Growth factor compositions for the treatment of eye diseases

Abstract

The present disclosure relates to pharmaceutical compositions comprising growth factors and methods of using the growth factors and compositions to treat or prevent ocular diseases.

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of U.S. Provisional Patent Application No. 63 / 188,816, filed May 14, 2021. The entire contents of the foregoing are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an ASCII text file named "Sequence_Listing.txt". The ASCII text file was created on May 10, 2022 and is 171 kilobytes in size. The material within the ASCII text file is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to pharmaceutical compositions comprising growth factors and methods of using the growth factors and compositions to treat or prevent ocular diseases. [Background technology]

[0004] Hepatocyte growth factor (HGF) is being investigated for the treatment of scarring corneal opacities. See U.S. Patent No. 10,449,234. Other eye injuries and diseases that may benefit from new therapies have been identified. For example, neurotrophic keratitis (also known as neurotrophic keratopathy (NK) or neuroparalytic keratitis) is a corneal disease resulting from either damage to the trigeminal nerve or its pathway to the cornea, or from an injured ocular surface, resulting in reduced or lost corneal sensation. NK was first recognized as a corneal disorder by Magendie in 1824 (Okada et al., 2010, Histol Histopathol, 25:771-780), and its molecular basis was subsequently established in animal models by Sigelman and Friedenwald in 1954 (Sigelman and Friedenwald, 1954, Arch Ophthalmol, 53:46-57). The cornea is the transparent "window" of the eye and, together with the sclera, forms the outer shell of the eyeball. As the cornea lacks a vascular supply, nutrients and oxygen are mainly supplied to this tissue anteriorly via the tear and limbal vessels and posteriorly via the aqueous humor. The corneal tissue is stratified into five layers: epithelium, Bowman's membrane, stroma, Desceme's membrane, and endothelium, with the epithelium acting as the main barrier protecting the underlying corneal stroma. The cornea is the most sensitive tissue in the body and is innervated by an extensive network of nerve fibers throughout the various layers and dense nerve endings. The sensory innervation of the cornea originates from the ophthalmic branch of the trigeminal nerve, which secretes growth factors essential for the survival of the epithelium. The trigeminal nerve maintains the stability of the tear film and further ensures the vitality of the corneal epithelium and stroma along with various physiologically active substances secreted by the epithelium (e.g. cytokines, proteases, and neuropeptides). Trigeminal nerve or epithelial disorders are most commonly caused by infection (e.g., herpes simplex or shingles infections), ophthalmic surgery (e.g., cataract surgery, corneal transplants, refractive surgery), other systemic diseases such as diabetes, leprosy, orbital tumors and inflammation, or physical trauma including chemical and thermal burns, and the use of contact lenses.

[0005] Thus, there is a continuing need for new treatments for ocular diseases such as NK, as well as new pharmaceutical formulations that can be administered to the eye. The methods described herein have been developed toward this end. Summary of the Invention

[0006] The present invention provides a method of treating or preventing neurotrophic keratitis in a subject in need thereof, the method comprising administering to the subject a growth factor, such as hepatocyte growth factor (HGF) or fibroblast growth factor (FGF).

[0007] The present invention further provides pharmaceutical compositions comprising HGF or FGF for the treatment of eye diseases.

[0008] Provided herein are methods for treating or preventing neurotrophic keratitis in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of hepatocyte growth factor (HGF) or fibroblast growth factor (FGF), e.g., as described herein.

[0009] In some embodiments, the HGF or FGF is purified.

[0010] In some embodiments, HGF or FGF is administered in combination with a corneal stromal permeability excipient.

[0011] In some embodiments, the HGF or FGF is formulated in a liquid pharmaceutical composition.

[0012] In some embodiments, HGF or FGF is administered to the eye. In some embodiments, HGF or FGF is administered topically to the eye. In some embodiments, HGF or FGF is administered to the eye by injection. In some embodiments, HGF or FGF is administered subconjunctivally. In some embodiments, HGF or FGF is administered into the anterior chamber.

[0013] In some embodiments, the liquid pharmaceutical composition comprises HGF or FGF at a concentration of about 0.01% (w / v) to about 1.0% (w / v). In some embodiments, the liquid pharmaceutical composition comprises HGF or FGF at a concentration of about 0.08% (w / v) to about 0.25% (w / v). In some embodiments, the liquid pharmaceutical composition comprises HGF or FGF at a concentration of about 0.1% (w / v). In some embodiments, the liquid pharmaceutical composition comprises HGF or FGF at a concentration of about 0.2% (w / v).

[0014] In some embodiments, HGF or FGF is administered in combination with an additional therapeutic agent, hi some embodiments, the additional therapeutic agent is an additional growth factor.

[0015] In some embodiments, HGF comprises a polypeptide sequence having any one of SEQ ID NOs: 1-27. In some embodiments, HGF comprises a polypeptide sequence having 95% sequence identity to SEQ ID NO: 1. In some embodiments, HGF comprises a polypeptide sequence having SEQ ID NO: 1.

[0016] The pharmaceutical compositions provided herein also comprise about 0.01% to about 1.0% (w / v) HGF, a buffer capable of maintaining the pH of the composition at about 5.8 to about 6.2, about 100 to about 300 mM of a stabilizer selected from trehalose, proline, sorbitol, and mixtures thereof, and optionally, an osmolality of the composition of about 250 mOsm / kg H 2 O~about 500mOsm / kg H 2 The composition further comprises a tonicity adjusting agent, which may be O, and, optionally, a surfactant.

[0017] In some embodiments, the pharmaceutical composition comprises about 0.05% to about 0.5% (w / v) HGF. In some embodiments, the pharmaceutical composition comprises about 0.08% to about 0.25% (w / v) HGF. In some embodiments, the pharmaceutical composition comprises about 0.1% (w / v) HGF. In some embodiments, the pharmaceutical composition comprises about 0.2% (w / v) HGF.

[0018] In some embodiments, the composition has a pH of about 6.0.

[0019] In some embodiments, the pharmaceutical composition comprises about 150 to about 250 mM of a stabilizer. In some embodiments, the pharmaceutical composition comprises about 200 mM of a stabilizer. In some embodiments, the stabilizer is trehalose or proline. In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is proline. In some embodiments, the stabilizer is sorbitol.

[0020] In some embodiments, the buffer is a citrate buffer. In some embodiments, the buffer is sodium citrate. In some embodiments, the pharmaceutical composition comprises about 10 to about 50 mM of the buffer.

[0021] In some embodiments, the osmolality of the composition is about 450 mOsm / kg H 2 It is O.

[0022] In some embodiments, the pharmaceutical composition does not include a tonicity agent. In some embodiments, the pharmaceutical composition includes a tonicity agent that is an alkali metal salt. In some embodiments, the pharmaceutical composition includes a tonicity agent that is sodium chloride.

[0023] In some embodiments, the pharmaceutical composition comprises a surfactant. In some embodiments, the surfactant is selected from polysorbate 80 (PS80), polaxomer 188, and polaxomer 407. In some embodiments, the surfactant is polysorbate 80 (PS80). In some embodiments, the surfactant is present in an amount of about 0.01% to about 0.1% (w / v). In some embodiments, the surfactant is present in an amount of about 0.02% to about 0.8% (w / v). In some embodiments, the surfactant is present in an amount of about 0.05% (w / v).

[0024] Also provided herein is an aqueous pharmaceutical composition comprising about 0.1% (w / v) HGF, about 20 mM sodium citrate, about 200 mM trehalose, proline, or sorbitol, about 0.05% (w / v) surfactant, wherein the pH of the composition is about 6.0 and the osmolality of the composition is about 350 mOsm / kg H 2 It is O.

[0025] In some embodiments, the aqueous pharmaceutical composition comprises about 0.1% (w / v) HGF, about 20 mM sodium citrate, about 200 mM trehalose, about 0.05% (w / v) polysorbate 80 (PS80), the pH of the composition is about 6.0, and the osmolality of the composition is about 350 mOsm / kg H 2 It is O.

[0026] In some embodiments, the aqueous pharmaceutical composition comprises about 0.1% (w / v) HGF, about 20 mM sodium citrate, about 200 mM proline, about 0.05% (w / v) polysorbate 80 (PS80), the pH of the composition is about 6.0, and the osmolality of the composition is about 350 mOsm / kg H 2 It is O.

[0027] In some embodiments, the aqueous pharmaceutical composition comprises about 0.1% (w / v) HGF, about 20 mM sodium citrate, about 200 mM sorbitol, about 0.05% (w / v) polysorbate 80 (PS80), the pH of the composition is about 6.0, and the osmolality of the composition is about 350 mOsm / kg H 2 It is O.

[0028] In some embodiments, the aqueous pharmaceutical composition comprises about 0.2% (w / v) HGF, about 20 mM sodium citrate, about 200 mM trehalose, proline, or sorbitol, The composition comprises about 0.05% (w / v) surfactant, the pH of the composition is about 6.0, and the osmolality of the composition is about 450 mOsm / kg H 2 It is O.

[0029] In some embodiments, the aqueous pharmaceutical composition comprises about 0.2% (w / v) HGF, about 20 mM sodium citrate, about 200 mM trehalose, about 0.05% (w / v) polysorbate 80 (PS80), the pH of the composition is about 6.0, and the osmolality of the composition is about 450 mOsm / kg H 2 It is O.

[0030] In some embodiments, the aqueous pharmaceutical composition comprises about 0.2% (w / v) HGF, about 20 mM sodium citrate, about 200 mM proline, about 0.05% (w / v) polysorbate 80 (PS80), the pH of the composition is about 6.0, and the osmolality of the composition is about 450 mOsm / kg H 2 It is O.

[0031] In some embodiments, the aqueous pharmaceutical composition comprises about 0.2% (w / v) HGF, about 20 mM sodium citrate, about 200 mM sorbitol, about 0.05% (w / v) polysorbate 80 (PS80), the pH of the composition is about 6.0, and the osmolality of the composition is about 450 mOsm / kg H 2 It is O.

[0032] In some embodiments, HGF comprises a polypeptide sequence of any one of SEQ ID NOs: 1-27. In some embodiments, HGF comprises a polypeptide sequence having 95% sequence identity to SEQ ID NO: 1. In some embodiments, HGF comprises a polypeptide sequence having SEQ ID NO: 1.

[0033] Also provided herein is a method of treating or preventing an ocular disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.

[0034] In some embodiments, the ocular disease is a corneal disease selected from neurotrophic keratitis, persistent corneal defects, corneal ulcers, dry eye disease, microbial keratitis, bacterial keratitis, viral keratitis, fungal keratitis, chemical burns, thermal burns, mechanical trauma, corneal abrasions, damaged endothelium, bullous keratopathy, Fuchs' corneal dystrophy, corneal scarring, Sjogren's syndrome, or post-operative complications. In some embodiments, the ocular disease is corneal opacity or scarring. In some embodiments, the ocular disease is neurotrophic keratitis. In some embodiments, the composition is administered in combination with a corneal stromal permeable excipient.

[0035] In some embodiments, the composition is administered ocularly. In some embodiments, the composition is administered topically to the eye. In some embodiments, the composition is administered by injection to the eye. In some embodiments, the composition is administered subconjunctivally. In some embodiments, the composition is administered intracamerally.

[0036] In some embodiments of the methods or compositions described herein, HGF is activated HGF. In some embodiments, activated HGF is activated dHGF.

[0037] In some embodiments of the methods or compositions described herein, HGF is (a) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO:2, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:2, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO:2, or (b) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 7, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 7, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 7, or (c) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 8, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 8, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 8, or (d) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 9, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 9, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 9, or (e) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 10, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 10, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 10, or (g) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 11, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 11, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 11, or (h) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 12, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 12, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 12, or (i) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 13, optionally including a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 13, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 13, or (j) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 14, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 14, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 14, or (k) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 15, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 15, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 15, or (l) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 16, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 16, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 16, or (m) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 17, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 17, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 17, or (n) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 18, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 18, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 18, or (o) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 19, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 19, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 19, or (p) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 20, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 20, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 20, or (q) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO:21, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:21, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO:21, or (r) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO:22, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:22, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO:22, or (s) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 23, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 23, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 23, or (t) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 24, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 24, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 24, or (u) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO: 25, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 25, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO: 25, or (v) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO:26, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:26, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO:26, or (w) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO:27, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:27, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO:27.

[0038] In some embodiments of the methods or compositions described herein, HGF is (a) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO:2, optionally including a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:2, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO:2; or (b) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO: 7, optionally including a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 7, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO: 7; or (c) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO:8, optionally including a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:8, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO:8; or (d) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO: 9, optionally including a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 9, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO: 9; or (e) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO: 10, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 10, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO: 10; or (g) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO:11, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:11, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO:11; or (h) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO: 12, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 12, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO: 12; or (i) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO: 13, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 13, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO: 13; or (j) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO: 14, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 14, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO: 14; or (k) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO: 15, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 15, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO: 15; or (l) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO: 16, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 16, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO: 16, or (m) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO: 17, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 17, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO: 17; or (n) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO: 18, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 18, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO: 18; or (o) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO: 19, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 19, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO: 19; or (p) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO: 20, optionally including a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 20, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO: 20, or (q) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO:21, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:21, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO:21; or (r) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO:22, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:22, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO:22; or (s) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO: 23, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO: 23, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO: 23, or (t) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO:24, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:24, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO:24, or (u) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO:25, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:25, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO:25; or (v) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO:26, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:26, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO:26; or (w) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO:27, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:27, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO:27.

[0039] In some embodiments of the methods or compositions described herein, the first polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:36 and the second polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:37.

[0040] In some embodiments of the methods or compositions described herein, the N-terminal amino acid of the first polypeptide is pyrrolidone carboxylic acid.

[0041] In some embodiments of the methods or compositions described herein, the first polypeptide and the second polypeptide are linked by one or more disulfide bonds.

[0042] In some embodiments of the methods or compositions described herein, HGF is capable of binding to c-MET and / or activating the MAPK pathway in epithelial cells. [Brief description of the drawings]

[0043] [Figure 1] Fluorescence microscopy images of slides containing serial sections of mouse optic nerves to assess corneal innervation 3 days after surgery. Tissue samples were stained for TUJ-1 (also known as beta III tubulin, circular area) to assess corneal innervation. Animals treated with control (PBS) showed potentially lower TUJ-1 staining (circular area) on day 3 compared to eyes that received 0.1% dHGF (SEQ ID NO: 1). DAPI was used as a counterstain to highlight all nuclei. [Diagram 2] Time to closure analysis of fluorescein-stained corneal surfaces of animals following surgery and routine treatment with PBS (vehicle control), 0.1% or 0.2% dHGF, 0.1% mNGF or 0.1% mHGF in a Phase 1 study in a mouse corneal mechanical injury model. [Figure 3A]Brightfield image analysis of the average scar area (in pixels) measured on the corneal surface of animals treated with PBS (control), 0.1% dHGF, or 0.2% dHGF on day 7 postoperatively in a mouse corneal mechanical injury model. [Figure 3B] Brightfield image analysis of the mean reduction in scar area (in pixels) measured on the corneal surface of animals treated with PBS (control), 0.1% mNGF, or 0.1% mHGF on day 7 postoperatively in a mouse corneal mechanical injury model. [Figure 4] Mean percentage scar reduction, standard error of measurement (SEM) and statistical comparisons from days 8 to 21 for animals treated with PBS (control), dHGF (0.1% and 0.2%), mHGF and mNGF in a mouse corneal mechanical injury model. [Diagram 5] Percent change in scar size at day 9 for animals treated with PBS (control), dHGF (0.1% and 0.2%), or mNGF in a mouse corneal E. coli LPS-induced keratitis model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] In some embodiments, the present invention provides, inter alia, a method for treating or preventing neurotrophic keratitis (NK) in a subject in need thereof, the method comprising administering hepatocyte growth factor (HGF) or fibroblast growth factor (FGF) to the subject.

[0045] Neurotrophic keratitis or alternatively, neurotrophic keratopathy (NK) is a degenerative condition of the cornea resulting from damage to the trigeminal nerve or its branches, or to an injured ocular surface, resulting in reduced corneal sensitivity or complete loss of corneal sensation. As used herein, the terms "disease", "condition", or "disorder" are used interchangeably and refer to any of a number of pathological conditions of an affected tissue or organ. Disorders of the trigeminal nerve or its branches are most commonly caused by infections (e.g., viral infections, including herpes simplex or varicella zoster infections, bacterial ulcers, late acanthamoeba ulcers), ophthalmic surgery (e.g., cataract surgery, corneal transplants, refractive surgery, and retinal surgery), diabetes, leprosy, or other systemic diseases such as orbital tumors and inflammation, fifth nerve palsies due to trigeminal nerve lesions in the posterior fossa, aneurysms, acoustic neuromas, meningiomas, or other physical damage to the corneal layers due to chemical and thermal burns. Other causes of NK include genetic disorders (e.g., familial corneal hypoesthesia, Goldenher-Gorlin syndrome, hereditary sensory and autonomic neuropathy types III, IV, or V, corneal dystrophies, and polyendocrine neoplasia IIb), use of certain medications (e.g., topical beta-blockers, topical nonsteroidal anti-inflammatory drugs (NSAIDs), local anesthetics), and contact lens use (for review, see Okada et al., supra).

[0046] NK can be treated or prevented according to the methods described herein by administration of hepatocyte growth factor (HGF) and / or fibroblast growth factor (FGF).

[0047] HGF (e.g., UniProt ID No. P14210) is a cMet kinase agonist that stimulates, inter alia, epithelial cell proliferation, motility, morphogenesis, and angiogenesis in various organs via tyrosine kinase signaling pathways and plays a key role in embryonic organ development and adult organ regeneration as well as wound healing. HGF is a protein of approximately 84 kDa that contains two subunits: an α subunit with an apparent molecular weight of 69 kDa and a β subunit with an apparent molecular weight of 34 kDa linked by a single disulfide bond.

[0048] HGF is produced by mesenchymal stromal cells (e.g., fibroblasts and macrophages) as a 728 amino acid pro-HGF molecule, with the first 1-31 amino acid residues corresponding to the secretory signaling sequence (Matsumoto and Nakamura, in Encyclopedia of Endocrine Diseases, Elsevier, 2004, 436-442). The primary amino acid sequence of pro-HGF is organized into a domain structure of four kringle (K) domains (Sigurdardottir, et al., 2015, Chem. Sci., 6:6147-6157). As used herein, a "kringle" domain refers to a polypeptide linear sequence folded into a triple-loop disulfide bridge domain (Simonneau, et al., 2015, Chem. Sci., 6:2110-2121). Kringle domains are present in a variety of polypeptides, including apolipoprotein A, blood clotting factor XII, plasminogen, and HGF. The name "kringle" comes from the Scandinavian pastry to which they resemble in structure. Kringle domains are believed to mediate binding interactions between proteins, membranes, and phospholipids. In some embodiments, the polypeptides of the present invention comprise at least one kringle domain.

[0049] Upon cleavage of the secretory signaling sequence, proteolytic cleavage at a trypsin-like site (R494-V495) between the fourth kringle and the C-terminal serine-proteinase homology (SPH) domain of pro-HGF results in mature activated HGF. The trypsin-like cleavage site is a peptide bond following a positively charged amino acid (e.g., lysine or arginine). Upon activation, HGF binds to and activates its receptor, cMet (also known as MET tyrosine kinase), resulting in tyrosine phosphorylation, which further affects the recruitment of various downstream adaptor molecules and the regulation of various intracellular pathways and biological activities collectively known as the invasive growth program (Nakamura, T., 1991, Prog. Growth Factor Res., 3:67-85; Bottaro, et al., 1991, Science 251:802-804). Both HGF and its receptor cMet are expressed in corneal epithelium, stromal cells, endothelium, and lacrimal glands, but the amount of HGF in human tears is very low, estimated to be approximately 500 pg / mL (Wilson et al., 1993, Invest. Ophthalmol. Vis. Sci., 34:2544-2561; Li et al., 1996, Invest. Ophthalmol. Vis. Sci., 37:727-739).

[0050] Six naturally occurring isoforms of HGF resulting from alternative splicing of HGF mRNA gene transcripts are known in the literature, of which the major isoforms are isoforms 1 (e.g., SEQ ID NO: 2) and 2 (e.g., SEQ ID NO: 3) (Bottaro, et al., 1991, Science, 251:802-804; Chan et al., 1991, Science, 254:1382-1385; Lokker and Godowski, 1992, EMBO J., 11:2503-2510; Cioce, et al., 1996, J. Biol. Chem., 271:13110-13115). As used herein, the term "isoform" refers to a protein resulting from alternative splicing of pre-mRNA encoding HGF. Isoform 1 mRNA transcript encodes the longest HGF gene sequence, containing 728 amino acid residues. The second major HGF isoform is encoded by an isoform 2 mRNA transcript that lacks multiple 3' exons but contains an alternative 3' exon relative to the isoform 1 transcript. The HGF protein encoded by the isoform 2 mRNA transcript contains 290 amino acid residues and is truncated after the second kringle domain compared to the isoform 1 HGF protein (Miyazawa, et al., 1991, Eur. J. Biochem., 197:15-22). The isoform 3 mRNA transcript encodes an HGF protein containing 723 amino acid residues (e.g., SEQ ID NO: 1) that lacks the in-frame coding segment present in isoform 1 and lacks the sequence "SFLPS" at positions 161-165 in the first kringle domain of isoform 1 (Rubin et al., 1991, Proc. Natl. Acad. Sci., 88:415-419). Isoform 4 HGF protein (eg, SEQ ID NO: 4) is a smaller molecule that includes residues 1-296, the amino acid sequence of which extends only through the second kringle domain (Chan et al., 1991, Science, 254:1382-1385).Isoform 5 HGF protein (e.g., SEQ ID NO: 5) is similar to isoform 2 HGF protein with an additional deletion of residues 161-165 (SFLPS) as isoform 3 HGF (Rubin et al., 1991, Proc. Natl. Acad. Sci., 88:415-419). Isoform 6 HGF protein (also known as NK1) (e.g., SEQ ID NO: 6) is the smallest of all HGF isoforms, containing only 210 amino acids (Cioce, et al., 1996, J. Bio. Chem., 271:13110-13115).

[0051] Both full-length and truncated isoforms of HGF have been shown to bind cMet, albeit with different potencies and interactions with heparin sulfate proteoglycans of the extracellular matrix, which serves to extend the circulatory half-life of HGF in vivo (Masumoto and Yamamoto, 1991, Biochem. Biophys. Res. Commun., 174:90-95; Montesano, et al., 1998, Cell Growth Differ., 9:355-365; Sakata et al., 1997, J. Biol. Chem., 272:9457-9463; Stahl et al., 1997, Biochem. J., 326:763-772). Isoforms 1 and 3 require proteolytic cleavage at R494-V495 to become biologically active, whereas truncated isoforms 2, 4, 5, and 6 do not have the R494-V495 cleavage site and therefore this activation step is not required for their biological activity. However, truncated isoforms 2, 4, 5, and 6 are known to be generally less potent than their full-length isoform 1 and 3 counterparts (Stahl et al., 1997; Montesano, et al., 1998, supra).

[0052] Fibroblast growth factors (FGFs) are a family of cell signaling proteins involved in a wide range of biological processes, including development. The human FGF family includes 22 members: FGF1, FGF2, FGF3 (INT2), FGF4, FGF5, FGF6, FGF7 (KGF), FGF8 (AIGF), FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

[0053] FGFs exert their biological effects by binding and activating fibroblast growth factor receptors (FGFRs). Activated FGFRs mediate signal transduction by recruiting specific molecules that bind to phosphorylated tyrosines in the cytoplasmic portion of the receptor, triggering several signaling pathways that lead to specific cellular responses. The human FGFR family includes four members: FGFR1, FGFR2, FGFR3, and FGFR4.

[0054] In some embodiments, HGF is a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-27, with or without a signal sequence (i.e., amino acids corresponding to amino acids 1-31 of SEQ ID NO: 1). In some embodiments, HGF is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, with or without a signal sequence (i.e., amino acids corresponding to amino acids 1-31 of SEQ ID NO: 1 or SEQ ID NO: 2). In some embodiments, HGF is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, with or without a signal sequence (i.e., amino acids corresponding to amino acids 1-31 of SEQ ID NO: 1). In some embodiments, HGF is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, with or without a signal sequence (i.e., amino acids corresponding to amino acids 1-31 of SEQ ID NO: 2). In some embodiments, HGF is a polypeptide comprising the activated form of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The term "activated form" refers, inter alia, to an HGF polypeptide in which the signal sequence (e.g., amino acids 1-31 of SEQ ID NO:1 or SEQ ID NO:2) has been cleaved and the HGF polypeptide has been cleaved between R489 and V490 of SEQ ID NO:1 or between R494 and V495 of SEQ ID NO:2 to form disulfide-bonded alpha and beta chains of HGF. In some embodiments, the N-terminal amino acid (e.g., amino acid 32 of SEQ ID NO:1 or SEQ ID NO:2) is a pyrrolidone carboxylic acid resulting, for example, from cleavage of the signal sequence. In some embodiments, the polypeptide of the present invention exists in an HGF precursor (pro-HGF) form in which the peptide bond between R489 and V490 of SEQ ID NO:1 or between R494 and V495 of SEQ ID NO:2 is intact. In some embodiments, the polypeptide of the present invention can be converted to an activated form by in vivo proteolytic cleavage of a trypsin-like cleavage site of the pro-HGF protein between the R489 and V490 amino acid residues of SEQ ID NO:1 or between the R494 and V495 amino acid residues of SEQ ID NO:2.In some embodiments, the polypeptide of the present invention can be converted to an active form by in vitro proteolytic cleavage of a trypsin-like cleavage site of the pro-HGF protein between the R489 and V490 amino acid residues of SEQ ID NO: 1, or between the R494 and V495 amino acid residues of SEQ ID NO: 2. In some embodiments, the HGF is a polypeptide comprising the amino acid sequence of amino acids 32-723 of the pro-HGF protein of SEQ ID NO: 1. In some embodiments, the HGF is a polypeptide comprising the amino acid sequence of amino acids 32-723 of the activated protein of SEQ ID NO: 1. In some embodiments, the HGF is a polypeptide comprising the amino acid sequence of amino acids 32-728 of the pro-HGF protein of SEQ ID NO: 2. In some embodiments, the HGF is a polypeptide comprising the amino acid sequence of amino acids 32-728 of the activated protein of SEQ ID NO: 2.

[0055] In some embodiments, HGF is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 4, 5, or 6, with or without a signal sequence (eg, amino acids corresponding to amino acids 1-31 of SEQ ID NO: 3, 4, 5, or 6).

[0056] In some embodiments, HGF is a polypeptide variant of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, with or without a signal sequence (e.g., amino acids corresponding to amino acids 1-31 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6). As used herein, the term "variant" is meant to refer to a polypeptide that differs from another polypeptide by a substitution, deletion, or insertion of one or more amino acids resulting from a mutation in a nucleic acid encoding the polypeptide. In some embodiments, HGF is a polypeptide that includes an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:6, with or without a signal sequence (e.g., amino acids corresponding to amino acids 1-31 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:6). In some embodiments, HGF is a polypeptide that includes an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:6, with or without a signal sequence (e.g., amino acids corresponding to amino acids 1-31 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:6). In some embodiments, HGF is a polypeptide comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:6, with or without a signal sequence (e.g., amino acids corresponding to amino acids 1-31 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:6). In some embodiments, HGF is a polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:6, with or without a signal sequence (e.g., amino acids corresponding to amino acids 1-31 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:6). In some embodiments, HGF variants are capable of (1) binding to c-MET and / or (2) activating the MAPK pathway in epithelial cells (e.g., human corneal epithelial cells).

[0057] In some embodiments, the HGF is a polypeptide comprising a mutation in the amino acid sequence of wild-type HGF isoform 1 (e.g., SEQ ID NO: 2), with or without a signal sequence (e.g., amino acids corresponding to amino acids 1-31 of SEQ ID NO: 2), at one or more of positions 62, 64, 77, 95, 125, 127, 130, 132, 137, 142, 148, 154, 170, 173, and 193. In some embodiments, the analogous position in wild-type HGF isoform 3 (e.g., SEQ ID NO: 1) is mutated. In some embodiments, the HGF is a polypeptide comprising a mutation in the amino acid sequence shown in Tables 1 and 2 below. Any combination of the mutations shown in Tables 1 and 2 may be included in the HGF polypeptide disclosed herein. [Table 1] [Table 2]

[0058] In some embodiments, HGF is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-27, with or without a signal sequence (e.g., amino acids corresponding to amino acids 1-31 of SEQ ID NOs: 7-27). In some embodiments, HGF is a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-27, with or without a signal sequence (e.g., amino acids corresponding to amino acids 1-31 of SEQ ID NOs: 7-27).

[0059] In some embodiments, the FGF is a polypeptide comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FGF is a polypeptide variant of SEQ ID NO: 28. In some embodiments, the FGF is a polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 28. In some embodiments, the FGF variant exhibits increased proteolytic stability compared to wild-type FGF1.

[0060] In some embodiments, the FGF is a polypeptide that includes a mutation in the amino acid sequence of wild-type FGF isoform 1 (e.g., SEQ ID NO: 28) at one or more of positions 28, 40, 47, 93, or 131. Any combination of mutations at positions 28, 40, 47, 93, and 131 may be included in the FGF polypeptides disclosed herein.

[0061] In some embodiments, the FGF1 variant comprises at least one amino acid substitution selected from the group consisting of D28N, Q40P, S47I, H93G, L131R, and L131K. In some embodiments, the FGF1 variant comprises the amino acid substitution L131R. In some embodiments, the FGF1 variant comprises the amino acid substitution L131K. In some embodiments, the variant comprises the amino acid substitutions D28N and L131R (e.g., SEQ ID NO: 29). In some embodiments, the variant comprises the amino acid substitutions D28N and L131K. In some embodiments, the variant comprises the amino acid substitutions Q40P, S47I, and H93G (e.g., SEQ ID NO: 30). In some embodiments, the variant comprises the amino acid substitutions Q40P, S47I, H93G, and L131R. In some embodiments, the variant comprises the amino acid substitutions Q40P, S47I, H93G, and L131K. In some embodiments, the variant comprises the amino acid substitutions D28N, Q40P, S47I, H93G, and L131R (e.g., SEQ ID NO: 31). In some embodiments, the variant comprises the amino acid substitutions D28N, Q40P, S47I, H93G, and L131K. In some embodiments, the FGF1 variant does not comprise the amino acid substitution L131A.

[0062] In some embodiments, the FGF is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 33. In some embodiments, the FGF is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 33.

[0063] The polypeptides disclosed herein (e.g., HGF polypeptides, FGF polypeptides) may be monomers or dimers. In some embodiments, HGF is a covalent dimer of any one of the polypeptides of SEQ ID NOs: 1-27, or a polypeptide variant thereof, with or without a signal sequence (e.g., amino acids corresponding to amino acids 1-31 of SEQ ID NOs: 1-27). In some embodiments, FGF is a covalent dimer of any one of the polypeptides of SEQ ID NOs: 29-33, or a polypeptide variant thereof. Covalent dimers of HGF or FGF polypeptides can be obtained by expressing any one of the HGF polypeptide sequences of SEQ ID NOs: 1-27, or a variant thereof, in a suitable expression system (e.g., yeast or E. coli), while a single amino acid residue, particularly the N-terminal amino acid residue, is replaced with a cysteine ​​residue. Expressed HGF polypeptide monomers, and for example, from which the signal sequence has been cleaved, or expressed FGF polypeptide monomers, can be induced to dimerize via formation of disulfide bonds between introduced cysteine ​​residues (see, e.g., Liu, et al., 2014, FEBS Letters, 588:4831-4837; Jones II, et al., 2011, Proc. Natl. Acad. Sci., 108:13035-13040, and USSN 15 / 365,514).

[0064] In some cases, an HGF polypeptide described herein includes one or more post-translational modifications, including, for example, one or more of the following: TIFF2024518178000003.tif179164

[0065] The polypeptides (e.g., HGF polypeptides, FGF polypeptides) disclosed herein can include substitutions with naturally occurring or non-naturally occurring amino acids, including, but not limited to, hydroxyproline (Hyp), beta-alanine, citrulline (Cit), ornithine (Orn), norleucine (Nle), 3-nitrotyrosine, nitroarginine, and pyroglutamic acid (Pyr). In some cases, the HGF polypeptides are post-translationally modified, for example, as shown in the table above.

[0066] As used herein, the term "sequence identity" refers to the percentage of identical residues between two polypeptide or nucleic acid sequences. One skilled in the art can easily determine sequence identity to amino acids of HGF or FGF, for example, by using the Basic Local Alignment Search Tool (BLAST), available online at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi, by inputting the amino acid (or nucleic acid) sequence of HGF or FGF and the polypeptide (or nucleic acid) in question. The program BLAST can be used to align two sequences (with default parameters), for example, as described by Tatiana A. Tatusova and Thomas L. Madden (1999), "Blast 2 sequences-a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250. Various other algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, the polypeptide of the present invention has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology with HGF, as determined, for example, by aligning the two sequences using the program BLAST (default parameters). In some cases, the sequence is substantially identical over the entire length of the compared sequence, for example, (i) the coding region of the nucleotide sequence or (ii) the amino acid sequence.

[0067] In some embodiments, HGF and / or FGF are isolated from biological sources such as amniotic membrane or amniotic fluid, cells or tissues that produce HGF and / or FGF (e.g., mesenchymal stromal cells or hepatic cells), or tears. In some embodiments, HGF is a recombinant protein expressed in a suitable host protein expression system such as, for example, E. coli, Saccharomyces cerevisiae, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK293) cells, or insect cell lines (e.g., Sf9, Sf21, or S2). In some embodiments, HGF is purified. As used herein, "purified" HGF refers to a polypeptide that has been processed to remove other undesired components or contaminants. In some embodiments, the FGF is a recombinant protein expressed in a suitable host protein expression system, such as, for example, E. coli, Saccharomyces cerevisiae, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK293) cells, or insect cell lines (e.g., Sf9, Sf21, or S2). In some embodiments, the FGF is purified. As used herein, "purified" FGF refers to a polypeptide that has been processed to remove other undesired components or contaminants.

[0068] Examples of undesirable components or contaminants removed during the purification process include cells, tissues, nucleic acids, polypeptides not related to HGF and / or FGF (e.g., host cell proteins from the expression system used, or abundant proteins from biological sources from which it is isolated, such as albumins and immunoglobulins), small fragments of HGF and / or FGF with less than 50% sequence homology, metals, and other inorganic salts. Examples of purification processes used by those skilled in the art include precipitation, flocculation, tangential flow filtration (TFF), ultrafiltration (UF), diafiltration (DF), dialysis, gel filtration chromatography (GFC), liquid chromatography (LC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and electrophoresis. In some embodiments, the purity of the purified HGF is at least about 50%. In some embodiments, the purity of the purified HGF is at least about 60%. In some embodiments, the purity of the purified HGF is about 75%. In some embodiments, the purity of the purified HGF is about 80%. In some embodiments, the purity of the purified HGF is about 85%. In some embodiments, the purity of the purified HGF is about 90%. In some embodiments, the purity of the purified HGF is about 95%. In a preferred embodiment, the purity of the purified HGF is about 97%. In some embodiments, the purity of the purified FGF is at least about 50%. In some embodiments, the purity of the purified FGF is at least about 60%. In some embodiments, the purity of the purified FGF is about 75%. In some embodiments, the purity of the purified FGF is about 80%. In some embodiments, the purity of the purified FGF is about 85%. In some embodiments, the purity of the purified FGF is about 90%. In some embodiments, the purity of the purified FGF is about 95%. In a preferred embodiment, the purity of the purified FGF is about 97%.

[0069] Determination of the purity and content of HGF and / or FGF can be readily accomplished by one of skill in the art using conventional analytical assays and methods, for example, by reverse phase high performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC), ion exchange chromatography (IEX), polyacrylamide gel electrophoresis (PAGE), capillary gel electrophoresis (CGE), and Western blotting.

[0070] As used herein, a "polypeptide" or "protein" is a polymer of amino acids, for example, having from 2 to about 1000 or more amino acid residues. In some embodiments, a "polypeptide" has from 10 to about 130 amino acids, from 10 to about 220 amino acids, from 10 to about 500 amino acids, or from 10 to about 730 amino acids. Any naturally occurring or synthetic amino acid can form a polypeptide. A polypeptide can also include modifications, such as glycosylation and other moieties. In some embodiments, a polypeptide of the present invention has the ability to selectively bind to a target polypeptide, for example, based on the amino acid sequence of the target, for example, the amino acid sequence of the N-terminus or C-terminus.

[0071] As used herein, the term "about" is used to mean approximately, roughly, or around the region. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value with a variance of 10%.

[0072] Therapeutic and Prophylactic Methods The present disclosure features a method for treating or preventing an ocular disease or disorder in a subject. In some embodiments, the ocular disease or disorder is a corneal disease. Non-limiting examples of corneal diseases include neurotrophic keratitis, persistent corneal defects, corneal ulcers, dry eye disease, microbial keratitis, bacterial keratitis, viral keratitis, fungal keratitis, chemical burns, thermal burns, mechanical trauma, corneal abrasions, damaged endothelium, bullous keratopathy, Fuchs corneal dystrophy, corneal scarring, Sjogren's syndrome, and post-operative complications. In some embodiments, the disease or disorder is corneal damage, including corneal opacity or scarring. In some embodiments, the disease is neurogenic keratitis. The method includes administering a therapeutically effective amount of an HGF and / or FGF polypeptide described herein to a subject. As used herein, "subject" or "patient," used interchangeably, includes mammals, such as humans, bovine, equine, canine, feline, porcine, and ovine animals. The subject is preferably a human.

[0073] In some embodiments, the subject has NK with damage to the trigeminal nerve or its branches. In certain cases, damage to the trigeminal nerve or its branches is caused by infection (e.g., bacterial or viral infection, such as herpes simplex or varicella zoster infection). In certain cases, damage to the trigeminal nerve or its branches is caused by eye surgery (e.g., cataract surgery, corneal transplant, refractive surgery). In certain cases, damage to the trigeminal nerve or its branches is caused by systemic diseases, such as diabetes, leprosy, orbital tumors, and inflammation. In some cases, damage to the trigeminal nerve or its branches is caused by physical trauma, including chemical burns and thermal burns. In other cases, damage to the trigeminal nerve or its branches is caused by the use of contact lenses. In some embodiments, the subject has NK with damage to the epithelium that is not associated with any perturbation to the trigeminal nerve pathway.

[0074] In some embodiments, HGF and / or FGF are administered to the eye of a subject. The route of administration of HGF and / or FGF can be performed according to known methods, for example, locally via eye drops or bandage contact lenses, local ocular injection (e.g., subconjunctival, intravitreal, retrobulbar, and intracameral), or by sustained release systems as described below. In some embodiments, HGF and / or FGF are administered locally to the eye. The term "topically" refers to applying HGF and / or FGF directly to the surface of the eye (i.e., the cornea). In some embodiments, HGF and / or FGF are administered to the eye by injection. In some embodiments, HGF and / or FGF are administered subconjunctivally. The term "subconjunctivally" refers to injection either under the bulbar conjunctiva (on the eye) or under the conjunctival lining of the eyelid (subpalpebral) (Stanley, R., 2008 "Ocular Clinical Pharmacology" Small Animal Clinical Pharmacology (2 nd Ed.). In some embodiments, HGF and / or FGF are administered intravitreally. The term "intravitreal" refers to injection directly into the vitreous cavity of the eye. In some embodiments, HGF and / or FGF are administered intracamerally. The term "intracameral" refers to injection directly into the anterior chamber of the eye. Therapeutic polypeptide compositions intended for injection are generally placed in a sterile container with an appropriate access port, e.g., a vial with a stopper that can be penetrated by a hypodermic needle. For topical administration, pharmaceutical compositions containing HGF and / or FGF can be placed in a squeezable eye drop container. In some embodiments, pharmaceutical compositions containing HGF and / or FGF are administered locally using an ophthalmic squeezable dispenser, e.g., a dispenser from Aptar Pharma.

[0075] Suitable examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, such as films, implants, or microcapsules.Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers, 22:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res., 15:267-277 and Langer, 1982, Chem. Tech., 12:98-105), ethylene vinyl acetate (Langer et al., supra) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). The sustained release composition also includes HGF, FGF, or both HGF and FGF entrapped in liposome.The liposome containing HGF, FGF, or both HGF and FGF can be prepared by the method known per se, DE3,218,121, Eppstein et al., 1985, Proc.Natl.Acad.Sci.USA, 82:3688-3692, Hwang et al., 1980, Proc.Natl.Acad.Sci.USA, 77:4030-4034, EP52,322, EP36,676, EP88,046, EP143,949, EP142,641, U.S. Patent No. 4,485,045 and 4,544,545, and EP102,324. Typically, the liposomes are small (about 200-800 angstroms), unilamellar type with lipid content greater than about 30 mole percent cholesterol, the selected ratio being adjusted for the most effective therapy.

[0076] The "effective amount" of HGF or HGF-containing composition and / or FGF or FGF-containing composition used therapeutically depends, for example, on the therapeutic purpose, route of administration, and condition of the subject. Thus, the therapist may need to titrate the dosage and modify the route of administration as necessary to obtain optimal therapeutic effect. Typically, the clinician administers HGF and / or FGF until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays and methods.

[0077] In the treatment and prevention of eye diseases and eye disorders such as corneal opacity or scarring, and NK, pharmaceutical compositions containing HGF and / or FGF can be formulated, dosed, and administered in a manner consistent with good medical practice.Factors to be considered in this context include the specific mammal to be treated, the clinical condition of the individual subject, the cause of the disorder, the method of administration, the schedule of administration, and other factors known to physicians.The "therapeutically effective amount" of HGF and / or FGF administered can be controlled by such considerations and is the minimum amount necessary to prevent, improve, or treat the symptoms of eye disease.Such an amount is preferably less than the amount that is toxic or causes significant adverse effects on the host.

[0078] As a general proposition, pharmaceutical compositions administered locally to a subject's eye include HGF and / or FGF at an initial concentration ranging from about 0.01% to about 1.0%, about 0.05% to about 0.5%, about 0.05% to about 0.4%, about 0.05% to about 0.3%, or about 0.08% to about 0.25% on a weight to volume (w / v) basis. In some embodiments, pharmaceutical compositions administered locally to a subject's eye include HGF at a concentration of about 0.1% (w / v). In some embodiments, pharmaceutical compositions administered locally to a subject's eye include HGF at a concentration of about 0.2% (w / v). In some embodiments, pharmaceutical compositions administered locally to a subject's eye include FGF at a concentration of about 0.1% (w / v). In some embodiments, pharmaceutical compositions administered locally to a subject's eye include FGF at a concentration of about 0.2% (w / v). In some embodiments, the pharmaceutical composition administered locally to the eye of a subject comprises both HGF and FGF at a concentration of about 0.1% (w / v) each. In some embodiments, the pharmaceutical composition administered locally to the eye of a subject comprises both HGF and FGF at a concentration of about 0.2% (w / v) each. In some embodiments, the pharmaceutical composition comprises HGF and is substantially free of FGF. In some embodiments, the pharmaceutical composition comprises FGF and is substantially free of HGF.

[0079] The present disclosure also features combination therapies. For example, a subject (e.g., a human) is administered an HGF as described herein in combination with a second therapeutic agent. In some embodiments, the subject is administered an HGF polypeptide simultaneously with the second therapeutic agent. As used herein, the term "simultaneously" means "present during the same period of time." In some embodiments, the HGF polypeptide and the second therapeutic agent are formulated in the same formulation. In some embodiments, the HGF polypeptide and the second therapeutic agent are formulated in separate formulations. In some embodiments, the HGF polypeptide and the second therapeutic agent are administered simultaneously. In some embodiments, the HGF polypeptide and the second therapeutic agent are administered sequentially. In some embodiments, the HGF polypeptide is administered before the second therapeutic agent is administered. In some embodiments, the HGF polypeptide is administered after the second therapeutic agent is administered. In some embodiments, the HGF polypeptide and the second therapeutic agent are administered 1 to 60 minutes apart, 5 to 30 minutes apart, or 10 to 20 minutes apart. In some embodiments, the HGF polypeptide and the second therapeutic agent are administered 5 minutes apart. In some embodiments, the HGF polypeptide and the second therapeutic agent are administered 10 minutes apart. In some embodiments, the HGF polypeptide and the second therapeutic agent are administered 15 minutes apart. In some embodiments, the HGF polypeptide and the second therapeutic agent are administered 30 minutes apart. In some embodiments, the HGF polypeptide and the second therapeutic agent are administered 1 hour apart. In some embodiments, an FGF polypeptide is the second therapeutic agent.

[0080] In some embodiments, the second therapeutic agent may induce the same biological and physiological effects as HGF, e.g., activation of similar intracellular pathways. In some embodiments, the second therapeutic agent may induce different biological and physiological effects as HGF. In some embodiments, the second therapeutic agent enhances the biological and physiological effects caused by HGF. In some embodiments, the second therapeutic agent is of the same class of molecule as HGF (i.e., both are polypeptides). In some embodiments, the second therapeutic agent is of a different class of molecule (e.g., small molecule or nucleic acid). Non-limiting examples of second therapeutic agents include small molecules, peptides, proteins, antibodies and antigen-binding fragments, nucleic acids, cell and tissue extracts, and amniotic fluid and other body fluids. The second therapeutic agent may be obtained via isolation from natural sources, synthetically produced, or obtained by cell culture. As used herein, the term "small molecule" refers to a low molecular weight organic compound less than 900 daltons. As used herein, the term "peptide" refers to a compound of 2 to about 50 subunits of amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine and both D or L optical isomers, amino acid analogs and peptidomimetics. As used herein, the term "antibody" includes polyclonal and monoclonal antibodies and fragments thereof. Antibodies include, but are not limited to, mouse, rat, rabbit, human, or chimeric antibodies. The term "antibody" also includes antibodies of all isotypes. As used herein, "nucleic acid" or "polynucleotide" refers to a polymeric form of nucleotides of any length or analogs thereof. Polynucleotides can contain deoxyribonucleotides, ribonucleotides, and / or their analogs. Nucleotides can have any three-dimensional structure and can perform any function, known or unknown. Nucleic acid molecules further include oligonucleotides, such as antisense molecules, probes, primers, etc.Oligonucleotides typically have from about 2 to about 100, 8 to about 30, or 10 to about 28 nucleotides or analogs thereof.

[0081] In some embodiments, the second therapeutic agent is an antibiotic. Non-limiting examples of antibiotics include trimethoprim, polymyxin B, azithromycin, gentamicin, besifloxacin, gatifloxacin, moxifloxacin, levofloxacin, ciprofloxacin, ofloxacin, and tobramycin. In some embodiments, the second therapeutic agent is a nonsteroidal anti-inflammatory drug (NSAID). Non-limiting examples of NSAIDs include aspirin, salsalate, celecoxib, diclofenac, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, and tolmetin. In some embodiments, the second therapeutic agent is an ophthalmic steroid. Non-limiting examples of ophthalmic steroids include dexamethasone ophthalmic (Maxidex®), difluprednate ophthalmic (Durezol®), fluorometholone ophthalmic (Flarex®, FML®, FML Liquifilm®, FML Forte®), Loteprednol etabonate ophthalmic (Alrex®, Lotemax®), prednisolone acetate ophthalmic (Omnipred®, Pred Forte®, Pred Mild®), prednisolone sodium phosphate ophthalmic, and rimexolone ophthalmic (Vexol®). In some embodiments, the second therapeutic agent is a local anesthetic. Non-limiting examples of local anesthetics include prilocaine, epinephrine, lidocaine, bupivacaine, lontocaine, novocaine, ropivacaine, procaine, amethocaine, cinchocaine, mepivacaine, and etidocaine. In some embodiments, the second therapeutic agent is an additional growth factor.Non-limiting examples of growth factors include epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF-1), platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF), transforming growth factor (TGF), vascular endothelial growth factor and granulocyte-macrophage colony-stimulating factor (GM-CSF), neurotrophins and nerve growth factor (NGF), tumor necrosis factor-alpha (TNF-α), and interleukins.

[0082] Pharmaceutical Compositions and Formulations Maintaining the stability of polypeptides against heat stress and minimizing the formation of visible particles and soluble aggregates is an important focus in the development of pharmaceutical compositions containing polypeptides, especially essentially water-soluble solutions that require long-term storage. See the following references in which HGF and related proteins are formulated with various stabilizers: WO90 / 10651 WO00 / 72873 (EP Patent No. 1180368), JP-A9-25241 (US Patent No. 7,173,008), WO2008 / 102849 (US Patent No. 8461112), and US Patent No. 10213485. Although the problems of protein aggregation and thermal stability can be avoided to some extent by using freeze-dried HGF formulations as described in the prior art, this requires a freeze-drying unit operation in the manufacture of pharmaceutical compositions containing HGF, which incurs additional manufacturing costs and complexity in the process. Furthermore, freeze-dried HGF formulations require reconstitution before dosing and administration. Therefore, a stable solution is advantageous.

[0083] In some embodiments of the present invention, HGF (and / or FGF) is formulated in a liquid pharmaceutical composition. In some embodiments, the pharmaceutical composition is an aqueous pharmaceutical composition. As used herein, "aqueous pharmaceutical formulation" refers to a water-based liquid. In some embodiments, the aqueous pharmaceutical composition comprises distilled water. In some embodiments, the aqueous pharmaceutical composition comprises deionized water. In some embodiments, the aqueous pharmaceutical composition comprises sterile water. In some embodiments, the aqueous pharmaceutical composition comprises water for injection (WFI). In some embodiments, the liquid pharmaceutical composition is formulated as eye drops. In some embodiments, the liquid pharmaceutical composition is formulated as a solution. In some embodiments, the liquid pharmaceutical composition is formulated as a suspension. In some embodiments, HGF is formulated with an additional therapeutic agent in a liquid pharmaceutical composition. In some embodiments, HGF is formulated with an additional therapeutic agent as eye drops. In some embodiments, HGF is formulated with an additional therapeutic agent as a solution.

[0084] Pharmaceutical formulations of the polypeptide or derivatives thereof of the present invention can be prepared for storage by mixing the polypeptide or derivatives thereof having the desired purity in the form of a lyophilized cake or aqueous solution with optional pharma- ceutically acceptable carriers, excipients, tonicity agents, or stabilizers (see, for example, Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed., Mack Publishing Co., Easton Pa. 18042, USA). Acceptable carriers, excipients, tonicity agents, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, and other organic acids, and the term "buffer" includes weak acids and their co-bases or vice versa used to maintain the pH of the solution approximately constant, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG), glycine, glycerol ... "sugar alcohol" refers to a mixture of amino acids such as glutamine, asparagine, arginine, histidine, proline, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, sucrose, trehalose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium or potassium; and / or non-ionic surfactants or co-solvents such as polysorbates and poloxamers; isotonicity agents such as sodium chloride, potassium chloride, mannitol, dextrose, glycerin, and magnesium chloride.

[0085] In some embodiments, the formulations of the invention are substantially free of tonicity agents, hi some embodiments, the formulations of the invention are substantially free of chloride salts, such as sodium chloride, potassium chloride, and magnesium chloride.

[0086] In some embodiments, the pH of the pharmaceutical formulation comprising HGF (and / or FGF) is about 5.5 to about 7.5. In some embodiments, the pH of the pharmaceutical formulation is about 5.5 to about 6.0. In some embodiments, the pH of the pharmaceutical formulation is about 5.8 to about 6.2. In some embodiments, the pH of the pharmaceutical formulation is about 6.0 to about 6.5. In some embodiments, the pH of the pharmaceutical formulation is about 6.5 to about 7.0. In some embodiments, the pH of the pharmaceutical formulation is about 7.0 to about 7.5. In some embodiments, the pH of the pharmaceutical formulation is about 6.0. In some embodiments, the pH of the pharmaceutical formulation is about 6.5. In some embodiments, the pH of the pharmaceutical formulation is about 7.0.

[0087] In some embodiments, the pharmaceutical formulation of the present invention further comprises a buffer. In some embodiments, the buffer is selected from acetate, citrate, glutamate, histidine, succinate, tartrate, and tris(hydroxymethyl)aminomethane (Tris). In some embodiments, the buffer is a citrate buffer, such as sodium citrate (e.g., sodium citrate dihydrate). In some embodiments, the buffer is an acetate buffer. In some embodiments, the buffer is a succinate buffer. In some embodiments, the buffer is a tartrate buffer. In some embodiments, the buffer is a glutamate buffer. In some embodiments, the buffer is Tris. In some embodiments, the buffer is present at about 10 mM to about 100 mM. In some embodiments, the buffer is present at about 20 mM to about 50 mM. In some embodiments, the buffer is present at about 20 mM.

[0088] In some embodiments, the pharmaceutical formulation of the present invention optionally further comprises an isotonicity agent. In some embodiments, the pharmaceutical formulation is substantially free of an isotonicity agent. In some embodiments, the isotonicity agent is an alkali metal salt, such as sodium chloride (NaCl) or potassium chloride (KCl). In some embodiments, the isotonicity agent is calcium chloride (CaCl 2 ). In some embodiments, the tonicity agent is mannitol. In some embodiments, the tonicity agent is trehalose (e.g., trehalose dihydrate). In some embodiments, the tonicity agent is present at about 0.1 to about 1.0 M, about 0.2 to about 0.8 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, or about 0.75 M. In some embodiments, the osmolality of the formulation is about 200 to about 500 mOsm / kg H 2 O, about 200~300mOsm / kg H 2 O, about 250~350mOsm / kg H 2 O, about 350~400mOsm / kg H 2 O, about 400~450mOsm / kg H 2 O, or about 450 mOsm / kg H 2 O~about 500mOsm / kg H 2 In some embodiments, the osmolality of the formulation is about 300 mOsm / kg H 2 In some embodiments, the osmolality of the formulation is about 350 mOsm / kg H 2 In some embodiments, the osmolality of the formulation is about 400 mOsm / kg H 2 In some embodiments, the osmolality of the formulation is about 425 mOsm / kg H 2 In some embodiments, the osmolality of the formulation is about 450 mOsm / kg H 2 In some embodiments, the osmolality of the formulation is about 475 mOsm / kg H 2 In some embodiments, the osmolality of the formulation is about 500 mOsm / kg H 2 It is O.

[0089] In some embodiments, the pharmaceutical formulation of the present invention further comprises one or more stabilizers. In some embodiments, the one or more stabilizers are selected from sorbitol, trehalose, sucrose, alanine, glycine, proline, glutamic acid, and arginine. In some embodiments, the one or more stabilizers are selected from sorbitol, proline, and trehalose. In some embodiments, the one or more stabilizers are selected from proline, arginine, and trehalose. In some embodiments, the one or more stabilizers are selected from glutamic acid, proline, and trehalose. In some embodiments, the one or more stabilizers are selected from arginine, glutamic acid, and trehalose. In some embodiments, the one or more stabilizers are selected from arginine, proline, and sorbitol. In some embodiments, the one or more stabilizers are selected from glutamic acid, proline, and sorbitol. In some embodiments, the one or more stabilizers are selected from arginine, glutamic acid, and sorbitol. In some embodiments, the one or more stabilizers are arginine and trehalose. In some embodiments, the one or more stabilizers are sorbital and trehalose. In some embodiments, the one or more stabilizers are proline and trehalose. In some embodiments, the one or more stabilizers are arginine and sorbitol. In some embodiments, the one or more stabilizers are glutamic acid and sorbitol. In some embodiments, the one or more stabilizers are proline and sorbitol. In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is proline. In some embodiments, the stabilizer is arginine. In some embodiments, the stabilizer is sorbitol.

[0090] In some embodiments, the pharmaceutical formulations of the present invention are substantially free of sucrose, alanine, and glycine. In some embodiments, the pharmaceutical formulations are substantially free of sucrose. In some embodiments, the pharmaceutical formulations are substantially free of alanine. In some embodiments, the pharmaceutical formulations are substantially free of glycine.

[0091] In some embodiments, the stabilizer is present in the formulation at a concentration of about 100 mM to about 500 mM. In some embodiments, the stabilizer is present at a concentration of about 150 mM to about 250 mM. In some embodiments, the stabilizer is present at a concentration of about 200 mM.

[0092] In some embodiments, the pharmaceutical formulation of the present invention optionally further comprises a surfactant. In some embodiments, the surfactant is selected from polysorbate 80 (PS80), polysorbate 20 (PS20), polaxamer 188 (P188), and polaxamer 407 (P407). In some embodiments, the surfactant is polysorbate 80. In some embodiments, the surfactant is present at about 0.02% to about 0.07% (w / v). In some embodiments, the surfactant is present at about 0.04% to about 0.06% (w / v). In some embodiments, the surfactant is present at about 0.05% (w / v).

[0093] In some embodiments, the pharmaceutical compositions of the present invention comprise (i) about 0.1% (w / v) to about 1.0% (w / v) HGF; (ii) a buffer capable of maintaining the pH of the composition at about 5.8 to about 6.2; (iii) about 100 to about 300 mM trehalose, proline, sorbitol, or a mixture thereof; (iv) optionally, increasing the osmolality of the composition to about 250 mOsm / kg H 2 O~about 500mOsm / kg H 2 and a tonicity adjusting agent capable of adjusting the (v) optionally, a surfactant.

[0094] In some embodiments, the pharmaceutical compositions of the present invention comprise (i) about 0.1% to about 0.5% (w / v) HGF; (ii) a buffer capable of maintaining a pH of the composition of about 5.8 to about 6.2; (iii) about 100 to about 300 mM trehalose, proline, sorbitol, or a mixture thereof; (iv) optionally, increasing the osmolality of the composition to about 250 mOsm / kg H 2 O~about 500mOsm / kg H 2 and a tonicity adjusting agent capable of adjusting the (v) optionally, a surfactant.

[0095] In some embodiments, the pharmaceutical compositions of the present invention comprise (i) about 0.1% (w / v) HGF; (ii) a buffer capable of maintaining a pH of the composition of about 5.8 to about 6.2; (iii) about 100 to about 300 mM trehalose, proline, sorbitol, or a mixture thereof; (iv) optionally, increasing the osmolality of the composition to about 250 mOsm / kg H 2 O~about 500mOsm / kg H 2 and a tonicity adjusting agent capable of adjusting the (v) optionally, a surfactant.

[0096] In some embodiments, the pharmaceutical compositions of the present invention comprise (i) about 0.2% (w / v) HGF; (ii) a buffer capable of maintaining a pH of the composition of about 5.8 to about 6.2; (iii) about 100 to about 300 mM trehalose, proline, sorbitol, or a mixture thereof; (iv) optionally, increasing the osmolality of the composition to about 250 mOsm / kg H 2 O~about 500mOsm / kg H2 and a tonicity adjusting agent capable of adjusting the (v) optionally, a surfactant.

[0097] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.1% (w / v) HGF, Sodium citrate at about 20 mM, about 200 mM trehalose, proline, or sorbitol; containing about 0.05% (w / v) surfactant, The pH of the composition is about 6.0.

[0098] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.1% (w / v) HGF, Sodium citrate at about 20 mM, Approximately 200 mM trehalose, Contains about 0.05% (w / v) polysorbate 80 (PS80), The pH of the composition is about 6.0.

[0099] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.1% (w / v) HGF, Sodium citrate at about 20 mM, Approximately 200 mM proline, Contains about 0.05% (w / v) polysorbate 80 (PS80), The pH of the composition is about 6.0.

[0100] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.1% (w / v) HGF, Sodium citrate at about 20 mM, About 200 mM sorbitol, Contains about 0.05% (w / v) polysorbate 80 (PS80), The pH of the composition is about 6.0.

[0101] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.2% (w / v) HGF, Sodium citrate at about 20 mM, about 200 mM trehalose, proline, or sorbitol; containing about 0.05% (w / v) surfactant, The pH of the composition is about 6.0.

[0102] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.2% (w / v) HGF, Sodium citrate at about 20 mM, Approximately 200 mM trehalose, Contains about 0.05% (w / v) polysorbate 80 (PS80), The pH of the composition is about 6.0.

[0103] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.2% (w / v) HGF, Sodium citrate at about 20 mM, Approximately 200 mM proline, Contains about 0.05% (w / v) polysorbate 80 (PS80), The pH of the composition is about 6.0.

[0104] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.2% (w / v) HGF, Sodium citrate at about 20 mM, About 200 mM sorbitol, Contains about 0.05% (w / v) polysorbate 80 (PS80), The pH of the composition is about 6.0.

[0105] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.1% (w / v) HGF, Sodium citrate at about 20 mM, about 200 mM trehalose, proline, or sorbitol; containing about 0.05% (w / v) surfactant, The pH of the composition is about 6.0 and the osmolality of the composition is about 350 mOsm / kg H 2 It is O.

[0106] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.1% (w / v) HGF, Sodium citrate at about 20 mM, Approximately 200 mM trehalose, Contains about 0.05% (w / v) polysorbate 80 (PS80), The pH of the composition is about 6.0 and the osmolality of the composition is about 350 mOsm / kg H 2 It is O.

[0107] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.1% (w / v) HGF, Sodium citrate at about 20 mM, Approximately 200 mM proline, Contains about 0.05% (w / v) polysorbate 80 (PS80), The pH of the composition is about 6.0 and the osmolality of the composition is about 350 mOsm / kg H 2 It is O.

[0108] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.1% (w / v) HGF, Sodium citrate at about 20 mM, About 200 mM sorbitol, Contains about 0.05% (w / v) polysorbate 80 (PS80), The pH of the composition is about 6.0 and the osmolality of the composition is about 350 mOsm / kg H 2 It is O.

[0109] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.2% (w / v) HGF, Sodium citrate at about 20 mM, about 200 mM trehalose, proline, or sorbitol; containing about 0.05% (w / v) surfactant, The pH of the composition is about 6.0 and the osmolality of the composition is about 450 mOsm / kg H 2 It is O.

[0110] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.2% (w / v) HGF, Sodium citrate at about 20 mM, Approximately 200 mM trehalose, Contains about 0.05% (w / v) polysorbate 80 (PS80), The pH of the composition is about 6.0 and the osmolality of the composition is about 450 mOsm / kg H 2 It is O.

[0111] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.2% (w / v) HGF, Sodium citrate at about 20 mM, Approximately 200 mM proline, Contains about 0.05% (w / v) polysorbate 80 (PS80), The pH of the composition is about 6.0 and the osmolality of the composition is about 450 mOsm / kg H 2 It is O.

[0112] In some embodiments, the pharmaceutical compositions of the present invention comprise Approximately 0.2% (w / v) HGF, Sodium citrate at about 20 mM, About 200 mM sorbitol, Contains about 0.05% (w / v) polysorbate 80 (PS80), The pH of the composition is about 6.0 and the osmolality of the composition is about 450 mOsm / kg H 2 It is O.

[0113] In some embodiments, HGF is administered in combination with a corneal stromal permeability excipient. A stromal permeability excipient is a compound that can enhance the delivery of a therapeutic agent across the corneal layers, mainly including the epithelium (Moiseev et al., 2019, Pharmaceutics, 11:321-354). These compounds, when included in a pharmaceutical composition and administered topically to the eye, can modify the tear film, mucus layer or ocular membrane, thereby increasing the corneal permeability of the therapeutic agent. Non-limiting examples of parenchymal permeable excipients include cyclodextrins (CDs) including α-, β-, and γ-CDs, chelating agents such as ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and ethylenediamine-N,N,N',N'-disuccinic acid (EDDS), crown ethers, surfactants, bile acids and bile salts, and cell-permeable peptides. HGF for in vivo administration is preferably sterile. This can be easily achieved by filtration of an aqueous solution of HGF through a sterile filtration membrane. HGF or its variants are usually stored in lyophilized form or in solution.

[0114] It will be appreciated that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. EXAMPLES

[0115] Example 1: Amino acid sequences of human HGF isoforms available in UniProtKB (uniprot.org / uniprot / P14210) SEQ ID NO:1. Amino acid sequence of Homo sapiens HGF isoform 3; (Identifier: P14210-3, Accession number NP_001010932.1) (dHGF) MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSYRGKDLQENYCRNPRGEEG GPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGESESPWCF TTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTPTIVNLDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEAW LGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS

[0116] SEQ ID NO:2. Amino acid sequence of Homo sapiens HGF isoform 1; (Identifier: P14210-1, Accession number: NP_000592.3) MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRG EEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGESESPW CFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTPTIVNLDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEA WLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS

[0117] SEQ ID NO: 3. Amino acid sequence of Homo sapiens HGF isoform 2 (identifier: P14210-2, accession number: NP_001010931.1): MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKS GIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCET

[0118] SEQ ID NO: 4. Amino acid sequence of Homo sapiens HGF isoform 4 (identifier: P14210-4): MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIK CQPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKNMRDITWALN

[0119] SEQ ID NO:5. Amino acid sequence of Homo sapiens HGF isoform 5 (identifier: P14210-5, NP_001010933.1): MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSI TKSGIKCQPWSSMIPHEHSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCET

[0120] SEQ ID NO:6. Amino acid sequence of Homo sapiens HGF isoform 6 (identifier: P14210-6, NP_001010934.1): MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMS SGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEGK

[0121] SEQ ID NO:7. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALEIK TKKANTADQCANRCTRSKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKDYIRDCIIGKGRSYRGTVSITKSGIKCQPWSAMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0122] Sequence number 8. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIK TEKANTADQCANRCIRNKGLPFTCKAFVFDKARKRCLWFPVNSMSSGVKKEFGHEFDLYE NKDYTRNCIVGNGRSYRGTVSTTKSGIKCQPWSAMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0123] Sequence number 9. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIK TEKANTADQCANRCTRSKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKDYIRDCIVGNGRSYRGTVSITKSGIECQPWSAMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDDPNIRVGYCSQIPNCDMHSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0124] SEQ ID NO: 10. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAKGQGKRRNTIHEFKKSAKTTLIKIDPALKIK TEKADTADQCANRCTRSKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYE NKDYIRDCIVGNGRSYRGTVSVTKSGIKCQPWSSMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0125] Sequence number 11. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNAIHEFKKSAKATLIKIDPALKIK TEKANTADQCANRCTRSKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKDYIRDCIVGNGRSYRGTVSITKSGIECQPWSSMIPHEHSFLPSSYRGEDLQENYCRNP WGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0126] Sequence number 12. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIK TEKANTADQCANRCTRSKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKDYTRNCIVGNGRSYRGTVSITKSGIECQPWSAMIPHEHSFLPSSYQGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDDPNIRVGYCSQIPNCDMHSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0127] SEQ ID NO: 13. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIK TKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYE NKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNP RGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0128] Sequence number 14. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRDAIHECKRSAKTTLIKIDPALKIK TEKANTADQCANRCTRNKGLPSTCKAFVFDKARKRRLRFPFNSMSSGVKKEFGHEFDLYE NKDYTRNCIVGKGRSYRGTVSTTKSGIKCQPWSAMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDDPNIRVGYCSQIPNCDMHSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0129] SEQ ID NO: 15. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQGKRRNTIHEFKKSAKTTLIKIDPALKIK TEKVNTADQCANRCTRSKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKDYIRDCIIGRGRSYRGTVSITKSGIKCQPWSAMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0130] SEQ ID NO: 16. HGF Variant: MWVTKLLPALLLQHVLLHLLLLPIAIPHAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIK TEKANTADQCANRCTRSKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKDYTRNCIVGNGRSYRGTVSTTKSGIKCQPWSAMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0131] SEQ ID NO: 17. HGF Variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIK TEKANTADQCANRCTRSRGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYE NKDYIRDCIIGNGRSYRGTVSVTKSGIKCQPWSAMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0132] SEQ ID NO: 18. HGF Variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIK TEKANTADQCANRCTRNKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKDYIRDCIVGNGRSYRGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0133] SEQ ID NO: 19. HGF Variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSVKTTLIKIDPALKIK TEKANTADQCANRCTRSKGLPFTCKAFVFDKARKRCLWFPVNSMSSGVKKESGHEFDLYE NKDYIRDCIVGNGRSYRGTVSTTKSGIKCQPWSAMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0134] Sequence number 20. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIK TEKANTADQCANRCTRSKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKDYIRDCIVGNGRSYRGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGEDLRENYCRNP WGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0135] SEQ ID NO: 21. HGF Variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALRIK TEKANTADQCANRCTRSRGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKDYIRDCIIGNGRSYRGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0136] Sequence number 22. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIK TEKANTADQCANRCTRSRRLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKDYIRDCIIGKGRSYRGTVSVTKSGIECQPWSAMIPHEHSFLPSNYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0137] Sequence number 23. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIK TEKANTADQCANRCTRSKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKDYIRDCIVGNGRSYRGTVSITKSGIECQPWSAMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDDPNIRVGYCSQIPNCDMHSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0138] SEQ ID NO:24. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIK TKKVDTADQCANRCTRNKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKDYIRDCIIGNGRSYRGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0139] Sequence number 25. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQGKRRNTIHEFKKSAKTTLIKIDPALRIK TEKANTADQCANRCTRSKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKAYIRDCIIGRGRNYRGTVSITKSGIKCQPWSAMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDDPNIRVGYCSQIPNCDMHSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0140] SEQ ID NO: 26. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAKGQRKRRNTIHEFKKSAKTTLIKIDPALEIK TEKVNTADQCANRCIRNKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKAYIRDCIIGRGRNYRGTVSITKSGIKCQPWSAMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDDPNIRVGYCSQIPNCDMHSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0141] SEQ ID NO:27. HGF variant: MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQGKRRNTIHEFKKSAKTTLIKIDPALKIK TEKVNTADQCANRCTRSKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYE NKDYIRDCIIGNGRSYRGTVSITKSGIKCQPWSAMIPHEHSFLPSSYRGEDLRENYCRNP RGEEGGPWCYTSDPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS ESPWCFTTDDPNIRVGYCSQIPNCDMHSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNME DLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTPTIVNL DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRD LKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAG AEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0142] SEQ ID NO:28. FGF1 wild type: FNLPPGNYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTE TGQYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEKNWFVGLKKNGSCKRG PRTHYGQKAILFLPLPVSSD

[0143] SEQ ID NO:29. FGF1 variant: FNLPPGNYKKPKLLYCSNGGHFLRILPNGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTE TGQYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEKNWFVGLKKNGSCKRG PRTHYGQKAIRFLPLPVSSD

[0144] SEQ ID NO: 30. FGF1 variant: FNLPPGNYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDPHIQLQLIAESVGEVYIKSTE TGQYLAMDTDGLLYGSQTPNEECLFLERLEENGYNTYISKKHAEKNWFVGLKKNGSCKRG PRTHYGQKAILFLPLPVSSD

[0145] SEQ ID NO: 31. FGF1 variant: FNLPPGNYKKPKLLYCSNGGHFLRILPNGTVDGTRDRSDPHIQLQLIAESVGEVYIKSTE TGQYLAMDTDGLLYGSQTPNEECLFLERLEENGYNTYISKKHAEKNWFVGLKKNGSCKRG PRTHYGQKAIRFLPLPVSSD

[0146] SEQ ID NO: 32. FGF1 variant: FNLPPGNYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTE TGQYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEKNWFVGLKKNGSCKRG PRTHYGQKAIRFLPLPVSSD

[0147] SEQ ID NO: 33. FGF1 variant: FNLPPGNYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTE TGQYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEKNWFVGLKKNGSCKRG PRTHYGQKAIKFLPLPVSSD

[0148] SEQ ID NO:34 Signal peptide of SEQ ID NO:1 MWVTKLLPALLLQHVLLHLLLLPIAIPYAEG

[0149] SEQ ID NO:35 Uncleaved alpha and beta chain peptide of SEQ ID NO:1 QRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEV ECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGSESPWCFTTDDPNIRVGYCSQIPN CDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLRVVNGIPRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEAWLGIHDVHG RGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS

[0150] SEQ ID NO:36 Alpha chain peptide of SEQ ID NO:1 QRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKS GIKCQPWSSMIPHEHSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDG QPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGSESPWCFTTDPNIRVGYCSQ IPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTPTIVNLDHPVISCAKTKQLR

[0151] SEQ ID NO:37 Beta chain peptide of SEQ ID NO:1 VVNGIPRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTS CSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS

[0152] Example 2: Formulation Design of Experiments (DoE) studies were performed to identify the optimal pH, buffer and stabilizers that could prevent aggregation of HGF in solution formulations and maintain the highest physicochemical stability. HGF protein (activated dHGF from SEQ ID NO: 1) was formulated at 34.67 mg / mL in 10 mM citrate, 1 M NaCl, 0.075% PS80, pH 6.0, and buffer exchanged into the formulations listed in Table 3 to a concentration of 1 mg / mL using an Amicon-15 concentrator unit (10 kDa MWCO). A volume of 340 μL of the 34.67 mg / mL solution was added to the pre-washed concentrator (with the appropriate buffer) and diluted to 15 mL in the appropriate formulation buffer (without surfactant). The sample was centrifuged at 3200 rcf until a volume of approximately 5 mL was achieved, and then diluted again to a total volume of approximately 15 mL with the appropriate formulation buffer. This buffer exchange process was repeated for 5 cycles with a total dilution of approximately 3610 times. A final centrifugation cycle reduced the sample volume to approximately 7 mL. Assuming a worst-case recovery of approximately 60%, the final protein concentration of each sample was approximately 1 mg / mL, with an estimated residual concentration of PS80 of approximately 0.004%.

[0153] Extinction coefficient 1.890mL mg -1 cm -1 Buffer exchanged samples were analyzed for protein content by UV-visible spectroscopy using the 10% PS80 in HGF formulation (w / v, USP grade PS80, prepared from JT Baker, Cat. No. 4117-04 or equivalent) was added to a target concentration of 0.05% PS80. The theoretical residual PS80 level at the end of the buffer exchange process (~0.004%) was not considered because the residual level of PS80 was minimal compared to the amount of spiked PS80.

[0154] After normalization for protein content and surfactant concentration, the formulations were sterile filtered using 0.22 μm sterile concentrators (Millipore Ultrafree-CL GV, P / N UFC40GV0S). Each formulation was sterile filtered into six sterile Type 1 borosilicate glass vials (2 mL, 13 mm, Catalog No. RTF8418, Afton) at 1.0 mL per vial. The remaining sample volume was transferred to 1 mL LDPE tubes (Cat. No. 03-439-61W Fisher) and used as the initial time point control (T0) for analytical testing. For center-point formulations, 200 μL was transferred from the T0 test vial to 1 mL LDPE tubes (Cat. No. 03-439-61W Fisher) and used for adsorption studies as described below. Sample transfer and sterile filtration procedures were performed in a sterile biosafety cabinet using aseptic technique. Vials were stoppered with sterile Fluorotec stoppers and capped with 13FO aluminum caps with buttons. Two vials of each formulation type were stored under the following storage conditions: 2-8°C for 4 weeks and 40°C for 4 weeks. Sample appearance was assessed for color, clarity, and particulates by visual inspection. Protein thermal stability was assessed by DSF / SLS. Additionally, turbidity measurements were performed using UV-visible spectroscopy by measuring absorbance at 340 nm wavelength. Protein conformational and aggregation stability were determined by DLS and SEC-HPLC, respectively. Samples were further analyzed using reduced and non-reduced capillary gel electrophoresis (CE-SDS) and isoelectric focusing (icIEF) gel electrophoresis. The presence of particulate matter was assessed using a HIAC particle counter, which reported the average number of particles >2 μm, ≥5 μm, ≥10 μm, and ≥25 μm in the samples. All DoE analyses were performed in a randomized order. Data from the TO and stability timepoints of all DoE formulations were evaluated using Design Expert Stat Ease® software to derive statistically significant trends.

[0155] Adsorption studies to evaluate non-specific HGF protein absorption to LDPE container surfaces Samples in LDPE tubes were stored at 2-8 °C for at least 5 days. Protein content, as determined by UV absorbance at 280 nm (A280), was measured for these samples after 5 days of storage and the values ​​were compared to the corresponding results at T0 to determine if there was any adsorption of protein to the LDPE container material. Minimal protein adsorption was observed when stored in LDPE containers, with the % difference in HGF concentration measured after 5 days being less than 4% for the majority of formulations tested, except for formulations containing sucrose and alanine, where an 8% decrease in HGF protein concentration was observed (Table 3). Results from this study support the use of LDPE tubes as a viable container for HGF formulations. [Table 3]

[0156] result In general, lower turbidity was observed in formulations at a higher pH range of 6.0-6.5 when stored at 40°C or 5°C for 1 month (Table 4). Formulations containing 200 mM trehalose showed low turbidity (less than 0.01) across the pH range of 5.0-6.5, and the formulation containing 200 mM trehalose at pH 6.0 showed the lowest turbidity when stored at 40°C for 1 month. [Table 4]

[0157] The presence of particulate matter upon storage at 5°C or 40°C for 1 month, as detected using HIAC, was observed in several formulations (Table 5). Formulations containing 200 mM sorbitol, 200 mM proline, or 200 mM trehalose at pH 5.0, or 200 mM trehalose or 0.08% glycine at pH 6.0, showed the lowest particle counts. However, other analytical data, including turbidity data shown in Table 4, pointed to an overall improved stability of HGF protein at pH 6.0, with only the 200 mM trehalose and 0.08% glycine formulations showing low particle counts upon storage. [Table 5]

[0158] Reducing capillary gel electrophoresis (R-CE-SDS) was used to further evaluate the chemical stability of HGF protein in different formulations. HGF is a heterodimer containing α and β chains linked via disulfide bonds. Under reducing conditions, two peaks were present in the electropherogram (% Main Peak 1 and % Main Peak 2 in Table 6), and the column labeled "% Other" represents potential degradation products of HGF protein. As shown in Table 6, HGF formulations at pH 6.0 generally exhibited better chemical stability during long-term storage at both 40°C and 5°C, with the highest % total main peak observed in formulations containing 200 mM trehalose or 200 mM proline at pH 6.0. HGF formulations containing 0.08% glycine, which showed good stability in terms of particle count and turbidity, were found to have poor chemical stability as revealed by R-CE-SDS. [Table 6]

[0159] Example 3: Bioassays to determine relative potency Formulations that exhibited the best overall attributes as identified in the formulation development experiments described in Example 2 were further evaluated for relative potency using a cell proliferation promoting activity potency assay with Mv.1.Lu cells (American mink lung cell line) in the presence of transforming growth factor beta (TGF-β). TGF-β inhibits cell proliferation in Mv.1.Lu cells, and the HGF formulations described herein can reverse this proliferation inhibition. Mv.1.Lu cells were thawed and the viability of the cell stock was assessed by trypan blue staining. After determination of viable cell density, an appropriate number of cells were transferred to flasks for initial culture. Cells were serially propagated and expanded in tissue culture flasks before plating for sample analysis.

[0160] Each reference standard and each test sample was diluted with assay medium to generate an 8-point dilution curve. The potency of each test sample was determined by comparison of the dilution curve generated by the test sample to the reference standard. Cells cultured from tissue culture flasks were trypsinized and plated at approximately 0.1 × 10 6 The cells were resuspended in 1000 cells / mL. 50 microliters of the cell suspension was plated into each well of a 96-well plate. The 96-well plate containing the cultured cells was incubated at 37°C / 5% CO 2 After incubation at 4°C for 1-4 hours, the reference standards and test samples were added. Test samples and reference standards were added in triplicate to separate wells and the cells were incubated for 72 hours.

[0161] After incubation, MTS proliferation reagent (Promega, Cat. No. G3582) or equivalent was added to each well and the 96-well plate was incubated at 37°C / 5% CO 2 The plates were incubated at 490 nm for 4 hours. After this incubation, the plates were placed in a UV-Vis plate reader and the absorbance was measured at 490 nm. Each reference standard and test sample was analyzed in triplicate and potency was determined by parallel line evaluation. Each sample was compared to the reference standard by a four-parameter constrained fit to determine relative potency.

[0162] Test sample (TS) pass criteria: 1.TS independent 4 parameter curve fit should show a dose-dependent relationship when plotting mean response vs log protein concentration. 2. TS independent 4-parameter fit R 2 The value must be ≥ 0.97. 3. All %CV values ​​for TS must be ≦20%. 4. System Suitability Sample potency is determined using a global fit model, and the F probability for each TS must be ≥ 0.01. 5. Test samples that do not pass all of the above criteria will be retested under the same conditions. 6. Assay plates that do not pass any of the above criteria must be retested once under the same conditions. 7. The %RP of the standard sample (SS) must be within the range of 50% to 200%.

[0163] Results from the in vitro relative potency bioassay of selected formulations are summarized in Table 7. It was determined that the HGF solution formulation containing 200 mM trehalose at pH 6.0 maintained excellent potency (99%) for HGF protein. [Table 7]

[0164] Example 4: Efficacy of topical formulations in a mouse corneal mechanical injury model The efficacy of a topical formulation of HGF for use in the treatment of NK was tested in a mouse corneal injury model. The efficacy study was conducted in two phases using male C57BL / 6 mice. In Phase I, a 3.5 μL volume of test sample was administered via pipette to one eye per each of 75 animals four times per day (QID), 2 h between doses on Day 0 (injury), and 3 h between doses for the remainder of the study (Day 8). Animals (approximately 10-20%) that did not respond to injury (i.e., had no corneal clouding / opacity on Day 3) were excluded from the study. The experimental design of the Phase I study is summarized below in Table 8. [Table 8] [Table 9]

[0165] In Phase 2 of the efficacy study, 85 animals were dosed on the injured cornea with a 3.5 μL volume of the test article four times per day (QID), 2 hours between doses on Day 11 (injury) and 3 hours between doses for the remainder of the study (Day 20). At the first treatment application (Day 11), the corneal epithelium was removed by gently applying the tip of an Algerbrush to the opaque area of ​​the cornea (marked with a 1 mm perforator). The corneal epithelium was removed a second time on Day 16. The Phase 2 study design is summarized in Table 10 below. [Table 10]

[0166] Corneal Injury (Phase 1 and 2): Prior to model introduction, test subjects received 0.01-0.05 mg / kg buprenorphine subcutaneously. Animals were also given a cocktail of tropicamide (1.0%) and phenylephrine (2.5%) topically to dilate and proptose the eye. Animals were then sedated for the surgical procedure with a ketamine / xylazine cocktail and one drop of 0.5% proparacaine HCl. Forceps (e.g., Dumont #4) were used to proptose the animal's eye, and a 2 mm perforator was placed over the central cornea using mild pressure. The perforator was rotated with gentle pressure for a full 3 hours to define the defect area. An Algerbrush II with a 0.5 mm burr (Alger Company Inc., Lago Vista, TX) was used to remove the corneal epithelium and anterior stroma within the perforator area. Damaged stroma was characterized by the appearance of stromal debris. Stromal debris was removed by irrigation with balanced salt saline (BSS). Animals were scanned by optical coherence tomography (OCT) and stained with fluorescein before applying the first dose of treatment to the injured eye. Topical antibiotic (Ofloxacin) was applied 15 min later, and animals were allowed to recover normally from surgery. Animals received a second dose of 0.01-0.05 mg / kg buprenorphine administered subcutaneously approximately 6-8 hours postoperatively, and then twice daily every 6-8 hours for 2 days postoperatively.

[0167] Corneal damage (epithelial removal, Phase 2 only): Prior to the start of phase 2, a pilot study was performed to determine the optimal number of epithelial scrapings after the initial injury, as described above for phase 1. Two scrapings were sufficient for the desired model induction. At the start of phase 2, animals were administered 0.01-0.05 mg / kg buprenorphine subcutaneously. Animals were also administered a cocktail of tropicamide (1.0%) and phenylphenidine (2.5%) topically to dilate and proptose the eye. Animals were then sedated for surgical procedures with a ketamine / xylazine cocktail and one drop of 0.5% proparacaine HCl was applied. On day 0, animals underwent surgical procedures as described above for phase 1. On days 11 and 16 after the initial injury and scar formation, the animal's eye was proptose using forceps (e.g., Dumont #4) and, using mild pressure, a 1 mm perforator was placed over the scarred area of ​​the cornea. The perforator was rotated with gentle pressure for 3 full hours to define the defect area. The corneal epithelium in the perforator area was removed using an Algerbrush II (Alger Company Inc., Lago Vista, TX) with a 0.5 mm burr. The ocular surface was then washed with BSS. The animals were scanned by optical coherence tomography (OCT), fluorescein staining, and photography before the first dose of treatment was applied to the injured eye. Topical antibiotics (Ofloxacin) were applied 15 minutes later, and the animals were allowed to recover normally from surgery. The animals received two doses of 0.01-0.05 mg / kg buprenorphine subcutaneously approximately 6-8 hours postoperatively, and twice daily (BID) every 6-8 hours for 2 days postoperatively.

[0168] Monitored parameters: (i) Examination and Weight: Mortality and morbidity were monitored twice daily with cage-side observations, with particular attention paid to both eyes. Due to a significant amount of handling during phase 1 of the study for dosing and imaging, n=18 animals died (group 1: 4 mice, group 2: 3 mice, group 3: 3 mice, group 4: 3 mice, group 5: 5 mice) or were euthanized prior to study termination. No mice died during the pilot phase 2. In phase 2, the number of imaging sessions was reduced and only n=3 mice died (group 1: 1 mouse, group 2: 1 mouse, group 4: 1 mouse). Body weights were measured prior to model introduction and at necropsy.

[0169] (ii) Ocular examination and brightfield imaging: Baseline ocular surface morphology was assessed by a veterinary ophthalmologist under a slit lamp biomicroscope prior to enrollment. Brightfield images were acquired for analysis of corneal opacity using Image J software at baseline and daily, as indicated in the study design.

[0170] (iii) Fluorescein staining: At the time points indicated in the experimental design table, animals underwent fluorescein ocular surface staining. Approximately 1.5 μl of 2.5% sodium fluorescein was applied to the corneal surface for 30 seconds, followed by rinsing with 1× PBS using a 1 ml syringe. Corneal staining was photographed under cobalt blue light using a Nikon digital SLR camera mounted on a tripod. The camera lens was placed in manual mode with the lens positioned 1 foot away. Images were collected with the animal's eye focused in the center of the viewing window and maintaining a consistent distance between the camera lens and the animal's eye. The area of ​​fluorescein staining (in pixels) for each eye at each time point was determined using Image J Software (NIH).

[0171] (iv) Optical Coherence Tomography (OCT): On the days indicated by the experimental design table, all animals underwent an anterior ocular OCT imaging procedure. Animals were anesthetized and the corneas were washed with PBS prior to OCT examination.

[0172] (v) Histology (Phase 1 only): On day 3, following final in-life measurements, a subset of animals were humanely euthanized by carbon dioxide asphyxiation followed by chest incision. The surgical eyes of the animals were harvested in 10% neutral buffered formalin. The following day, eyes were placed in 70% ethanol, embedded in paraffin wax, and sectioned sagittally. Three slides containing serial sections including the optic nerve were stained with hemotoxylin and eosin (H&E) for morphological analysis. Additionally, blocks remaining after H&E were further sectioned and subjected to immunohistochemistry for TUJ-1 staining (also known as beta III tubulin) to assess corneal innervation. DAPI (blue) was used as a counterstain to highlight all nuclei. Slides were analyzed by a veterinary ophthalmologist.

[0173] (vii) Results: 1) Accelerated healing (fluorescein staining) More TUJ01 staining was observed in tissue samples from animals treated with 0.1% dHGF 3 days after surgery compared to animals treated with PBS vehicle (Figure 1), indicating corneal innervation in animals treated with 0.1% dHGF. Epithelial healing time was faster with 0.1% dHGF (p=0.041) and 0.2% dHGF compared to PBS control and 0.1% mNGF (Figure 2). The time to complete healing of 50% of the eyes was 3 days in the 0.1% dHGF group, half the time of the PBS group (6 days, p=0.41). Both the 0.2% dHGF and 0.1% mHGF groups healed at two-thirds the rate of the PBS group (4 days vs. 6 days). Furthermore, 0.1% mHGF was significantly faster than mNGF (p=0.053) and PBS (p=0.0018). Animals receiving 0.1% dHGF initiated closure sooner, with >75% reaching closure by day 4 after treatment, and a similar effect was seen with 0.1% mHGF (Figure 2). In comparison, only 50% of PBS-treated animals reached complete wound closure, and this was not achieved until day 6.

[0174] 2) Scar prevention (bright field image analysis) Following surgery and daily treatment with PBS (control), 0.1% or 0.2% dHGF, 0.1% mNGF, or 0.1% mHGF, all test samples except the PBS control effectively reduced the average scar size by day 7 (Figure 3). Quantification of the effect of the test samples on scar formation revealed that 0.1% mHGF had the greatest effect, followed by 0.1% dHGF. The dHGF groups had significantly smaller scars than the PBS control (p=0.0439 for the 0.1% group and p=0.0126 for the 0.2% group, respectively), but were not significantly different from each other (p=0.7406). The average area of ​​scar in the 0.1% mHGF group was significantly smaller at day 7 than the control group (p=0.0001) and 0.1% mNGF (p=0.0029). The mean scar size in the control and 0.1% mNGF groups was not significantly different from each other (p=0.2481).

[0175] The effect size driven by HGF is reflected in the time to closure analysis (see section vii.1). The reduction in scar formation with 0.1% mHGF was approximately 9-fold greater than that observed in the control group, while the percent reduction with 0.1% and 0.2% dHGF was more than 5-fold greater than control. The percent reduction with 0.1% mNGF was approximately half that of the dHGF group, resulting in a numerically greater reduction in scar size than the PBS control group, but this was not statistically significant.

[0176] 3) Scar reversal (bright-field image analysis) After surgery to remove the corneal epithelium and anterior stroma, and two repeated scrapes to remove only the epithelium, PBS-treated animals showed little change in scar size, whereas animals receiving 0.1% dHGF, 0.2% dHGF, or 0.1% mHGF showed a decrease in corneal scar size. In the PBS control group, the average scar size continued to increase by more than 20% from baseline (day 8) to day 21. In contrast, the size of the scars in the three HGF groups decreased by more than 35% from baseline to day 21, with an overall difference in average size of 55% compared to the PBS group. As shown in Figure 4, the difference between 0.1% dHGF and 0.2% dHGF compared to the PBS control was statistically significant (p=0.0054 and p=0.0015, respectively). The average scar size of the 0.1% mNGF group decreased from day 8 to day 21, but was not significantly different from the PBS control (p=0.0859). Results from treatment with either 0.1% or 0.2% dHGF were not significantly different (p=0.7558).

[0177] Based on the results of this study, 0.1% dHGF treatment following a single epithelial and anterior stromal removal (Phase 1) or multiple epithelial abrasions (Phase 2) resulted in faster wound closure (Phase 1) and a greater reduction in scar size (Phase 2) when administered four times daily starting on the date of the initial surgical procedure (Phase 1) or on day 11 (Phase 2).

[0178] In phase 1, all animals underwent a single surgical procedure to remove the corneal epithelium and anterior stroma within a 2 mm area. All animals received 4 doses per day after treatment. Approximately 50% of PBS-treated animals (vehicle control) were healed by postoperative day 7, whereas >75% of animals receiving 0.1% dHGF or 0.1% mHGF were healed by day 7 (Figures 2 and 3). Furthermore, a greater proportion of corneas in these animals were completely healed by postoperative day 3 compared to those treated with PBS (>60% vs. 30%, respectively). Analysis of corneal innervation on day 3 by immunofluorescence revealed increased TUJ-1 staining in 0.1% dHGF-treated animals (Figure 1).

[0179] In phase 2, animals underwent the same surgical procedures as in phase 1, but then underwent two additional procedures that removed only the epithelium. In this experiment, animals began receiving treatment after the initial epithelial abrasion. Under these conditions, animals receiving HGF (0.1% dHGF, 0.2% dHGF, or 0.1% mHGF) showed a substantial reduction in scar size compared to PBS-treated eyes, with scar size being reduced by >35% in all HGF-treated eyes (Figure 4).

[0180] The results from this study indicate that topical administration of HGF following corneal epithelial injury resulted in a faster healing rate and reduced scar size in single and multiple injury models.

[0181] Example 5: Efficacy of topical formulations in a mouse corneal bacterial LPS-induced keratitis model The efficacy of a topical formulation of HGF for use in the treatment of NK was tested in a murine corneal bacterial LPS-induced keratitis model. Efficacy testing was performed in male C57BL / 6 mice. Test article was administered via pipette in a 3.5 μL volume to one eye per each of 40 animals, 3 h, four times per day (QID) between dosing days 4-9 post-wound. Injured eyes received test article or control as indicated in the experimental design (Table 11). A stock solution of lipopolysaccharide (LPS) from E. coli O111:B4 (Invivogen) was diluted in 1 mL of sterile water, aliquoted, and stored according to the manufacturer's protocol. On the day of dosing, the diluted LPS solution was further diluted 1:1 (v / v) with PBS, and approximately 5 μg of LPS material was delivered in a 2.0 μL injection. [Table 11]

[0182] Corneal injury using LPS from E. coli O111:B4 Prior to model induction, test subjects received 0.01-0.05 mg / kg buprenorphine subcutaneously. Animals were also given a cocktail of tropicamide (1.0%) and phenylephrine (2.5%) topically to dilate and proptose the eyes. Animals were then sedated for the surgical procedure with a ketamine / xylazine cocktail and one drop of 0.5% proparacaine HCl. Forceps (e.g., Dumont #4) were used to proptose the animal's eyes, and approximately 5 μg of LPS (E. coli O111:B4) was injected in a volume of 2.0 μL using a 34 G needle attached to a 2.5 μL syringe. LPS injections were performed on days 0 and 4. The first dose of treatment was applied to the injured eye after the day 4 injection of LPS. A topical antibiotic (Ofloxacin) was applied 15 minutes later, and the animals were allowed to recover normally from surgery. Animals received a second dose of 0.01-0.05 mg / kg buprenorphine administered subcutaneously approximately 6-8 hours after surgery, and then twice daily every 6-8 hours for 2 days after surgery.

[0183] Monitored parameters (i) Examination and Weight: Mortality and morbidity were monitored twice daily with cage side observations. Body weights were measured before model administration and at necropsy.

[0184] (ii) Ocular examination and brightfield images: Prior to enrollment, ocular surface morphology was assessed by a veterinary ophthalmologist under a slit-lamp biomicroscope. At baseline and on the days indicated in the study design, bright-field images were acquired for analysis of corneal opacity using Image J software.

[0185] (iii) Fluorescein staining: At the time points indicated in the experimental design table, animals underwent fluorescein ocular surface staining. Approximately 1.5 μl of 2.5% sodium fluorescein was applied to the corneal surface for 30 seconds, followed by rinsing with 1× PBS using a 1 ml syringe. Corneal staining was photographed under cobalt blue light using a Nikon digital SLR camera mounted on a tripod. The camera lens was placed in manual mode and set 1 foot away. Images were collected with the animal's eye focused in the center of the viewing window, maintaining a consistent distance between the camera lens and the animal's eye. The area of ​​fluorescein staining (in pixels) for each eye at each time point was determined using Image J Software (NIH).

[0186] (iv) Optical Coherence Tomography (OCT): On the days indicated in the experimental design table, all animals underwent an anterior ocular OCT imaging procedure. Animals were anesthetized and the corneas were washed with PBS prior to OCT examination.

[0187] (v) Results: Following induction of the model and treatment four times daily with PBS (control), 0.1% or 0.2% dHGF, or 0.1% mNGF, groups 2 and 3 (0.1% and 0.2% dHGF, respectively) showed the greatest reduction in scar size by day 9. Normalized to group 1, groups 2 and 3 reduced scar size by nearly 60%, while the test sample in group 4 reduced scar size by approximately 25% (Figure 5). The mean scar area was significantly smaller with 0.1% dHGF (p=0.0110) and 0.2% dHGF (p=0.0028) compared to the PBS control. Scars were approximately 5-fold larger in the PBS control compared to the 0.1% and 0.2% HGF groups, and approximately 2.5-fold larger in PBS than the 0.1% mNGF group (p=0.0417). The resulting mean scar sizes in the two dHGF groups were not significantly different from each other (p=0.6176) (FIG. 5).

[0188] In summary, the results from this study suggest that treatment of injured eyes with HGF following corneal epithelial injury induced by stromal administration of E. coli LPS led to improved healing rates.

[0189] Various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each of the references cited in this application, including all patents, patent applications, and publications, is hereby incorporated by reference in its entirety.

Claims

1. 1. An aqueous pharmaceutical composition comprising: About 0.01% to about 1.0% (w / v) HGF; a buffer capable of maintaining the pH of the composition at about 5.8 to about 6.2; and about 100 to about 300 mM of a stabilizer selected from trehalose, proline, sorbitol, and mixtures thereof.

2. 10. The aqueous pharmaceutical composition of claim 1, comprising about 0.05% to about 0.5% (w / v) HGF.

3. 10. The aqueous pharmaceutical composition of claim 1, comprising about 0.08% to about 0.25% (w / v) HGF.

4. 10. The aqueous pharmaceutical composition of claim 1, comprising about 0.1% (w / v) HGF.

5. 10. The aqueous pharmaceutical composition of claim 1, comprising about 0.2% (w / v) HGF.

6. 10. The aqueous pharmaceutical composition of claim 1, wherein the composition has a pH of about 6.

0.

7. 10. The aqueous pharmaceutical composition of claim 1, comprising about 150 to about 250 mM of a stabilizer.

8. 10. The aqueous pharmaceutical composition of claim 1, comprising about 200 mM of a stabilizer.

9. 2. The aqueous pharmaceutical composition of claim 1, wherein the stabilizer is trehalose or proline.

10. 2. The aqueous pharmaceutical composition of claim 1, wherein the stabilizer is trehalose.

11. 2. The aqueous pharmaceutical composition of claim 1, wherein the stabilizer is proline.

12. 2. The aqueous pharmaceutical composition of claim 1, wherein the stabilizer is sorbitol.

13. 2. The aqueous pharmaceutical composition of claim 1, wherein the buffer is a citrate buffer.

14. 2. The aqueous pharmaceutical composition of claim 1, wherein the buffer is sodium citrate.

15. 10. The aqueous pharmaceutical composition of claim 1, comprising about 10 to about 50 mM buffer.

16. The composition has an osmolality of about 450 mOsm / kg H ２ 2. The aqueous pharmaceutical composition of claim 1, wherein

17. 10. The aqueous pharmaceutical composition of claim 1, which does not contain a tonicity agent.

18. 10. The aqueous pharmaceutical composition of claim 1, comprising a tonicity agent that is an alkali metal salt.

19. 10. The aqueous pharmaceutical composition of claim 1, comprising a tonicity agent that is sodium chloride.

20. 10. The aqueous pharmaceutical composition of claim 1, comprising a surfactant.

21. 21. The aqueous pharmaceutical composition of claim 20, wherein the surfactant is selected from polysorbate 80 (PS80), polaxomer 188, and polaxomer 407.

22. 21. The aqueous pharmaceutical composition of claim 20, wherein the surfactant is polysorbate 80 (PS80).

23. 21. The aqueous pharmaceutical composition of claim 20, wherein the surfactant is present in an amount of about 0.01% to about 0.1% (w / v).

24. 21. The aqueous pharmaceutical composition of claim 20, wherein the surfactant is present in an amount of about 0.02% to about 0.8% (w / v).

25. 21. The aqueous pharmaceutical composition of claim 20, wherein the surfactant is present in an amount of about 0.05% (w / v).

26. 1. An aqueous pharmaceutical composition comprising: About 0.1% (w / v) HGF; About 20 mM sodium citrate; about 200 mM trehalose, proline, or sorbitol; about 0.05% (w / v) surfactant; The aqueous pharmaceutical composition, wherein the pH of the composition is about 6.

0.

27. About 0.1% (w / v) HGF; About 20 mM sodium citrate; About 200 mM trehalose; and about 0.05% (w / v) polysorbate 80 (PS80); 27. The aqueous pharmaceutical composition of claim 26, wherein the pH of the composition is about 6.

0.

28. About 0.1% (w / v) HGF; About 20 mM sodium citrate; About 200 mM proline; and about 0.05% (w / v) polysorbate 80 (PS80); 27. The aqueous pharmaceutical composition of claim 26, wherein the pH of the composition is about 6.

0.

29. About 0.1% (w / v) HGF; About 20 mM sodium citrate; About 200 mM sorbitol; and about 0.05% (w / v) polysorbate 80 (PS80); 27. The aqueous pharmaceutical composition of claim 26, wherein the pH of the composition is about 6.

0.

30. 1. An aqueous pharmaceutical composition comprising: About 0.2% (w / v) HGF; About 20 mM sodium citrate; about 200 mM trehalose, proline, or sorbitol; about 0.05% (w / v) surfactant; The aqueous pharmaceutical composition, wherein the pH of the composition is about 6.

0.

31. About 0.2% (w / v) HGF; About 20 mM sodium citrate; About 200 mM trehalose; and about 0.05% (w / v) polysorbate 80 (PS80); 31. The aqueous pharmaceutical composition of claim 30, wherein the pH of the composition is about 6.

0.

32. About 0.2% (w / v) HGF; About 20 mM sodium citrate; About 200 mM proline; and about 0.05% (w / v) polysorbate 80 (PS80); 31. The aqueous pharmaceutical composition of claim 30, wherein the pH of the composition is about 6.

0.

33. About 0.2% (w / v) HGF; About 20 mM sodium citrate; About 200 mM sorbitol; and about 0.05% (w / v) polysorbate 80 (PS80); 31. The aqueous pharmaceutical composition of claim 30, wherein the pH of the composition is about 6.

0.

34. 27. The aqueous pharmaceutical composition of claim 26, wherein the trehalose, proline, or sorbitol is trehalose, and the surfactant is polysorbate 80.

35. 31. The aqueous pharmaceutical composition of claim 30, wherein the trehalose, proline, or sorbitol is trehalose, and the surfactant is polysorbate 80.

36. 1. An aqueous pharmaceutical composition comprising: About 0.1% (w / v) HGF; About 20 mM sodium citrate; and about 200 mM trehalose.

37. 37. The aqueous pharmaceutical composition of claim 36, wherein the HGF is activated HGF.

38. 38. The aqueous pharmaceutical composition of claim 37, wherein the activated HGF is an activated dHGF.

39. 37. The aqueous pharmaceutical composition of claim 36, having a pH of about 5.8 to about 6.

2.

40. 37. The aqueous pharmaceutical composition of claim 36, having a pH of about 6.

0.

41. 37. The aqueous pharmaceutical composition of claim 36, further comprising about 0.05% (w / v) of a surfactant.

42. 42. The aqueous pharmaceutical composition of claim 41, wherein the surfactant is polysorbate 80.

43. Osmolality is about 200 to about 500 mOsm / kg H ２ 37. The aqueous pharmaceutical composition of claim 36, wherein

44. 1. An aqueous pharmaceutical composition comprising: About 0.1% (w / v) HGF; About 20 mM sodium citrate; and about 200 mM proline.

45. 45. The aqueous pharmaceutical composition of claim 44, wherein the HGF is activated HGF.

46. 46. ​​The aqueous pharmaceutical composition of claim 45, wherein the activated HGF is an activated dHGF.

47. 45. The aqueous pharmaceutical composition of claim 44, having a pH of about 5.8 to about 6.

2.

48. 45. The aqueous pharmaceutical composition of claim 44, having a pH of about 6.

0.

49. 45. The aqueous pharmaceutical composition of claim 44, further comprising about 0.05% (w / v) of a surfactant.

50. 50. The aqueous pharmaceutical composition of claim 49, wherein the surfactant is polysorbate 80.

51. Osmolality is about 200 to about 500 mOsm / kg H ２ 45. The aqueous pharmaceutical composition of claim 44, wherein

52. 1. An aqueous pharmaceutical composition comprising: About 0.1% (w / v) HGF; About 20 mM sodium citrate; and about 200 mM sorbitol.

53. 53. The aqueous pharmaceutical composition of claim 52, wherein the HGF is activated HGF.

54. 54. The aqueous pharmaceutical composition of claim 53, wherein the activated HGF is an activated dHGF.

55. 53. The aqueous pharmaceutical composition of claim 52, having a pH of about 5.8 to about 6.

2.

56. 53. The aqueous pharmaceutical composition of claim 52, having a pH of about 6.

0.

57. 53. The aqueous pharmaceutical composition of claim 52, further comprising about 0.05% (w / v) of a surfactant.

58. 58. The aqueous pharmaceutical composition of claim 57, wherein the surfactant is polysorbate 80.

59. Osmolality is about 200 to about 500 mOsm / kg H ２ 53. The aqueous pharmaceutical composition of claim 52, wherein

60. 60. The aqueous pharmaceutical composition of any one of claims 1 to 59, wherein the HGF is activated HGF.

61. 61. The aqueous pharmaceutical composition of claim 60, wherein the activated HGF is an activated dHGF.

62. The HGF (a) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:2, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:2, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:2, or (b) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:7, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:7, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:7, or (c) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:8, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:8, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:8, or (d) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:9, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:9, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:9, or (e) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:10, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:10, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:10, or (g) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:11, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:11, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:11, or (h) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:12, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:12, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:12, or (i) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO: 13, optionally comprising a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO: 13, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO: 13, or (j) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:14, optionally comprising a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:14, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:14, or (k) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:15, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:15, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:15; or (l) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:16, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:16, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:16, or (m) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:17, optionally comprising a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:17, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:17; or (n) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:18, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:18, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:18; or (o) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:19, optionally comprising a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:19, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:19, or (p) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:20, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:20, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:20, or (q) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:21, optionally comprising a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:21, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:21, or (r) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:22, optionally comprising a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:22, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:22, or (s) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:23, optionally comprising a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:23, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:23, or (t) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:24, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:24, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:24; or (u) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:25, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:25, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:25, or (v) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32-494 of SEQ ID NO:26, optionally comprising a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:26, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495-728 of SEQ ID NO:26, or 60. The aqueous pharmaceutical composition of any one of claims 1 to 59, comprising: (w) a first polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 32 to 494 of SEQ ID NO:27, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:27, and a second polypeptide comprising an amino acid sequence having at least 80%, preferably 90%, identity to amino acids 495 to 728 of SEQ ID NO:

27.

63. The HGF (a) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:2, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:2, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:2; or (b) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:7, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:7, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:7; or (c) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:8, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:8, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:8; or (d) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:9, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:9, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:9; or (e) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:10, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:10, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:10; or (g) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:11, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:11, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:11; or (h) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:12, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:12, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:12; or (i) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO: 13, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO: 13, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO: 13; or (j) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:14, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:14, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:14; or (k) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:15, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:15, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:15; or (l) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:16, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:16, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:16; or (m) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:17, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:17, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:17; or (n) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:18, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:18, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:18; or (o) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:19, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:19, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:19; or (p) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:20, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:20, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:20; or (q) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:21, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:21, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:21; or (r) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:22, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:22, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:22; or (s) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:23, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:23, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:23; or (t) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:24, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:24, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:24; or (u) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:25, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:25, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:25; or (v) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32-494 of SEQ ID NO:26, optionally including a deletion of amino acids corresponding to amino acids 161-165 of SEQ ID NO:26, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495-728 of SEQ ID NO:26; or 60. The aqueous pharmaceutical composition of any one of claims 1 to 59, comprising: (w) a first polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 32 to 494 of SEQ ID NO:27, optionally comprising a deletion of amino acids corresponding to amino acids 161 to 165 of SEQ ID NO:27, and a second polypeptide comprising or consisting of an amino acid sequence corresponding to amino acids 495 to 728 of SEQ ID NO:

27.

64. 63. The aqueous pharmaceutical composition of claim 62, wherein the first polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:36 and the second polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:

37.

65. 63. The aqueous pharmaceutical composition of claim 62, wherein the N-terminal amino acid of the first polypeptide is pyrrolidone carboxylic acid.

66. 63. The aqueous pharmaceutical composition of claim 62, wherein the first polypeptide and the second polypeptide are linked by one or more disulfide bonds.

67. 61. The aqueous pharmaceutical composition of claim 60, wherein the HGF is capable of binding to c-MET and / or activating the MAPK pathway in epithelial cells.

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