Compositions and methods for prevention and treatment of corneal haze and scarring

Abstract

To provide methods for treatment and prevention of corneal haze and scarring and ophthalmic formulations.SOLUTION: Methods for treatment and prevention of corneal haze and scarring include the step of administering to a subject a hepatocyte growth factor receptor (HGFR)-binding composition comprising at least one purified hepatocyte growth factor (HGF) agent.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 217,611, filed September 11, 2015, the contents of which are incorporated herein by reference in their entirety and for all purposes.

[0002] Field of Disclosure The present invention relates to compositions and methods for treating and preventing corneal opacification and scarring.

[0003] INCORPORATION BY REFERENCE OF SEQUENCE LISTING The contents of the text file named 36770-548001WO_ST25.TXT, created on September 7, 2016, and having a size of 6,610 bytes, are hereby incorporated by reference in their entirety.

[0004] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under Grant No. W81XWH-11-1-0477 awarded by the Department of Defense. The government has certain rights in this invention. [Background technology]

[0005] Background to the disclosure Corneal disease and injury are the second leading cause of non-refractive blindness, affecting over 10 million people worldwide. Several pathological conditions can lead to corneal scarring, including injury (e.g., chemical burns / occupational accidents); infection (e.g., contact lens-associated infection or ocular herpes); and laser-assisted vision correction (PRK). Ninety percent of blindness is permanent due to scarring and neovascularization. Scarring induced via a fibrotic cell response heals the tissue but fails to restore transparency.

[0006] There is a need to prevent or treat the serious effects of corneal opacification and scarring caused by disease or injury to prevent deterioration of vision, including blindness. Summary of the Invention

[0007] Disclosure Overview Prior to the compositions and methods described herein, treatments for corneal opacification and scarring were often associated with non-selective effects, adverse immunosuppression, and / or secondary infections. The invention described herein provides solutions to these and other problems in the field of corneal opacification and scarring. The invention described herein also relates to pharmaceutical formulations for use in the treatment and prevention of corneal opacification and scarring caused by disease or injury. The invention also provides methods for the treatment and prevention of corneal opacification or scarring in a subject in need of such treatment by administering the formulations of the invention directly to the subject's eye, e.g., onto the surface of the cornea, or to an area of ​​the eye, e.g., an area adjacent to the cornea (e.g., topically or subconjunctivally). The subject is preferably a mammal in need of such treatment, e.g., a subject diagnosed with, or predisposed to, corneal opacification or scarring. For example, the subject has suffered from one or more injuries (e.g., chemical burns / work-related accidents), infections (e.g., contact lens-related infections or ocular herpes), and / or has undergone or is expected to undergo laser keratinocyte replacement (PRK) surgery. The mammal can be any mammal, such as humans, primates, mice, rats, dogs, cats, horses, and livestock or animals raised for food consumption, such as cows, sheep, pigs, chickens, and goats. In a preferred embodiment, the mammal is a human.

[0008] Corneal opacity or scarring is the clouding or loss of transparency of the cornea. It can be a side effect of ocular disease, injury, or surgery, for example, as a result of an aggressive wound response. Corneal opacity or scarring describes the cloudy or opaque appearance of the cornea. The cornea is normally clear, so corneal opacity can significantly impair vision. Although opacity or scarring can occur in any part of the cornea, it is most commonly found in the thicker middle layer of the cornea, called the stroma. Corneal opacity or scarring is most commonly caused by inflammatory cells and other debris activated during trauma, infection, or surgery. Corneal opacity or scarring can be graded on a scale of 1 to 4, as shown in Table 1 below. Thus, in some embodiments, identifying a subject in need of treatment includes determining or calculating the subject's corneal clarity rating (e.g., slight, mild, moderate, or severe).

[0009] Table 1. Corneal opacity / scarring grading scale TIFF2025181979000002.tif74148

[0010] A method for treating corneal opacity or scarring involves identifying a subject who has experienced or is identified as having a predisposition to corneal opacity or scarring, and administering to ocular or adnexal tissues a composition containing an effective amount of a purified hepatocyte growth factor (HGF) agent or compound that binds to HGF or cMET and / or induces HGF- or cMET-mediated signaling. In this context, HGF agents can include HGF, or truncations, variants, mimetics, agonists, or analogs thereof. HGF agents can induce HGF-mediated signaling through the HGF receptor (HGFR or cMET) and can include, without limitation, natural proteins, recombinant proteins or peptides, and fusion or chimeric proteins that can bind to HGFR and biological or chemical small molecule agonists of HGFR. A small molecule is a compound with a mass of less than 2,000 daltons. The molecular weight of the small molecule is preferably less than 1000 daltons, more preferably less than 600 daltons, for example, the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons. HGF agents can be used alone or in combination with additional HGF agents or other therapies. However, HGF agents do not include other growth factors, including epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), and synthetic or naturally occurring mixtures thereof, including growth factor-rich plasma (PRGF).

[0011] The pharmaceutical formulations (e.g., HGF agents) of the present invention are formulated for ocular delivery, for example, corneal delivery or administration, so as to penetrate the corneal stroma. Keratocytes are physiologically and phenotypically different from epithelial cells, and they are located in the stroma, not in any other layer of the cornea, where opacity and scarring are found. For example, the pharmaceutical composition is formulated for subconjunctival administration. In embodiments, administration includes contacting the composition described herein with the corneal stroma or keratocytes in a subject in need thereof. Alternatively, the pharmaceutical composition is formulated for topical administration to the eye or eye area. For example, the formulation may include one or more tear substitutes. Alternatively, the formulation may include an ocular lubricant.

[0012] The pH of the formulation is 5.5 to 7.5 (e.g., about 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5). For example, the pH of the formulation is about 7.4. The formulation is in the form of a single-dose unit or a multi-dose system.

[0013] Suitable forms of the composition include solids, pastes, ointments, gels, liquids, aerosols, sprays, polymers, films, emulsions, or suspensions. In some cases, the composition is incorporated into or coated onto contact lenses. Preferably, the formulation is an aqueous formulation. The term "aqueous" typically refers to aqueous compositions in which the carrier is water to an extent of >50%, more preferably >75%, and especially >90% by weight.

[0014] Polynucleotides, polypeptides, or other agents are purified and / or isolated. Specifically, as used herein, an "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, or protein is substantially free of other cellular material, or culture medium if produced by recombinant techniques, or chemical precursors or other chemicals if chemically synthesized. Similarly, a cell population is substantially free of other cellular material or culture medium. A purified compound is at least 60% by weight (dry weight) of the compound of interest. Preferably, the preparation is at least 75% by weight, more preferably at least 90%, and most preferably at least 99% by weight of the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) of the desired compound. Purity is measured by any appropriate standard method, such as column chromatography, thin-layer chromatography, or high-performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally occurring state. A purified or isolated polypeptide is free of the amino acids or sequences that flank it in its naturally occurring state. Purified also defines a degree of sterility that makes it safe for administration to human subjects, e.g., lacking infectious or toxic agents.

[0015] In some cases, the method further includes administration of a second therapeutic agent, for example, a steroidal, other biologic, or small molecule-based anti-inflammatory therapy (e.g., cytokine targeting).

[0016] HGF agent is administered at a frequency that provides optimal effectiveness.For example, HGF agent is administered every 72 hours, every 48 hours, every 24 hours, every 12 hours, every 6 hours, every 3 hours, every hour, or any other suitable interval.HGF agent is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 30 days, 60 days, 90 days, or 120 days.Administration can be after injury or damage to cornea, or can be administered before surgery to prevent corneal opacity and scarring.Injury or damage to cornea can be the result of injury (for example, chemical burns / work accidents); infection (for example, contact lens-related infection or ocular herpes); and laser correction of vision (PRK). Alternatively, the HGF agent is administered for long-term use, i.e., for a period of more than 120 days, more than 150 days, more than 180 days, more than 210 days, more than 240 days, more than 270 days, more than 300 days, more than 330 days, or more than 360 days.

[0017] Also provided is a method for preventing corneal opacity or scarring, comprising identifying a subject at risk of developing corneal opacity or scarring, and administering to ocular or adnexal tissue a composition comprising an effective amount of an HGF agent.In some cases, the subject is asymptomatic, but at high risk of developing corneal opacity or scarring.

[0018] Optionally, the method further comprises administering a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" refers to compositions, polymers, and other materials and / or dosage forms that are recognized in the art and are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. The method may further comprise administering an "ophthalmologically acceptable" carrier.

[0019] The phrase "pharmaceutically acceptable carrier" is art-recognized and refers to a pharmaceutically acceptable material, composition, or vehicle, such as, for example, a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting any additive or composition, or components thereof, from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the additive and not harmful to the patient. Optionally, a pharmaceutically acceptable carrier is non-pyrogenic. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol. (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer; (21) liposome-based excipients; (22) cyclodextrins; (23) nanoparticle-based excipients; and (24) other non-toxic compatible materials employed in pharmaceutical formulations.

[0020] The excipient for topical administration of the compositions of the present invention preferentially promotes drug penetration through the ocular surface and corneal epithelium and into the corneal stroma. Exemplary excipients suitable for preferential drug delivery to the corneal stroma, for example, at least 10%, 25%, 50%, 75%, 2-fold, 5-fold, 10-fold, or more, compared to other anatomical locations in the eye, include polysaccharides and their derivatives (e.g., chitosan, n-carboxymethylchitosan, chitosan HCL, N-trimethylchitosan, xyloglucan, hyaluronic acid, alginic acid, gellan gum, cyclodextrin, etc.); nanoparticles (e.g., nanoparticles conjugated, for example, covalently linked, to the drug of the present invention); liposomes, for example, those having compounds / therapeutic agents bound thereto or linked thereto; surfactants; benzalkonium chloride; and EDTA. Liposomes are spherical containers having at least one lipid bilayer. Nanoparticles are microscopic particles having at least one dimension less than 100 nm. These excipients can be used as single excipients or in combination.

[0021] The phrase "ophthalmically acceptable" refers to a composition containing an excipient, emulsifier, wetting agent, carrier, or filler suitable for application to the eye and eye-area tissues. Such ophthalmically acceptable compositions may contain, for example, polyethylene glycols designated 200, 300, 400, and 600, or carbowaxes designated 1000, 1500, 4000, 6000, and 10000; complexing agents such as EDTA-disodium or EDTA; antioxidants such as ascorbic acid, acetylcysteine, cysteine, sodium bisulfite, butyl-hydroxyanisole, butyl-hydroxytoluene; stabilizers such as thiourea, thiosorbitol, dioctyl sodium sulfosuccinate, or monothioglycerol; or other excipients such as sorbitol laurate, triethanolamine oleate, or palmitate.

[0022] Other carriers and excipients, for example, Kreuter, J., "Nanoparticles," Colloidal Drug Delivery Systems, edited by Jork Kreuter and Marcel Dekker, New York, NY (USA), Chapter 5, p. 219 (1994); Gurny, R., "Ocular therapy with nanoparticles," Polymeric Nanoparticles and Microspheres, edited by P. Guiot and P. Couvreur, Boca Raton, Fla. (USA): CRC Press, p. 127 (1986); Gurny, R., "Preliminary study of a prolonged-acting drug delivery system for the treatment of glaucoma," Pharm Acta Helv., Vol. 56, p. 130 (1981); Zimmer, et al., "Microspheres and nanoparticles used in ocular delivery systems," Advanced Drug Delivery Reviews, Vol. 16, No. 1, pp. 61-73 (1995); Zambito, et al., "Polysaccharides as excipients for Ocular Topical Formulations," Biomaterials Applications for Nanomedicine, Prof. Rosario Pignatello (ed.), ISBN: 978-953-307-661-4, InTech, available at http: / / www.intechopen.com / books / biomaterials-applications-for-nanomedicine / polysaccharidesas-excipients-for-ocular-topical-formulations, pp. 253-284; Kompella, et al."Recent Advances in Ophthalmic Drug Delivery," Ther Deliv, 2010 September 1, 1(3): 435-456; McCann, J., "Advances and Challenges in Topical Ocular Medications," Advanced Ocular Care, March 2011, pp. 23-25; and Calvo, et al., "Comparative in vitro evaluation of several colloidal systems, nanoparticles, nanocapsules, and nanoemulsions, as ocular drug carriers," J Pharm. Sci, Vol. 85, No. 5, pp. 530-536 (May 1996), are known in the art. These references are incorporated herein by reference in their entirety.

[0023] As used herein, the term "tear substitute" refers to a molecule or composition that, when administered to the eye, lubricates, "moists," approximates the consistency of endogenous tears, helps increase natural tear production, or provides temporary relief of dry eye symptoms and conditions.

[0024] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids. The terms also apply to naturally occurring and non-naturally occurring amino acid polymers.

[0025] Similarly, by "substantially pure" is meant a nucleotide or polypeptide that has been separated from components that naturally accompany it. Typically, nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99-100% by weight free from the proteins and naturally-occurring organic molecules with which they are naturally associated.

[0026] "Conservatively modified variations" of a particular polynucleotide sequence refer to polynucleotides that encode identical or essentially identical amino acid sequences, or essentially identical sequences if the polynucleotide does not encode an amino acid sequence. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode any given polypeptide. For example, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, wherever arginine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent substitutions" or "silent variations," which are a type of "conservatively modified variations." Any polynucleotide sequence described herein that encodes a polypeptide also describes any possible silent variations, unless otherwise noted. Thus, silent substitutions are an implicit feature of any nucleic acid sequence that encodes an amino acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is normally the only codon for methionine) can be altered by standard techniques to produce a functionally identical molecule.

[0027] Similarly, "conservative amino acid substitutions" of one or several amino acids in an amino acid sequence are substituted with different amino acids having very similar properties and are easily identified as being very similar to a specific amino acid sequence or a specific nucleic acid sequence encoding the amino acids. Such conservatively substituted variations of any specific sequence are a feature of the present invention. Individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in the encoded sequence are "conservatively modified variations" if the change results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. See, for example, Creighton (1984) Proteins, W.H. Freeman and Company, incorporated herein by reference. Table 2 below provides exemplary amino acid substitutions.

[0028] Table 2: Conservative amino acid substitutions TIFF2025181979000003.tif108148

[0029] By "isolated nucleic acid" is meant a nucleic acid that is free of the genes that flank it in the naturally occurring genome of the organism from which it is derived. The term encompasses, for example: (a) DNA that is part of a naturally occurring genomic DNA molecule but is not flanked by both of the nucleic acid sequences that flank that portion of the molecule in the genome of the naturally occurring organism; (b) a nucleic acid that has been incorporated into a vector or into the genomic DNA of a prokaryotic or eukaryotic organism in such a manner that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) an isolated molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene (i.e., a gene encoding a fusion protein). Isolated nucleic acid molecules according to the present invention further include synthetically produced molecules and any nucleic acid that has been chemically modified and / or has a modified backbone. For example, an isolated nucleic acid is a purified cDNA or RNA polynucleotide.

[0030] The term "percent sequence identity" or "percent sequence identity" refers to the sequence overlap in amino acid or nucleic acid sequences. As used herein, the term "identity" or "percent identity" refers to the subunit sequence similarity between two polymer molecules, such as two polynucleotides or two polypeptides. If a subunit position in both molecules is occupied by the same monomer subunit, for example, if a position in each of two peptides is occupied by serine, they are identical at that position. The identity between two sequences is a direct function of the number of matching or identical positions; for example, if half of the positions in two peptide or compound sequences (for example, 5 positions in a polymer of 10 subunits in length) are identical, the two sequences are 50% identical; if 90% of the positions, for example, 9 out of 10, are identical, the two sequences share 90% sequence identity. The identity between two sequences is a direct function of the number of matching or identical positions. Therefore, if a part of a reference sequence is missing in a particular peptide, the missing part is not counted for the purpose of calculating sequence identity. Identity is often measured using sequence analysis software, such as BLASTN or BLASTP (available at the World Wide Web ("www") of the National Center for Biotechnology Information ("ncbi") of the National Institutes of Health ("nih") of the U.S. government (".gov") in the "Blast" directory (" / BLAST / "). The default parameters for comparing two sequences (e.g., "Blast" two sequences against each other) by BLASTN (for nucleotide sequences) are given as follows: match=1, penalty for mismatch=-2, open gap=5, and extension gap=2. When using BLASTP for protein sequences, the default parameters are given as follows: match=0, penalty for mismatch=0, open gap=11, and extension gap=1. In addition, computer programs for determining identity are known in the art.

[0031] "Similarity" or "percent similarity" in the context of two or more polypeptide sequences refers to two or more sequences or subsequences that are the same, or two or more sequences or subsequences that have a specified percentage of amino acid residues or conservative substitutions thereof that are the same when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0032] HGF agents of the present invention may include polypeptides having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to human HGF (SEQ ID NO:1).

[0033] The phrase "nucleic acid molecule" primarily refers to the physical nucleic acid, and the phrase "nucleic acid sequence" refers to the linear listing of nucleotides in a nucleic acid molecule, although the two terms can be used interchangeably.

[0034] The terms "effective amount" and "therapeutically effective amount" of a formulation or formulation component refer to a sufficient amount of the formulation or component, alone or in combination, to provide the desired effect. For example, an "effective amount" refers to the amount of a compound, alone or in combination, required to reduce or prevent corneal opacity or scarring in a mammal. In some cases, an effective amount is an amount sufficient to inhibit corneal fibroblast differentiation by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more upon treatment with a composition described herein compared to levels without treatment with a composition described herein. In some cases, an effective amount is an amount sufficient to inhibit α-smooth muscle actin (αSMA) expression by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more upon treatment with a composition described herein compared to levels without treatment with a composition described herein. In some cases, an effective amount is an amount sufficient to increase corneal epithelial cell stratification by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more upon treatment with a composition described herein, compared to levels without treatment with a composition described herein. In some cases, an effective amount is an amount sufficient to increase c-met expression by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more upon treatment with a composition described herein, compared to levels without treatment with a composition described herein. In some cases, an effective amount is an amount sufficient to restore damaged corneal thickness to about 50%, 60%, 70%, 80%, 90%, 95%, or more of the thickness of a normal (i.e., healthy) cornea. Ultimately, the appropriate amount and dosage regimen will be determined by the attending physician or veterinarian.

[0035] As used herein, the terms "treat" and "treatment" refer to the administration of an agent or formulation to a clinically symptomatic individual suffering from a deleterious condition, disorder, or disease, such as corneal opacification or scarring, to result in a reduction in the severity and / or frequency of symptoms, to eliminate symptoms and / or their underlying causes, and / or to promote amelioration or repair of damage.

[0036] The terms "prevent" and "prevention" refer to the administration of an agent or composition to a clinically asymptomatic individual who is susceptible to or predisposed to a particular adverse condition, disorder, or disease, and thus relates to the prevention of the occurrence of the condition and / or its underlying cause, e.g., those characterized as PRK patients, chemical burn victims, or ocular injury victims.

[0037] The transitional phrase "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps, and those that do not "materially affect the basic and novel characteristics" of the claimed invention.

[0038] Human hepatocyte growth factor (GenBank accession number: P14210.2) has the following amino acid sequence (SEQ ID NO: 1): TIFF2025181979000004.tif59128

[0039] The HGF agent may include full-length HGF (SEQ ID NO: 1). The HGF agent may also include a truncated HGF or a specific domain of the full-length peptide, for example, a fragment of the full-length or parent protein, for example, HGF. As used herein, the term "fragment" refers to a portion of a polypeptide or polynucleotide that is less than the entire polypeptide or polynucleotide. As used herein, a "functional fragment" of a reference protein, for example, HGF, is a fragment of a polypeptide that is shorter than the full-length, immature, or mature polypeptide and has at least 25% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 100% or more) of the activity of the full-length mature reference protein. The activity may be, for example, the activity of reducing corneal opacity or scarring. Methods for verifying whether an HGF fragment is functional / active are known in the art, for example, as determined by the criteria set forth in Table 1. For example, fragments of interest can be produced either by recombinant, synthetic, or proteolytic digestion methods, and then isolated and tested for their ability to costimulate T cells by the procedures described herein.

[0040] For example, the mature protein includes amino acids 32-494 (underlined). Other examples include amino acids 126-207, 208-289, 302-384, and 388-470, each of which contains a kringle domain, each of which may be individually involved in binding to a mediator.

[0041] Thus, an HGF agent described herein may comprise a polypeptide having the amino acid sequence of SEQ ID NO:1. In embodiments, an HGF agent described herein may comprise a polypeptide having the amino acid sequence of residues 32-494 of SEQ ID NO:1. In embodiments, an HGF agent described herein may comprise a polypeptide having the amino acid sequence of residues 126-207 of SEQ ID NO:1. In embodiments, an HGF agent described herein may comprise a polypeptide having the amino acid sequence of residues 208-289 of SEQ ID NO:1. In embodiments, an HGF agent described herein may comprise a polypeptide having the amino acid sequence of residues 302-384 of SEQ ID NO:1. In embodiments, an HGF agent described herein may comprise a polypeptide having the amino acid sequence of residues 388-470 of SEQ ID NO:1.

[0042] The HGF agents described herein can include fragments of SEQ ID NO: 1. Fragments can be 3-10 amino acids, 10-20 amino acids, 20-40 amino acids, 40-56 amino acids in length, or even longer. Amino acid sequences having at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% identity, and most preferably 95% identity to the fragments described herein are also included within the scope of the invention described herein.

[0043] [The present invention 1001] identifying a subject in need of treatment or prevention of corneal opacification or scarring; administering to the subject a hepatocyte growth factor receptor (HGFR) binding composition comprising at least one purified hepatocyte growth factor (HGF) agent. 10. A method for treating or preventing corneal opacification or scarring, comprising: [The present invention 1002] 1001. The method of claim 1001, wherein the HGF agent comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof. [The present invention 1003] 1002. The method of claim 1002, wherein the HGF agent comprises a polypeptide comprising an amino acid sequence having 70% sequence identity to SEQ ID NO:1. [The present invention 1004] 1001. The method of claim 1001, wherein the HGF agent comprises an agonist of HGF. [The present invention 1005] 1001. The method of claim 1001, wherein the composition comprising at least one purified HGF agent further comprises a corneal stroma-penetrating excipient. [The present invention 1006] 1001. The method of claim 1001, wherein said composition acts to inhibit alpha-smooth muscle actin (alpha-SMA) in the cornea. [The present invention 1007] The method of claim 1001, wherein the purified HGF agent is present at a concentration of 0.001% to 1% w / v. [The present invention 1008] The method of claim 1007, wherein the purified HGF agent is present at a concentration of 0.005% to 0.05% w / v. [The present invention 1009] The method of claim 1008, wherein the purified HGF agent is present at a concentration of about 0.01% w / v. [The present invention 1010] 1001. The method of claim 1001, wherein said composition is administered daily for 5 days after injury to the cornea. [The present invention 1011] 1001. The method of claim 1001, wherein said composition is administered daily for three days after injury to the cornea. [The present invention 1012] 1001. The method of claim 1001, wherein said composition is administered daily for one day after injury to the cornea. [The present invention 1013] A composition for the treatment of corneal opacity or scarring comprising at least one HGF agent. [The present invention 1014] The composition of claim 1013, wherein at least one HGF agent comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof. [The present invention 1015] The composition of claim 1013, wherein the at least one HGF agent comprises a polypeptide comprising an amino acid sequence having 70% sequence identity to SEQ ID NO:1. [The present invention 1016] The composition of claim 1013, wherein the at least one HGF agent comprises an agonist of HGF. [The present invention 1017] The composition of claim 1013, further comprising a corneal stroma-penetrating excipient. [The present invention 1018] The composition of the present invention 1013, wherein at least one HGF agent is present at a concentration of 0.001% to 1% w / v. [The present invention 1019] The composition of the present invention 1018, wherein the at least one HGF agent is present at a concentration of 0.005% to 0.05% w / v. [The present invention 1020] The composition of the present invention 1019, wherein the at least one HGF agent is present at a concentration of about 0.01% w / v. [The present invention 1021] The composition of claim 1013, wherein the at least one HGF agent is present in a concentration effective to restore corneal thickness to normal corneal thickness. [The present invention 1022] The composition of claim 1013, wherein at least one HGF agent is present in a concentration effective to inhibit the trafficking of inflammatory leukocytes to the injured cornea. [The present invention 1023] The composition of claim 1013, wherein the at least one HGF agent is present in a concentration effective to inhibit α-SMA expression in the cornea. [The present invention 1024] 1001. The method of claim 1001, wherein the identifying step comprises calculating a rating scale of the subject's corneal transparency. [The present invention 1025] 1001. The method of claim 1001, wherein the administering step comprises topical administration or subconjunctival administration. [The present invention 1026] 1001. The method of claim 1001, wherein the administering step comprises contacting the composition with the corneal stroma of the subject. [The present invention 1027] The method of claim 1001, wherein said composition is formulated as an eye drop. [The present invention 1028] The method of claim 1001, wherein the purified HGF agent comprises a polypeptide comprising the amino acid sequence of residues 32 to 494 of SEQ ID NO:1. [The present invention 1029] The method of claim 1001, wherein the purified HGF agent comprises a polypeptide comprising the amino acid sequence of residues 126-207 of SEQ ID NO:1. [The present invention 1030] The method of claim 1001, wherein the purified HGF agent comprises a polypeptide comprising the amino acid sequence of residues 208-289 of SEQ ID NO:1. [The present invention 1031] The method of claim 1001, wherein the purified HGF agent comprises a polypeptide comprising the amino acid sequence of residues 302-384 of SEQ ID NO:1. [The present invention 1032] 1001. The method of claim 1001, wherein the purified HGF agent comprises a polypeptide comprising the amino acid sequence of residues 388-470 of SEQ ID NO:1. Other features and advantages of the present invention will be apparent from the following description of the preferred embodiments thereof and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in practicing or testing the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated by reference. Genbank and NCBI deposits identified by accession numbers cited herein are incorporated by reference. All other published references, documents, manuscripts, and scientific papers cited herein are incorporated by reference. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are merely illustrative and are not intended to be limiting. [Brief explanation of the drawings]

[0044] [Figure 1] FIG. 1 is a diagram of the corneal anatomy, revealing the layers of the cornea. [Figure 2A] Representative slit lamp (bright field and fluorescein) biomicroscopy images of injured corneas are shown. Images of corneal injury are shown at 1, 3, 5, and 7 days after injury. Photographs of injured corneas were taken with slit lamp biomicroscopy, with or without fluorescein (green) staining. [Figure 2B] Fig. 1 shows a bar graph presenting quantification of corneal opacity (clouding / scarring) using Image J software, demonstrating a significant reduction in the progression of corneal opacity in HGF-treated mice compared to albumin-treated controls. Quantification of corneal damage at 1, 3, 5, and 7 days post-injury is shown. [Figure 2C]This is a bar graph showing quantification of fluorescein-stained area using Image J software, demonstrating a significant reduction in fluorescein staining (i.e., damaged area) in HGF-treated mice compared to the control group. (N=5-6 mice / group) Quantification of corneal damage at 1, 3, 5, and 7 days after injury is shown. [Figure 3] Figure 3A is a bar graph demonstrating that HGF inhibits the expression of α-smooth muscle actin (αSMA, a factor that causes scarring) by corneal keratocytes, and Figure 3B is a series of photographs. In vitro analysis of mouse corneal keratocytes (MK / T1) demonstrates that HGF significantly inhibits TGFp-induced expression of αSMA in keratocytes, as measured by real-time PCR (Figure 3A) and immunohistochemistry (Figure 3B). [Figure 4] Confocal microscopy images showing immunostaining for the pan-leukocyte marker CD45 in corneas excised 3 days after injury. Corneal injury was induced in C57BL6 mice by mechanical removal of the complete corneal epithelium using ALGBERRUSH-II™. Mouse recombinant HGF was then topically applied to the injured eye twice daily (dose: 3 μl of 0.01% HGF in PBS per eye). A control group received a similar dose of mouse serum albumin. Blue staining indicates DAPI staining of cell nuclei; green staining indicates the presence of CD45, a pan-leukocyte marker. [Figure 5A] Representative photomicrograph images showing the histology of normal, injured control, and HGF-treated corneas. [Figure 5B] 1 is a bar graph of cumulative data showing that HGF-treated corneas recover their thickness to a similar level as normal corneas. Injured control corneas show a significant increase in thickness compared to normal and HGF-treated corneas. (N=5 mice / group). [Figure 6]This is a bar graph showing that HGF inhibits the differentiation of human corneal fibroblasts into myofibroblasts. Human corneal fibroblasts were stimulated with human recombinant TGFβ1 (100 ng / ml, Peprotech) for 24 hours in the presence or absence of rhHGF (10 ng / ml, R&D Systems). αSMA expression (normalized to the internal control, GAPDH) was assessed using real-time PCR. Values ​​shown are the mean ± SD from three independent experiments performed in triplicate (error bars); *p<0.02, **p<0.001). [Figure 7A] (A series of images shows that HGF increases epithelial cell stratification after corneal injury.) Corneas were excised from normal, albumin-treated, and HGF-treated mice 7 days after injury. Corneal cross sections were stained with the nuclear stain DAPI to visualize the corneal epithelial cell layer using a confocal microscope (400x). [Figure 7B] 1 is a bar graph showing that HGF increases epithelial cell stratification after corneal injury. Bar chart showing the thickness (μm) of the epithelial cell layer in normal corneas (white bars), injured control corneas, and HGF-treated injured corneas (black bars). Values ​​shown are mean ± SD (error bars); n = 5 mice / group. [Figure 8] This is a bar graph showing that HGF promotes HGF-R (c-met) expression in the injured cornea. Corneas were excised from the normal group (checkered bars), the mouse albumin-treated injured control group (white bars), and the HGF-treated injured group (black bars) on days 3 and 7 after injury. Total RNA was isolated from the excised corneas. HGF-R mRNA expression was quantified using real-time PCR. GAPDH was used as an internal control. Values ​​shown are mean ± SD, and each group consisted of n = 6 mice. *p < 0.03, **p < 0.01. DETAILED DESCRIPTION OF THE INVENTION

[0045] Detailed Description of the Invention The present invention provides compositions, methods, and treatments for corneal opacification and scarring. The methods for treating and preventing corneal opacification and scarring in humans involve the therapeutic administration of HGF or its fragments or agonists onto or into the cornea, or in combination with a pharmaceutically suitable vehicle or another therapeutic agent. HGF agents include HGF or agents capable of inducing HGF-mediated signal transduction through the HGF receptor (HGFR or cMET), and may include, without limitation, natural proteins, recombinant proteins, or peptides, and fusion or chimeric proteins capable of binding to HGFR and biological or chemical small molecule agonists of HGFR.

[0046] Corneal Anatomy The cornea is composed of multiple layers of varying thickness, cellular composition, and function. Corneal layers include the epithelium, Bowman's membrane or layer, stroma, Descemet's membrane or layer, and endothelium. Each of the layers is illustrated in Figure 1.

[0047] The epithelium is the layer of cells that covers the surface of the cornea. It is only about 5 to 6 cell layers thick and regenerates quickly if the cornea is injured. If the injury penetrates deeper into the cornea, it can leave a scar. Scars leave opaque areas, causing the cornea to lose its clarity and brightness.

[0048] Bowman's membrane lies just below the epithelium. This layer is very tough and impermeable, protecting the cornea from damage.

[0049] The stroma is the thickest layer and lies just below Bowman's membrane. The stroma is composed of fine collagen fibers that run parallel to each other. This special configuration of collagen fibers gives the cornea its clarity. HGF agents act in the corneal stroma to prevent and treat corneal opacity or scarring. HGF agents inhibit the expression of α-smooth muscle actin in keratocytes of the corneal stroma, suppressing keratocyte function and the migration of inflammatory cells into the corneal stroma, preventing the development of corneal opacity and scarring. These keratocytes are not present in other corneal layers.

[0050] Corneal opacification is not an epithelial or endothelial cell event. Corneal opacification and scarring are stromal pathologies that primarily result from dysfunction of stromal components, including excessive expression of actin and collagen fibers by keratocytes, infiltration of inflammatory cells, and differentiation of keratocytes into myofibroblasts. These cellular processes are distinct from corneal epithelial cell proliferation.

[0051] Descemet's membrane lies between the stroma and the endothelium.

[0052] The endothelium is just below Descemet's and is only one cell layer thick. This layer draws moisture from the cornea, keeping it clear. In the event of damage or disease, these cells are not thought to regenerate.

[0053] Previous methodologies have utilized steroid therapy for the treatment of corneal opacity and scarring. Table 3 below details the improvements of the HGF therapy described herein compared to steroid therapy.

[0054] (Table 3) TIFF2025181979000005.tif57152

[0055] The mechanism of action of HGF is cell-specific in that it targets HGF-R-expressing cells.Steroids have a broad spectrum and non-selective action, which often leads to non-specific immunosuppression and secondary infection.As shown in Table 3, the risk of secondary infection is reduced using the compositions and methods described herein, and cell proliferation is increased compared to treatment with steroids, leading to reduced side effects and an increased speed and degree of healing and prevention.

[0056] How to use Provided herein is a method for treating or preventing corneal opacity or scarring by identifying a subject in need thereof and administering to the subject an HGFR-binding composition comprising at least one purified HGF agent.In an embodiment, the administering step comprises contacting the composition with the subject's corneal stroma.In an embodiment, the identifying step comprises calculating the subject's corneal transparency rating scale as described in Table 1.

[0057] Topical ophthalmic preparations are useful for treating corneal opacity or scarring.Therefore, the present invention also provides a method for treating corneal opacity or scarring in a subject who needs such treatment by directly administering the composition described herein (for example, the ophthalmic preparation of the present invention) to the eye or eye area of ​​the subject.For example, the administering step can include contacting the ophthalmic preparation described herein with corneal stroma or keratocytes.

[0058] The pharmaceutical preparations containing the HGF agent fragments of the present invention or its agonists can be used to treat corneal opacity or scarring.For example, the pharmaceutical composition is formulated for local administration to the eye (e.g., subconjunctival administration; eye drops).Optionally, the pharmaceutical composition can further comprise a tear substitute.Suitable tear substitutes can include glycerin, propylene glycol, HPMC (hydroxypropylmethylcellulose, hypromellose), dextran 70, mineral oil, petrolatum, Carbopol 980, povidone, CMC (sodium carboxymethylcellulose), PVA (polyvinyl alcohol), or other active and inactive ingredients.

[0059] Also provided are methods for treating or preventing corneal opacification or scarring in a subject in need thereof, comprising administering to the ocular surface of the subject a pharmaceutical composition comprising an effective amount of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.) HGF agent. Optionally, administration of an HGF agent to the eye of a subject in need of treatment or prevention of corneal opacification or scarring is also effective in alleviating or reducing one or more symptoms associated with a disease or condition on the corneal surface. An effective amount is an amount that reduces the opacification / scarring score by at least 1 unit, e.g., a unit shown in Table 1. For example, an effective amount reduces the score from "+3" to "+2." The subject is preferably a human, but may be another mammal, such as a dog, cat, rabbit, mouse, rat, or non-human primate.

[0060] The formulation may contain an effective amount of HGF agent and, optionally, one or more additional active ingredients that are effective for intended use.Specific dosages are also selected based on several factors, including the age, sex, species, and medical condition of the subject.Effective amounts can also be estimated from dose-response curves derived from in vitro test systems or animal models.The term "effective amount" refers to the amount of HGF agent that is sufficient to prevent, eliminate, or reduce corneal opacity or scarring.

[0061] An effective amount is an amount sufficient to treat or prevent corneal opacification or scarring. In this context, "treatment" refers to reducing or improving at least one symptom resulting from corneal opacification or scarring. In this context, "prevention" refers to reducing the frequency or delaying the onset of symptoms associated with a disease or condition compared to a subject not administered the composition. In some cases, the methods described herein inhibit corneal fibroblast differentiation by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more when administered with a composition described herein, compared to the level without administration of the composition described herein. In some cases, the methods described herein inhibit alpha-smooth muscle actin (αSMA) expression by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, when administered with a composition described herein, compared to levels without administration of a composition described herein. In some cases, the methods described herein increase corneal epithelial cell stratification by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, when administered with a composition described herein, compared to levels without administration of a composition described herein. In some cases, the methods described herein increase c-met expression by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, when administered with a composition described herein, compared to levels without administration of a composition described herein. In some cases, the methods described herein restore the thickness of the damaged cornea to about 50%, 60%, 70%, 80%, 90%, 95%, or a higher percentage of that of a normal (i.e., healthy) cornea upon administration of the compositions described herein.In some cases, the methods described herein inhibit the trafficking of inflammatory leukocytes to the damaged cornea by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more when administering a composition described herein compared to the level without administration of a composition described herein.

[0062] The present invention features a method for treating corneal opacity or scarring in a subject, comprising using the above-described preparation.For example, the method for treating corneal opacity or scarring can comprise administering to the eye surface of a subject a pharmaceutical composition comprising an effective amount of at least one HGF agent and tear substitute in a pharmaceutically acceptable carrier.

[0063] Ophthalmic preparations HGF agent can be formulated in combination with suitable pharmaceutical carrier.Such formulation comprises a therapeutically effective amount of HGF agent and a pharmaceutically acceptable carrier (excipient).Such carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.Formulation should be suitable for the mode of administration, which is well within the skill of the art.

[0064] For example, the pharmaceutical composition of the present invention can comprise at least one (for example, 1, 2, 3, 4, 5, 6, etc.) combination of HGF agents.In some embodiments, the pharmaceutical composition is formulated for subconjunctival administration.For example, the pharmaceutical composition is formulated for topical administration to the eye (for example, subconjunctival administration; eye drops).The pharmaceutical composition can further comprise tear substitute.

[0065] The concentration of the HGF agent is about 0.001% to about 10.0% (w / v), e.g., about 0.001% to about 5%, about 0.001% to about 2.5%, about 0.001% to about 1%, about 0.001% to about 0.5%, about 0.005% to about 0.5%, about 0.005% to about 0.05%, or about 0.01%. For example, the concentration of the HGF agent is effective for restoring corneal thickness to normal corneal thickness and / or inhibiting the transport of inflammatory leukocytes to the damaged cornea.

[0066] Preferably, pharmaceutical compositions according to the present invention are formulated as solutions, suspensions, and other dosage forms for topical administration. Aqueous solutions are generally preferred due to ease of formulation and the ease with which patients can administer such compositions by instilling one or two drops of the solution into the affected eye. However, the compositions may also be suspensions, viscous or semi-viscous gels, or other types of solid or semi-solid compositions.

[0067] Any of a variety of carriers can be used in the formulations of the present invention, including water, mixtures of water with water-miscible solvents such as C1-C7 alkanols, vegetable or mineral oils containing 0.5-5% non-toxic water-soluble polymers, natural products such as gelatin, alginate, pectin, tragacanth, karaya gum, xanthan gum, carrageenan, agar, and acacia, starch derivatives such as starch acetate and hydroxypropyl starch, and other synthetic products such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, preferably cross-linked polyacrylic acid such as neutral carbopol, or mixtures of these polymers. The concentration of the carrier is typically 1-100,000 times that of the active ingredient. Additional ingredients that may be included in the formulation include tonicity-enhancing agents, preservatives, stabilizers, non-toxic excipients, demulcents, sequestering agents, pH adjusters, cosolvents, and viscosity-increasing agents.

[0068] Buffers can be particularly useful for adjusting the pH, preferably to a physiological pH. The pH of the solution can be about 4.0 to 8.0 (e.g., about 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8), more preferably ... The pH should be maintained within the range of about 4.0 to 6.0 (e.g., about 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6), more preferably about 6.5 to 7.8 (e.g., about 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8). Suitable buffers may be added, such as boric acid, sodium borate, potassium citrate, citric acid, sodium bicarbonate, TRIS, and various mixed phosphate buffers (including combinations of NaHPO, NaHPO, and KHPO), as well as mixtures thereof. Borate buffers are preferred. Generally, buffers are used in amounts ranging from about 0.05 to 10 percent by weight.

[0069] Tonicity is typically adjusted, if necessary, with a tonicity enhancer. Such agents can be, for example, ionic and / or nonionic. Examples of ionic tonicity enhancers include alkali metal or earth metal halides, such as CaCl2, KBr, KCl, LiCl, NaCl, NaBr, or NaCl, Na2SO4, or boric acid. Nonionic tonicity enhancers include, for example, urea, glycerol, sorbitol, mannitol, propylene glycol, or dextrose. Aqueous solutions of the present invention are typically adjusted with a tonicity enhancer to approximate the osmolality of normal tears, which is equivalent to a 0.9%±0.1% sodium chloride solution or a 2.5%±0.3% glycerol solution. An osmolality of approximately 225-400 mOsm / kg is preferred, with a range of 280-320 mOsm being more preferred.

[0070] The at least one HGF agent may be administered by use of or in the form of a hydrogel, a drug-eluting contact lens, and a nanosystem (liposome system, dendrimers, solid biodegradable nanoparticles, nanogels), and / or an irrigation solution.

[0071] Ophthalmic formulations, eye ointments, creams, salves, powders, solutions and the like, are also contemplated as being within the scope of the present invention.

[0072] eye drops Eye drops can be formulated with or without one or more tear substitutes.Also provided is a pharmaceutical composition comprising an effective amount of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.) HGF agents and tear substitutes in a pharmaceutically acceptable carrier for the treatment of corneal opacity or scarring.The HGF agent and tear substitute can act synergistically to provide the HGF agent with a longer residence time on the cornea, thereby increasing the duration and efficacy of action.

[0073] A variety of tear substitutes are known in the art, including, but not limited to, monomeric polyols such as glycerol, propylene glycol, and ethylene glycol; polymeric polyols such as polyethylene glycol; cellulose esters such as hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and hydroxypropylcellulose; dextrans such as dextran 70; water-soluble proteins such as gelatin; vinyl polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and povidone; and carbomers such as carbomer 934P, carbomer 941, carbomer 940, and carbomer 974P. Many such tear substitutes are commercially available, including, but not limited to, cellulose esters such as Bion Tears®, Celluvisc®, Genteal®, OccuCoat®, Refresh®, Teargen II®, Tears Naturale®, Tears Natural II®, Tears Naturale Free®, and TheraTears®; and polyvinyl alcohols such as Akwa Tears®, HypoTears®, Moisture Eyes®, Murine Lubricating®, and Visine Tears®. Tear substitutes can also be composed of paraffin, such as the commercially available Lacri-Lube® ointment. Other commercially available ointments used as tear substitutes include Lubrifresh PM®, Moisture Eyes PM®, and Refresh PM®.

[0074] In one aspect, the tear substitute contains hydroxypropyl methylcellulose. The tear substitute is GenTeal® lubricating eye drops. GenTeal® (CibaVision--Novartis) is a sterile lubricating eye drop containing 3 mg / g hydroxypropyl methylcellulose and preserved with sodium perborate.

[0075] Pharmaceutical compositions of the present invention may include a combination of one or more HGF agents and one or more tear substitutes.

[0076] Therapeutic Administration The effective amount of active agent in formulation depends on the absorption rate, inactivation rate and excretion rate of drug and the delivery rate of compound from formulation.It should be noted that dosage value can also vary according to the severity of the condition that is to be alleviated.It should further be understood that for any specific subject, specific dosage regimen should be adjusted over time according to individual need and the professional judgment of the person who manages or supervises the administration of composition.Typically, dosage is determined by the technique known to those skilled in the art.

[0077] The dosage of any compound of the present invention varies according to the condition, age and other physical characteristics of patient, the nature and severity of the disorder to be treated or prevented, the desired comfort level, route of administration and the form of additive.Any of the subject preparations can be administered in single dose or divided doses.The dosage of the preparation of the present invention can be easily determined by the technique known to those skilled in the art or as taught herein.

[0078] Effective dose or amount and any possible effect of the timing of administration of preparation may need to be identified for any specific preparation of the present invention.This can be achieved by routine experiment as described herein.The effectiveness of any preparation and treatment or prevention method can be assessed by administering preparation and measuring one or more indicators related to the efficacy of active agent and the degree of patient comfort as described herein, and comparing the post-treatment value of these indicators with the value of the same indicator before treatment, or comparing the post-treatment value of these indicators with the value of the same indicator using different preparations.

[0079] The exact time of administration and amount of any particular formulation that will result in the most effective treatment in a given patient will depend on the activity, pharmacokinetics, and bioavailability of the particular compound, the patient's physiological condition (including age, sex, type and stage of disease, general physical condition, responsiveness to a given dosage, and type of medication), route of administration, etc. Using the guidance provided herein, treatment can be optimized, e.g., the optimal time and / or amount of administration can be determined, which will require only routine experimentation consisting of monitoring the subject and adjusting dosage and / or timing.

[0080] Because the onset and duration of effect of the various components are complimentary, the combined use of several active agents formulated in the compositions of the present invention can reduce the required dosage of any individual component. In such combined therapy, the various active agents can be delivered together or separately, and simultaneously or at different times of the day.

[0081] Packaging The formulations of the present invention can be packaged as either single-dose or multi-dose products. Single-dose products are sterile before opening the package, and all of the composition in the package is intended to be consumed in a single application to one or both eyes of a patient. The use of antimicrobial preservatives to maintain the sterility of the composition after opening the package is generally not necessary.

[0082] Multi-dose products are also sterile before the package is opened. However, because a container for a composition can be opened multiple times before all of the composition in the container is consumed, multi-dose products must have sufficient antimicrobial activity to ensure that the composition is not contaminated by microorganisms as a result of repeated opening and handling of the container. The level of antimicrobial activity required for this purpose is well known to those skilled in the art and is specified in official publications such as the United States Pharmacopeia ("USP") and corresponding publications in other countries. Detailed descriptions of specifications for preserving ophthalmic pharmaceutical products against microbial contamination and procedures for evaluating the preservative effectiveness of specific formulations are provided in these publications. In the United States, preservative effectiveness standards are commonly referred to as "USP PET" requirements (the acronym "PET" stands for "Preservative Effectiveness Test Method").

[0083] kit The present invention provides a kit for packaging and / or storing and / or using the formulations described herein, and a kit for practicing the methods described herein.Thus, for example, the kit can include one or more containers containing one or more ophthalmic solutions, tablets or capsules of the present invention.The kit can be designed to facilitate one or more aspects of delivery, use and storage.

[0084] Kits may optionally include instructions containing instructions (i.e., protocols) disclosing means of using the formulations provided therein. The instructions typically include written or printed material, but are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROMs), and the like. Such media may include an address to an internet site providing such instructions. [Example]

[0085] Example 1: Topical HGF inhibits the development of corneal opacity and scarring and promotes wound healing Corneal injury was induced in C57131_6 mice by mechanical removal of the complete corneal epithelium using an Algerbrush-II. Complete removal of the corneal epithelium results in damage to the underlying layers, including the corneal stroma. Under a dissecting microscope, the central area of ​​the cornea was defined with a 3 mm punch and gently rotated to incise the stroma. A circular area was drawn with sharp surgical forceps, and then the corneal epithelium and basement membrane, including the anterior part of the stroma, were removed using a handheld ALGERBRUSH II™ (Alger Equipment Co., Texas). This type of wound results in a bare stroma with the epithelium and basement membrane removed, leading to a significant inflammatory response. After injury, the cornea was flushed with sterile saline followed by HGF treatment or control treatment. Mouse recombinant HGF was then topically applied to the injured eye twice daily for up to 7 days after injury (dose: 3 μl of 0.01% HGF in PBS per eye). The control group received a similar dose of mouse serum albumin. Photographs of the injured corneas (with or without fluorescein (green) staining) were taken using slit-lamp biomicroscopy on days 1, 3, 5, and 7 after injury. A smaller area of ​​fluorescein (green) staining represents more rapid repair of the corneal injury. Figure 2A shows the reduction in opacity and increased wound healing over the days after injury in treated animals compared to controls. The reduction in opacity in treated animals is statistically significant on day 5 after injury (Figure 2B). The reduction in wound area, indicated by green staining, is statistically reduced on days 1, 3, and 5 after injury (Figure 2C).

[0086] Example 2: HGF inhibits the expression of α-smooth muscle actin (αSMA: a factor that causes scarring) by corneal keratocytes In vitro analysis of mouse keratocytes (MK / T1) demonstrates that HGF significantly inhibits TGFβ-induced expression of αSMA in keratocytes, as measured by real-time PCR (Figure 3A) and immunohistochemistry (Figure 3B). These keratocytes reside in the corneal stroma. HGF action in these cell types demonstrates HGF function in specialized cells of the corneal stroma, a corneal layer essential for visual clarity.

[0087] Corneal opacification and scarring in the stromal layer of the cornea can lead to vision loss and blindness. Preventing, inhibiting, or reducing scarring in these tissues through inhibition of α-SMA is effective in treating corneal opacification and scarring, thereby helping to improve vision.

[0088] Example 3: Topical HGF treatment inhibits trafficking and homing of inflammatory leukocytes to the injured cornea Corneal injury was induced in C57BL6 mice by mechanical removal of the intact corneal epithelium using ALGERBRUSH-II™. Mouse recombinant HGF was then topically applied to the injured eye twice daily (dose: 3 μl of 0.01% HGF in PBS per eye). A control group received a similar dose of mouse serum albumin. Three days after injury, corneas were excised, immunostained for the pan-leukocyte marker CD45, and examined using a confocal microscope (N = 5 mice per group). The results are shown in Figure 4: blue color indicates DAPI staining of cell nuclei, and green color indicates staining for the pan-leukocyte marker CD45.

[0089] Example 4: Topical HGF treatment restores the structure and thickness of injured corneal tissue Corneal injury was induced in C57BL6 mice by mechanical removal of the intact corneal epithelium using ALGERBRUSH-II™. Mouse recombinant HGF was then topically applied to the injured eye twice daily (dose: 3 μl of 0.01% HGF in PBS per eye). The control group received a similar dose of mouse serum albumin. Seven days after injury, corneas were excised and cross-sections were stained with hematoxylin and eosin (H&E). Figure 5A shows representative photomicrographs illustrating the histology of normal, injured, and HGF-treated corneas. Figure 5B presents cumulative data showing that HGF-treated corneas recover their thickness to a similar extent as normal corneas. Injured control corneas show a significant increase in thickness compared to normal and HGF-treated corneas (N = 5 mice per group).

[0090] Example 5: HGF treatment increases epithelial cell stratification after corneal injury As shown in Figures 7A and 7B, HGF increases epithelial cell stratification after corneal injury. Seven days after injury, corneas were excised from normal, albumin-treated, and HGF-treated mice. Corneal cross-sections were stained with the nuclear stain DAPI, and the corneal epithelial cell layer was visualized using a confocal microscope (400x) (Figure 7A). The bar chart in Figure 7B shows the thickness (μm) of the epithelial cell layer in normal corneas (white bars), injured control corneas, and HGF-treated injured corneas (black bars). Values ​​shown are the mean ± SD (error bars); n = 5 mice per group.

[0091] Example 6: HGF promotes HGF-R (c-met) expression As shown in Figure 8, HGF promotes HGF-R (c-met) expression in the injured cornea. Corneas were excised from the normal group (checkered bars), the mouse albumin-treated injured control group (white bars), and the HGF-treated injured group (black bars) on days 3 and 7 after injury. Total RNA was isolated from the excised corneas. HGF-R mRNA expression was quantified using real-time PCR. GAPDH was used as an internal control. Values ​​shown are means ± SD, and each group consisted of n = 6 mice.* p<0.03, ** p<0.01.

[0092] Other Aspects While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative of the invention, not limiting, its scope, as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

[0093] The patents and scientific papers referred to in this specification establish knowledge that is available to those skilled in the art.All US patents and published or unpublished US patent applications cited in this specification are incorporated by reference.All published foreign patents and patent applications cited in this specification are incorporated by reference.Genbank and NCBI deposits indicated by accession numbers cited in this specification are incorporated by reference.All other published references, documents, manuscripts and scientific papers cited in this specification are incorporated by reference.

[0094] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the scope of the invention as encompassed by the appended claims.

[0095] Sequence information SEQUENCE LISTING <110> MASSACHUSETTS EYE AND EAR INFIRMARY <120> COMPOSITIONS AND METHODS FOR PREVENTION AND TREATMENT OF CORNEAL HAZE AND SCARRING <150> US 62 / 217,611 <151> 2015-09-11 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 728 <212> PRT <213> Homo sapiens <400> 1 Met Trp Val Thr Lys Leu Pro Ala Leu Leu Gln His Val Leu 1 5 10 15 Leu His Leu Leu Leu Pro Ile Wing Ile Pro Tyr Ala Glu Gly Gln 20 25 30 Arg Lys Arg Arg Asn Thr With Glue Phe Lys Thr Served Ala Lys Thr 35 40 45 Thr Leu Lys Ile Asp Pro Ala Leu Lys Ile Lys Lys Val 50 55 60 Asn Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu 65 70 75 80 Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys 85 90 95 Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe 100 105 110 Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 115 120 125 Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys 130 135 140 Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His 145 150 155 160 Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr 165 170 175 Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser 180 185 190 Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu 195 200 205 Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp 210 215 220 His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro 225 230 235 240 His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp 245 250 255 Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr 260 265 270 Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys 275 280 285 Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu 290 295 300 Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile 305 310 315 320 Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu 325 330 335 His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn 340 345 350 Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr 355 360 365 Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp 370 375 380 Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met 385 390 395 400 Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp 405 410 415 Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala 420 425 430 Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His 435 440 445 Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys 450 455 460 Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu 465 470 475 480 Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val 485 490 495 Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg 500 505 510 Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp 515 520 525 Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr 530 535 540 Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu Lys 545 550 555 560 Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly 565 570 575 Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp 580 585 590 Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu 595 600 605 Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn 610 615 620 Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu 625 630 635 640 Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu 645 650 655 Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp 660 665 670 Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu 675 680 685 Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly 690 695 700 Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile 705 710 715 720 Leu Thr Tyr Lys Val Pro Gln Ser 725

Claims

1. identifying a subject in need of treatment or prevention of corneal opacification or scarring; administering to the subject a hepatocyte growth factor receptor (HGFR) binding composition comprising at least one purified hepatocyte growth factor (HGF) agent.

10. A method for treating or preventing corneal opacification or scarring, comprising:

2. 10. The method of claim 1, wherein the HGF agent comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof.

3. 3. The method of claim 2, wherein the HGF agent comprises a polypeptide comprising an amino acid sequence having 70% sequence identity to SEQ ID NO:

1.

4. 10. The method of claim 1, wherein the HGF agent comprises an agonist of HGF.

5. 10. The method of claim 1, wherein the composition comprising at least one purified HGF agent further comprises a corneal stroma penetrating excipient.

6. The method of claim 1, wherein the composition acts to inhibit alpha-smooth muscle actin (alpha-SMA) in the cornea.

7. 10. The method of claim 1, wherein the purified HGF agent is present at a concentration of 0.001% to 1% w / v.

8. 8. The method of claim 7, wherein the purified HGF agent is present at a concentration of 0.005% to 0.05% w / v.

9. 10. The method of claim 8, wherein the purified HGF agent is present at a concentration of about 0.01% w / v.

10. 10. The method of claim 1, wherein the composition is administered daily for 5 days after injury to the cornea.

11. 10. The method of claim 1, wherein the composition is administered daily for three days after injury to the cornea.

12. 10. The method of claim 1, wherein the composition is administered daily for one day after injury to the cornea.

13. A composition for the treatment of corneal opacity or scarring comprising at least one HGF agent.

14. 14. The composition of claim 13, wherein the at least one HGF agent comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof.

15. 14. The composition of claim 13, wherein the at least one HGF agent comprises a polypeptide comprising an amino acid sequence having 70% sequence identity to SEQ ID NO:

1.

16. 14. The composition of claim 13, wherein the at least one HGF agent comprises an agonist of HGF.

17. 14. The composition of claim 13, further comprising a corneal stromal penetrating excipient.

18. 14. The composition of claim 13, wherein the at least one HGF agent is present at a concentration of 0.001% to 1% w / v.

19. 20. The composition of claim 18, wherein the at least one HGF agent is present at a concentration of 0.005% to 0.05% w / v.

20. 20. The composition of claim 19, wherein the at least one HGF agent is present at a concentration of about 0.01% w / v.

21. 14. The composition of claim 13, wherein the at least one HGF agent is present in a concentration effective to restore corneal thickness to normal corneal thickness.

22. 14. The composition of claim 13, wherein the at least one HGF agent is present in a concentration effective to inhibit the trafficking of inflammatory leukocytes to the injured cornea.

23. 14. The composition of claim 13, wherein the at least one HGF agent is present at a concentration effective to inhibit α-SMA expression in the cornea.

24. 10. The method of claim 1, wherein the identifying step comprises calculating a rating scale of the subject's corneal clarity.

25. 10. The method of claim 1, wherein the administering step comprises topical administration or subconjunctival administration.

26. The method of claim 1, wherein the administering step comprises contacting the composition with the corneal stroma of the subject.

27. The method of claim 1 , wherein the composition is formulated as an eye drop.

28. 10. The method of claim 1, wherein the purified HGF agent comprises a polypeptide comprising the amino acid sequence of residues 32 to 494 of SEQ ID NO:

1.

29. 10. The method of claim 1, wherein the purified HGF agent comprises a polypeptide comprising the amino acid sequence of residues 126-207 of SEQ ID NO:

1.

30. 10. The method of claim 1, wherein the purified HGF agent comprises a polypeptide comprising the amino acid sequence of residues 208-289 of SEQ ID NO:

1.

31. 10. The method of claim 1, wherein the purified HGF agent comprises a polypeptide comprising the amino acid sequence of residues 302-384 of SEQ ID NO:

1.

32. 10. The method of claim 1, wherein the purified HGF agent comprises a polypeptide comprising the amino acid sequence of residues 388-470 of SEQ ID NO:1.

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