceponate, hydrocortisone buteprate, hydrocortisone butyrate, loteprednol, medrysone, meprednisone, methylprednisolone, methylprednisolone aceponate, mometasone furoate, paramethasone, prednicarbate, prednisone/prednisolone, rimexolone, tixocortol, triamcinolone, andulobetasol.

[00191] Other agents used as anti-inflammatories include those disclosed in U.S. patent publication 2005/0227929, herein incorporated by reference.

[00192] Some commercially available anti-inflammatories include, but are not limited to: Arthrotec® (diclofenac and misoprostol), Asacol®(5-aminosalicyclic acid), Salofalk®(5- aminosalicyclic acid), Auralgan® (antipyrine and benzocaine), Azulfidine® (sulfasalazine), Daypro® (oxaprozin), Lodine®(etodolac), Ponstan® (mefenamic acid), Solumedrol® (methylprednisolone), Bayer®(aspirin), Bufferin® (aspirin), Indocin® (indomethacin), Vioxx® (rofecoxib), Celebrex® (celecoxib), Bextra® (valdecoxib), Arcoxia® (etoricoxib), Prexige® (lumiracoxib), Advil®, Motrin® (ibuprofen), Voltaren®(diclofenac), Orudis®(ketoprofen), Mobic®(meloxicam), Relafen® (nabum etone), Aleve®, Naprosyn® (naproxen), Feldene® (piroxicam).

[00193] In one embodiment, compositions described herein are administered with leukotriene receptor antagonists including, but are not limited to, BAY u9773 (see EP 00791576; published 27 Aug 1997), DUO-LT (Tsuji et al, Org. Biomol. Chem., 1, 3139-3141, 2003), zafirlukast (Accolate®), montelukast (Singulair®), prankulast (Onon®), and derivatives or analogs thereof. [00194] In some embodiments, the modified FGF-1 polypeptides as described herein or pharmaceutical compositions comprising the same are administered in combination with a novel low molecular weight dextran-sulphate, ILB to reduce IOP.

[00195] In some embodiments, the modified FGF-1 polypeptides as described herein or pharmaceutical compositions comprising the same are administered in combination with prostaglandins. These can increase the outflow of the fluid in eye (aqueous humor), thereby reducing eye pressure. Some commercially available prostaglandins include, but are not limited to: latanoprost (Xalatan), travoprost (Travatan Z), tafluprost (Zioptan), bimatoprost (Lumigan) and latanoprostene bunod (Vyzulta).

[00196] In some embodiments, the modified FGF-1 polypeptides as described herein or pharmaceutical compositions comprising the same are administered in combination with beta blockers. These can reduce the production of fluid in eye, thereby lowering the pressure in eye (intraocular pressure). Some commercially available beta blockers include, but are not limited to: timolol (Betimol, Istalol, Timoptic) and betaxolol (Betoptic).

[00197] In some embodiments, the modified FGF-1 polypeptides as described herein or pharmaceutical compositions comprising the same are administered in combination with alpha- adrenergic agonists. These can reduce the production of aqueous humor and increase outflow of the fluid in eye. Some commercially available alpha-adrenergic agonists include, but are not limited to: apraclonidine (lopidine) and brimonidine (Alphagan P, Qoliana).

[00198] In some embodiments, the modified FGF-1 polypeptides as described herein or pharmaceutical compositions comprising the same are administered in combination with carbonic anhydrase inhibitors. These medicines can reduce the production of fluid in eye. Some commercially available carbonic anhydrase inhibitors include, but are not limited to: dorzolamide (Trusopt) and brinzolamide (Azopt).

[00199] In some embodiments, the modified FGF-1 polypeptides as described herein or pharmaceutical compositions comprising the same are administered in combination with miotic or cholinergic agents. These can increase the outflow of fluid from eye. Some commercially available miotic or cholinergic agents include, but are not limited to: pilocarpine (Isopto Carpine).

[00200] In some embodiments, the modified FGF-1 polypeptides as described herein or pharmaceutical compositions comprising the same are administered in combination with nitric oxides.

[00201] In some embodiments, the modified FGF-1 polypeptides as described herein or pharmaceutical compositions comprising the same are administered in combination with other IOP lowering agents selected from the group comprising prostaglandin analogs (PAs) — latanoprost, travoprost, bimatoprost, unoprostone, latanoprostene bunod; beta-adrenergic blockers (BBs) — timolol, betaxolol, levobunolol, metipranolol, carteolol; adrenergic agonists (AAs) — clonidine, apraclonidine, brimonidine; carbonic anhydrase inhibitors (CAIs) — brinzolamide, dorzolamide; miotics — pilocarpine, carbachol, acetylcholine, demecarium bromide, echothiopate iodide; and rho-kinase inhibitors (RKIs) — netarsudil. These can reduce IOP by increasing the aqueous humor flow or by reducing its production.

[00202] In some embodiments, the modified FGF-1 polypeptides as described herein or pharmaceutical compositions comprising the same are administered after surgical approaches including laser treatments as well as more invasive surgical techniques. In some embodiments, the laser treatment comprises treatment using femtosecond pulsed laser, selective laser trabeculoplasty (SLT), diode laser trabeculoplasty (DLT), micro-pulsed diode laser trabeculoplasty (MDLT), or ab-interno excimer laser trabeculotomy (ELT).

[00203] In some embodiments, the modified FGF-1 polypeptides as described herein or pharmaceutical compositions comprising the same are administered in combination with rehabilitation therapy. In some embodiments, the rehabilitation therapy comprises epi -retinal implants, sub-retinal implants, trans-choroidal implants, stimulation of optic nerve, or cortical implants.

[00204] In some embodiments, the modified FGF-1 polypeptides as described herein or pharmaceutical compositions comprising the same are administered in combination with optical coherence tomography, detection of apoptotic retinal cells, or telemetric contact lenses.

[00205] In some embodiments, the modified FGF-1 polypeptides as described herein or pharmaceutical compositions comprising the same are administered in combination with a drug that is FDA-approved fortreatment of idiopathic pulmonary fibrosis (IPF) including, e.g., nintedanib (Ofev®) and pirfenidone (Esbriet®).

[00206] In some embodiments, the modified FGF-1 polypeptides as described herein or pharmaceutical compositions comprising the same are administered with one or more Rho kinase inhibitors. In some embodiments, the modified FGF-1 polypeptides as described herein or pharmaceutical compositions comprising the same are administered with one or more additional growth factors, including, but not limited to epidermal growth factor (EGF) and nerve growth factor (NGF). See, e.g., see Joyce etal. (2009) Invest Ophthalmol. VisSci. 50:2116- 2122, vascular endothelial growth factor (VEGF), transforming growth factor alpha and beta (TGF-alpha and TFG-beta), platelet-derived endothelial growth factor (PD-ECGF), platelet- derived growth factor (PDGF), tumor necrosis factor alpha (TNF-alpha), hepatocyte growth factor (HGF), insulin like growth factor (IGF), erythropoietin, colony stimulating factor (CSF), macrophage-CSF (M-CSF), granulocyte/macrophage CSF (GM-CSF) and nitric oxide synthase (NOS).

Kits/Articles of Manufacture

[00207] For use in the therapeutic applications described herein, kits and articles of manufacture are also provided herein. Such kits include, in certain embodiments, a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) including one of the separate elements to be used in a method described herein.

[00208] Also provided herein are kits comprising a pharmaceutical composition comprising a modified FGF-1 polypeptide provided herein, and a container. In some embodiments, the kit comprises a pharmaceutical composition comprising a modified FGF-1 polypeptide having a sequence comprising one or more mutations relative to the wild-type FGF-1 sequence of SEQ ID NO: 1, and a container.

[00209] Suitable containers include, for example, bottles, vials, eye droppers, eye drop containers, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic.

[00210] In certain embodiments, the container is a bottle. In some embodiments, the bottle is a unit-dose bottle. In some embodiments, the bottle comprises a containing space, a nozzle, a dropper tip, a screw cap, a temper-evident overcap, and a base support. In certain embodiments, the container is a vial. In some embodiments, the container is an eye dropper. In another embodiments, the container is an eye drop container. In other embodiments, the container is a syringe. In other embodiments, the container is a test tube. Specific examples of the container in such a form include a container for injection, a container for inhalation, a container for spray, a bottle-shaped container, a tube-shaped container, an eye drop container, a nasal drop container, an ear drop container, a bag shaped container and the like.

[00211] In some embodiments, the container is made of glass, including type 1 glass, as is commonly used for long-term storage of drugs andbiologics. In some embodiments, the container is made of non-leachable plastic materials that are used for long-term storage of drugs or biologies, such as cyclic olefin polymer (such as Crystal Zenith®), cyclic olefin co-polymer and the like.

[00212] In some embodiments, the container is made of plastic. In some embodiments, the plastic is a thermoplastic resin including a natural resin and a synthetic resin. In certain embodiments, the resin is a rigid, high-strength, semi-tough, transparent plastic with high heat resistance and hydrolytic stability . Nonlimiting examples of the resin include a polyolefin resin, a polyester resin, a poly sulfone resin, a polyamide resin, a polycarbonate resin, a polyphenylene ether resin, a polyvinyl chloride resin, a styrene resin, and combinations thereof. In certain embodiments, the resin comprises a polyolefin resin, a polyester resin, or combinations thereof. In some embodiments, the polyolefin resin is selected from the group consisting of : low density polyethylene, high density polyethylene, medium density polyethylene, polypropylene, cyclic polyolefin, poly(4-methylpentene), polytetrafluoroethylene, an ethylene-propylene copolymer, an ethylene-a-olefin copolymer, an ethylene-acrylic acid copolymer, an ethylene-methacrylic acid copolymer, an ethylene-vinyl acetate copolymer, and an ethylene-ethyl acrylate copolymer, and combinations thereof. In certain embodiments, the resin comprises cyclic polyolefin. [00213] In some embodiments, the container is Ready -to-Use" (pre-sterilized) non-glass vial. In some embodiments, the container is a Crystal Zenith®vial. In some embodiments, the container is a Crystal Zenith® syringe. In some embodiments, the container is a Crystal Zenith® insert needle syringe. In some embodiments, the container has lower particle levels. In some embodiments, the container is break resistant.

[00214] In some embodiments, the polyester resin is selected from the group consisting of: homopolymer comprising polyalkylene terephthalate, polyalkylene naphthalate, poly cycloalkylene terephthalate or polyarylate; copolyester comprising units of the homopolyester; a copolymer of the homopolyester, and combinations thereof.

[00215] In certain cases, the container blocks a light. In some embodiments, the container blocks a light having a wavelength of from 320 to 380 nm. In some embodiments, the resin is blended with an UV light absorbing agent or an UV light scattering agent. In some cases, the inside of the container is visible to the naked eye or the like. In some embodiments, the container is compatible with high and low pH conditions. In some embodiments, the container comprises a low protein and peptide adsorption. In some embodiments, the container reduces the risk of breakage within cold storage environments that are commonly associated with glass. In some embodiments, the vials comprise a Crystal Zenith® vial.

[00216] In some embodiments, the container (preferably, a container whose inside is visible) comprises a polyolefin resin (preferably, polypropylene) kneaded with a substance which interferes with transmission of ultraviolet light (more preferably one or more substances selected from the group consisting of an ultraviolet scattering agent and an ultraviolet absorbing agent; particularly preferably one or more substances selected from the group consisting of zinc oxide, titanium oxide and a benzotriazole-based ultraviolet absorbing agent).

[00217] In some embodiments, the container (preferably, a container whose inside is visible) comprises a polyolefin resin (preferably, polypropylene) having a member (preferably, a heat- shrinkable film (a shrink film)) kneaded with a substance which interferes with transmission of ultraviolet light (more preferably one or more substances selected from the group consisting of an ultraviolet scattering agent and an ultraviolet absorbing agent; particularly preferably one or more substances selected from the group consisting of zinc oxide, titanium oxide and a benzotriazole-based ultraviolet absorbing agent), the member being wound around on a side surface of the container.

[00218] The articles of manufacture provided herein contain packaging materials. Packaging materials foruse in packaging pharmaceutical products include, e.g., U.S. PatentNos. 5,323,907, 5,052,558 and 5,033,252,EP 3590513Al, EP 3590514A1, EP 3590515A1, and EP3603642A1, each of which is incorporated by reference herein. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. A wide array of ophthalmic formulations of the modified FGF-1 polypeptides and pharmaceutical compositions provided herein are contemplated as are a variety of treatments for any ocular disease, disorder, or condition that would benefit by administration of a modified FGF ore pharmaceutical composition described herein.

[00219] For example, the container(s) can include a modified FGF such as a modified FGF-1 having a sequence of SEQ ID NO: 2. The container(s) optionally have a sterile access port. Such kits optionally comprising compounds with an identifying descriptions or labels or instructions relating to their use in the methods described herein.

[00220] In some embodiments, a kit may be suitable for or designed to be suitable for an injectable liquid formulation for intraocular delivery. The kit may be designed as a low-volume vial and may comprise a conical insert. In some embodiments, the kit is the dropper bottle. In some embodiments, the dropper bottle may be enabled to provide at least on dose of modified FGF-1 in the injectable formulation. In some embodiments, the dropper bottle further comprises a sterile filter. In some embodiments, the container comprises the syringe. In some embodiments, the syringe comprises a material selected from the group consisting of tuberculin polypropylene and glass. In some embodiments, the syringe is prefilled with an injectable formulation. In some embodiments, the kit may further comprise an electronic control unit. In some embodiments, the electronic control unit enables control of administration of a volume of an injectable formulation according to that described in the preceding sections, wherein the volume is from at least about 10 microliters to about 100 microliters. In some embodiments, the dropper bottle of the kit is enabled to provide at least on dose of modified FGF-1 in the injectable formulation of any one of embodiments described above, or the pharmaceutical composition described anywhere in the disclosure. In some embodiments, the dropper bottle may further comprise a sterile filter. In some embodiments, the container comprises the syringe. In some embodiments, the syringe comprises a material selected from the group consisting of tuberculin polypropylene and glass. In some embodiments, the syringe is prefilled with an injectable formulation according to any one of embodiments described above, or the pharmaceutical composition described anywhere in the disclosure. The kit may further comprise an electronic control unit. In some embodiments, the electronic control unit enables control of administration of a volume of an injectable formulation or, a pharmaceutical composition, wherein the volume is from at least about 10 pL to about 100 pL.

[00221] In some embodiments, a kit includes one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and/or devices) desirable from a commercial and user standpoint for use of a modified FGF described herein. Non-limiting examples of such materials include, but not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[00222] A label can be on or associated with the container. A label can be on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. A label can be used to indicate thatthe contents are to be used for a specific therapeutic application. The label can also indicate directions for use of the contents, such as in the methods described herein.

[00223] In certain embodiments, a modified FGF polypeptide containing pharmaceutical composition is presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein. The pack can for example contain metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration. The pack or dispenser can also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, can be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions containing a modified FGF provided herein formulated in a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container, and lab eled for treatment of an indicated condition. EXAMPLES

[00224] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein. The starting materials and reagents usedin the examples described herein may be synthesized or can be obtained from commercial sources.

Example 1: Therapeutic effect of modified FGF-1 polypeptides on glaucoma induced- elevated IOP

[00225] The efficacy of modified FGF-1 polypeptides in treating elevated intra ocular pressure (IOP) associated glaucoma was evaluated.

[00226] The effect of administering a composition comprising the modified FGF-1 polypeptide of sequence of SEQ ID NO: 2 (N-Met-TTHXl 114) was tested in this example. The modified FGF-1 polypeptide N-Met-TTHXl 114 comprising the mutations Cysl6Ser, Ala66Cys, and Cysl 17 Vai was administered three times daily (t.i.d.) to one eye of New Zealand White (NZW) rabbits (n=3 per dose group) for 7 days. The exemplary formulation used had 10 mM Histidine, pH=5.8, 5% sorbitol, 0.1% polysorbate 80. To evaluate the effect ofthe amount of dose, three different doses of N-Met-TTHXl 114, i.e., 0.3 pg/eye/dose, 0.9 pg/eye/dose, and 3 pg/eye/dose, were separately administered to one eye of the rabbits. At the end of the 7^th day, the IOP of each tested NZW rabbit was measured using rebound tonometry. Statistical test was two sample t-test assuming equal variances without correction for multiple observations. As shown in FIG. 1, the results showed that the dose of 3 pg/eye/dose decreased IOP for about 14% as compared to the reference, i.e., the IOP of non -treated NZW rabbit eye, and the dose of 0.9 pg/eye/dose did not reduced the IOP of the NZW rabbit eye (FIG. 1).

Example 2: Evaluation of treatment with modified FGF-1 polypeptides on profound lung fibrosis

[00227] Direct delivery of bleomycin to the lung (e.g. , intratracheal) causes direct damage to alveolar epithelial cells and induces rapidly developing fibrosis after a single administration. This is followed by the development of neutrophilic and lymphocytic alveolitis within the first week. After the first week, alveolar inflammatory cells are cleared, fibroblast proliferation is initiated, with synthesis of extracellular matrix. The development of fibrosis in this model can be seen biochemically and histologically by day 14 with maximal responses observed between21- 28 days.

[00228] The study is directed towards the effect of administering a composition comprising modified FGF-1 polypeptide of SEQ ID NO: 2 (N-Met-TTHXl 114) on treatment of idiopathic pulmonary fibrosis, in a pulmonary fibrosis model. Bleomycin induced lung fibrosis is used as an animal model of induction of lung fibrosis. Male C57B1/6 mice (N=10) are used. [00229] Bodyweight changes: Body weights of all animals are recorded on a weekly basis. Compared to animals of Sham/Vehicle group, animals of Bleomycin group show significantly reduced body weights since week 1. Data are mean ± SEM and analyzed by T-test as applicable. [00230] Lung hydroxyproline: On day 21, all animals are euthanized fortissue collection. One side of lung is dissected and used for Hydroxyproline level analysis using Hydroxyproline Colorimetric Assay Kit. Compared to the Sham/Vehicle group, animals of Bleomycin group show significantly increased hydroxy proline level in lung, demonstrating that pulmonary fibrosis has formed.

[00231] H&E, PSR Staining and Histopathology Score: Hematoxylin and eosin (H&E) and Picrosirius Red (PSR) staining are commonly used histopathology techniques to visualize inflammation and collagen deposit in tissue sections. Pathological alterations are evaluated by an ACVP Board Certified Toxicology Pathologist in blind manner. Interstitial Fibrosis is assessed based on the increase of PSR staining. The subacute interstitial inflammation and Agonal Hemorrhage are evaluated mainly based on the H&E staining. Compared to the lung samples from Sham/Vehicle group, significant increases in both interstitial fibrosis and inflammation are observed in the lung samples of Bleomycin/Vehicle group.

[00232] Treatment using a composition comprising the modified FGF-1 polypeptide of SEQ ID NO: 2 (N-Met-TTHXl 114) is seen to significantly improve interstitial fibrosis induced by Bleomycin.

Table 1: SEQUENCES

Claims

WHAT IS CLAIMED IS: . A method of treating glaucoma, or preventing or delaying the onset or progression of glaucoma, the method comprising: administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide, wherein the modified FGF-1 polypeptide comprises a sequence comprising one or more mutations relative to the wild-type FGF-1 sequence of SEQ ID NO: 1. . The method of claim 1, wherein the modified FGF-1 comprises at least two mutations with respectto the wild type FGF-1 of SEQ ID NO: 1. . The method of claim 1 or 2, wherein the modified FGF-1 polypeptide comprises an N- terminal methionine upstream of the first residue of SEQ ID NO: 1. . The method of any one of claims 1-3, wherein the modified FGF-1 comprises an amino acid sequence of that is at least about 80% identical to the sequence of SEQ ID NO: 2.. The method of any one of claims 1 -4, wherein the glaucoma is open-angle glaucoma.. The method of any one of claims 1 -4, wherein the glaucoma is angle-closure glaucoma. . The method of any one of claims 1-5, wherein the subject has an elevated intra ocular pressure (IOP), increased aqueous humor production, impaired outflow/deflow of aqueous humor, or any combinations thereof. . The method of any one of claims 1 -7, wherein the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide is administered to the subject at an early stage of glaucoma. . The method of claim 8, wherein the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide is administered to the subject prior to scleral canal wall expansion and/or posterior deformation of the lamina cribrosa. 0. The method of any one of claims 1 -9, wherein the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide is administered to the subject at an advanced stage of the glaucoma, wherein the advanced stage of the glaucoma is characterized by loss of retinal ganglion cells (RGC) in the subject. 1. The method of any one of claims 1-10, wherein the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide is administered to the subject at a stage prior to trabeculectomy.

- 89 - The method of any one of claims 1-11, wherein administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide prevents or delays the onset or progression of glaucoma. The method of any one of claims 1-12, wherein administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide prevents or decreases the need for surgical intervention. The method of any one of claims 1-13, wherein administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide reduces at least one clinical symptom associated with glaucoma. The method of any one of claims 1-14, wherein administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide results in at least one of: reduced IOP, reduced aqueous humor production, increased outflow/deflow of aqueous humor, or any combinations thereof. The method of any one of claims 1-15, wherein administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide reduces or eliminates epithelial-mesenchymal like transition of trabecular meshwork (TM) cells. The method of any one of claims 1-16, wherein administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide reduces macrophage infiltration in ocular tissue as observed by scanning electron microscopy. The method of any one of claims 1-17, wherein administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide reduces at least one of: eye pain, headache, nausea, poor vision, optic nerve damage, alteration in corneal thickness, narrowed drainage angle and loss of vision associated with glaucoma. The method of any one of claims 1-18, wherein the pharmaceutical composition is formulated for ophthalmic administration. The method of any one of claims 1-19, wherein the pharmaceutical composition comprises a formulation for ocular delivery. The method of claim 20, wherein the formulation for ocular delivery comprises a humectant at a concentration of at least about 5%. The method of claim 21, wherein the humectant is selected from the group consisting of : Glycerin, Maltitol, Propylene Glycol, Sorbitol, and Triacetin.

- 90 - The method of any one of claims 1 -22, wherein the formulation comprises at least about 0.1% (w/v) of a surfactant. The method of claim 23, wherein the surfactant is polysorbate 80 or polysorbate 20. The method of any one of claims 1-24, wherein the pharmaceutical composition comprises: about 10 mM citrate or Histidine, about 5% sorbitol, and about 0.1% polysorbate 80, and has a pH of about 5.8. The method of any one of claims 1-25, wherein the pharmaceutical composition comprises phosphate buffered saline, and about 0.1% polysorbate 80, and has a pH of about 7.4. The method of any one of claims 19-26, wherein the pharmaceutical composition is administered at a dose comprising about 10 ng to about 1000 ng of the modified FGF-1 polypeptide per eye per administration, wherein the administration is intracameral. The method of any one of claims 19-26, wherein the pharmaceutical composition is administered at a dosage comprising the modified FGF-1 polypeptide at a concentration of about 0.1 to 10 pg/ml, wherein the administration is topical. The method of any one of claims 19-26, wherein the pharmaceutical composition is administered at a dose comprising about 0.3 mg/kg to about 10 mg/kg of the modified FGF-1 polypeptide. The method of any one of claims 19-26, wherein the pharmaceutical composition is administered at a dose comprising about 0.3 pg/eye to about 3 pg/eye of the modified FGF-1 polypeptide, per administration. The method of any one of claims 19-30, wherein the pharmaceutical composition is administered one to five times a day. The method of any one of claims 19-30, wherein the pharmaceutical composition is administered about two times a day. The method of any one of claims 19-30, wherein the pharmaceutical composition is administered about three times a day. The method of any one of claims 19-33, wherein the pharmaceutical composition is administered for at least five consecutive days. The method of any one of claims 19-33, wherein the pharmaceutical composition is administered for at least seven consecutive days. The method of any one of claims 19-33, wherein the pharmaceutical composition is administered at least for 15 days, 21 days, 24 days, 28 days, 30 days. The method of any one of claims 19-33, wherein the pharmaceutical composition is administered via intracameral or intravitreal injection every 7 to 30 days.

- 91 - The method of any one of claims 19-37, wherein administering the pharmaceutical composition for about 7 days reduces the IOP in the eye by at least about 1.12%. A method of treating an ocular disease, disorder or a condition associated with a fibrotic or profibrotic induction in a tissue, or a condition associated with a risk of a fibrotic induction in a tissue, the method comprising: administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide, wherein the modified FGF-1 polypeptide comprises a sequence comprising an N-terminal methionine and a sequence comprising one or more mutations relative to the wild-type FGF-1 sequence of SEQ ID NO: 1. The method of claim 39, wherein the disease, disorder or condition is Geographic atrophy (GA). The method of claims 39 or 40, wherein administering the pharmaceutical composition comprising a therapeutically effective amount of the modified FGF-1 polypeptide reduces at least one symptom associated with GA, wherein the at least one symptom is selected from poor vision, loss of melanin from the retina, GA lesions, central RPE loss, choriocapillaiy thinning and choroidal enhancement, and RPE cell migration. The method of claim 41, wherein the RPE cell migration occurs from a native loc tion of the RPE cells in the outer retinal layer to ectopic locations in the inner retinal layers. The method of claim 39, wherein the disease, disorder or condition is drusen. The method of claim 39, wherein the disease, disorder or condition is Stevens-Johnson Syndrome (SJS). The method of claim 39, wherein the disease, disorder or condition is Graves’ orbitopathy. The method of claim 39, wherein the disease, disorder or condition is pingueculae or pterygia. The method of claim 39, wherein the disease, disorder or condition is Limbal epithelial stem cell deficiency (LSCD). The method of claim 39, wherein the disease, disorder or condition is chronic cicatrizing conjunctivitis. The method of any one of claims 39-48, wherein the pharmaceutical composition comprises a formulation for ocular delivery. The method of claim 49, wherein the formulation comprises a humectant at a concentration of at least about 5% concentration. The method of claim 50, wherein the humectantis selected from the group consisting of : Glycerin, Maltitol, Propylene Glycol, Sorbitol, and Triacetin.

- 92 - The method of any one of claims 39-51, wherein the formulation comprises at least about 0.1% of a surfactant. The method of claim 52, wherein the surfactant comprises polysorbate 80 or polysorbate 20. The method of any one of claims 39-53, wherein the pharmaceutical composition comprises: about lOmM citrate or Histidine, about 5% sorbitol, about 0.1% polysorbate 80, and has a pH of about 5.8. The method of any one of claims 39-53, wherein the pharmaceutical composition comprises: wherein the pharmaceutical composition comprises phosphate buffered saline, about 0.1% polysorbate 80, and has a pH of about 7.4. A method of treating a disease, disorder or a condition resulting from a fibrotic or profibrotic induction in a tissue, the method comprising: administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide, wherein the modified FGF-1 polypeptide comprises a sequence comprising the mutations Cysl6Ser, Ala66Cys, and Cysl 17Val relative to the wild-type FGF-1 sequence of SEQ ID NO: 1, and wherein the disease, disorder, or a condition is not an ocular disease, disorder, or condition. The method of claim 54, wherein the disease, disorder or condition is a fibrotic disease, disorder or condition of the lung. The method of claim 54, wherein the disease, disorder or condition is idiopathic pulmonary fibrosis. The method of claim 54, wherein the disease, disorder or condition is a fibrotic disease, disorder or condition of the cardiac tissue. The method of claim 54, wherein the disease, disorder or condition is myocardial infarction. The method of claim 54, wherein the disease, disorder or condition is a fibrotic disease, disorder or condition of the renal tissue. The method of claim 54, wherein the disease is selected from anti-glomerular basement membrane glomerulonephritis, lupus nephritis, antigen-induced immune complex glomerulonephritis, renal allograft injury, ischemia reperfusion injury, and nephropathy. The method of any one of claims 54-60, wherein the disease, disorder or condition results from pneumonia, rheumatoid arthritis, mixed connective tissue disease, systemic erythematosus lupus, scleroderma, dermatomyositis, polymyositis, systemic sclerosis or sarcoidosis.

- 93 - The method of any one of claims 39-63, wherein the disease is associated with an elevated expression of TGF-beta, TNF-alpha, IFN-gamma, IL-6, alpha-SMA, vimentin, fibronectin, ZEB-1 or ZEB-2, Slug, Snail or Twist, and administering the pharmaceutical composition reduces the elevated expression levels of any one or more of TGF-beta, TNF-alpha, IFN- gamma, IL-6, alpha-SMA, vimentin, fibronectin, ZEB-1 or ZEB-2, Slug, Snail and Twist. The method of any one of claims 39-64, wherein administering the pharmaceutical composition reduces alpha-smooth muscle actin (a-SMA) upregulation in TM cells and/or RPE cells. A method of treating a disease, disorder or a condition associated with activation of myeloid cells, the method comprising: administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide, wherein the modified FGF-1 polypeptide comprises a sequence comprising the mutations Cysl6Ser, Ala66Cys, and Cysl 17Val relative to the wild -type FGF-1 sequence of SEQ ID NO: 1, and wherein the disease, disorder, or a condition is not an ocular disease, disorder, or condition. The method of claim 66, wherein the myeloid cell is an antigen presenting cell, a macrophage, a dendritic cell, or a neutrophil. The method of claim 66 or 67, wherein the myeloid cell is macrophage. The method of 66, wherein the disease disorder or condition is characterized by macrophage infiltration in an affected tissue of the subject. The method of claim 68 or 69 wherein administering the pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide alters at least one gene expression signature associated with macrophage infiltration in the affected tissue of the subject. The method of any one of claims 66-70, wherein the myeloid cell is an M2 polarized macrophage. The method of any one of claims 66-71, wherein administering the pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide increases the expression of a M2 polarized macrophage specific biological marker in an affected tissue of the subject. The method of claim 72, wherein the M2 macrophage specific biological marker is CD163, and administering the pharmaceutical composition reduces the number of CD163+ cells in the ocular tissue.

- 94 - The method of any one of claims 66-73, wherein the disease disorder or condition is characterized by macrophage to myofibroblast transformation. The method of claim 74, wherein administering the pharmaceutical composition reduces at least one gene expression signature associated with macrophage to myofibroblast transformation. The method of any one of claims 66-73, wherein the disease disorder or condition is characterized by increased inflammatory gene expression. The method of any one of claims 66-73, wherein the disease disorder or condition is characterized by increased expression ofLy6C, Ibal, and/or Argl, and administering the pharmaceutical composition reduces the expression of one or more of Ly6C, Ibal , and/or Argl . The method of any one of claims 66-73, wherein the disease disorder or condition is characterized by increased expression of TGF-beta, TNF-alpha, IFN-gamma, IL-6, alpha- SMA, vimentin, fibronectin, ZEB- 1 or ZEB-2, Slug, Snail or Twist. The method of any one of claims 66-73, wherein the disease disorder or condition is characterized by increased expression of genes related to vasculogenesis or fibrogenesis. The method of any one of claims 66-73, wherein the disease, disorder or condition is characterized by increased VEGF, PDGF, peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), matrix metalloproteinases, a disintegrin and metalloprotease (ADAM), caspases, or components of the renin-angiotensin-aldo sterone system (ANG II). The method of claim 80, wherein administering the pharmaceutical composition comprising a therapeutically effective amount of a modifiedFGF-1 polypeptide reduces the gene expression of one or more of VEGF, PDGF, peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), matrix metalloproteinases, a disintegrin and metalloprotease (ADAM), caspases, or components of the renin-angiotensin- aldosterone system (ANG II). The method of any one of claims 66-73, wherein the disease, disorder or condition is characterized by altered gene expression of a cytokine or a chemokine gene. The method of claim 82, wherein the cytokine or chemokine is IL4, IL-13, IL-21, TGF- H, MCP-1, orMIP-1 ?. The method of claim 82 or 83, wherein administering the pharmaceutical composition reverses the alteration of gene expression of one or more of IL4, IL-13, IL-21, TGF- H, MCP- 1 and MIP- 1 fl induced by the disease, disorder or condition.

- 95 - The method of any one ofclaims 76-84, wherein the gene expression is detected by RT- PCR of a biological sample from the subject. The method of claim 85, wherein the biological sample is obtained from a tissue or cells from the subject that is affected by the disease, disorder or condition. The method of claim 86, wherein the biological sample is obtained from peripheral blood. The method of any one of claims 66-87, wherein, the disease, disorder or condition is associated with aberrant myeloid cell activation and is further characterized by activation of an epithelial or endothelial cell. The method of any one of claims 66-88, wherein the modified FGF-1 comprises a modified human FGF-1. The method of any one of claims 66-89, wherein the modified FGF-1 comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 2. The method of any one of claims 1-90, wherein the subject is human. A kit comprising (a) a pharmaceutical composition comprising a modified FGF-1 polypeptide, and (b) a container; wherein the modifiedFGF-1 polypeptide comprises a sequence comprisingthe mutations Cysl6Ser, Ala66Cys, and Cysl 17 Vai relative to the wild-type FGF-1 sequence of SEQ ID NO: 1, and wherein the disease, disorder, or a condition is notan ocular disease, disorder, or condition. The kit of claim 92, wherein the container is an eye dropper or a vial. The kit of claim 92 or 93, wherein the container is a glass container or a non-glass (e.g., resin) container. The kit of any one of claim 92-94, wherein the container comprises a polyolefin resin, a polyester resin, a polysulfone resin, a polyamide resin, a polycarbonate resin, a polyphenylene ether resin, a polyvinyl chloride resin, a styrene resin, or combinations thereof. The kit of any one of claim 92-95, wherein the container comprises a polyolefin resin. The kit of any one of claim 92-96, wherein the container consists essentially of a polyolefin resin. The kit of any one of claim 92-97, wherein the container is made of a polyolefin resin. The kit of any one of claim 96-98, wherein the polyolefin resin is a cyclic polyolefin resin. The kit of any one of claim 95-99 , wherein the container comprises a resin and an UV light absorbing agent or an UV light scattering agent.

- 96 - The kit of any one of claim 92-100, wherein the container is compatible with high and low pH conditions. The kit of any one of claim 92-100, wherein the container comprises a low protein and peptide adsorption. The kit of any one of claim 92-99, wherein the container is a Crystal Zenith vial or Crystal Zenith syringe. The kit of claim 103, wherein the container is a Crystal Zenith vial.