Recombinant modified fibroblast growth factor and its therapeutic use

Abstract

This specification describes modified fibroblast growth factor (FGF) polypeptides, pharmaceutical compositions and drugs comprising such modified FGF polypeptides, and methods of using such modified FGF polypeptides to treat or prevent diseases for which FGF administration is beneficial.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 492,570, filed Mar. 8, 2023, which is incorporated herein by reference in its entirety.

[0002] Reference to Electronically Submitted Sequence Listing This application incorporates by reference a sequence listing submitted herewith as a text file entitled “45341-713_601_SL.xml,” created on Mar. 18, 2024, and having a size of 227,701 bytes.

Background Art

[0003] Descemetorhexis without Endothelial Keratoplasty (DWEK), also known as Descemet Stripping Only (DSO), is a procedure for patients with Fuchs endothelial corneal dystrophy (FECD) in which the central region of Descemet's membrane is surgically removed and endothelial keratoplasty is not performed. Corneal endothelial cells (CECs) in the peripheral region of the cornea migrate to the central region of the cornea and become dense again. A potential advantage of DWEK / DSO is that it does not involve transplanted tissue, thereby avoiding related problems regarding tissue availability and possible rejection reactions. Fibroblast growth factor 1 (FGF-1) is known to be the most potent CEC proliferation stimulator. However, FGF-1 in its native or wild-type form (wtFGF-1) is susceptible to both proteolysis and cysteine oxidation, creating a very short biological half-life and thus being unsuitable as a therapy. Despite continued efforts to accelerate patient recovery from DWEK / DSO, the need for an approach that overcomes the inherent limitations of conventional methods for effectively stimulating CEC proliferation and migration remains unmet. [Overview of the project]

[0004] This specification provides methods for treating or preventing corneal diseases or disorders in subjects. In some embodiments, the method comprises the step of intrachorally administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide having at least 90% sequence identity with the wild-type FGF-1 amino acid sequence of SEQ ID NO: 1 and having Cys16Ser, Ala66Cys, and Cys117Val mutations. In some embodiments, the corneal disease or disorder includes Descemet's membrane detachment surgery without endothelial keratoplasty (DWEK) or Descemet's membrane detachment alone (DSO).

[0005] In some embodiments, the modified FGF-1 polypeptide contains methionine upstream of the first residue of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide contains an amino acid sequence that is at least about 90% identical to the sequence of SEQ ID NO: 2. In some embodiments, the modified FGF-1 polypeptide contains an amino acid sequence that is at least about 95% identical to the sequence of SEQ ID NO: 2. In some embodiments, the modified FGF-1 polypeptide contains an amino acid sequence that is at least about 99% identical to the sequence of SEQ ID NO: 2. In some embodiments, the modified FGF-1 polypeptide contains the amino acid sequence of SEQ ID NO: 2.

[0006] In some embodiments, the subject undergoes cataract surgery before the administration step, simultaneously with the administration step, or after the administration step. In some embodiments, the corneal disease or illness includes corneal endothelial dystrophy or corneal endothelial injury.

[0007] In some embodiments, the subject undergoes cataract surgery before the administration step, simultaneously with the administration step, or after the administration step, and the corneal disease or illness includes corneal endothelial dystrophy or corneal endothelial injury.

[0008] In some embodiments, corneal endothelial dystrophy or corneal endothelial injury includes Fuchs dystrophy, bullous keratopathy, congenital hereditary endothelial dystrophy 1, congenital hereditary endothelial dystrophy 2, posterior polymorphic corneal dystrophy, corneal endothelial injury following ophthalmic surgery, corneal endothelial injury following cataract surgery, or corneal endothelial injury following oxidative stress.

[0009] In some embodiments, subjects have risk factors for corneal endothelial injury induced by ophthalmic surgery. In some embodiments, risk factors include diabetes mellitus, low corneal endothelial cell density, corneal endothelial dystrophy, small pupil, shallow anterior chamber, mature cataract or brunescent cataract, or a combination thereof.

[0010] In some embodiments, the subject undergoes cataract surgery before, simultaneously with, or after the administration step, and the corneal disease or condition includes Fuchs dystrophy. In some embodiments, DWEK or DSO is performed before, simultaneously with, or after cataract surgery. In some embodiments, corneal endothelial dystrophy or corneal endothelial injury is caused by cataract surgery. In some embodiments, corneal endothelial dystrophy or corneal endothelial injury is not caused by cataract surgery.

[0011] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition comprises a liquid formulation. In some embodiments, the pharmaceutically acceptable carrier, excipient, or diluent comprises physiological saline. In some embodiments, the physiological saline comprises phosphate-buffered saline (PBS). In some embodiments, PBS comprises one or more salts of sodium ions or potassium ions. In some embodiments, one or more salts comprises sodium chloride, potassium chloride, monosodium phosphate, potassium dihydrogen phosphate monobasic, disodium hydrogen phosphate anhydride, or disodium hydrogen phosphate anhydride. In some embodiments, one or more salts comprises sodium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate anhydride. In some embodiments, the pharmaceutically acceptable carrier, excipient, or diluent comprises a surfactant. In some embodiments, the surfactant comprises polysorbate, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil, or equivalents thereof. In some embodiments, the polysorbate comprises polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. In some embodiments, the polysorbate comprises polysorbate 80. In some embodiments, the pharmaceutically acceptable carrier, excipient, or diluent comprises albumin. In some embodiments, the albumin comprises human serum albumin. In some embodiments, the albumin comprises recombinant human serum albumin. In some embodiments, the pharmaceutically acceptable carrier, excipient, or diluent comprises (i) PBS comprising sodium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate anhydrous, (ii) polysorbate 80, and (iii) recombinant human serum albumin.

[0012] In some embodiments, the concentration of polysorbate is at least about 0.001% (w / v), 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), or 0.1% (w / v). In some embodiments, the concentration of polysorbate is about 0.01% (w / v).

[0013] In some embodiments, the pharmaceutical composition contains at least about 0.1 ng / μl, at least about 0.5 ng / μl, at least about 1 ng / μl, at least about 5 ng / μl, or at least about 10 ng / μl of modified FGF-1 polypeptide. In some embodiments, the pharmaceutical composition contains about 1 ng / μl of modified FGF-1 polypeptide.

[0014] In some embodiments, the pharmaceutical composition has a pH of about 7.2 to about 7.6. In some embodiments, the pharmaceutical composition does not contain heparin.

[0015] In some embodiments, the administration step includes administering a modified FGF-1 polypeptide in a dose of at least about 1 ng, at least about 3 ng, at least about 5 ng, at least about 10 ng, at least about 30 ng, at least about 50 ng, at least about 100 ng, at least about 150 ng, or at least about 200 ng. In some embodiments, the administration step includes administering a modified FGF-1 polypeptide in a dose of up to about 1 ng, up to about 3 ng, up to about 5 ng, up to about 10 ng, up to about 30 ng, up to about 50 ng, up to about 100 ng, up to about 150 ng, or up to about 200 ng. In some embodiments, the administration step includes administering a modified FGF-1 polypeptide in a dose of about 1 ng, about 3 ng, about 5 ng, about 10 ng, about 30 ng, about 50 ng, about 100 ng, about 150 ng, or about 200 ng.

[0016] In some embodiments, the administration step includes administering a first dose and a second dose, the second dose being less than or equal to the first dose, and the first or second dose being at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of modified FGF-1 polypeptide. In some embodiments, the administration step includes administering a first dose and a second dose, the second dose being greater than or equal to the first dose, and the first or second dose being at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of modified FGF-1 polypeptide.

[0017] In some embodiments, the administration step includes administering a first dose, a second dose, and a third dose, where the second dose is less than or equal to the first dose, the third dose is less than or equal to the second dose, and the first dose, second dose, or third dose is at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of modified FGF-1 polypeptide. In some embodiments, the administration step includes administering a first dose, a second dose, a third dose, and a fourth dose, where the first dose, second dose, third dose, or fourth dose is at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of modified FGF-1 polypeptide. In some embodiments, the administration step includes administering a first dose, a second dose, a third dose, a fourth dose, and a fifth dose, where the first dose, second dose, third dose, fourth dose, or fifth dose is at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of modified FGF-1 polypeptide. In some embodiments, the administration step comprises administering at least five doses, each of which is at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of modified FGF-1 polypeptide.

[0018] In some embodiments, the administration step includes administering a certain dose of a modified FGF-1 polypeptide before, simultaneously with, or after the DWEK or DSO treatment.

[0019] In some embodiments, the administration step includes administering a certain dose of modified FGF-1 polypeptide at least 1, 2, 3, 4, 5, 6, 7, 2, 3, or 4 weeks prior to the DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of modified FGF-1 polypeptide up to 1, 2, 3, 4, 5, 6, 7, 2, 3, or 4 weeks prior to the DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of modified FGF-1 polypeptide 2 to 3 days prior to the DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of modified FGF-1 polypeptide on the same day as the DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of a modified FGF-1 polypeptide at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of a modified FGF-1 polypeptide at a maximum of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of a modified FGF-1 polypeptide weekly at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after treatment with DWEK or DSO. In some embodiments, the administration step includes administering a certain dose of a modified FGF-1 polypeptide weekly at a maximum of 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after treatment with DWEK or DSO.In some embodiments, the administration step includes administering a certain dose of modified FGF-1 polypeptide weekly, three weeks after DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of modified FGF-1 polypeptide on days 7, 14, and 21 after DWEK or DSO treatment.

[0020] In some embodiments, the administration step involves administering at least about 10 microliters (mcL), 30 mcL, 50 mcL, or 100 mcL of a pharmaceutical composition having a modified FGF-1 polypeptide at a concentration of 1 ng / mcL.

[0021] In some embodiments, the administration step includes administering the drug one to three times a day. In some embodiments, the administration step includes administering the drug once a day. In some embodiments, the administration step includes administering the drug twice a day. In some embodiments, the administration step includes administering the drug three times a day. In some embodiments, the administration step includes administering the drug one to three times a week.

[0022] In some embodiments, the pharmaceutical composition is administered by microneedles.

[0023] In some embodiments, the administration step includes administering to one eye. In some embodiments, the administration step includes administering to both eyes.

[0024] In some embodiments, DWEK or DSO is performed on one eye. In some embodiments, DWEK or DSO is performed on both eyes. In some embodiments, cataract surgery is performed on one eye. In some embodiments, cataract surgery is performed on both eyes.

[0025] In some embodiments, the administering step results in a therapeutic effect including one or more selected from the group consisting of a decrease in corneal endothelial damage, an increase in corneal endothelial cell migration, an increase in corneal endothelial cell proliferation, a decrease in corneal edema, an improvement in best corrected visual acuity (BCVA), a decrease in corneal thickness, a decrease in central corneal thickness, an improvement in the measurement of visual function and corneal health state (V-FUCHS), a decrease in intraocular pressure, an improvement in slit lamp examination, an improvement in central corneal endothelial cell count, an improvement in peripheral corneal endothelial cell count, or a shortening of the time to achieve any of them.

[0026] In some embodiments, the decrease in corneal endothelial damage includes a decrease in cell death, a decrease in dysfunction, or both.

[0027] In some embodiments, the administering step results in a therapeutic effect at most 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the administering step. In some embodiments, the administering step results in a therapeutic effect at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the administering step.

[0028] In some embodiments, BCVA is measured using an Early Treatment Diabetic Retinopathy Scale (ETDRS) scoring system having a score of at least 69 letters and at least 20 / 40.

[0029] In some embodiments, the administration step includes administering to both eyes, and the administration results in a therapeutic effect in both eyes that is similar or the same between the two eyes. In some embodiments, the administration step includes administering to both eyes, and the administration results in an improvement in BCVA that is similar or the same between the two eyes. In some embodiments, the administration step results in a dose-dependent therapeutic effect. In some embodiments, the therapeutic effect increases with the dose of the modified FGF-1 polypeptide administered. In some embodiments, the administration step results in a therapeutic effect that is not dose-dependent. In some embodiments, the administration step results in a therapeutic effect in subjects with or without cataract surgery, and the therapeutic effect is similar or the same between subjects with and without cataract surgery. In some embodiments, the administration step results in an improvement in corneal edema or central corneal thickness in subjects with or without cataract surgery, and the improvement in corneal edema or central corneal thickness is similar or the same between subjects with and without cataract surgery. In some embodiments, the administration step results in an improvement in BCVA. In some embodiments, the administration step results in a reduction or elimination of corneal edema. In some embodiments, the administration step results in an accelerated reduction or elimination of corneal edema.

[0030] In some embodiments, the method includes a treatment course of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after DWEK or DSO treatment. In some embodiments, the method includes a treatment course of up to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after DWEK or DSO treatment.

[0031] In some embodiments, the administration step includes administration to both eyes, and the administration results in an improvement in BCVA that is similar or the same between the two eyes. In some embodiments, the administration step results in a dose-dependent therapeutic effect. In some embodiments, the therapeutic effect increases with the dose of the modified FGF-1 polypeptide administered. In some embodiments, the administration step results in a therapeutic effect that is not dose-dependent. In some embodiments, the administration step results in a therapeutic effect in subjects with or without cataract surgery, and the therapeutic effect is similar or the same between subjects with and without cataract surgery. In some embodiments, the administration step results in an improvement in corneal edema or central corneal thickness in subjects with or without cataract surgery, and the improvement in corneal edema or central corneal thickness is similar or the same between subjects with and without cataract surgery. In some embodiments, the administration step results in an improvement in BCVA. In some embodiments, the administration step results in a reduction or elimination of corneal edema. In some embodiments, the administration step results in an accelerated reduction or elimination of corneal edema.

[0032] In some embodiments, the method includes a treatment course of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after DWEK or DSO treatment. In some embodiments, the method includes a treatment course of up to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after DWEK or DSO treatment.

[0033] In some embodiments, DWEK / DSO involves a central descemetorhexis of approximately 4 mm to 5 mm. In some embodiments, reducing the size of the DSO results in a reduction in the time to achieve therapeutic effect in the subject.

[0034] In some embodiments, the therapeutic effect includes faster improvement to BCVA 20 / 40, accelerated improvement to BCVA 20 / 40, a reduction in the time to BCVA 20 / 40, or a reduction in corneal edema.

[0035] In some embodiments, the subject has diabetes. In some embodiments, the administration step results in a therapeutic effect including a reduction in corneal thickness or corneal edema.

[0036] In some embodiments, the corneal disease or illness includes Fuchs endothelial cell dystrophy (FECD) with central guttata as a contributing factor to the visual symptoms. In some embodiments, the visual symptoms include decreased visual acuity from the cornea, morning blur, or central corneal edema. In some embodiments, the subject has a clear peripheral cornea with adequate corneal endothelial cell (CEC) reserve capacity. In some embodiments, the subject does not have subepithelial opacity or fibrosis. In some embodiments, the subject does not exhibit secondary corneal pathology, refractive surgery of the eye, or extraocular inflammation caused by non-infectious or infectious pathogens of the eye. In some embodiments, the subject is human.

[0037] Further aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, which only illustrates the exemplary embodiments of the present disclosure. As will be understood, other embodiments and different embodiments are possible, and some of their details can be modified in various obvious ways without departing from the present disclosure. Accordingly, the drawings and description should be considered illustrative and not restrictive.

[0038] Built-in by reference All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually incorporated by reference. [Brief explanation of the drawing]

[0039] Novel features of the present invention are described in detail in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of this disclosure are utilized, and to the appended drawings (also referred to herein as (FIGURE.) or (FIGURES.)). [Figure 1] The complete test treatment schemes of groups 1 to 4 relating to one or more embodiments of this disclosure are shown. [Figure 2] The diagrams of clinical trials involving doses, administration dates, and hospital visits for evaluation, relating to one or more embodiments of this disclosure, are shown. [Figure 3] This shows an intracameral injection entry point according to one or more embodiments of the present disclosure. [Figure 4] This shows an analysis of the proportion of all eyes in pooled groups 3 and 4 for eyes that underwent Descemet's membrane stripping only (DSO) (dashed line) or DSO + cataract surgery (solid line) at each visit, according to one or more embodiments of this disclosure. [Figure 5] This document presents an analysis of the proportion of all eyes in groups 2, and groups 3 and 4, pooled for eyes that underwent DSO only or DSO + cataract surgery at each visit, according to one or more embodiments of this disclosure. [Figure 6]This shows the proportion of subjects / eyes in each group with central corneal thickness (CCT) less than or equal to 10% above baseline at each visit (central corneal thickness (CCT) less than or equal to 110% of baseline). The difference between group 2 (low dose) and groups 3 and 4 (high dose) is statistically significant, with p<0.05 at day 28 by Fisher's exact test according to one or more embodiments of this disclosure. [Figure 7] This shows the proportion of subjects / eyes in each group with central corneal thickness (CCT) less than 10% above baseline at each visit. The difference between group 2 (low dose) and groups 3 and 4 (high dose) is statistically significant, with p<0.05 at day 28 by Fisher's exact test according to one or more embodiments of this disclosure. [Figure 8] This shows the percentage of subjects / eyes in each group that had complete resolution of corneal edema (central corneal thickness below baseline) at each visit. P-values ​​and Fisher's exact test for one or more embodiments of this disclosure. [Figure 9] This represents the percentage of subjects / eyes in each group who have a best corrected visual acuity (BCVA) of 69 letters (20 / 40) or better in all eyes, according to one or more embodiments of the present disclosure, at each visit. [Figure 10] The proportion of subjects / eyes with a 0.6 logMAR (6 lines, 30 characters) increase in BCVA from the first postoperative visit in each group is shown for each visit. A comparison of group 2 vs. group 3 by Fisher's exact test according to one or more embodiments of this disclosure. [Figure 11] The average corneal thickness (+ / -SD) of groups 2 or 3 and 4, pooled for each visit, is shown. According to one or more embodiments of this disclosure, solid bars represent eyes that have received DSO only, and dotted bars represent eyes that have received DSO + cataract surgery. [Figure 12] The average corneal thickness of groups 3 and 4 pooled at each visit is shown. According to one or more embodiments of this disclosure, the light bars include eyes that have received DSO only, and the dark bars include eyes that have received DSO + cataract surgery. [Figure 13]This shows the percentage of subjects / eyes in each group that had complete resolution of corneal edema (central corneal thickness below baseline) at each visit. According to one or more embodiments of this disclosure, solid bars represent eyes that received DSO only, and dotted bars represent eyes that received DSO + cataract surgery. [Figure 14] The average corneal thicknesses of groups 2 and 3 and 4, pooled at each visit, are shown. According to one or more embodiments of this disclosure, the solid line represents eyes that received DSO only, and the dotted line represents eyes that received DSO + cataract surgery. [Figure 15] The mean Early Treatment Diabetic Retinopathy Scale (ETDRS) letter scores for the first and second eyes on days 14, 21, 28, 56, and 84 are shown according to one or more embodiments of the present disclosure. [Figure 16] The median ETDRS character scores for the first and second eyes on days 14, 21, 28, 56, and 84 are shown according to one or more embodiments of the present disclosure. [Figure 17] This document presents an analysis of the proportion of first eye (solid line) and second eye (dashed line) in 13 subjects who underwent bilateral DSO with TTHX1114, measured using an ETDRS scoring system in which ≥69 characters = ≥20 / 40 BCVA, according to one or more embodiments of the present disclosure. [Figure 18] One or more embodiments of this disclosure show that the time to 20 / 40 (weeks) was faster for subjects with smaller DSO (4mm = 4.5, >4.5 to ≤4.5mm = 6.7, ≥5mm = 6.6). [Figure 19] One or more embodiments of this disclosure show that the time to 20 / 40 (weeks) was slower in subjects with diabetes (7.7 compared to 5.2 in subjects without diabetes). [Modes for carrying out the invention]

[0040] Corneal endothelial cell hypocellarization, induced by trauma, surgical injury, or corneal endothelial cell (CEC) loss due to corneal endothelial dystrophy (CED), can have a significant impact on vision and quality of life. Corneal endothelial cell loss or low CEC count contributes to poor outcomes in ophthalmic surgeries, including cataract surgery. Fuchs corneal endothelial dystrophy (FECD) is the most common driver of corneal transplantation. Corneal endothelial hypocellarization secondary to surgery or trauma is a rare but serious complication that can lead to corneal transplantation and contribute to visual impairment.

[0041] Aside from symptomatic treatment using various pharmacological therapies, the only treatment option for patients with CED is CEC transplantation as a whole corneal transplant or as endothelial layer transplantation using various endothelial-keratoplasty procedures, including Descemet's membrane dissection endothelial-keratoplasty (DSEK) and Descemet's membrane endothelial-keratoplasty (DMEK). Even after transplantation, the number of CECs in the transplant continues to decrease, and repeated transplants may be necessary.

[0042] While CECs do not re-densify or regenerate in either normal eyes or FECD patients, there is evidence suggesting that cells derived from the peripheral corneal endothelium can migrate to the center under certain conditions, and that cells from the peripheral corneal endothelium or trabecular meshwork (TM) may contain progenitor cell populations.

[0043] Cataract surgery almost always causes some degree of cerebrovascular center (CEC) loss. In patients with low endothelial cell density or other risk factors for CEC loss, cataract surgery can cause significant CEC loss and loss of CEC function, leading to edema and vision loss.

[0044] Descemetorhexis without Endothelial Keratoplasty (DWEK), sometimes also called Descemet Stripping Only (DSO), is a procedure for FECD patients in which the central region of Descemet's membrane (along with FECD-associated guttata) is surgically removed without endothelial keratoplasty. The corneal ectopic vascular tissue (CEC) in the peripheral region of the cornea migrates to the central region of the cornea and becomes dense again. A potential advantage of DWEK / DSO is that it does not involve transplanted tissue, thereby avoiding associated issues related to tissue availability and potential rejection.

[0045] Fibroblast growth factor (FGF) is a large family of proteins that provide protection from injury and cell death and stimulate the proliferation and migration of a wide variety of cells. FGF is an important developmental growth factor in many tissues, including the cornea. Fibroblast growth factor-1 (FGF-1) is the most potent stimulant of corneal endothelial cell (CEC) proliferation known and is the rationale for applying FGF-1 to CED. Its native or wild-type form, FGF-1 (wtFGF-1), is sensitive to both proteolysis and cysteine ​​oxidation, resulting in a very short biological half-life, which therefore makes it unsuitable as a treatment.

[0046] Pharmaceutical compositions are provided herein that contain a therapeutically effective amount of modified FGF-1 polypeptide having at least 90% sequence identity with the wild-type FGF-1 amino acid sequence of SEQ ID NO: 1 and having mutations at Cys16Ser, Ala66Cys, and Cys117Val. In some embodiments, the modified FGF-1 polypeptide contains methionine upstream of the first residue of SEQ ID NO: 1. In some embodiments, the pharmaceutical composition is for treating or preventing corneal diseases or disorders in a subject. In some embodiments, the corneal diseases or disorders include Descemet's membrane detachment without endothelial keratoplasty (DWEK) or Descemet's membrane detachment alone (DSO).

[0047] In some embodiments described herein, where the modified FGF-1 polypeptide is expressed with an N-terminal methionine (N-Met) residue, the polypeptide is subsequently purified without a step requiring proteolytic cleavage for the removal of the N-terminal peptide. Therefore, in some embodiments, the disclosure provides a modified FGF-1 polypeptide prepared by a rapid purification method without including a proteolytic cleavage step for the removal of the N-terminal peptide. This is particularly advantageous for the production of modified FGF-1 polypeptides compliant with good manufacturing practice (GMP) guidelines. The advantages include the absence of a cleavage step, including the elimination of the need for subsequent purification of the cleaved product and removal of reagents used for cleavage. Further advantages include increased yield due to a reduction in the number of operations and reduced need to test residual cleavage reagents and contaminants introduced for cleavage and subsequent separation of cleaved material from uncleaved material.

[0048] The modified FGF-1 polypeptides described herein may have increased stability (e.g., thermal stability), a reduced number of embedded free thiols, and / or increased effective heparan sulfate proteoglycan (HSPG) affinity.

[0049] Several other advantages relate to the use of modified FGF-1 polypeptides in the methods described herein. For example, the modified FGF-1 polypeptides described herein can be administered without heparin in their pharmaceutical compositions or formulations (e.g., ophthalmic formulations), avoiding potential safety issues associated with their biological origin. Furthermore, the avoidance of heparin allows for the use of higher doses of modified FGF-1 polypeptides without local heparin-induced adverse events or complications resulting from pre-existing anti-heparin antibodies. In addition, in the absence of heparin, the immediate binding of modified FGF to tissues is maximized, and systemic distribution is significantly reduced. The modified FGF-1 polypeptides described herein also have the advantage of having enhanced local sequestration and reduced redistribution kinetics, thus increasing the efflux half-life and mean residence time (MRT) at the site of delivery, and allowing for reduced administration frequency. This may be a result of the modified FGF-1 polypeptides described herein having increased stability (e.g., thermal stability), a reduced number of embedded free thiols, and / or increased effective heparan sulfate proteoglycan (HSPG) affinity.

[0050] The FGF-1 polypeptides of this disclosure, in various embodiments, include modifications at the N-terminus of the polypeptide, such as addition, cleavage, or a combination of addition and cleavage. In some embodiments, the modification is the addition of a single N-terminal methionine residue. In some embodiments, the modification is the addition of an elongated peptide. In some embodiments, the modification is the cleavage of one or more of the first five residues of the FGF-1 polypeptide. In some embodiments, the FGF-1 polypeptide includes a sequence such as that shown in SEQ ID NO: 1, with one or more mutations in addition to the N-terminal modification.

[0051] Some examples of modified FGF-1 polypeptides disclosed herein include an N-terminal methionine (N-Met) residue in the mature form of the polypeptide. The retention of biological activity when an amino acid is added to the N-terminus of a protein is unpredictable. Some proteins are tolerant of this, while others are not, and the possibility of retention of biological activity and changes in stability is determined only empirically. This disclosure identifies that the addition of an N-terminal Met residue is tolerable while retaining biological activity and stability.

[0052] Expression and maturation forms of modified FGF-1 polypeptides FGF stimulates a family of seven FGF receptor isoforms, and each FGF stimulates a different pattern of receptors to achieve its specific effect. See, for example, Ornitz et al. (1996) The Journal of Biological Chemistry, 1996, 271(25):15292-7; Zhang et al. (2006) The Journal of Biological Chemistry, 2006, 281(23):15694-700). In some embodiments, modified FGF-1 polypeptides are preferred because they bind to and stimulate all seven FGF receptor isoforms. See Ornitz et al. (1996) The Journal of Biological Chemistry, 1996, 271(25):15292-7.

[0053] The embodiments disclosed herein relate to modified FGF-1 polypeptides or pharmaceutical compositions (e.g., ophthalmic formulations) comprising modified FGF-1 polypeptides. The embodiments disclosed herein further relate to methods for treating chemical injury or blistering drug injury by administering modified FGF-1 polypeptides or pharmaceutical compositions (e.g., ophthalmic formulations) comprising modified FGF polypeptides or pharmaceutical compositions (e.g., ophthalmic formulations). Modified FGF polypeptide, as used herein, refers to recombinant FGF comprising substitutions or mutations of one or more different amino acid residues of SEQ ID NO: 1, and / or deletions of one or more amino acid residues, and / or additions of one or more amino acid residues.

[0054] This specification provides, in a first embodiment, a modified FGF-1 polypeptide comprising a sequence described as SEQ ID NO: 1 having one or more mutations, wherein the modified polypeptide further comprises a methionine residue upstream of the first residue of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide comprising an N-terminal methionine (N-Met) residue is the mature form of the polypeptide. In some examples, the modified FGF-1 polypeptide according to the first embodiment comprises one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide is expressed in host cells having a methionine residue upstream of the first residue of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide does not undergo N-terminal processing to remove the N-Met residue during maturation. Thus, in some embodiments, the mature form of modified FGF-1 comprises an N-Met residue and one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. An exemplary modified FGF-1 sequence containing an N-Met residue is disclosed as Sequence ID No. 2.

[0055] This disclosure identifies that the modified FGF-1 described herein, which contains the mature form of the N-Met residue, has similar biological activity to the version without the N-Met residue. N-terminal methionine removal or excision is a co-translational process that occurs as soon as the polypeptide emerges from the ribosome. N-terminal methionine removal involves the substrate specificity of the cleaving enzyme methionine aminopeptidase (metAP), which recognizes the methionine residue and then recognizes amino acid residues with small side chains, such as alanine, glycine, proline, serine, threonine, or valine. Due to this substrate sequence specificity, the modified FGF-1 of the first embodiment, which contains the N-Met residue followed by phenylalanine (see position 1 of SEQ ID NO: 1), is not processed by metAP. Therefore, by expressing a modified FGF-1 having a methionine residue immediately upstream of SEQ ID NO: 1, a mature modified FGF-1 containing methionine as its N-terminal residue can be obtained. In some embodiments, the modified FGF-1 according to the first embodiment is not expressed with the N-terminal peptide and therefore does not undergo proteolytic cleavage for the removal of the N-terminal peptide during subsequent purification.

[0056] This specification provides a modified FGF-1 polypeptide comprising, in a second embodiment, a sequence described as SEQ ID NO: 1 having one or more mutations, the modified polypeptide further comprising a methionine residue upstream of the first residue of SEQ ID NO: 1 and one or more amino acids of the peptide described as SEQ ID NO: 3. The peptide comprising one or more residues of SEQ ID NO: 3 is referred to herein as the “elongated peptide”. Thus, the modified FGF-1 according to the second embodiment comprises the sequence described as SEQ ID NO: 1 having one or more mutations, a methionine residue upstream of the first residue of SEQ ID NO: 1, and an elongated peptide located between the methionine residue and the first residue of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide comprising an N-terminal methionine and an elongated peptide located between the methionine residue and the first residue of SEQ ID NO: 1 is the mature form of the polypeptide. In some embodiments, the modified FGF-1 polypeptide contains one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1, and this polypeptide is expressed in a host cell having a methionine residue upstream of the first residue of SEQ ID NO: 1, and an elongation peptide located between the methionine residue and the first residue of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide according to the second embodiment is expressed together with an elongation peptide containing five residues of SEQ ID NO: 3 located between the methionine residue and the first residue of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide according to the second embodiment is expressed together with four residues of SEQ ID NO: 3 located between the methionine residue and the first residue of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide according to the second embodiment is expressed together with three residues of SEQ ID NO: 3 located between the methionine residue and the first residue of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide according to the second embodiment is expressed together with two residues of SEQ ID NO: 3 located between the methionine residue and the first residue of SEQ ID NO: 1.In some embodiments, the modified FGF-1 polypeptide according to the second embodiment is expressed with one residue of SEQ ID NO: 3 located between the methionine residue and the first residue of SEQ ID NO: 1. Exemplary sequences of the elongated peptide include SEQ ID NOs: 4-8.

[0057] In some examples, the modified FGF-1 polypeptide of the second embodiment, which includes an elongated peptide and an N-terminal methionine residue, does not undergo N-terminal processing for the removal of the methionine residue, while in some examples, the methionine is cleaved by a cleaving enzyme. Typically, the cleaving enzyme is methionine aminopeptidase (metAP). Thus, in some examples, the mature form of the modified FGF-1 polypeptide according to the second embodiment includes an N-Met residue followed by the elongated peptide described herein. Exemplary sequences of the mature form of the modified FGF-1 polypeptide according to the second embodiment, which includes an N-terminal methionine and one or more residues of the elongated peptide located between the methionine residue and the first residue of SEQ ID NO: 1, are described as SEQ ID NOs: 9-13, and the sequences further include one or more mutations in the amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. Further exemplary sequences of the mature modified FGF-1 polypeptide including the N-terminal methionine and the elongated peptide are described as SEQ ID NOs: 14-18. In some other examples, the mature form of the modified FGF-1 polypeptide according to the second embodiment does not contain an N-Met residue but contains only the elongation peptide. Exemplary sequences of the mature form of the modified FGF-1 polypeptide according to the second embodiment, which contains the elongation peptide located upstream of the first residue of SEQ ID NO: 1, are shown as SEQ ID NOs: 19-23, and the sequences further include one or more mutations in the amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. Further exemplary sequences of the mature modified FGF-1 polypeptide containing one or more residues of the elongation peptide are described as SEQ ID NOs: 24-28. In some embodiments, the methionine residue is cleaved by metAP when the elongation peptide begins with alanine (as in SEQ ID NO: 4) or threonine (as in SEQ ID NO: 5). In such examples, the mature FGF-1 polypeptide does not contain an N-terminal methionine residue, e.g., SEQ ID NOs: 19, 21, 24, and 26.

[0058] This specification provides, in a third embodiment, a modified FGF-1 polypeptide comprising a sequence described as SEQ ID NO: 1 having one or more mutations, wherein the modified polypeptide further comprises an elongation peptide located upstream of the first residue of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide comprising the elongation peptide is the mature form of the polypeptide. In some embodiments, a modified FGF-1 polypeptide comprising one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1, wherein the polypeptide is expressed in a host cell together with one or more amino acid residues of the elongation peptide located upstream of the first residue of SEQ ID NO: 1. Exemplary sequences of modified FGF-1 polypeptides comprising an elongation peptide expressed without an N-terminal methionine residue are shown as SEQ ID NOs: 19-23, wherein the sequences further comprise one or more mutations at the amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. Further exemplary sequences of mature, modified FGF-1 polypeptides expressed containing one or more residues of the elongated peptide but without the N-terminal methionine residue are described as SEQ ID NOs: 24–28.

[0059] This specification provides, in a fourth embodiment, a modified FGF-1 polypeptide comprising a sequence described as SEQ ID NO: 1 having one or more mutations, wherein the modified polypeptide further comprises one or more cleavages of the first five residues of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide comprising one or more cleavages of the first five residues of SEQ ID NO: 1 is the mature form of the polypeptide. In some embodiments, the modified FGF-1 polypeptide comprises one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1, with one or more deletions of the first five residues of SEQ ID NO: 1. Optionally, the modified FGF-1 polypeptide comprising cleavages is expressed together with an N-terminal methionine residue. For example, the modified FGF-1 polypeptide according to the fourth embodiment may have a sequence in which an N-Met residue is followed by asparagine, the second residue of SEQ ID NO: 1. Optionally, the modified FGF-1 polypeptide comprises an N-Met residue followed by leucine, the third residue of SEQ ID NO: 1. In some cases, the modified FGF-1 polypeptide contains an N-Met residue followed by proline, which is the fourth residue in SEQ ID NO: 1. In some cases, the modified FGF-1 polypeptide contains an N-Met residue followed by proline, which is the fifth residue in SEQ ID NO: 1. The elongated peptide can be located between the N-Met residue and the first, second, third, fourth, or fifth residue in SEQ ID NO: 1. An example of a mature form of the modified FGF-1 polypeptide according to the fourth embodiment, in which the N-Met residue is followed by the second, third, fourth, or fifth residue in SEQ ID NO: 1, is shown in SEQ ID NOs. 37-40, and the sequence further contains one or more mutations in the amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. Further examples of modified FGF-1 polypeptides containing cleavage and N-Met residues are provided in SEQ ID NOs. 41-44.

[0060] This disclosure further relates to a modified FGF-1 polypeptide containing one or more mutations in SEQ ID NO: 1, wherein the polypeptide is expressed with an N-Met residue followed by an elongation peptide, followed by a cleavage of one or more of the first five residues of SEQ ID NO: 1. In some embodiments, the modified FGF-1 polypeptide contains one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1, wherein the polypeptide is expressed with an N-Met residue followed by an elongation peptide, followed by a cleavage of one or more of the first five residues of SEQ ID NO: 1. Examples of such sequences expressed with an N-Met residue followed by an elongation peptide (followed by a cleavage of one or more of the first five residues of SEQ ID NO: 1) are disclosed as SEQ ID NOs: 45-68, which further contain one or more mutations in the amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. In some examples, the N-terminal methionine is cleaved by N-terminal processing, and therefore the mature form of the modified FGF-1 polypeptide contains only one or more residues of the leader fragment, followed by cleavage of one or more of the first five residues of SEQ ID NO: 1, as exemplified in SEQ ID NOs. 69–92. The exemplary sequences further contain one or more mutations in the amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. Further examples of sequences that do not have an N-Met residue but contain cleavage of the elongated peptide and N-terminal residue are provided in SEQ ID NOs. 93–117.

[0061] In some examples, the N-Met residue is retained in the mature, modified FGF-1 polypeptide sequence, and therefore the mature form includes the sequences exemplified in SEQ ID NOs. 45–68, further including one or more mutations in the amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO. 1. Further examples of sequences containing the N-Met residue, elongated peptide, and cleavage of the N-terminal residue are provided in SEQ ID NOs. 118–141.

[0062] In the fifth embodiment, a cleaved form of a modified FGF-1 polypeptide containing one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1 is expressed without the N-terminal methionine residue and without the elongated peptide. In some examples, the mature modified FGF-1 polypeptide according to the fifth embodiment contains the sequences described in SEQ ID NOs: 29-32, which further contain one or more mutations at the amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. In some examples, the modified FGF-1 polypeptide according to the fifth embodiment contains sequences selected from the group consisting of SEQ ID NOs: 33-36.

[0063] In cases where a modified FGF-1 polypeptide or a cleaved version thereof, containing one or more mutations at positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1, is expressed with N-terminal methionine followed by an elongated peptide, the methionine residue is either retained during polypeptide maturation after expression or cleaved from the N-terminus. In some cases, when a modified FGF-1 polypeptide is expressed with alanine adjacent to the N-Met residue (e.g., SEQ ID NO: 14), the methionine is cleaved to produce a mature FGF-1 polypeptide without the N-Met residue, e.g., SEQ ID NO: 19. In some cases, when a modified FGF-1 polypeptide is expressed with threonine adjacent to the N-Met residue (e.g., SEQ ID NO: 16), the methionine is cleaved to produce a mature FGF-1 polypeptide without the N-Met residue, e.g., SEQ ID NO: 20. In some cases, when a modified FGF-1 polypeptide is expressed with glutamic acid adjacent to the N-Met residue (e.g., SEQ ID NO: 17), the methionine is not cleaved, and mature FGF-1 is produced that contains the N-terminal methionine and has the same sequence as the expressed form.

[0064] In a sixth embodiment, a modified FGF-1 polypeptide comprising the sequence described as SEQ ID NO: 1, including a mutation at position 67, is provided herein. In some embodiments, the modified FGF-1 polypeptide is expressed with an N-Met residue, including a mutation at position 67 of SEQ ID NO: 1, and one or more further mutations at positions 12, 16, 66, 117, and 134. The internal methionine at position 67 can be replaced, for example, with an alanine residue. If internal methionine is absent at position 67, the N-terminal methionine of the modified FGF-1 polypeptide can be cleaved after expression using cyanogen bromide (CNBr), an agent that specifically cleaves the amide bond after the methionine residue. Optionally, the modified FGF-1 polypeptide according to the sixth embodiment is expressed with an elongated peptide. In some other cases, the modified FGF-1 polypeptide according to the sixth embodiment is expressed in a form that includes a cleavage of one or more of the first five residues of SEQ ID NO: 1, as exemplified by SEQ ID NOs: 142-149, and the sequence further includes one or more mutations in the amino acids corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO: 1. In yet another example, the modified FGF-1 polypeptide according to the sixth embodiment is expressed in a form that includes an elongated peptide and a cleavage of one or more of the first five residues of SEQ ID NO: 1, as exemplified by SEQ ID NOs: 151-175. Further examples of the modified FGF-1 polypeptide according to the sixth embodiment are described in their mature forms in SEQ ID NOs: 174-204. In modified FGF-1 polypeptides expressed in forms containing internal methionine mutations, where the polypeptide is expressed with an N-terminal methionine, followed by an alanine or threonine residue from the elongated peptide (e.g., SEQ ID NO: 175 and SEQ ID NO: 177, respectively), the N-terminal methionine may be cleaved during polypeptide maturation by metAP or CNBr.

[0065] In a seventh embodiment, a modified FGF-1 polypeptide comprising the sequence described as SEQ ID NO: 205 is provided herein for use in the method described herein. In an eighth embodiment, a modified FGF-1 polypeptide comprising the sequence described as SEQ ID NO: 206 is provided herein for use in the method described herein.

[0066] This disclosure further relates to modified FGF-1 polypeptides including any combination of deletions, insertions, and substitutions of SEQ ID NO: 1, provided that the modified polypeptides include one or more mutations of SEQ ID NO: 1. Amino acid substitutions may be introduced into modified FGF-1 polypeptides, and the products may be screened for desired activity, e.g., retention / improvement of effect in the treatment of eye disorders, increased potency in the treatment of Fuchs dystrophy, or improvement in the treatment of mustard gas keratopathy. Amino acid substitutions may further be introduced into modified FGF-1 polypeptides, and the products may be screened for desired physicochemical properties, e.g., a tendency to be less aggregated, improved solubility, extended half-life, ease of formulation as an ophthalmic drug, enhanced stability, or improved shelf life. Both conservative and non-conservative amino acid substitutions are intended.

[0067] The modified FGF-1 polypeptide is expressed in a form containing at least 136 amino acids, as in any of the embodiments described above. In some embodiments, the modified FGF-1 polypeptide is expressed in a form containing 137 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form containing 138 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form containing 139 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form containing 140 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form containing 141 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form containing 142 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form containing 143 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form containing 144 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form containing 145 amino acids. In some embodiments, the modified FGF-1 polypeptide is expressed in a form containing 146 amino acids.

[0068] The modified FGF-1 polypeptide contains at least 136 amino acids in its mature form, as in any of the embodiments described above. In some examples, the modified FGF-1 polypeptide contains 137 amino acids in its mature form. In some examples, the modified FGF-1 polypeptide contains 138 amino acids in its mature form. In some examples, the modified FGF-1 polypeptide contains 139 amino acids in its mature form. In some examples, the modified FGF-1 polypeptide contains 140 amino acids in its mature form. In some examples, the modified FGF-1 polypeptide contains 141 amino acids in its mature form. In some examples, the modified FGF-1 polypeptide contains 142 amino acids in its mature form. In some examples, the modified FGF-1 polypeptide contains 143 amino acids in its mature form. In some examples, the modified FGF-1 polypeptide contains 144 amino acids in its mature form. In some examples, the modified FGF-1 polypeptide contains 145 amino acids in its mature form. In some cases, modified FGF-1 polypeptides contain 146 amino acids in their mature form.

[0069] In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 1, provided that the polypeptide contains an N-Met residue in its mature form. In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences selected from SEQ ID NOs. 1, provided that the polypeptide contains an N-Met residue in its mature form, and the polypeptide contains one or more mutations at amino acid positions corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO. 1. In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences selected from SEQ ID NOs. 14-18, provided that the polypeptide contains an N-Met residue in its mature form. In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences selected from SEQ ID NOs. 19-23, provided that the polypeptide does not contain an N-Met residue in its mature form, and the polypeptide contains one or more mutations at amino acid positions corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO. 1.In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences selected from SEQ ID NOs. 24-28, provided that the polypeptide does not contain an N-Met residue in its mature form. In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences selected from SEQ ID NOs. 19-23, provided that the polypeptide does not contain an N-Met residue in its mature form, and the polypeptide contains one or more mutations at amino acid positions corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO. 1. In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences selected from SEQ ID NOs. 37-40, provided that the polypeptide contains an N-Met residue in its mature form, and the polypeptide contains one or more mutations at amino acid positions corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO. 1. In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences selected from SEQ ID NOs. 41-44, provided that the polypeptide contains an N-Met residue in its mature form.In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any sequence selected from SEQ ID NOs. 45 to 68, provided that the polypeptide contains one or more mutations at amino acid positions corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO. 1, and the polypeptide does not contain an N-Met residue in its mature form. In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any sequence selected from SEQ ID NOs. 69 to 92, and contains one or more mutations at amino acid positions corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO. 1, and the polypeptide contains an N-Met residue in its mature form. In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any sequence selected from SEQ ID NOs. 93 to 117, provided that the polypeptide does not contain an N-Met residue in its mature form. In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences selected from SEQ ID NOs. 118-141, provided that the polypeptide contains an N-Met residue in its mature form.In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences selected from SEQ ID NOs. 29 to 32, provided that the polypeptide contains one or more mutations at amino acid positions corresponding to positions 12, 16, 66, 117, and 134 of SEQ ID NO. 1. In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences selected from SEQ ID NOs.

[0070] In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences selected from SEQ ID NOs.

[0071] In some embodiments, the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 1 mutated at position 12, for example, having mutant Lys12Val, and the modified FGF-1 polypeptide contains an N-terminal methionine in its mature form. In some embodiments, the modified FGF-1 polypeptide contains a sequence having a mutation at position 12 of SEQ ID NO: 1, for example, mutant Lys12Val, and a cleavage of one or more of the first five residues of SEQ ID NO: 1, and the modified FGF-1 polypeptide contains an N-Met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence having a mutation at position 12 of SEQ ID NO: 1, for example, mutant Lys12Val, an elongated peptide, and a cleavage of one or more of the first five residues of SEQ ID NO: 1, wherein the modified FGF-1 polypeptide contains an N-met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a mutation at position 12 of SEQ ID NO: 1, for example, mutant Lys12Val, and the polypeptide further comprises a methionine mutation at position 67 of SEQ ID NO: 1, expressed together with methionine at the N-terminus, and the methionine is cleaved from the polypeptide in its mature form.

[0072] In some embodiments, the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 1 mutated at position 16, for example, having a mutant Cys16Ser, and the modified FGF-1 polypeptide contains an N-met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide contains a sequence having a mutation at position 16 of SEQ ID NO: 1, for example, a mutant Cys16Ser, and a cleavage of one or more of the first five residues of SEQ ID NO: 1, and the modified FGF-1 polypeptide contains an N-met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence having a mutation at position 16 of SEQ ID NO: 16, for example, a mutant Cys16Ser, an elongated peptide, and a cleavage of one or more of the first five residues of SEQ ID NO: 1, wherein the modified FGF-1 polypeptide comprises an N-met residue. In some embodiments, the modified FGF-1 polypeptide comprises a mutation at position 16 of SEQ ID NO: 1, for example, a mutant Cys16Ser, and the polypeptide further comprises a methionine mutation at position 67 of SEQ ID NO: 1, expressed together with methionine at the N-terminus, and the methionine is cleaved from the polypeptide in its mature form.

[0073] In some embodiments, the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 1 mutated at position 66, for example, having mutant Ala66Cys, and the modified FGF-1 polypeptide contains an N-terminal methionine in its mature form. In some embodiments, the modified FGF-1 polypeptide contains a sequence having a mutation at position 66 of SEQ ID NO: 1, for example, mutant Ala66Cys, and a cleavage of one or more of the first five residues of SEQ ID NO: 1, and the modified FGF-1 polypeptide contains an N-met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence having a mutation at position 66 of SEQ ID NO: 1, for example, mutant Ala66Cys, an elongated peptide, and a cleavage of one or more of the first five residues of SEQ ID NO: 1, and the modified FGF-1 polypeptide is expressed together with an N-Met residue. In some embodiments, the modified FGF-1 polypeptide comprises a mutation at position 66 of SEQ ID NO: 1, for example, mutant Ala66Cys, and the polypeptide further comprises a methionine mutation at position 67 of SEQ ID NO: 1, and is expressed together with methionine at the N-terminus, and the methionine is cleaved from the polypeptide in its mature form.

[0074] In some embodiments, the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 1 mutated at position 117, for example, having the mutant Cys117Val, and the modified FGF-1 polypeptide contains an N-met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide contains a sequence having a mutation at position 117 of SEQ ID NO: 1, for example, the mutant Cys117Val, and a cleavage of one or more of the first five residues of SEQ ID NO: 1, and the modified FGF-1 polypeptide contains an N-met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence having a mutation at position 117 of SEQ ID NO: 1, for example, mutant Cys117Val, an elongated peptide, and a cleavage of one or more of the first five residues of SEQ ID NO: 1, wherein the modified FGF-1 polypeptide contains an N-met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a mutation at position 117 of SEQ ID NO: 1, for example, mutant Cys117Val, and the polypeptide further comprises a methionine mutation at position 67 of SEQ ID NO: 1, expressed together with methionine at the N-terminus, and the methionine is cleaved from the polypeptide in its mature form.

[0075] In some embodiments, the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 1 mutated at position 134, for example, having the mutant Pro134Val, and the modified FGF-1 polypeptide contains an N-terminal methionine in its mature form. In some embodiments, the modified FGF-1 polypeptide contains a sequence having a mutation at position 134 of SEQ ID NO: 1, for example, the mutant Pro134Val, and a cleavage of one or more of the first five residues of SEQ ID NO: 1, and the modified FGF-1 polypeptide contains an N-met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence having a mutation at position 134 of SEQ ID NO: 1, for example, the mutant Pro134Val, an elongated peptide, and a cleavage of one or more of the first five residues of SEQ ID NO: 1, wherein the modified FGF-1 polypeptide contains an N-met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a mutation at position 134 of SEQ ID NO: 1, for example, the mutant Pro134Val, and the polypeptide further comprises a methionine mutation at position 67 of SEQ ID NO: 1, expressed together with methionine at the N-terminus, and the methionine is cleaved from the polypeptide in its mature form.

[0076] In some embodiments, the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 1 mutated at positions 16, 66, and 117, for example, having mutant Cys16Ser, Ala66Cys, and Cys117Val, and the modified FGF-1 polypeptide contains an N-met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence having mutations at positions 16, 66, and 117 of SEQ ID NO: 1, for example, mutant Cys16Ser, Ala66Cys, and Cys117Val, and a cleavage of one or more of the first five residues of SEQ ID NO: 1, and the modified FGF-1 polypeptide contains an N-met residue in its mature form. In some embodiments, the modified FGF-1 polypeptide comprises a sequence having mutations at positions 16, 66, and 117 of SEQ ID NO: 1, for example, mutant Cys16Ser, Ala66Cys, and Cys117Val, an elongated peptide, and a cleavage of one or more of the first five residues of SEQ ID NO: 1, and the modified FGF-1 polypeptide contains an N-met residue. In some embodiments, the modified FGF-1 polypeptide comprises a sequence having mutations at positions 16, 66, and 117 of SEQ ID NO: 1, e.g., mutations Cys16Ser, Ala66Cys, and Cys117Val, and the polypeptide further comprises a methionine mutation at position 67 of SEQ ID NO: 1, which is expressed together with methionine at the N-terminus, and the methionine is cleaved from the polypeptide in its mature form.

[0077] In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs. In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 205 or 206.

[0078] In some embodiments, the modified FGF-1 polypeptide is thermally stable. As used herein, thermally stable FGF (e.g., thermally stable FGF-1) refers to an FGF having a modified amino acid sequence compared to SEQ ID NO: 1, which is further more stable than the polypeptide of SEQ ID NO: 1 under the same conditions. Examples of mutations that can confer thermal stability to FGF (e.g., FGF-1) and methods for evaluating thermal stability are described, for example, in U.S. Patents 7,790,682, 7,595,296, 7,696,171, 7,776,825, 7,659,379, 8,119,776, 8,153,770, 8,153,771, and 8,461,111, U.S. Patent Application Publication 2011 / 0224404, and 2013 / 0130983, and Xia et al. PloS one. (2012) 7(11):e48210. In some embodiments, mutations are made in FGF-1 at positions 12 and / or 134 to produce a modified FGF-1 that is thermally stable.

[0079] In some embodiments, the modified FGF-1 polypeptide includes one or more modifications that reduce the number of reactive thiols (e.g., free cysteine). Examples of such modifications in FGF-1 are described, for example, in U.S. Patents 7,790,682, 7,595,296, 7,696,171, 7,776,825, 7,659,379, 8,119,776, 8,153,770, 8,153,771, and 8,461,111, U.S. Patent Application Publication 2011 / 0224404, and 2013 / 0130983, as well as in Xia et al. PloS one. (2012) 7(11):e48210. In some embodiments, modified FGF-1 is produced by mutating the 83rd and / or 117th positions in SEQ ID NO: 1, thereby reducing the number of reactive thiols.

[0080] In some embodiments, the modified FGF includes one or more modifications that enable the formation of an internal disulfide bond. In some embodiments, a modified FGF-1 containing an internal disulfide bond is produced by mutating the 66 position in SEQ ID NO: 1.

[0081] In some embodiments, the modified FGF-1 polypeptides described herein can be administered without exogenous heparin in formulations for stability, and they can be formulated and applied without heparin, and therefore can bind better to tissue heparan. Such modified FGF-1 polypeptides have high affinity for tissue heparan to which they are exposed in surgical, traumatic, or dystrophic and disease states, and therefore bind to diseased tissue upon application. Furthermore, the more thermally stable modified FGF-1 polypeptides are suitable for formulation and storage at room temperature. Due to their stability, the modified FGF-1 polypeptides are also suitable for administration in both solution (e.g., immediate-release) and sustained-release formulations.

[0082] In some embodiments, the modified FGF-1 polypeptide is SEQ ID NO: 1 modified at one or more positions 12, 16, 66, 117, and 134. In some embodiments, the modified FGF is SEQ ID NO: 1 modified at positions 16, 66, and 117. The amino acid positions can be substituted with, for example, Ser, Cys, Val, or other amino acids to create disulfide bonds between the modified amino acids and the wild-type amino acids. In some embodiments, the modified FGF contains the amino acid sequence of SEQ ID NO: 2, also known as N-Met THX1114. In some embodiments, the modified FGF-1 polypeptide contains one or more mutations selected from the group consisting of Lys12Val, Pro134Val, Ala66Cys, Cys117Val, and Pro134Val. In some embodiments, the modified FGF-1 polypeptide contains the sequence of SEQ ID NO: 2.

[0083] In some embodiments, the modified FGF-1 polypeptides or compositions described herein may be prepared as prodrugs. “Prodrug” refers to a drug that is converted to a parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than parent drugs. They may be bioavailable, for example, by oral administration, whereas parent drugs are not. Prodrugs may also have improved solubility in pharmaceutical compositions than parent drugs.

[0084] The modified FGF-1 polypeptides described herein may be labeled isotopically (e.g., using radioactive isotopes) or by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, photoactivatable labels or chemiluminescent labels.

[0085] The disclosure further relates to modified FGF polypeptides including N-terminal modifications, wherein the modified FGF polypeptides may be any member of the FGF family, including FGF-1 (SEQ ID NO: 1), FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, and FGF-23 and FGF-24.

[0086] In some embodiments, the synthesis of modified FGF-1 polypeptides as described herein is achieved by means of means described in the Art, by methods described herein, or by a combination thereof.

[0087] In some embodiments, the modified FGF sequence has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 1 mutated at one or more positions, positions 16, 66, and 117, for example, having mutant Cys16Ser, Ala66Cys, and Cys117Val. In some embodiments, the modified FGF comprises a wild-type human FGF-1 sequence having mutations at positions 16, 66, and 117, for example, mutant Cys16Ser, Ala66Cys, and Cys117Val.

[0088] In some embodiments, the sequence of the modified FGF-1 polypeptide has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 50% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 55% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 60% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 65% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 70% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 75% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 80% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 85% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 86% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 87% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 88% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 89% sequence identity with the sequence selected from SEQ ID NO: 2.In some embodiments, the sequence of the modified FGF-1 polypeptide has 90% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 91% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 92% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 93% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 94% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 95% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 96% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 97% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 98% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 99% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 100% sequence identity with the sequence selected from SEQ ID NO: 2.

[0089] Recombinant technology for the preparation of modified FGF-1 polypeptides Various host expression vector systems can be used to produce the modified FGF-1 polypeptides provided herein. Such host expression systems represent vehicles from which the modified FGF-1 polypeptides can be produced and subsequently purified, but also represent cells that may exhibit the modified gene product in situ when transformed or transfected with an appropriate nucleotide coding sequence. Examples of host expression systems include, but are not limited to, insect cell lines infected with recombinant viral expression vectors (e.g., baculovirus) containing nucleotide sequences encoding modified FGF-1 polypeptides, plant cell lines infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing coding sequences of modified FGF-1 polypeptides, or human cell lines, such as mammalian cell lines containing HT1080, COS, CHO, BHK, 293, 3T3, and other derivatives thereof, that have recombinant expression constructs. Examples of bacterial expression systems include, but are not limited to, E. coli plasmids (e.g., pBR322, pBR325, pUC12, pUC13, and pET-3) and Bacillus subtilis plasmids (e.g., PUB110, pTP5, and pC194).

[0090] In some embodiments, host cell lines are selected to regulate the expression of an inserted sequence or to modify and process gene products in a specific manner of desire. Such modification and processing of protein products may be important for protein function. Different host cells have specific mechanisms for post-translational processing and modification of proteins and gene products. By selecting an appropriate cell line or host system, precise modification and processing of expressed foreign proteins can be ensured. For this purpose, eukaryotic host cells with cellular mechanisms for proper processing of primary transcripts, glycosylation of gene products, and phosphorylation may be used. Such mammalian host cells, including human host cells, include, but are not limited to, HT1080, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.

[0091] For long-term, high-yield production of recombinant peptides, stable expression is desirable. For example, cell lines that stably express recombinantly modified FGF-1 polypeptides can be manipulated. In some embodiments, instead of using expression vectors containing viral replication origins, host cells can be transformed with DNA controlled by appropriate expression regulatory elements, such as promoters, enhancers, sequences, transcription terminators, polyadenylation sites, and selection markers. After introduction of the foreign DNA, the manipulated cells can be grown in concentrated medium for 1-2 days and then switched to selective medium. The selection marker in the recombinant plasmid confers resistance to selection, allowing the cell to stably incorporate the plasmid into its chromosomes, grow, and form lesions, which can then be cloned and grown into a cell line. In some examples, this method can be advantageously used to manipulate cell lines expressing modified FGF-1 polypeptide products. Such manipulated cell lines can be particularly useful in screening and evaluation of compounds that biologically affect gene products.

[0092] Disulfide bond formation in modified FGF-1 polypeptides In some embodiments, the modified FGF-1 polypeptides of this disclosure include the following mutations in SEQ ID NO: 1—Cys16Ser, Ala66Cys, and Cys117Val—and the polypeptide contains an internal disulfide bond between cysteine ​​residues at positions 66 and 83. For many recombinant proteins, the correct formation of disulfide bonds is essential to achieving their biologically active three-dimensional conformation. Incorrect disulfide bond formation can lead to protein misfolding and aggregation into inclusion bodies. In E. coli, cysteine ​​oxidation typically occurs in the periplasm, where disulfide bonds are formed primarily from the Dsb family in disulfide exchange reactions catalyzed by numerous enzymes (Rosano, GL, & Ceccarelli, EA (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology, 5, 172). In contrast, disulfide bond formation in the cytoplasm is rare. This situation affects the production of recombinant proteins containing disulfide bonds produced in the cytoplasm, such as modified FGF-1 polypeptides containing an internal disulfide bond between Cys66 and Cys83. Therefore, in some cases, engineered E. coli strains with an oxidative cytoplasmic environment favorable to disulfide bond formation are selected as host cells for the expression of modified FGF-1 polypeptides (Rosano, GL, & Ceccarelli, EA (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology, 5, 172).Examples of such strains include, but are not limited to, Origami (Novagen) with the trxB-gor-genotype on a K-12 background, and SHuffle® T7 Express strain (NEB) with the trxB-gor-genotype on a BL21 (DE3) background, constitutively expressing a chromosomal copy of the disulfide bond isomerase DsbC. DsbC has also been shown to be a chaperone that promotes the correction of mis-oxidized proteins to the correct form and can further assist in the folding of proteins that do not require disulfide bonds. While not bound by any particular theory, it is intended that the action of DsbC reduces the aggregation of target proteins, such as modified FGF-1 polypeptides containing an internal disulfide bond between Cys66 and Cys83, into inclusion bodies. Therefore, in certain embodiments, this disclosure identifies an improved method for cytoplasmic production of modified FGF-1 polypeptides containing an internal disulfide bond between Cys16 and Cys83.

[0093] In some embodiments in which the modified FGF-1 polypeptide is expressed with an N-Met residue, the polypeptide is subsequently purified without a step requiring proteolytic cleavage to remove the N-terminal peptide. Thus, in some embodiments, the present disclosure provides a method for rapidly purifying the modified FGF-1 polypeptide described herein without a proteolytic cleavage step to remove the N-terminal peptide. This is particularly advantageous for the production of modified FGF-1 polypeptides that comply with good manufacturing practice (GMP) guidelines. The advantages include the absence of a cleavage step, including the elimination of the need for subsequent purification of the cleaved product and removal of reagents used for cleavage. Further advantages of this include increased yield due to a reduction in the number of operations and reduced need to test residual cleavage reagents and contaminants introduced for cleavage and subsequent separation of cleaved material from uncleaved material.

[0094] How to use This disclosure provides a method for treating or preventing a corneal disease or illness in a subject, comprising the step of intrachorally administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide having at least 90% sequence identity to the wild-type FGF-1 amino acid sequence of SEQ ID NO: 1 and having mutations at Cys16Ser, Ala66Cys, and Cys117Val, wherein the corneal disease or illness includes Descemet's membrane detachment without endothelial keratoplasty (DWEK) or Descemet's membrane detachment alone (DSO). In some embodiments, the modified FGF-1 polypeptide contains methionine upstream of the first residue of SEQ ID NO: 1.

[0095] In some embodiments, the sequence of the modified FGF-1 polypeptide has 80% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 85% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 86% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 87% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 88% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 89% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 90% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 91% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 92% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 93% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 94% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 95% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 96% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 97% sequence identity with the sequence selected from SEQ ID NO: 2.In some embodiments, the sequence of the modified FGF-1 polypeptide has 98% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 99% sequence identity with the sequence selected from SEQ ID NO: 2. In some embodiments, the sequence of the modified FGF-1 polypeptide has 100% sequence identity with the sequence selected from SEQ ID NO: 2.

[0096] Depending on the circumstances, the subjects may undergo cataract surgery before, simultaneously with, or after the administration process. Depending on the circumstances, corneal diseases or conditions may include corneal endothelial dystrophy or corneal endothelial injury.

[0097] Depending on the circumstances, (i) the subject undergoes cataract surgery before, simultaneously with, or after the administration step, and (ii) the corneal disease or condition includes corneal endothelial dystrophy or corneal endothelial injury.

[0098] In some embodiments, corneal endothelial dystrophy or corneal endothelial injury includes Fuchs dystrophy, bullous keratopathy, congenital hereditary endothelial dystrophy 1, congenital hereditary endothelial dystrophy 2, posterior polymorphic corneal dystrophy, corneal endothelial injury following ophthalmic surgery, corneal endothelial injury following cataract surgery, or corneal endothelial injury following oxidative stress.

[0099] In some embodiments, subjects have risk factors for corneal endothelial injury induced by ophthalmic surgery. In some embodiments, risk factors include diabetes mellitus, low corneal endothelial cell density, corneal endothelial dystrophy, small pupil, shallow anterior chamber, mature cataract or brunescent cataract, or a combination thereof.

[0100] In some embodiments, the subject undergoes cataract surgery prior to the administration step, and the corneal disease or illness includes Fuchs dystrophy. In some embodiments, the subject undergoes cataract surgery concurrently with the administration step, and the corneal disease or illness includes Fuchs dystrophy. In some embodiments, the subject undergoes cataract surgery after the administration step, and the corneal disease or illness includes Fuchs dystrophy. In some embodiments, DWEK or DSO is performed before cataract surgery. In some embodiments, DWEK or DSO is performed concurrently with cataract surgery. In some embodiments, DWEK or DSO is performed after cataract surgery.

[0101] In some embodiments, corneal endothelial dystrophy or corneal endothelial injury is caused by cataract surgery. In some embodiments, corneal endothelial dystrophy or corneal endothelial injury is not caused by cataract surgery.

[0102] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition comprises a liquid formulation. In some embodiments, the pharmaceutically acceptable carrier, excipient, or diluent comprises physiological saline. In some embodiments, the physiological saline comprises phosphate-buffered saline (PBS). In some embodiments, PBS comprises one or more salts of sodium ions or potassium ions. In some embodiments, one or more salts comprises sodium chloride, potassium chloride, monosodium phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate anhydride, or disodium hydrogen phosphate anhydride. In some embodiments, one or more salts comprises sodium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate anhydride. In some embodiments, the pharmaceutical composition comprises sodium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate anhydride. In some embodiments, the pharmaceutically acceptable carrier, excipient, or diluent comprises sodium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate anhydride.

[0103] In some embodiments, the pharmaceutically acceptable carrier, excipient, or diluent includes a surfactant. In some embodiments, the surfactant includes polysorbate, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil, or equivalents thereof. In some embodiments, the pharmaceutically acceptable carrier, excipient, or diluent includes polysorbate. In some embodiments, the polysorbate includes polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. In some embodiments, the polysorbate includes polysorbate 20. In some embodiments, the polysorbate includes polysorbate 40. In some embodiments, the polysorbate includes polysorbate 60. In some embodiments, the polysorbate includes polysorbate 80. In some embodiments, the pharmaceutical composition includes polysorbate 80. In some embodiments, the pharmaceutically acceptable carrier, excipient, or diluent includes polysorbate 80.

[0104] In some embodiments, the pharmaceutically acceptable carrier, excipient, or diluent comprises albumin. In some embodiments, the albumin comprises human serum albumin. In some embodiments, the albumin comprises recombinant human serum albumin. In some embodiments, the pharmaceutical composition comprises recombinant human serum albumin. In some embodiments, the pharmaceutically acceptable carrier, excipient, or diluent comprises recombinant human serum albumin.

[0105] In some embodiments, the pharmaceutical composition comprises (i) PBS containing sodium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate anhydrous, (ii) polysorbate 80, and (iii) recombinant human serum albumin. In some embodiments, the pharmaceutically acceptable carrier, excipient, or diluent comprises (i) PBS containing sodium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate anhydrous, (ii) polysorbate 80, and (iii) recombinant human serum albumin.

[0106] In some embodiments, the concentration (w / v) of the polysorbate is at least about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%. In some embodiments, the concentration (w / v) of the polysorbate is up to about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%. In some embodiments, the polysorbate concentration (w / v) is approximately 0.001% to 0.1%, 0.003% to 0.09%, 0.005% to 0.08%, 0.007% to 0.07%, 0.01% to 0.06%, 0.02% to 0.05%, or 0.03% to 0.04%. In some embodiments, the polysorbate concentration is approximately 0.01% (w / v). In some embodiments, the concentration (w / v) of polysorbate 80 is at least about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%. In some embodiments, the concentration (w / v) of polysorbate 80 is up to about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%. In some embodiments, the concentration (w / v) of polysorbate 80 is approximately 0.001% to 0.1%, 0.003% to 0.09%, 0.005% to 0.08%, 0.007% to 0.07%, 0.01% to 0.06%, 0.02% to 0.05%, or 0.03% to 0.04%. In some embodiments, the concentration of polysorbate is approximately 0.01% (w / v).

[0107] In some embodiments, the pharmaceutical composition contains at least about 0.1 ng / μl, 0.2 ng / μl, 0.3 ng / μl, 0.4 ng / μl, 0.5 ng / μl, 0.6 ng / μl, 0.7 ng / μl, 0.8 ng / μl, 0.9 ng / μl, 1 ng / μl, 2 ng / μl, 3 ng / μl, 4 ng / μl, 5 ng / μl, 6 ng / μl, 7 ng / μl, 8 ng / μl, 9 ng / μl, 10 ng / μl, 12 ng / μl, 15 ng / μl, or 20 ng / μl of modified FGF-1 polypeptide. In some embodiments, the pharmaceutical composition contains a modified FGF-1 polypeptide in a maximum of approximately 0.1 ng / μl, 0.2 ng / μl, 0.3 ng / μl, 0.4 ng / μl, 0.5 ng / μl, 0.6 ng / μl, 0.7 ng / μl, 0.8 ng / μl, 0.9 ng / μl, 1 ng / μl, 2 ng / μl, 3 ng / μl, 4 ng / μl, 5 ng / μl, 6 ng / μl, 7 ng / μl, 8 ng / μl, 9 ng / μl, 10 ng / μl, 12 ng / μl, 15 ng / μl, or 20 ng / μl. In some embodiments, the pharmaceutical composition contains approximately 0.1 ng / μl to 20 ng / μl, 0.2 ng / μl to 15 ng / μl, 0.3 ng / μl to 12 ng / μl, 0.4 ng / μl to 10 ng / μl, 0.5 ng / μl to 9 ng / μl, 0.6 ng / μl to 8 ng / μl, 0.7 ng / μl to 7 ng / μl, 0.8 ng / μl to 6 ng / μl, 0.9 ng / μl to 5 ng / μl, 1 ng / μl to 4 ng / μl, or 2 ng / μl to 3 ng / μl of modified FGF-1 polypeptide. In some embodiments, the pharmaceutical composition contains approximately 1 ng / μl of modified FGF-1 polypeptide.

[0108] In some embodiments, the pH of the pharmaceutical composition is at least about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the pH of the pharmaceutical composition is at most about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the pH of the pharmaceutical composition is about 7.0–8.0, 7.1–7.9, 7.2–7.8, 7.3–7.7, 7.4–7.6, or 7.2–7.6. In some embodiments, the pH of the pharmaceutical composition is about 7.4.

[0109] In some embodiments, the pharmaceutical composition does not contain heparin.

[0110] In some embodiments, the administration step includes administering a modified FGF-1 polypeptide in a dose of at least about 1 ng, at least about 2 ng, at least about 3 ng, at least about 4 ng, at least about 5 ng, at least about 6 ng, at least about 7 ng, at least about 8 ng, at least about 9 ng, at least about 10 ng, at least about 12 ng, at least about 15 ng, at least about 18 ng, at least about 20 ng, at least about 30 ng, at least about 40 ng, at least about 50 ng, at least about 60 ng, at least about 70 ng, at least about 80 ng, at least about 90 ng, at least about 100 ng, at least about 120 ng, at least about 150 ng, at least about 180 ng, at least about 200 ng, at least about 300 ng, at least about 400 ng, or at least about 500 ng. In some embodiments, the administration step includes administering a modified FGF-1 polypeptide in doses of up to approximately 1 ng, up to approximately 2 ng, up to approximately 3 ng, up to approximately 4 ng, up to approximately 5 ng, up to approximately 6 ng, up to approximately 7 ng, up to approximately 8 ng, up to approximately 9 ng, up to approximately 10 ng, up to approximately 12 ng, up to approximately 15 ng, up to approximately 18 ng, up to approximately 20 ng, up to approximately 30 ng, up to approximately 40 ng, up to approximately 50 ng, up to approximately 60 ng, up to approximately 70 ng, up to approximately 80 ng, up to approximately 90 ng, up to approximately 100 ng, up to approximately 120 ng, up to approximately 150 ng, up to approximately 180 ng, up to approximately 200 ng, up to approximately 300 ng, up to approximately 400 ng, or up to approximately 500 ng. In some embodiments, the administration step includes administering modified FGF-1 polypeptide in doses of approximately 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, 12 ng, 15 ng, 18 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 120 ng, 150 ng, 180 ng, 200 ng, 300 ng, 400 ng, and 500 ng.In some embodiments, the administration step includes administering a modified FGF-1 polypeptide in doses of approximately 1 ng to 200 ng, approximately 3 ng to 180 ng, approximately 5 ng to 150 ng, approximately 10 ng to 120 ng, approximately 20 ng to 100 ng, approximately 30 ng to 90 ng, approximately 40 ng to 70 ng, or approximately 50 ng to 200 ng.

[0111] In some embodiments, the administration step includes administering a first dose and a second dose, where the second dose is equal to the first dose. In some embodiments, the first or second dose includes the doses disclosed herein. In some embodiments, the first or second dose is at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of modified FGF-1 polypeptide. In some embodiments, the administration step includes administering a first dose and a second dose, where the second dose is less than the first dose. In some embodiments, the first or second dose includes the doses disclosed herein. In some embodiments, the first or second dose is at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of modified FGF-1 polypeptide. In some embodiments, the administration step includes administering a first dose and a second dose, wherein the second dose is greater than the first dose. In some embodiments, the first dose or the second dose includes the doses disclosed herein. In some embodiments, the first dose or the second dose is at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of modified FGF-1 polypeptide.

[0112] In some embodiments, the administration step includes administering a first dose, a second dose, and a third dose, where the second dose is less than or equal to the first dose, and the third dose is less than or equal to the second dose. In some embodiments, the first dose, the second dose, or the third dose includes the doses disclosed herein. In some embodiments, the first dose, the second dose, or the third dose is at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of modified FGF-1 polypeptide.

[0113] In some embodiments, the administration step includes administering a first dose, a second dose, and a third dose, where the second dose is greater than or equal to the first dose, and the third dose is greater than or equal to the second dose. In some embodiments, the first dose, the second dose, or the third dose includes the doses disclosed herein. In some embodiments, the first dose, the second dose, or the third dose is at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of modified FGF-1 polypeptide.

[0114] In some embodiments, the administration step includes administering a first dose, a second dose, a third dose, and a fourth dose. In some embodiments, the first dose, the second dose, the third dose, or the fourth dose includes the doses disclosed herein. In some embodiments, the first dose, the second dose, the third dose, or the fourth dose is at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of modified FGF-1 polypeptide. In some embodiments, the administration step comprises administering a first dose, a second dose, a third dose, a fourth dose, and a fifth dose, wherein the first, second, third, fourth, or fifth dose comprises doses disclosed herein or at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of modified FGF-1 polypeptide. In some embodiments, the administration step comprises administering at least five doses, each of which at least five doses comprises at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of modified FGF-1 polypeptide.

[0115] In some embodiments, the administration step includes administering a certain dose of modified FGF-1 polypeptide before treatment with DWEK or DSO. In some embodiments, the administration step includes administering a certain dose of modified FGF-1 polypeptide concurrently with treatment with DWEK or DSO. In some embodiments, the administration step includes administering a certain dose of modified FGF-1 polypeptide after treatment with DWEK or DSO.

[0116] In some embodiments, the administration step includes administering a certain dose of modified FGF-1 polypeptide at least 1, 2, 3, 4, 5, 6, 7, 2, 3, or 4 weeks prior to the DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of modified FGF-1 polypeptide up to 1, 2, 3, 4, 5, 6, 7, 2, 3, or 4 weeks prior to the DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of modified FGF-1 polypeptide 2 to 3 days prior to the DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of modified FGF-1 polypeptide on the same day as the DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of a modified FGF-1 polypeptide at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of a modified FGF-1 polypeptide at a maximum of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the DWEK or DSO treatment.

[0117] In some embodiments, the administration step includes administering a certain dose of a modified FGF-1 polypeptide weekly at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of a modified FGF-1 polypeptide weekly at up to 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of a modified FGF-1 polypeptide weekly 3 weeks after the DWEK or DSO treatment. In some embodiments, the administration step includes administering a certain dose of a modified FGF-1 polypeptide on days 7, 14, and 21 following treatment with DWEK or DSO.

[0118] In some embodiments, the administration step includes administering at least about 10 microliters (mcL), 20 mcL, 30 mcL, 40 mcL, 50 mcL, 60 mcL, 70 mcL, 80 mcL, 90 mcL, or 100 mcL of the pharmaceutical composition. In some embodiments, the pharmaceutical composition has a modified FGF-1 polypeptide at a concentration of about 1 ng / mcL. In some embodiments, the administration step includes administering up to about 10 microliters (mcL), 20 mcL, 30 mcL, 40 mcL, 50 mcL, 60 mcL, 70 mcL, 80 mcL, 90 mcL, or 100 mcL of the pharmaceutical composition. In some embodiments, the pharmaceutical composition has a modified FGF-1 polypeptide at a concentration of about 1 ng / mcL. In some embodiments, the administration step involves administering about 10 microliters (mcL), 20 mcL, 30 mcL, 40 mcL, 50 mcL, 60 mcL, 70 mcL, 80 mcL, 90 mcL, or 100 mcL of a pharmaceutical composition having a modified FGF-1 polypeptide at a concentration of about 1 ng / mcL.

[0119] In some embodiments, the administration step includes administering the drug 1 to 3 times a day. In some embodiments, the administration step includes administering the drug once a day. In some embodiments, the administration step includes administering the drug twice a day. In some embodiments, the administration step includes administering the drug three times a day. In some embodiments, the administration step includes administering the drug 1 to 3 times a week. In some embodiments, the administration step includes administering the drug once a week. In some embodiments, the administration step includes administering the drug twice a week. In some embodiments, the administration step includes administering the drug three times a week.

[0120] In some embodiments, the pharmaceutical composition is administered by microneedles.

[0121] In some embodiments, the administration step includes administering to one eye. In some embodiments, the administration step includes administering to both eyes. In some embodiments, DWEK or DSO is performed on one eye. In some embodiments, DWEK or DSO is performed on both eyes. In some embodiments, cataract surgery is performed on one eye. In some embodiments, cataract surgery is performed on both eyes. In some embodiments, cataract surgery and DWEK or DSO are performed simultaneously (in the same surgical session) on one or both eyes.

[0122] In some embodiments, the administration step results in one or more therapeutic effects selected from the group including reduced corneal endothelial damage, increased corneal endothelial cell migration, increased corneal endothelial cell proliferation, reduced corneal edema, improved best-corrected visual acuity (BCVA), reduced corneal thickness, reduced central corneal thickness, improved visual function and corneal health (V-FUCHS) measurements, reduced intraocular pressure, improved slit-lamp examination, improved central corneal endothelial cell count, improved peripheral corneal endothelial cell count, or a reduction in the time to achieve any of these. In some embodiments, the administration step reduces corneal endothelial damage. In some embodiments, the administration step increases corneal endothelial cell migration. In some embodiments, the administration step increases corneal endothelial cell proliferation. In some embodiments, the administration step reduces corneal edema. In some embodiments, the administration step improves best-corrected visual acuity (BCVA). In some embodiments, the administration step reduces corneal thickness. In some embodiments, the administration step reduces central corneal thickness. In some embodiments, the administration step improves the measurement of visual function and corneal health (V-FUCHS). In some embodiments, the administration step reduces intraocular pressure. In some embodiments, the administration step improves slit-lamp examination. In some embodiments, the administration step improves central corneal endothelial cell count. In some embodiments, the administration step improves peripheral corneal endothelial cell count. In some embodiments, the administration step reduces the time to achieve any of those disclosed above.

[0123] In some embodiments, a reduction in corneal endothelial damage includes a reduction in cell death. In some embodiments, a reduction in corneal endothelial damage includes a reduction in functional impairment. In some embodiments, a reduction in corneal endothelial damage includes a reduction in cell death and a reduction in functional impairment.

[0124] In some embodiments, the administration step produces a therapeutic effect at a maximum of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the administration step. In some embodiments, the administration step produces a therapeutic effect at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the administration step.

[0125] In some embodiments, BCVA is measured using the Early Treatment Diabetic Retinopathy Scale (ETDRS) scoring system, which has at least 69 letters and at least 20 / 40 scores.

[0126] In some embodiments, the administration step includes administering to both eyes, and the administration step brings about a therapeutic effect in both eyes that is similar or the same between the two eyes as disclosed herein.

[0127] In some embodiments, the administration step includes administering to both eyes, and the administration results in an improvement in BCVA that is similar or the same between the two eyes.

[0128] In some embodiments, the administration step results in a dose-dependent therapeutic effect. In some embodiments, the therapeutic effect increases with the dose of the modified FGF-1 polypeptide administered. In some embodiments, the administration step results in a dose-independent therapeutic effect.

[0129] In some embodiments, the administration step produces a therapeutic effect in subjects with or without cataract surgery, and the therapeutic effect is similar or the same between subjects with and without cataract surgery.

[0130] In some embodiments, the administration step results in an improvement in corneal edema or central corneal thickness in subjects with or without cataract surgery, and the improvement in corneal edema or central corneal thickness is similar or the same in subjects with and without cataract surgery. In some embodiments, the administration step results in an improvement in BCVA. In some embodiments, the administration step results in a reduction or elimination of corneal edema. In some embodiments, the administration step results in an accelerated reduction or elimination of corneal edema.

[0131] In some embodiments, the method includes a treatment course of at least approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after DWEK or DSO treatment. In some embodiments, the method includes a treatment course of up to approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after DWEK or DSO treatment. In some embodiments, the method includes a treatment course of approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after DWEK or DSO treatment.

[0132] In some embodiments, DWEK / DSO includes a central descemetorhexis of approximately 4mm to 5mm. In some embodiments, DWEK / DSO includes a central descemetorhexis of at least approximately 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, or 8mm. In some embodiments, DWEK / DSO includes a central descemetorhexis of up to approximately 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, or 8mm. In some embodiments, DWEK / DSO includes a central descemetorhexis of approximately 2mm to 8mm, 3mm to 7mm, 4mm to 6mm, or 4mm to 8mm.

[0133] In some embodiments, a reduction in DSO size results in a reduction in the time to achieve a therapeutic effect in the subject. In some embodiments, the therapeutic effect includes faster improvement to BCVA 20 / 40, accelerated improvement to BCVA 20 / 40, a reduction in the time to BCVA 20 / 40, or a reduction in corneal edema. In some embodiments, the therapeutic effect includes faster improvement to BCVA 20 / 40. In some embodiments, the therapeutic effect includes accelerated improvement to BCVA 20 / 40. In some embodiments, the therapeutic effect includes a reduction in the time to BCVA 20 / 40. In some embodiments, the therapeutic effect includes a reduction in corneal edema.

[0134] In some embodiments, the subject has diabetes. In some embodiments, the administration step results in a therapeutic effect including a reduction in corneal thickness or corneal edema. In some embodiments, the corneal disease or illness includes Fuchs endothelial cell dystrophy (FECD) with central guttata as a contributing factor to visual symptoms.

[0135] In some embodiments, visual symptoms include decreased visual acuity from the cornea, morning blur, or central corneal edema. In some embodiments, the subject has a clear peripheral cornea with adequate corneal endothelial cell (CEC) reserve capacity. In some embodiments, the subject does not have subepithelial opacity or fibrosis. In some embodiments, the subject does not exhibit secondary corneal pathology, refractive surgery of the eye, or extraocular inflammation caused by non-infectious or infectious pathogens of the eye. In some embodiments, the subject is human.

[0136] Pharmaceutical composition Pharmaceutical compositions comprising modified FGF-polypeptides described herein may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and adjuvants that facilitate the processing of the active compound into a pharmaceutically usable preparation. The appropriate formulation depends on the selected route of administration. Further details regarding appropriate excipients for the pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), the above literature is incorporated herein by reference with respect to such disclosures.

[0137] When used herein, a pharmaceutical composition refers to a mixture of modified FGF with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspenders, thickeners, and / or excipients, and optionally, other therapeutic and / or prophylactic components. A pharmaceutical composition facilitates the administration of modified FGF to an organism. When performing a treatment or method of use provided herein, a therapeutically effective amount of modified FGF-1 polypeptide described herein, in the pharmaceutical composition, is administered to a mammal with an eye disease, disorder, or illness being treated. In some embodiments, the mammal is a human. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors. A pharmaceutically acceptable or suitable composition includes an ophthalmologically suitable or acceptable composition.

[0138] Pharmaceutical compositions (for example, for delivery by injection or for application as eye drops) may be in liquid or solid form. Liquid pharmaceutical compositions may include, for example: a sterile diluent, e.g., water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, a non-volatile oil that can function as a solvent or suspension medium, polyethylene glycol, glycerin, propylene glycol, or other solvent, an antimicrobial agent, an antioxidant, a chelating agent, a buffer for adjusting isotonicity, and one or more agents, e.g., sodium chloride or dextrose. Parenteral preparations may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials. Physiological saline is usually used as an excipient, and compositions that are injectable pharmaceutical compositions or compositions delivered to the eye (for example, as eye drops) are preferably sterile.

[0139] The modified FGF polypeptides or pharmaceutical compositions described herein can be delivered to a target by any suitable means, including, for example, topical, intraocular, intrachorally, orally, parenterally, intravenously, intraperitoneally, or intranasally (or by other delivery methods to the mucous membranes of the nose, throat, and bronchi), or by topical administration to the eye, or by intraocular or periorbital devices. Examples of topical administration include topical application, eye drops, intraocular injection, or periorbital injection. Periorbital injection typically involves injection of the compound subconjunctivally or into Tenon's space (below the fibrous tissue covering the eye). Intraocular injection typically involves injection of the modified FGF or pharmaceutical composition into the vitreous humor. In certain embodiments, administration is non-invasive, such as by topical application or eye drops. In some embodiments, administration is by a combination of topical and intrachorbital methods.

[0140] The modified FGF or pharmaceutical compositions described herein can be formulated for administration using pharmaceutically acceptable (suitable) carriers or vehicles and techniques routinely used in the art. Pharmaceutically acceptable or suitable carriers include ophthalmologically suitable or acceptable carriers. Carriers are selected according to the solubility of the particular modified FGF. Suitable ophthalmic compositions and formulations include those that can be administered topically to the eye by eye drops, injections, etc. In the case of eye drops, formulations may further optionally include ophthalmologically suitable agents, such as isotonic agents such as sodium chloride and concentrated glycerin; buffers, such as sodium phosphate and sodium acetate; surfactants, such as polyoxyethylene sorbitan monooleate (also known as polysorbate 80), polyoxyl stearate 40, and polyoxyethylene hydrogenated castor oil; stabilizers, such as sodium citrate and sodium edate; preservatives, such as benzalkonium chloride and parabens; and other components. Preservatives can be used, for example, at a level of approximately 0.001 to 1.0% by weight / volume. The pH of the formulation is usually within an acceptable range for ophthalmic formulations, for example, within a range of approximately pH 4 to 8.

[0141] In some embodiments, the pharmaceutical compositions disclosed herein include sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate anhydrous, recombinant human albumin solution, or polysorbate 80.

[0142] For injection, modified FGF or pharmaceutical compositions can be provided in injection-grade saline solutions in the form of liposome solutions for injection, sustained-release polymer systems, etc. Intraocular and periorbital injections are known to those skilled in the art and are described in many publications, for example, Spaeth, Ed., Ophthalmic Surgery: Principles of Practice, WBSanders Co., Philadelphia, Pa., 85-87, 1990.

[0143] In some embodiments, modified FGF or pharmaceutical composition (e.g., ophthalmic formulations) is administered to the cornea via microneedles (Jiang et al. (2007). Invest Ophthalmol Vis Sci 48(9):4038-4043). A microneedle array is coated with the modified FGF or pharmaceutical composition and pressed against the cornea so that the microneedles penetrate the corneal stroma but not the entire cornea. It is then removed, leaving the modified FGF or pharmaceutical composition in the corneal stroma. This modified FGF or pharmaceutical composition can stimulate corneal cells to proliferate and migrate, suppressing the scarring response normally present in stromal cells.

[0144] For the delivery of a composition comprising at least one of the modified FGF-1 polypeptides described herein via mucosal pathways, including delivery to the nasal passages, throat, and airways, the composition may be delivered in aerosol form. The compound may be in liquid or powder form for intramucosal delivery. For example, the composition may be delivered via a pressurized aerosol container containing a suitable spray agent, such as a hydrocarbon spray agent (e.g., propane, butane, isobutene). The composition may be delivered via a non-pressurized delivery system, such as a nebulizer or atomizer.

[0145] Suitable oral dosage forms include, for example, tablets, pills, sachets, or capsules made of hard or soft gelatin, methylcellulose, or other suitable material that readily dissolves in the gastrointestinal tract. Suitable non-toxic solid carriers can be used, for example, those containing pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, or magnesium carbonate. (See, for example, Remington: The Science and Practice of Pharmacy (Gennaro, 21st Ed. Mack Pub. Co., Easton, PA (2005))).

[0146] The modified FGF-1 polypeptides or pharmaceutical compositions described herein may be formulated for sustained release or prolonged release. Such compositions may generally be prepared using well-known techniques and may be administered, for example, by periorbital, intraocular, rectal, oral, or subcutaneous implantation, or by implantation into a desired target site, or by topical application. Sustained-release formulations may contain the agent dispersed in a carrier matrix and / or contained in a reservoir surrounded by a rate-limiting membrane. Excipients for use in such formulations may be biocompatible and biodegradable, and preferably the formulation results in the release of a relatively constant level of the active ingredient. The amount of the active compound contained in a sustained-release formulation depends on the implantation site, the rate of release and the expected duration, as well as the nature of the disease being treated or prevented.

[0147] Systemic drug absorption of drugs or compositions administered via the ocular route is known to those skilled in the art (see, for example, Lee et al., Int. J. Pharm. 233:1-18 (2002)). In one embodiment, the compounds described herein are delivered by topical ocular delivery methods (see, for example, Curr. Drug Metab. 4:213-22 (2003)). The compositions may be in the form of eye drops, salves, or ointments, e.g., aqueous eye drops, aqueous ophthalmic suspensions, non-aqueous eye drops, and non-aqueous ophthalmic suspensions, gels, ophthalmic ointments, etc. For the preparation of gels, for example, carboxyvinyl polymers, methylcellulose, sodium alginate, hydroxypropylcellulose, ethylene maleic anhydride polymer, etc., may be used.

[0148] In another embodiment, the modified FGF solution or pharmaceutical composition (e.g., an ophthalmic formulation) contains hyaluronic acid, carboxymethylcellulose, or other polysaccharides that increase ocular tolerance, viscosity, and osmotic pressure to produce a comfortable ophthalmic solution.

[0149] Pharmaceutical compositions may be administered in a manner appropriate to the disease, disorder, or illness being treated (or prevented), as determined by those skilled in the medical field. The appropriate dose and the appropriate duration and frequency of administration depend on factors such as the patient's condition, the type and severity of the patient's disease, disorder, or illness, the specific form of the active ingredient, and the method of administration. Generally, an appropriate dose and treatment regimen provides a composition in an amount sufficient to provide therapeutic and / or prophylactic benefits (e.g., improved clinical outcomes such as more frequent complete or partial remission, longer periods of disease-free life, or reduced severity of symptoms). For prophylactic use, the dose must be sufficient to prevent, delay the onset of, or reduce the severity of an eye disease, disorder, or illness. The optimal dose can generally be determined using experimental models and / or clinical trials. The optimal dose may depend on the patient's BMI, weight, or blood volume.

[0150] Kit / Product Kits and products are also provided herein for use in the therapeutic applications described herein. Such kits may include a carrier, package, or container partitioned to receive one or more containers such as vials, tubes, etc., each container containing one of the separate elements used in the manner described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from a variety of materials such as glass or plastic.

[0151] The products provided herein include packaging materials. Examples of packaging materials for use in packaging pharmaceuticals include U.S. Patents No. 5,323,907, 5,052,558, and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging materials suitable for the selected formulation and the intended mode of administration and treatment. A wide variety of ophthalmic formulations of modified FGF-1 polypeptides and pharmaceutical compositions provided herein are intended as treatments for various eye diseases, disorders, or conditions that would benefit from the administration of the modified FGF or pharmaceutical compositions described herein.

[0152] For example, the container may contain modified FGF, such as modified FGF-1 having the sequence of SEQ ID NO: 2. The container may optionally have a sterile access port. Such kits may optionally include the compound along with a description, label, or instruction for specific use in the methods described herein.

[0153] The kit may typically include one or more additional containers, each containing one or more of the various materials (such as reagents, optionally concentrated forms, and / or devices) that are commercially and user-desirable for the use of the modified FGF described herein. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes, carriers, packages, containers, vials, and / or instructions for use, as well as tube labels listing the contents and / or package inserts together with the instructions for use. A set of instructions is also typically included.

[0154] Labels may be on the container or attached to the container. Labels may be on the container when the letters, numbers, or other symbols forming the label are attached, molded, or etched onto the container itself, or they may be attached to the container, for example, as a package insert, if they reside within a receptacle or carrier that holds the container. Labels may be used to indicate that the contents are for use in a specific therapeutic application. Labels may further indicate instructions for using the contents, such as in the methods described herein.

[0155] In certain embodiments, modified FGF pharmaceutical compositions may be provided in packs or dispenser devices that can contain one or more unit dosage forms containing the compounds provided herein. Packs may include, for example, metal or plastic foil such as blister packs. Instructions for administration may be attached to the packs or dispenser devices. The packs or dispensers may also be attached to the containers with notices in the form prescribed by government agencies that regulate the manufacture, use, or sale of pharmaceuticals, and these notices reflect agency approval of the form of the drug for human or veterinary administration. Such notices may be, for example, labels or approved product inserts approved by the U.S. Food and Drug Administration for prescription drugs. Compositions containing modified FGF provided herein, formulated in a suitable pharmaceutical carrier, may further be prepared for the treatment of a indicated condition, placed in appropriate containers, and labeled.

[0156] definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which the claimed subject matter belongs. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or for immediate reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from those commonly understood in the art.

[0157] Throughout this application, various embodiments may be presented in range format. It should be understood that the use of range format is for convenience and brevity only and should not be interpreted as an inflexible limitation to the scope of this disclosure. Therefore, range descriptions should be considered to have all specifically disclosed subranges and, similarly, individual numerical values ​​within those ranges. For example, a range description such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and, similarly, individual numerical values ​​within those ranges such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0158] As used herein and in the claims, the singular forms “a,” “and,” and “the” include plural references unless otherwise clearly indicated in the content. For example, the term “sample” includes multiple samples, and also includes mixtures thereof.

[0159] As used herein, the term “percent (%) amino acid sequence identity” with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular sequence, after aligning the sequences and introducing gaps as necessary to achieve maximum percent sequence identity, and without considering conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A person skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. Alignment for the purpose of determining percent amino acid sequence identity can be achieved, for example, using the publicly available sequence comparison computer program ALIGN-2. The source code for the ALIGN-2 sequence comparison computer program is available with user documentation from the U.S. Copyright Office (Washington, DC, 20559) and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program can be compiled for use on UNIX® operating systems such as Digital UNIX® V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0160] Standard definitions of chemical terms, though not limited, can be found in references including Carey and Sundberg, “ADVANCED ORGANIC CHEMISTRY 4TH ED.” Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwise indicated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are included.

[0161] Unless otherwise specified, the nomenclature used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as their experimental procedures and techniques, are recognized in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and for the treatment of patients. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Reaction and purification techniques can be carried out, for example, using kits specified by the manufacturer, or as commonly achieved in the art, or as described herein. The aforementioned techniques and procedures can generally be carried out by conventional methods and as described in the various general and more specific references cited and discussed throughout this specification.

[0162] The methods and compositions described herein are, but are not limited to, specific methodologies, protocols, cell lines, constructs, and reagents described herein, and should therefore be understood to be subject to change. The terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the methods, compounds, and compositions described herein.

[0163] The terms “to treat,” “to treat,” or “treatment” include, but are not limited to, relieving, reducing, or improving the symptoms of a disease, disorder, or illness; preventing further symptoms; improving or preventing the underlying metabolic causes of symptoms; inhibiting a disease, disorder, or illness, for example, preventing the onset of a disease, disorder, or illness; reducing a disease, disorder, or illness; causing regression of a disease, disorder, or illness; improving a condition caused by a disease, disorder, or illness; or cessating the symptoms of a disease, disorder, or illness. The terms “to treat,” “to treat,” or “treatment” include, but are not limited to, preventive and / or therapeutic treatments.

[0164] With respect to formulations, compositions, or components, the terms “acceptable” or “pharmaceutically acceptable” mean that they do not have a sustained adverse effect on the systemic health of the subject being treated, nor do they inhibit the biological activity or properties of the modified FGF described herein, and are relatively non-toxic.

[0165] The term “improvement” of symptoms of a particular disease, disorder, or illness by administration of a particular modified FGF or pharmaceutical composition means any reduction in severity, delay in onset, delay in progression, or reduction in duration, whether permanent or temporary, sustained or transient, that may be caused by or associated with the administration of the modified FGF or pharmaceutical composition.

[0166] As used herein, the terms “combination” or “pharmaceutical combination” mean a product resulting from a mixture or combination of one or more active ingredients, and include both fixed and unfixed combinations of active ingredients. The term “fixed combination” means that both one active ingredient (e.g., modified FGF) and an adjuvant are administered to the patient simultaneously in the form of a single entity or dosage. The term “unfixed combination” means that one active ingredient (e.g., modified FGF) and an adjuvant are administered to the patient simultaneously, in parallel, or sequentially as separate entities without specific intervening time limitations, and such administration provides two effective levels of the drug in the patient’s body. The latter also applies to cocktail therapies, e.g., administration of three or more active ingredients.

[0167] As used herein, the term “pharmaceutical composition” refers to one or more modified FGF-1 polypeptides having one or more other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. Pharmaceutical compositions facilitate the administration of modified FGF-1 polypeptides to living organisms. Multiple techniques for administering modified FGF-1 polypeptides exist in the art, but are not limited to topical, ocular, intraocular, periocular, intravenous, oral, aerosol, parenteral, and administration methods.

[0168] As used herein, the term “carrier” refers to a relatively non-toxic chemical compound or drug that facilitates the uptake of a drug of interest (e.g., modified FGF) into a cell or tissue.

[0169] The term "diluent" refers to a chemical compound used to dilute a drug of interest (e.g., modified FGF) before delivery. Diluents can also be used to stabilize drugs, as they can provide a more stable environment. Salts dissolved in buffer solutions (which can also provide pH control or maintenance) are used as diluents in the art, but are not limited to phosphate-buffered saline.

[0170] Terms such as "concurrent administration" mean the administration of selected drugs (e.g., modified FGF or its compositions and adjuvants) to a single patient, and are intended to include treatment regimens in which the drugs are administered by the same or different routes of administration, or at the same or different times.

[0171] The terms “effective dose” or “therapeutic effective dose” refer to a sufficient amount of the modified FGF-1 polypeptide, drug, combination, or pharmaceutical composition described herein administered that would, to some extent, alleviate one or more symptoms of the disease, disorder, or illness being treated. The result may be a reduction and / or mitigation of the signs, symptoms, or causes of the disease, or any other desired change in the biological system. For example, “effective dose” for therapeutic use is the amount of modified FGF, drug, combination, or pharmaceutical composition required to provide the desired pharmacological effect, therapeutic improvement, or clinically significant reduction of disease symptoms without excessive adverse side effects. The appropriate “effective dose” in any individual case may be determined using techniques such as dose escalation studies. The term “therapeutic effective dose” includes, for example, a prophylactic effective dose. It is understood that “effective dose” may vary from subject to subject due to variations in the metabolism of the modified FGF, combination, or pharmaceutical composition, the subject’s age, weight, general condition, the disease being treated, the severity of the disease being treated, and the prescribing physician’s judgment. As just one example, the therapeutically effective dose can be determined by routine experiments, including dose-escalation clinical trials, but is not limited to these.

[0172] The term "preventive effective dose" refers to the amount of the modified FGF, compound, drug, combination, or pharmaceutical composition described herein that would, to some extent, alleviate one or more symptoms of the disease, illness, or disorder being treated. In such preventive applications, such amounts may depend on the patient's health status, weight, etc. Determining such a preventive effective dose through routine experiments, including but not limited to dose-escalation clinical trials, is considered to be within the scope of the skill of those skilled in the art.

[0173] As used herein, the terms “subject” or “patient” refer to an animal being treated, observed, or experimented on. For example, a subject may be a mammal, including, but is not limited to, a human.

[0174] The terms “enhance” or “to augment” mean to increase or extend a desired effect in terms of either potency or duration. For example, “enhancing” the effect of a therapeutic agent, either alone or in combination, refers to the ability to increase or extend the potency, duration, and / or magnitude of the drug’s effect on treating a disease, disorder, or illness. When used in a patient, the effective dose for this use depends on the severity and course of the disease, disorder, or illness, previous treatments, the patient’s health status and response to the drug, and the judgment of the treating physician.

[0175] The term "modulate" means interacting with a target (e.g., an FGF receptor) directly or indirectly to alter the activity of the target, including, but not limited to, enhancing, inhibiting, or antagonizing the activity of the target, or extending the activity of the target. In some embodiments, the modified FGF-1 polypeptides and pharmaceutical compositions described herein can modulate the activity of one or more respective targets (e.g., one or more FGF receptors). In some embodiments, the modified FGF-1 polypeptides described herein modulate (e.g., increase) the activity of one or more FGF receptors on a cell (e.g., a corneal endothelial cell), thereby causing, for example, cell migration and / or cell proliferation.

[0176] As used herein, the term “target” refers to a biological molecule (e.g., a target protein or protein complex), such as an FGF receptor, or a portion of a biological molecule that can be conjugated by a selective binder (e.g., modified FGF) or a pharmaceutical composition described herein. As used herein, the term “non-target” refers to a biological molecule or portion of a biological molecule that is not selectively conjugated by a selective binder or pharmaceutical composition described herein.

[0177] The terms “target activity” or “cellular response” refer to any biological activity that can be regulated by modified FGF, or any cellular response resulting from the binding of modified FGF to FGF receptors. Specific exemplary targeted activities and cellular responses include, but are not limited to, binding affinity, signaling, gene expression, cell migration, cell proliferation, cell differentiation, and improvement of one or more symptoms associated with eye diseases, disorders, or illnesses.

[0178] Whenever “at least,” “greater than,” or “greater than or equal to” precedes the first number in a set of two or more numbers, the terms “at least,” “greater than,” or “greater than or equal to” apply to each of the numbers in the set. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0179] Whenever "nothing less than," "less than," or "less than or equal to" precedes the first number in a set of two or more numbers, "nothing less than," "less than," or "less than or equal to" applies to each of the numbers in that set. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0180] The expressions "at least one of A and B" and "at least one of A or B" may be interpreted as meaning at least A, at least B, or at least A and B (i.e., a set including A and B, which may include one or more additional elements). The term "A and / or B" may be interpreted as meaning A only, B only, or both A and B.

[0181] The phrases "at least about A, B, and C" and "at least about A, B, or C" may be interpreted as meaning at least about A, at least about B, or at least about C. The phrases "up to about A, B, and C" and "up to about A, B, or C" may be interpreted as meaning up to about A, up to about B, or up to about C.

[0182] The expression "approximately between A and B, C and D, and E and F" can be interpreted as meaning approximately between A and B, approximately between C and D, and approximately between E and F. The expression "approximately between A and B, C and D, or E and F" can be interpreted as meaning approximately between A and B, approximately between C and D, or approximately between E and F.

[0183] The expression "approximately A to B and C to D" can be interpreted as meaning approximately A to approximately B and approximately C to approximately D. The expression "approximately A to B or C to D" can be interpreted as meaning approximately A to approximately B or approximately C to approximately D.

[0184] As used herein, the term “exemplary” means “serving as an example, case, or illustration.” No embodiment described herein as “exemplary” should be construed as preferable or advantageous to any other embodiment.

[0185] The terms “determining,” “measuring,” “evaluating,” “assessing,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The above terms include determining whether an element is present or not (e.g., detection). These terms may include quantitative, qualitative, or both quantitative and qualitative determinations. Evaluation may be relative or absolute. “Detecting the presence of ~” may include determining the quantity of something that is present, in addition to determining whether something is present or not, depending on the context.

[0186] The terms “subject,” “individual,” and “patient” are often used interchangeably herein. A “subject” may be a biological entity containing expressed genetic material. A biological entity may be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protists. A subject may be a tissue, cell, or offspring of a biological entity obtained in vivo or cultured in vitro. A subject may be a mammal. A mammal may be a human. A subject may be diagnosed or suspected of being at high risk for a disease. In some cases, a subject may not necessarily be diagnosed or suspected of being at high risk for a disease.

[0187] The term "in vivo" is used to describe events that occur within the body of a subject.

[0188] The term "ex vivo" is used to describe events that occur outside the body of a subject. Ex vivo assays are not performed on the subject; rather, they are performed on a sample isolated from the subject. An example of an ex vivo assay performed on a sample is an "in vitro" assay.

[0189] The term "in vitro" is used to describe events that occur when materials are contained within a container for holding laboratory reagents, so as to be separated from the biological source from which the material is obtained. In vitro assays can include cell-based assays in which living or dead cells are used. In vitro assays can further include cell-free assays in which intact cells are not used.

[0190] As used herein, a number preceded by the term "approximately" refers to a number within plus or minus 10% of that number. A range preceded by the term "approximately" refers to a range within minus 10% of the minimum value and plus 10% of the maximum value.

[0191] The paragraph headings used in this specification are for organizational purposes only and should not be construed as restricting the subject matter described herein. [Examples]

[0192] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0193] Example 1: Clinical effects of modified FGF-1 polypeptide (TTHX114)

[0194] Nonclinical trials

[0195] The nonclinical program for TTHX1114 had several objectives: (1) to understand the in vitro, ex vivo, and in vivo pharmacodynamics of TTHX1114; (2) to evaluate the ocular pharmacokinetics (PK) after a single intraocular (IC) injection in rabbits; and (3) to evaluate the ocular and systemic safety, toxicokinetics (TK), and immunogenicity of TTHX1114 after repeated administration for up to one month by weekly IC injections in rabbits and dogs and every other day intravenous (IV) injections in rats.

[0196] The safety pharmacological parameters evaluated in toxicity studies demonstrated that TTHX1114 did not cause any adverse effects on central nervous system function in a 1-month IV toxicity study in rats, and did not cause any effects on respiratory or cardiovascular function in a 1-month IC toxicity study in dogs.

[0197] In a critical 1-month IV toxicity study in rats administered TTHX1114 at a dose of 50 μg / kg or less every other day, TK evaluation showed that systemic exposure (C) max and AUC last It was demonstrated that the amount increased proportionally to the dose, with no gender difference, and repeated administration resulted in a 2-4 times accumulation.

[0198] In Phase 1 / Phase 2 clinical trials, the absence of systemic effects and systemic exposure in 1-month IC toxicity studies in animals at ocular doses that were 880 and 88 times the planned starting and maximum ocular doses, respectively, was 7.6 times lower than the systemic exposure of LLOQ used in human PK compared to LOEL in rats, and ED in the NIH 3T3 cell proliferation assay. 50 Combined with the fact that it was 17 times lower than the potency value, this indicated a high ocular safety margin, difficulty in detecting systemic exposure, and that LLOQ represented negligible systemic exposure in humans.

[0199] The THX1114 preclinical program supports repeated administration of TTHX114 for a total of up to five times.

[0200] Potential risks and benefits

[0201] Anticipated hypothetical adverse events included, but were not limited to, anterior chamber flare, conjunctivitis, endophthalmitis, and inflammation of the anterior chamber (e.g., cornea and / or iris).

[0202] Complications of DWEK / DSO included descemetorhexis decentration, descemetor detachment, posterior interstitial opacity, abnormal corneal topography, and persistent corneal edema.

[0203] Theoretical basis for dose selection

[0204] Dose levels up to 1000 ng have been evaluated in non-clinical studies, and the maximum dose planned in this study was far below the highest non-clinical dose level evaluated.

[0205] The previous trial of TTHX-001 was designed to determine the recommended Phase 2 dose of TTHX1114 and evaluated dose levels of 1 ng, 3 ng, and 10 ng. The maximum tolerated dose (MTD) was not exceeded. All doses in the TTHX-001 trial were administered via 10 microliters (mcL) IC injection.

[0206] A normal adult eye contains approximately 300 mcL of aqueous humor in the anterior chamber. Since TTHX1114 was provided at a concentration of 1000 ng / mL, increasing the dose required increasing the volume. The turnover of aqueous humor (AqHu) in the anterior chamber of the eye was approximately 90 minutes, during which any significant increase in IOP was normalized via drainage through the trabecular meshwork. Patients with uncontrolled glaucoma were excluded from this study.

[0207] Dose-limiting toxicity

[0208] Potential dose-limiting toxicity (DLT) is defined as a suspected adverse reaction, which is (1) a post-injection IOP measurement >20% from before injection that persists for more than 90 minutes, or (2) an IOP that remains above 32 mmHg and is unresponsive to treatment.

[0209] If any subject experienced a potential DLT, further experimental treatment was performed, and the Medical Monitor was contacted to consider possible treatments and medical management of the subject, including further unplanned experimental evaluations.

[0210] 10 ng (10 mcL volume): Study TTHX-001 evaluated the safety and tolerability of a maximum TTHX1114 dose level of 10 ng administered weekly to non-surgical eyes. The 10 mcL volume IC injection appeared to be well-tolerated, with no clinically significant changes in reported IOPs or any IOP-related adverse events.

[0211] 30 ng (30 mcL volume): IC injections of 10 mcL volume appeared to be well-tolerated, and the 30 mcL volume injection showed the potential to increase fluid volume in the anterior chamber by approximately 10%. The 30 mcL volume injection was used as a "step-up" in all subjects. Following demonstrated tolerance (no DLT) of the 30 mcL volume injection, subjects were administered 50 ng / 50 mcL doses of TTHX1114. After at least 10 IC injections of 30 mcL were safely tolerated, the "step-up" dose was removed, and subjects were administered 50 ng / 50 mcL on non-operative days.

[0212] 50 ng (50 mcL volume): Infusion of 50 mcL volume showed the potential to increase fluid volume in the anterior chamber by approximately 17%. Intra-subject dose escalation occurred after subjects demonstrated tolerance to 30 mcL volume injections (without DLT). After at least 10 IC injections of 30 mcL were safely tolerated, the “escalation” dose was removed, and subjects were administered 50 ng / 50 mcL on non-surgical days.

[0213] 100 ng (100 mcL volume): During DWEK / DSO, the anterior ventricle was surgically opened, resulting in some loss of aqueous humor. The 100 mcL dose volume did not restore the entire preoperative volume, but the postoperative eye was placed in the desired semi-constricted state. As described above, no concerns were raised regarding a potential increase in IOP after the administration of the 50 mcL volume and the injection of the 100 mcL volume on the day of surgery.

[0214] Test group

[0215] The test group consisted of men and women aged 18 and over who were diagnosed with FECD and were taking DWEK / DSO.

[0216] Test design

[0217] This was an open-label, multicenter trial enrolling up to approximately 50 FECD patients scheduled for DWEK / DSO. Participants were assigned to the test treatment group based on the publicly available group at the time of treatment of the primary (first) eye. If a participant chose to treat an eligible second (contralateral) eye, that eye was treated based on the publicly available group at that time. The number of participants per group and per test treatment in the trial is summarized in Table 1 and Figure 1 below.

[0218] This test allowed for treatment of the other eye. To avoid ambiguity, the eye treated first is called the test eye (SE), and the other eye is called the other eye (FE).

[0219] [Table 1]

[0220] Participants agreed to participate during the screening period prior to the scheduled DWEK / DSO procedure and were screened for eligibility.

[0221] The test schema is shown in Figures 1 and 2.

[0222] Eligible subjects who did not wish to receive the investigational drug or who did not have a qualifying eye were assigned to treatment group 1.

[0223] Following a preliminary safety and tolerability assessment of 50 ng IC injection in Group 2, it was chosen to publish Groups 3 and 4.

[0224] In Group 3, eligible subjects were given a "one-and-done" dosing regimen in which TTHX1114 was administered as an injection of 100 ng / 100 mcL at the time of DWEK / DSO.

[0225] In Group 4, eligible subjects received a 100 ng / 100 mcL injection at the time of DWEK / DSO, followed by weekly injections of 30 or 50 ng (30 or 50 mcL) x 3. Subjects in Group 4 who had not fully recovered by day 28 were given an additional dose of TTHX1114 after consultation with the medical monitor.

[0226] Study Treatment Group 1: Subjects in Group 1 underwent Descemet's membrane decompression without endothelial keratoplasty (DWEK / DSO) on day 0 of the study and received no TTHX1114. These subjects served as the control group and were followed up on days 1, 14, 28, and 56 after DWEK / DSO. Follow-up evaluations included routine eye assessments.

[0227] [Table 2]

[0228] Study treatment group 2: Approximately 48–72 hours prior to DWEK / DSO (-3 days), subjects were administered a "conditioning dose" (10 ng [10 mcL]) of TTHX1114. The conditioning dose was intended to provide anti-apoptotic protection to existing cells. On day 0, subjects underwent DWEK / DSO and were administered TTHX1114 (50 ng [50 mcL]) at the time of the surgical procedure. As mentioned above, the anterior chamber immediately after surgery should readily accommodate additional volume. Subjects returned for follow-up on day 1 (the day after DWEK / DSO) and for subsequent administrations of the study treatment on days 7, 14, and 21. Follow-up visits were made on days 28 and 56.

[0229] [Table 3]

[0230] In the 3rd treatment group, on day 0, subjects underwent DWEK / DSO and were administered TTHX1114 (100 ng [100 mcL]) during the surgical procedure. As mentioned above, the anterior chamber should readily accommodate additional volume immediately after surgery. Subjects returned for follow-up on day 1 (the day after DSO), and subsequent follow-up visits were made on days 7, 14, 21, 28, and 56.

[0231] [Table 4]

[0232] In the 4-day study group, subjects underwent DWEK / DSO on day 0 and received TTHX1114 (100 ng [100 mcL]) at the time of the surgical procedure, followed by injections three times weekly. Subjects who had not fully recovered by day 28 received an additional dose of TTHX1114 after consultation with the medical monitor. Subjects returned for follow-up on day 1 (the day after DWEK / DSO) and for subsequent administrations of the study procedure on days 7, 14, and 21. Follow-up visits were on day 28. * This was done on the 56th day. The subjects who had not fully recovered by the 28th day... * After consultation with the medical monitor, an additional dose of TTHX1114 was administered. The study subjects received their fifth dose of TTHX1114, repeated all evaluations scheduled for day 21 on day 28, and made unscheduled visits on or around day 35, after all evaluations scheduled for day 28 had been completed.

[0233] [Table 5]

[0234] Long-term follow-up (all treatment groups): Participants underwent long-term follow-up for up to one year after the first administration of DWEK / DSO or TTHX1114. Long-term follow-up visits were made on days 84, 168, and 336 / end of study visit. For participants who had both eyes treated during the study, the visits on days 168 and 336 for both eyes were combined based on the date of surgery for the second eye. Day 336 was considered the end of the study visit and included a comprehensive eye examination.

[0235] End-of-Trial Visit: End-of-Trial visits (EOS) were performed for all subjects. EOS were performed at any point after day 168, during which the EOS evaluation scheduled for day 336 was performed and plasma samples were collected for anti-drug antibody (ADA) analysis. If a subject had already completed the trial, they must be contacted to request the provision of a plasma sample for ADA analysis. Subjects for whom an evaluable mirror image could not be obtained at the most recent visit must be re-acquired to obtain an evaluable image. It is important that an evaluable mirror image was collected for all subjects at their final visit.

[0236] Efficacy endpoint

[0237] The efficacy endpoints in this study include (1) the number of subjects in each group with a best corrected visual acuity (BCVA) of 20 / 40 or better on day 28, and (2) central corneal thickness on day 28.

[0238] The following additional endpoints were also evaluated: (1) the number of subjects in each group with a BCVA of 20 / 40 or better on days 21, 56, and 84 (Figures 4 and 5), (2) Early Treatment Diabetic Retinopathy Study (ETDRS) letter scores on days 21, 28, 56, and 84, (3) time to BCVA of 20 / 40 throughout the study, (4) central corneal thickness on days 21, 56, and 84 (Figure 6), (5) visual function and corneal health (V-FUCHS) on days 28 and 56, (6) change in intraocular pressure from baseline throughout the study, (7) change in intraocular pressure from before to after injection on all injection days, (8) slit-lamp examination on days 28, 56, and 84, and (9) central and peripheral CEC numbers throughout the study. Figure 10 shows the proportion of subjects / eyes in each group with a 0.6 logMAR (6 lines, 30 characters) increase in BCVA from the first postoperative visit at each visit. Fisher's exact test compares group 2 vs group 3.

[0239] Figure 4 shows an analysis of the proportion of all eyes in groups 3 and 4, pooled for eyes that received Descemet's membrane stripping only (DSO) (dashed line) or DSO + cataract surgery (solid line) at each visit. Accelerated recovery of visual acuity was maintained despite cataract surgery. Figure 5 shows an analysis of the proportion of all eyes in group 2, as well as groups 3 and 4, pooled for eyes that received DSO only or DSO + cataract surgery at each visit. Visual outcomes in high-risk patients improved regardless of cataract surgery.

[0240] This test allowed for treatment of the opposite eye. To avoid ambiguity, the eye treated first was called the test eye (SE), and the opposite eye was called the other eye (FE).

[0241] Measurements performed to minimize / avoid bias

[0242] All adverse events were documented, regardless of any questionable causal relationship. Furthermore, subjects were assigned to publicly available treatment groups at the time of eligibility determination based on their desire to receive the investigational drug. All subjects in all treatment groups were required to meet all eligibility criteria. All primary and secondary efficacy endpoints were source data validated or provided by an independent third party (e.g., a central reader).

[0243] Test treatment: The test treatment (TTHX1114) was administered as an anterior chamber injection to subjects in treatment group 2.

[0244] TTHX1114 Drug Product

[0245] The TTHX1114 drug product is a solution formulated in phosphate-buffered saline manufactured in a sterile filling / finishing facility contracted in accordance with cGMP regulations. TTHX1114 is supplied at a concentration of 1.0 ng / mcL.

[0246] TTHX1114 was supplied in a carton of 5 vials. The kit's outer box contained the kit number, storage instructions, and the following statement: "WARNING: NEW DIRECTORY - For investigational use only, as required by federal law (or U.S. law)." Each vial was labeled with a lot number.

[0247] Save TTHX1114

[0248] TTHX1114 was frozen (with dry ice) for transport and stored in a safe area at -20°C. Deviations from the temperature limit were reported to Trefoil (or designated person) to determine whether the affected vial was usable. Access to the investigational drug was limited to individuals authorized by the principal investigator to administer the study procedure.

[0249] TTHX1114 preparation

[0250] The vials were thawed and gently mixed by inversion before administering the test treatment. Preparation for the test treatment was started after the subjects arrived at the clinic to receive the test treatment (i.e., thawed at room temperature; NB: no external heat was applied). All test treatment injections were administered within 8 hours of starting the thawing of the test treatment vials.

[0251] Low-volume syringes were supplied for administering each test procedure. To ensure accurate administration of the correct volume of the test procedure, a JuvaPen® device for administering doses of less than 30 mcL was provided.

[0252] Period of testing and testing procedures

[0253] Each participant was expected to be in the testing phase for approximately one year after completing DWEK / DSO.

[0254] [Table 6]

[0255] Termination Criteria: The termination criterion for the study was an unacceptable incidence of toxicity. This was an open-label study, and the frequency and severity of all adverse events were continuously monitored.

[0256] Accountability for investigational drugs

[0257] In accordance with 21 CFR 312.61 and 21 CFR 312.62(a), the Principal Investigator (1) administered the drug only to subjects under the personal supervision of the Principal Investigator or under the supervision of the Principal Investigator who is liable to the Principal Investigator; (2) supplied the investigational drug to any person not authorized under this party to administer the investigational drug; and (3) maintained proper records of the prescription of the investigational drug, including the date, quantity, and use by subject.

[0258] If the clinical trial ends, is suspended, discontinued, or completed, the principal investigator returns any unused drug supplies to the sponsor or otherwise disposes of the unused drug supplies under 21 CFR 312.59.

[0259] Data entered into the database

[0260] The majority of the collected data was entered into the database via the EDC system. Data from third parties (e.g., central research institutes or reading facilities) was imported into the clinical database, and this data included (1) the number and characteristics of endothelial cells assessed by the central reading center, (2) pharmacokinetic measurements, (3) anti-drug antibody assay results, and (4) clinical laboratory results.

[0261] The imported data was compared with the clinical database (e.g., subject ID, date, time, point in time), and the results were uploaded to the database under agreed-upon quality and data transfer specifications. The uploaded data underwent 100% quality checks against the source data.

[0262] Other data and results generated by the clinical trial sites were entered directly into the electronic data capture (EDC) system. All data in the EDC was monitored, and critical variables were 100% source-validated.

[0263] Target selection and departure

[0264] Potential subjects were identified based on a confirmed diagnosis of Fuchs endothelial corneal dystrophy (FECD) and their suitability as candidates for DWEK / DSO. Most potential subjects were obtained directly from patients receiving care at the clinical trial sites. All subjects were suitable for the DWEK / DSO procedure independently of the clinical trial.

[0265] Inclusion criteria: (1) Male or female aged 18 years or older, (2) Planned for DWEK / DSO with a planned central descemetorhexis of approximately 4-5 mm, (3) Female subjects of childbearing potential (WOCBP) must be treated with an acceptable method of childbirth management, (4) The following criteria: a. Fuchs endothelial cell dystrophy with central guttata (FECD) considered to be a contributing factor to visual symptoms (i.e., decreased visual acuity of corneal origin, such as morning blur and / or central corneal edema). Non-confluent peripheral guttata is not exclusive: b. Clear peripheral cornea with adequate CEC reserve or based on other criteria such as CEC characteristics; c. Absence of subepithelial opacity / fibrosis that would interfere with postoperative visual acuity; d. No history of extraocular inflammation from any non-infectious or infectious cause (bacterial, viral, or fungal) in the test eye within 6 months prior to day 0 of the test (Note: Mild blepharitis and / or dry eye-related inflammation are acceptable); e. Absence of any symptoms that would impair examination of the anterior chamber structure; (5) Subjects administered TTHX1114 must have another eye with adequate function (i.e., visual acuity is 20 / 100 or better).

[0266] To be eligible for the optional extended administration period (OEDP), study subjects must have visible central endothelium on day 168 as determined by microscopic examination.

[0267] Exclusion Criteria: (1) Planned use of postoperative rho kinase inhibitors; (2) Eye cancer (including melanoma), corneal herpes, demonstrated and repeated IOP elevation in either eye (Note: well-controlled glaucoma is acceptable), posterior polymorphic corneal dystrophy (PPCD, also known as Schlichting dystrophy), uveitis, or intolerance, hypersensitivity, or significant allergy to any drug compound, food, or other substance (Note: this includes all components and excipients of the study drug); (3) Current or recent (e.g., 28 days prior to day 0 of the study) participation in any other interventional clinical study; (4) Use of systemic or dermatological cytotoxic chemotherapy or rho kinase inhibitors within 3 months prior to day 0 of the study; (5) Use of cyclosporine ophthalmic emulsion (e.g., RESTASIS®), Xiidra® within 1 month (28 days) prior to day 0 of the study. (6) Use of any systemic (intranasal, inhaled, oral, parenteral, or topical) corticosteroid (Refitegrast ophthalmic solution), or any other systemic (intranasal, inhaled, oral, parenteral, or topical) corticosteroid (Note: Potential subjects with stable, chronic low doses that are not expected to change over the three months following day 0 of the study may be considered for the study after approval by the medical monitor), (6) Use of hypertonic saline eye drops (e.g., Muro128) four days prior to day 0 of the study, (7) Women who are currently pregnant or of childbearing potential, breastfeeding, planning to become pregnant during the study, or who do not intend to use highly effective means of childbirth management, (8) On-site employees or their immediate family members who are directly involved in the administration, operation, or support of this study, (9) Any other reason that, in the opinion of the principal investigator, could increase the risk to the subject, interfere with the interpretation of the study results, or affect the subject's ability to provide informed consent or to participate in the study (e.g., a serious systemic disease or an uncontrolled medical condition).

[0268] Guidelines for eliminating intolerance, hypersensitivity, or significant allergies:

[0269] Patients with a history of allergy and / or hyperimmune response were not included in this study. However, potential subjects with a history of mild, non-significant allergies (e.g., antibiotic allergies without respiratory components) were permitted at the discretion of the principal investigator of the clinical trial. The determination of "significant" was intentionally left to the discretion of the principal investigator of the clinical trial. To provide additional guidance specific to the definition of "significant", use the NCI CTCAE criteria of grade 3 or higher as "significant".

[0270] Grade 1 represents a systemic intervention not shown.

[0271] Grade 2 represents an oral intervention shown.

[0272] Grade 3 represents bronchospasm. Clinical sequelae require hospitalization and intravenous intervention.

[0273] Grade 4 represents life-threatening consequences. Emergency intervention is required.

[0274] Therefore, those with an allergic response to any substance that caused bronchospasm, hospitalization, and / or parenteral intervention were excluded from this study. Known allergy sensitivity (regardless of severity) to any component of TTHX1114 was considered exclusionary.

[0275] As a reminder, the TTHX1114 components include sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate anhydrous, recombinant human albumin solution, or polysorbate 80.

[0276] Subject Withdrawal Criteria

[0277] The following: intolerance side effects, non-compliance, or pregnancy, if any occurred and the principal investigator of the clinical trial determined it to be in the best interest of the subject, the subject discontinued the ongoing trial treatment.

[0278] The subjects whose trial treatment was discontinued were expected to remain in the trial and complete all appropriate safety and efficacy evaluations.

[0279] Participants may withdraw from the clinical trial at any time for any reason.

[0280] If a participant withdrew from the trial, an attempt was made to contact them to determine the reason for withdrawal, while respecting their privacy. All procedures and assessments required for the final trial visit were completed in the case of early withdrawal. All participants who discontinued the trial due to adverse events were followed until the event resolved, returned to baseline, or stabilized (if resolution was not expected).

[0281] Target treatment

[0282] Target screening and registration:

[0283] Potential trial participants were asked to sign an Informed Consent Form (ICF) before any trial evaluation. Screening was completed within approximately 28 days of trial day 0 (unless otherwise approved by the medical monitor). Non-eligibility screening evaluations (e.g., slit-lamp biomicroscopy) and screening evaluations outside this window were repeated. Screening evaluations included trial eligibility assessment, medical and ophthalmic history, BCVA assessment, comprehensive ophthalmological examination (including slit-lamp biomicroscopy and dilated pupillary examination of the lens, fundus, and vitreous humor), and slit-lamp biomicroscopy (including central and peripheral corneal imaging).

[0284] Descemet's membrane decomposition without endothelial keratoplasty (DWEK) / Descemet's membrane decomposition only (DSO): Patients with FECD who underwent the planned DWEK / DSO or DMEK procedure were evaluated for eligibility in this study. Since the DWEK / DSO procedure was not a study procedure, DWEK / DSO was performed according to the institutional policy of a 4-5 mm Descemet's membrane decomposition area. Surgical methods included the use of a Sinskey Hook (with or without forceps) or an irrigation / aspiration (I&A) tip.

[0285] Principal investigators were selected based on their demonstrated expertise (e.g., number of procedures performed and relative success rate). The lead investigator assisted in site selection and investigator screening. Only qualified and reviewed ophthalmologists performed surgical procedures on subjects enrolled in this study to minimize operator variability. Subjects with a Descemet's membrane decompression area >5 mm were excluded from the efficacy analysis but included in the safety analysis.

[0286] AS-OCT was performed at least once after DSO (e.g., on day 28) to confirm the size of the Descemet's membrane dissection area, especially in patients with delayed recovery.

[0287] Test eye (SE): Because eligibility for the trial required a planned DWEK / DSO procedure designed for the medical management of the patient, the test eye (SE) was determined by the principal investigator prior to the clinical trial. An SE was defined as the eye selected by the physician to be treated for the DWEK / DSO surgical intervention and the eye to which TTHX1114 was administered (if applicable).

[0288] If a subject wished to receive DWEK / DSO in the other eye after SE had recovered, the subject was re-enrolled in group 3 or group 4 and the other eye was treated. "Recovery" was at the discretion of the investigator after the assessment of the primary efficacy endpoint at day 28 (e.g., having a measured CCT of at least 20 / 40 BCVA or less than 10% increase from baseline).

[0289] Test treatment administration / intraocular injection:

[0290] This procedure was performed by a qualified trained ophthalmologist proficient in DWEK / DSO and trained in the administration of TTHX1114.

[0291] Materials: (1) Standard eyelid speculum, (2) Povidone iodine solution (5% or 10%), (3) Balanced salt solution (BSS) flush, (4) Topical fluoroquinolone antibiotic, e.g., gatifloxacin eye drops 0.5% and moxifloxacin hydrochloride eye drops 0.5%. If allergic or contraindicated to fluoroquinolone, Polytrim® (polymyxin B sulfate and trimethoprim eye drops, USP) may be used, (5) Topical anesthetic (at the discretion of the study physician), (6) 30-gauge needle, (7) 1 mL syringe (provided by the company), (8) Test treatment (TTHX1114), (9) Sterile cotton tip applicator, (9) Additional materials if required by the facility protocol.

[0292] Procedure - Non-surgical day: After appropriate sterile prep and drape, 1. Apply topical antibiotic to the test eye 2. Instill topical anesthetic 3. Wash the periorbital area (including the lashes and lid margins) with povidone-iodine (5% or 10%) 4. Irrigate the eye with povidone-iodine (5% or 10%). a. (At the discretion of the study physician) Irrigate with balanced salt solution (BSS) after povidone-iodine. 5. Ensure adequate anesthesia 6. Insert the eyelid speculum. 7. Stabilize the eye. 8. Administer the test treatment (Figure 3) a. Insert the needle into the limbus with the bevel side down. i. The 3 o'clock and 9 o'clock positions are recommended for optimal access to the cornea (especially in patients with deep orbits, prominent eyebrows, and / or a prominent nose that may interfere with access). ii. Avoid areas of previous corneal / scleral or puncture sites. b. To avoid risk to the iris and / or lens, slowly advance the needle with its bevel facing downwards, parallel to the iris plane. c. The anterior chamber stops when it enters the vicinity of the periphery. i. If possible, avoid the center of the eye. d. Deliver the dose for the test procedure using the syringe holder lever. e. Once the test procedure has been delivered, remove the needle from the eye. 9. Apply a sterile cotton tip applicator to the injection site. a. Avoid extensive massage of the eyelids or the surface of the eye. 10. Monitor IOP 30–60 minutes after IC injection to ensure that the post-treatment IOP is no more than 5 mmHg higher than the pre-injection IOP measurement. Continue monitoring at least every 30 minutes until the post-injection IOP is within 5 mmHg of the pre-injection IOP measurement. 11. Apply a topical antibiotic to the test eye before discharge. 12. Instruct the subjects to self-administer topical antibiotics to the test eye at least twice at home (for example, at 6 p.m. and bedtime).

[0293] Procedure Day 0 of the exam:

[0294] Following surgical closure of the eye, the eye did not fully expand, and intraocular pressure was low, possibly unmeasurable. After completion of the DWEK / DSO procedure, TTHX1114 was administered as soon as possible (as described in step 8 above), and standard postoperative care, including steps 9-12 above, was performed as appropriate.

[0295] The principal investigator administered / prescribed post-treatment steroid eye drops (e.g., prednisolone acetate [1% ophthalmic suspension], qid) when deemed clinically necessary or at the principal investigator's discretion.

[0296] Timing and schedule of TTHX1114 administration:

[0297] For Group 2: The injection on day 3 was administered within approximately 48-72 hours before the planned day 0 (DWEK / DSO day).

[0298] For groups 2, 3, and 4, the injection on day 0 (DWEK / DSO day) was administered at any time after the completion of DWEK / DSO.

[0299] For groups 1a and 3a, (1) the Day 0 injection was administered approximately 56 days after the DWEK / DSO day, after consultation with and approval from the medical monitor (or designated person), and (2) the initiation of TTHX1114 was brought forward based on the time to response observed in subjects in group 2.

[0300] Subsequent administration of TTHX1114:

[0301] Following the initial administration of TTHX1114 on day 0, subsequent weekly doses were administered approximately every 7 ± 1 days. If a study treatment could not be administered within 6-8 days of the previous dose, the study treatment was delayed, and the schedule for all remaining doses was adjusted to ensure that TTHX1114 was not administered less than 5 days after the previous dose.

[0302] Concomitant medications and treatments:

[0303] Concomitant medications: Participants were expected to receive concomitant medications for the management of concurrent medical conditions. Medications taken from 28 days prior to 28 days after the last study treatment were recorded. Artificial tears were permitted during the study, but other eye medications were discussed with a blinded medical monitor.

[0304] Prohibited Drugs: The following drugs are prohibited unless approved by the medical monitor: (1) Rho kinase inhibitors (topical or systemic; e.g., fasudil, netalusdil [Rhopressa]), (2) systemic or dermatological cytotoxic chemotherapy, (3) cyclosporine ophthalmic emulsion, or (4) Xiidra® (Livitegrast ophthalmic solution). Patients requiring the use of prohibited drugs must consult with the medical monitor.

[0305] Pregnancy Testing and Contraception: Women of childbearing potential (WOCBP) are defined as premenopausal women who could become pregnant. Women who are not WOCBP are those who have not had a menstrual cycle for at least two years, or who have undergone a hysterectomy, tubal ligation, or bilateral oophorectomy. Sexually active female subjects who are WOCBP must use at least one highly effective method of contraception or at least two less effective methods of contraception (including one barrier method) starting at least one month before day 0, and sexually active male subjects with women of childbearing potential must agree to use a double barrier method. According to ICH M3(R2) Non-Clinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals, a highly effective method is defined as "a single or combination method that, when used consistently and accurately, results in a low failure rate (i.e., less than 1% per year)."

[0306] Test procedure:

[0307] Demographics: Demographic information, including age, sex, race, and ethnicity, was recorded. Height and weight of participants were measured and recorded.

[0308] Medical History and Eye History: During the screening / pre-treatment period, the principal investigator (or designated investigator) reviewed the medical history of all potential subjects, documenting all relevant clinically significant baseline medical conditions, concomitant medication use, and determining study eligibility. The history assessment included a review of all available medical records and patient interviews. Assessment of all eligibility criteria was documented in the study records. The history included a detailed history of onset, severity, and prior treatment history.

[0309] In addition to data collected in demographics and medical history, specific information regarding each subject's eye medical history was collected, including (1) current status of duration (date of onset / age), involvement of monocular or bilateral eyes, and associated symptoms; (2) other corneal dystrophy; (3) contact lens use and history; (4) past ophthalmic surgery (including in vivo laser refractive surgery (LASIK)); (5) history of eye trauma; (6) family history of eye symptoms; (7) allergies; (8) diabetes; (9) hypertension; and (10) autoimmune diseases. Additional relevant medical history was also recorded.

[0310] Comprehensive ophthalmological examination: The comprehensive ophthalmological examination included slit-lamp biomicroscopy and dilated pupillary examination of the lens, fundus (retina, optic disc, macula, fovea, and posterior pole), and vitreous humor. The comprehensive ophthalmological examination further included baseline macular optical coherence tomography (Mac-OCT). Since BCVA was the primary efficacy measure in this study, it was important to identify any macular pathologies or other confounding comorbidities.

[0311] Slit-lamp biomicroscopy: Slit-lamp biomicroscopy was performed using a 1.0 mm high and 1.0 mm wide beam with maximum brightness, and a high-power lens. Subjects were seated during the examination. This procedure was performed in the same manner for all subjects and included evaluation of the following: (1) lens, (2) corneal edema (evaluated in the central region), (3) size of the corneal edema area (in mm), (4) corneal opacity, (5) corneal stromatous transparency (according to the CPTS grading scale), (6) conjunctival hyperemia, (7) scleral-ciliary hyperemia, (8) anterior chamber cells, and (9) anterior chamber flare. These evaluations were graded according to the criteria listed in Table 9.

[0312] [Table 7]

[0313] Mirror microscopy: Mirror microscopy images were obtained from the peripheral and central regions of the cornea. The central region of the cornea was defined as the area of ​​the cornea above the pupil. If the subject's pupil was dilated upon arrival, mirror microscopy was performed before dilation. Five acceptable images were taken at each necessary time point to account for differences in image position within a given region of the cornea. A marginal image was collected at the screening visit. Central images were collected before and after the procedure whenever possible. Due to the nature of corneal edema after DSO, mirror microscopy images could not be obtained at all postoperative time points before corneal disturbance. Attempts were made to collect images at all time points. If an image could not be obtained, the reason was recorded. If a mirror microscopy image was obtainable, it was expected that the image would remain obtainable at all subsequent time points. Before the end of each mirror microscopy session, the technician reviewed the images to ensure that the obtained images were of the highest possible quality. Subjects with unsuitable images were requested to return to the clinical trial site for repeated imaging as an unscheduled visit. Endothelial cell density, percent hexagonality, and coefficient of variation were evaluated from mirror microscope images. The Cornea Image Analysis Reading Center (CIARC) at University Hospitals Cleveland Medical Center served as the central reading center, responsible for equipment certification, technician training, and image reading and analysis. Where more than one mirror microscope was installed at the implementation site, the same microscope was used for all visits of a given patient. Baseline and central images during the trial were sent to CIARC. Baseline images were archived, and images during the trial were evaluated for efficacy.

[0314] Evaluation of central corneal thickness: Anterior segment optical coherence tomography (AS-OCT) is the preferred method for measuring corneal thickness. When AS-OCT was unavailable, Pentacam or ultrasound pachymetry was used to measure central corneal thickness (CCT). Other reliable methods for measuring CCT (e.g., LENSTAR Optical Biometer) were used when approved by the medical monitor. When ultrasound pachymetry was used as the measurement method, the measurements were obtained using a pachymeter approved by Trefoil (or the designated). Automated "pachymetry" readings obtained using a mirror microscope were not acceptable. The same methodology for evaluating CCT was used consistently within the subject whenever possible. When alternative methods were used, the reasons for the alternative methods were reported. CCT was measured by several modalities at each time point (Figures 6–8 and 11–14).

[0315] Figure 6 shows the proportion of subjects / eyes in each group with central corneal thickness (CCT) less than 10% above baseline at each visit (CCT less than 110% of baseline). The difference between group 2 (low dose) and groups 3 and 4 (high dose) was statistically significant, with Fisher's exact test showing p<0.05 at day 28. TTHX1144 accelerates edema resolution after endothelial injury (DSO). Figure 7 shows the proportion of subjects / eyes in each group with central corneal thickness (CCT less than 110% of baseline) less than 10% above baseline at each visit. The difference between group 2 (low dose) and groups 3 and 4 (high dose) was statistically significant, with Fisher's exact test showing p<0.05 at day 28. This shows the dose response to central corneal thickness. Figure 8 shows the proportion of subjects / eyes in each group with complete resolution of corneal edema (central corneal thickness below baseline) at each visit. P-values ​​and Fisher's exact test were used to show the dose-response relationship to central corneal thickness.

[0316] Figure 11 shows the mean corneal thickness (+ / -SD) of groups 2 or 3 and 4 pooled at each visit. Solid bars include eyes that received DSO only, and dotted bars include eyes that received DSO + cataract surgery. Figure 12 shows the mean corneal thickness of groups 3 and 4 pooled at each visit. Bright bars include eyes that received DSO only, and dark bars include eyes that received DSO + cataract surgery. Figure 13 shows the percentage of subjects / eyes with complete resolution of corneal edema (central corneal thickness below baseline) at each visit in each group. Solid bars include eyes that received DSO only, and dotted bars include eyes that received DSO + cataract surgery. Figure 14 shows the mean corneal thickness of groups 2 and 3 and 4 pooled at each visit. Solid lines include eyes that received DSO only, and dotted lines include eyes that received DSO + cataract surgery.

[0317] Best Corrected Visual Acuity (BCVA): Best corrected visual acuity (BCVA) was measured using an ETDRS (Early Treatment Diabetic Retinopathy Study) chart under normal brightness lighting, whenever possible (unless otherwise instructed), between 7:00 AM and 11:00 AM, in accordance with institutional practice. The BCVA test was performed prior to any examination requiring eye contact, including IOP measurement, and before the administration of any dyes or drops to dilate or anesthetic the eye. Refraction was performed before BCVA measurement, and appropriate corrective devices were fitted to the test frame during visual acuity testing. If a subject did not have a BCVA of ≥20 / 40 (>68 ETDRS letter score) on day 28 or day 56, further methods of BCVA were used (i.e., hard lens and / or pinhole refraction) to determine whether the continued visual acuity decline was due to factors other than corneal edema. Unscheduled visits were made at the discretion of the principal investigator prior to the next scheduled visit (Figures 9 and 10).

[0318] Figure 9 shows the proportion of subjects / eyes in each group with a best corrected visual acuity (BCVA) of 69 characters (20 / 40) or better in all eyes at each visit. Figure 10 shows the proportion of subjects / eyes in each group with an increase of 0.6 logMAR (6 lines, 30 characters) in BCVA from the first postoperative visit at each visit. Fisher's exact test compares groups 2 vs. 3.

[0319] Intraocular pressure measurement: Intraocular pressure (IOP) was measured by Goldmann applanation tonometry. At least two measurements were obtained and recorded (a third measurement was obtained if there was a difference of ≥3 mmHg between the first two measurements). After each TTHX1114 injection (except after DWEK / DSO), IOP was measured at least every 30–60 minutes until the IOP was at least 5 mmHg higher than the pre-injection IOP. The mean IOP measurement was reported at each time point.

[0320] Urine pregnancy testing: WOCBP was tested before and after the start and completion of the test procedure to confirm a continued non-pregnancy state. Pregnancy tests using commercially available urine test strips were acceptable.

[0321] Delayed recovery: Patients with delayed recovery underwent further ocular evaluation on day 28, day 56, or during unscheduled visits. These evaluations included (1) further BCVA methods, (2) central corneal thickness (primary or alternative methods), (3) AS-OCT, and (4) Pentacam. Further ocular evaluations were reported as unscheduled visits.

[0322] Clinical Laboratory Evaluation: Samples for clinical laboratory evaluation were obtained and transmitted to the central laboratory. The laboratory parameters to be tested are summarized in Table 10 below. All clinically significant laboratory results outside the normal range were reported as adverse events (AEs). Abnormal laboratory values ​​were considered clinically significant if any of the following conditions were met: (1) the abnormality was determined by the principal investigator and, at the request of the principal investigator and in consultation with a medical monitor specialist, suggested a novel disease and / or organ toxicity that had worsened from baseline; and (2) the abnormality required further active management, such as dose change, drug discontinuation, close monitoring, more frequent follow-up assessment, or further diagnostic investigation. Therefore, clinically significant laboratory values ​​indicated a new disease process, exacerbation or worsening of an existing condition, or required further action. If clinically significant laboratory findings were found in the test results (e.g., hematology), the principal investigator determined whether the underlying condition was a reportable AE instead of the individual laboratory parameters (e.g., anemia instead of decreased red blood cell count, hematocrit, reticulocytes, and hemoglobin levels).

[0323] [Table 8]

[0324] Plasma for PK and ADA: Blood samples were collected for pharmacokinetic (PK) and anti-drug antibody (ADA) analysis at selected sites. Samples were centrifuged and / or prepared according to instructions from the analytical laboratory. Pre-infusion samples were collected, if possible, within 24 hours prior to administration of the test procedure. PK samples were collected within ±5 minutes of the nominal time or as soon as feasible. After evaluation of systemic exposure (pharmacokinetics) at a 50 ng dose level, collection of PK samples at dose levels ≤50 ng was discontinued. Pre- and post-exposure ADA samples were continued to be collected regardless of observed PK exposure. Plasma samples were obtained at the end of the study for ADA analysis.

[0325] Endothelial tissue collection: Study subjects were asked to provide tissue excised for research purposes. Specific research consent was included in the Informed Consent Form (ICF). As soon as possible after the procedure on day 0 of the study, the tissue was placed in the provided transport tubes (without supernatant) and either frozen or sent to the central laboratory. These samples were collected in subsets of the study sites.

[0326] Aqueous humor collection: Participants were asked to provide aqueous humor samples obtained during the DWEK / DSO procedure on day 0 of the study. The collected aqueous humor was frozen or sent to the central laboratory. Where possible, the aqueous humor samples were divided into two equal aliquots. These samples were evaluated for research purposes, including endogenous levels of FGF-1. These samples were collected from subsets of the test sites.

[0327] Visual Function and Corneal Health (V-FUCHS): The V-FUCHS is a 15-item questionnaire designed to measure patient-reported visual impairment in Fuchs endothelial corneal dystrophy (FECD). Upon completion of the questionnaire, the study coordinator checked it for completeness. Any omissions or ambiguous responses were clarified by the subject before leaving the clinic. The V-FUCHS was self-administered; however, subjects unable to complete the questionnaire themselves had it administered by trained site staff. V-FUCHS collection was performed prior to any examinations requiring eye contact, including IOP measurement, and before the administration of any dyes or drops to dilate or anesthetic the eye (if applicable).

[0328] Voluntary blood samples for genetic testing: Participants were asked to provide voluntary blood samples for genetic testing. Participants were explicitly asked to consent to this voluntary genetic testing. The samples were completely despecified before being sent to the testing facility. This sample was voluntary, and participation in the study was not conditional on consenting to provide this sample for genetic testing. Voluntary blood samples for genetic testing were collected at any point during the study.

[0329] Trial visit:

[0330] All trial visits / assesses were performed by qualified site staff. Trial visits / assesses were conducted within the protocol-specified window for each visit / assessment. If an assessment / visit could not be collected / performed within the protocol-specified window, it was collected / performed as soon as possible, with the actual date / time recorded. Comparative assessments were collected simultaneously at each point in time where possible (unless otherwise instructed). If site visits were not possible, as many assessments as possible were performed remotely.

[0331] Screening Period / Baseline Assessment: Screening / baseline assessments were performed at any point during the screening period unless otherwise approved by the medical monitor, except for informed consent (which must be obtained before any protocol-specific assessments).

[0332] Intra-study visits: The frequency of intra-study visits varied depending on the treatment group to which the study subject belonged. All investigational drug administration days were (where applicable) at least 5 days after the previous investigational drug administration day. All other visits were scheduled based on the number of days after day 0. If investigational drug administration could not be administered within the protocol window, it was administered as soon as possible. Each treatment group was provided with a worksheet to record which assessments were required at each visit.

[0333] Long-term follow-up: After the 56-day evaluation, all subjects were continued to be tracked for long-term efficacy, persistence of response, and SAE / SAR resolution. Follow-up visits were made approximately 3, 6, and 12 months after the DWEK / DSO procedure, or until subsequent treatment in the FECD test eye. Subjects were also considered part of routine health checkups, and evaluations of all relevant eyes collected during this period were recorded.

[0334] Early Termination Visit: If a subject discontinued the study treatment early or withdrew from the study, the subject was evaluated approximately 28 days after the last administration of the study treatment. If a subject could not be evaluated approximately 28 days after the last administration of the study treatment, the subject was evaluated as quickly as possible (at least by telephone). All evaluations scheduled for day 28 were performed at the early termination visit if the day 28 visit had not yet been completed; otherwise, evaluations scheduled for day 56 had to be collected.

[0335] Unscheduled visits: At the discretion of the principal investigator, the subject was observed and any relevant clinical trial evaluations were performed at any point between scheduled clinical trial visits. The date and reason for the unscheduled visit, as well as the results of the relevant evaluations, were recorded.

[0336] End-of-Trial Visit: End-of-Trial visits (EOS) were performed for all subjects. EOS were performed at any point after day 168, during which the EOS evaluation scheduled for day 336 was performed and plasma samples for ADA analysis were collected. If a subject had already completed the trial, they were contacted to request the provision of plasma samples for ADA. Any subjects for whom evaluable mirror images were not obtained at the most recent visit were re-imaging to obtain evaluable images. (1) Comprehensive ophthalmological examination, (2) mirror image acquisition, (3) corneal thickness assessment, (4) best corrected visual acuity assessment, and (5) plasma samples collected for ADA.

[0337] Pregnancy: Participants were instructed to notify the principal investigator as soon as possible after becoming pregnant or after learning of their partner's pregnancy. If a participant or their partner became pregnant during treatment or within 120 days of the last dose of the investigational drug, the principal investigator was instructed to notify Trefoil (or the designated person) within 24 hours of learning of the pregnancy. If a participant became pregnant while receiving the investigational drug, the investigational drug was permanently discontinued. Procedures requiring protocols for trial discontinuation and follow-up were to be performed for participants unless contraindicated by pregnancy. Other appropriate pregnancy follow-up procedures were considered as needed. If a participant's partner became pregnant, the principal investigator was instructed to obtain a Pregnant Partner Release Form from the pregnant partner and to collect relevant information about the partner and the pregnancy. The principal investigator discussed the risks and concerns of exposure of the developing fetus to the investigational drug and counseled the participant and / or their pregnant partner (or ensured that such counseling was provided). Pregnancies were followed throughout the pregnancy outcomes. Newborns had to be followed for a minimum of 8 weeks. The principal investigator completed the pregnancy monitoring form and reported information on the pregnancy, prognosis, and neonatal status as needed.

[0338] Evaluation of effectiveness

[0339] The efficacy endpoints were visual acuity and endothelial cell count / density as measured by slit-lamp biomicroscopy. Other efficacy assessments included (1) slit-lamp biomicroscopy, (2) central corneal thickness, and (3) IOP. The primary efficacy time point was day 28.

[0340] Safety evaluation

[0341] Adverse events:

[0342] The principal investigator collected information related to adverse events (AEs) throughout this clinical trial. Terms and definitions were consistent with the Guidance for Industry and Investigators Safety Reporting Requirements for INDs and BA / BE Studies, FDA 2012.

[0343] All adverse events (AEs) that occurred in all subjects were reported from the time of exposure to the test treatment until 28 days after each test treatment.

[0344] AEs and SAEs that the principal investigator (SAR) considered to be related to the study procedure were followed until they resolved, returned to baseline, or stabilized (if resolution was not expected).

[0345] Changes in the subjects' medical condition prior to their initial exposure to the test procedure were reported as part of their medical history. At follow-up visits after each procedure, subjects were non-leadingly questioned about all possible adverse events. One example of a non-leading method for eliciting AE information was, "Have you experienced any changes in your health since your last visit?" Subjects were also asked about the severity and / or persistence of any AEs that were ongoing at the time of their final visit.

[0346] Definition of adverse events:

[0347] Adverse Events (21 CFR 312.32(a)): An adverse event (AE) is defined as any adverse medical event in a human being associated with the use of a drug, whether or not it is considered drug-related. An AE (also called an adverse experience) may be any undesirable and unintended sign (e.g., an abnormal laboratory finding), symptom, or illness temporarily associated with the use of a drug and does not imply any judgment of causality. Adverse events may occur with any use of a drug (e.g., off-label use, use in combination with another drug), and with any route of administration, formulation, or dose, including overdose.

[0348] Suspected adverse reaction (21 CFR 312.32(a)): A suspected adverse reaction refers to any adverse event that has a reasonable possibility of being caused by the drug. For the purposes of IND safety reporting, "reasonable possibility" means that there is evidence suggesting a causal relationship between the drug and the adverse event. A suspected adverse reaction implicitly means a lower degree of certainty regarding the cause than an adverse reaction, meaning any adverse event caused by the drug.

[0349] Unexpected (21 CFR 312.32(a)): An adverse event or suspected adverse reaction is considered “unexpected” if it is not listed in the Investigational Brochure or is not listed by observed specificity or severity, or if, where the Investigational Brochure is not required or available, it does not align with the risk information described in the General Knowledge Plan or elsewhere in this Application, as modified. For example, under this definition, if the Investigational Brochure refers only to elevated liver enzymes or hepatitis, hepatic necrosis would be unexpected (due to its higher severity). Similarly, cerebral thromboembolism and cerebrovascular vasculitis would be unexpected (due to their higher specificity) if the Investigational Brochure lists only cerebrovascular events. As used in this definition, “unexpected” further refers to an adverse event or suspected adverse reaction that is mentioned in the Investigational Brochure as something that occurs with a class of drugs or as expected from the pharmacological properties of the drug, but is not specifically mentioned as something that occurs with the particular drug under investigation.

[0350] Serious adverse events (21 CFR 312.32(a)): An adverse event or suspected adverse reaction is considered “serious” if, from the perspective of either the principal investigator or the sponsor, it results in any of the following: death, a life-threatening adverse event, hospitalization of a patient or prolongation of an existing hospitalization, persistent or significant impairment or substantial disruption of the ability to perform normal daily activities, or a birth defect / defect.

[0351] Important medical events that may not result in death, may not be life-threatening, or may not require hospitalization may be considered serious if, based on appropriate medical judgment, they may endanger the patient or subject and require medical or surgical intervention to prevent one of the consequences listed in this definition. Examples of such medical events include allergic bronchospasm requiring intensive care in the emergency room or at home, a blood disorder or seizure in a hospitalized patient that does not lead to hospitalization, or the onset of drug dependence or abuse.

[0352] Adverse event report:

[0353] Adverse Event Terminology: Adverse events must be reported using standard medical terminology. The use of abbreviations (standard and non-standard) should be avoided to help ensure a clear understanding of the event. An example of a standard abbreviation that may have several meanings is "MI," which may mean "myocardial infarction" or "mitral regurgitation." All AE terms are encoded using a standardized dictionary (i.e., the Medical Dictionary for Regulatory Activities [MedDRA]).

[0354] Generally, when reporting a well-known and understood condition, it is preferable to report the overall diagnosis rather than individual signs and symptoms. The exception to this rule in this study is when a subject experiences an injection site reaction.

[0355] The term "intermittent" should be avoided because the duration and incidence of events are helpful in understanding the safety profile of the investigational drug.

[0356] Adverse event severity: Adverse events are reported at the highest level of experience. The severity of adverse events is graded according to the criteria described in Table 11.

[0357] [Table 9]

[0358] Adverse event duration: Record the start date (the date the event was first reported) and the end date (the date the event completely resolved or returned to baseline). If the exact date is unknown, the best estimate should be reported.

[0359] Causal relationship of adverse events: If the investigator's assessment of the relationship between an adverse event (AE) and the investigational drug depends on medical judgment, that decision must be made with the appropriate involvement of the investigator, or, if the investigator is not a physician, with the appropriate involvement of a designated subordinate investigator who is a physician. The investigator shall use the following criteria to assess whether the study procedure (drug or procedure) caused or reasonably likely contributed to the AE.

[0360] Relevant: There is a “reasonable possibility” based on evidence that there is a causal relationship between the test treatment and the adverse event. The principal investigator must use the following criteria when evaluating causality. • Is the AE a known side effect / adverse reaction to the test procedure or other treatments in this class of treatments? Is there a reasonable time relationship between the initiation of an adverse event (AE) and the administration of the test treatment? Did the adverse event (AE) improve when the test procedure was stopped? If applicable, did the adverse event (AE) recur when the test treatment was resumed? • Can adverse events (AEs) easily be caused by comorbidities or concomitant medications / treatments?

[0361] Unrelated: There is no “reasonable possibility” based on evidence to suggest a causal relationship between the test treatment and the adverse event.

[0362] Severity of adverse events: SAE criteria have the following guidelines: (1) Death: Report whether death is suspected to be a result of the adverse event, including the date if known; (2) Life-threatening: Report whether the patient was at substantial risk of death at the time of the adverse event, or whether the use or continued use of the device or other medical supplies is suspected to have caused the patient's death; (3) Hospitalization (initial or prolonged): Report whether hospitalization or prolonged hospitalization was a result of the adverse event. Emergency room visits that do not result in hospitalization must be evaluated for one of the other serious consequences (e.g., life-threatening; intervention required to prevent permanent disability or injury; other serious medically important events), (4) disability or permanent injury: report whether the adverse event resulted in substantial disruption of the person's ability to perform normal life functions, i.e., whether the adverse event resulted in significant, persistent or permanent change, disability, injury or disruption in the patient's physical function / structure, physical activity and / or quality of life, (5) birth defects / absences: report whether it is suspected that pre-conception or pregnancy exposure to a drug may have resulted in an adverse outcome for the child, (6) other serious (important medical events): report when the event does not fit into the other outcomes but the event may endanger the patient and may require medical or surgical intervention (treatment) to prevent one of the other outcomes. Examples include allergic bronchospasm (a serious respiratory problem) requiring emergency room treatment, severe blood cachexia (a blood disorder), or seizures / convulsions that do not require hospitalization. The onset of drug dependence or abuse can also be an example of a significant medical event.

[0363] Reporting of Serious Adverse Events: Adverse events that meet the definition of serious require prompt reporting. The principal investigator must report all serious adverse events (SAEs) regardless of direct causal relationship (within 24 hours of becoming aware of the event). If an adverse event of special interest (AESI) is identified during the study, Trefoil will notify the principal investigator and provide instructions for reporting. Deaths and AESIs occurring within one year of the study procedure are reported to Trefoil within the same timeframe as SAEs.

[0364] Adverse events of special interest: (1) Adverse events of special interest (AESIs) are reported regardless of the investigator's assessment of causality and / or the date of onset. AESIs include neoplasms, (2) exacerbation or new onset of glaucoma, and (3) all clinically significant ocular adverse events (e.g., need for capsulotomy). AESIs are reported as non-severe AEs and are reviewed regularly by medical monitors.

[0365] statistics

[0366] Sample size: The sample size for this study was determined by clinical and practical considerations rather than statistical considerations. Sixteen subjects in the active treatment group were planned during the study to provide preliminary estimates of safety and efficacy, compared to eight subjects in the untreated control group.

[0367] Analysis: The study included a concurrent untreated control group. The active treatment group was compared to the control group. In addition, other efficacy variables and safety outcomes were summarized in tables using descriptive statistics. Baseline and demographic characteristics were presented. Continuous variables were summarized using descriptive statistics (sample size, mean / standard deviation, median, minimum, and maximum). Discrete variables were summarized by frequency and percentage. Eye AEs involved in the test eye were summarized by presenting the number and percentage of subjects with AEs in any eye. Any other information collected (e.g., severity or relationship between the investigational drug and anterior chamber injection) was listed as appropriate.

[0368] Analysis of primary efficacy variables: A complete statistical analysis plan was created before locking the database. All post-hoc analyses were identified and described in the clinical trial reports. Statistical tests were applied where possible.

[0369] Analysis Population and Set: The analysis sets for this clinical trial are defined according to ICH E9 Guidance for Industry Statistical Principles for Clinical Trials. The maximum analysis set was intended to be as complete as possible and as close as possible to the ideal population for treatment intent. This was an open-label, non-randomized trial, and therefore the maximum analysis set included only subjects who received any study treatment and was the same as the safety population. The analysis sets are listed in Table 12 below.

[0370] [Table 10]

[0371] Primary and secondary efficacy endpoints were evaluated using a protocol-compliant population compared to the control group.

[0372] The safety population set included all participants in any group receiving TTHX1114 and was the population for safety outcomes. This population was compared to the control group. AEs of the eye involved in the test eye were presented in a table. AEs of the contralateral eye and non-ocular eyes were provided as lists.

[0373] clinical results

[0374] Positive predictive value of first eye recovery in patients who received Descemet's membrane detachment only (DSO) using TTHX1114

[0375] Objective: Fuchs endothelial corneal dystrophy (FECD) is a genetic condition, and therefore has a bilateral nature. DSO is an important surgical intervention for some patients with FECD. As DSO is to be further investigated as an effective intervention, the potential confounding contribution of including a second eye must be considered in efficacy analyses. We examined data from the STORM Study (TTHX-002) and compared outcomes in patients who underwent bilateral DSO with those in TTHX-1114.

[0376] Methods: Study TTHX-002 was an open-label study of TTHX1114 (manipulated FGF1) administered as an adjunct to DSO containing three active regimens. Participants were assigned to treatment at the discretion of the principal investigator. A dose-response was observed between the low-dose group and the two high-dose groups. Efficacy was similar in the two high-dose groups, and 49 patients were enrolled. Of these, 45 were evaluable in the protocol-compliant population, and 13 of these received bilateral DSO with TTHX1114. Not all participants were eligible for bilateral treatment. The primary efficacy variable was best corrected visual acuity (BCVA), measured using the Early Treatment Diabetic Retinopathy Scale (ETDRS) scoring system, where ≥69 letters = ≥20 / 40 BCVA.

[0377] Results: Mean and median ETDRS character scores were similar for both eye sets at all time points, as were the overall responder rates, as shown in Figures 15–17.

[0378] It is important to note that one subject who did not respond to an ETDRS character score of ≥69 characters was the same subject in both the first and second eye groups. In this subject, the first eye recovered to 78 characters at day 168, and the second eye recovered to 80 characters at the visit on day 168.

[0379] Conclusion: Of the 13 subjects who underwent DSO of the second eye using TTHX1114, all had a positive outcome from the first eye procedure. Data regarding the decision to proceed with (or not proceed with) the second eye surgery were not collected. The potential negative predictive value for poor outcomes from the first eye surgery cannot be determined from this dataset.

[0380] Factors affecting recovery after Descemet's membrane delamination alone (DSO) by TTHX1114

[0381] Objective: Recovery after DSO can be influenced by many variables, including, but not limited to, the size of the Descemet's membrane dissection site, comorbid diabetes, and concurrent cataract treatment. We examined data from the STORM trial (TTHX-002) to determine the influence of these variables on the time to recovery to 20 / 40 or better BCVA.

[0382] Methods: Study TTHX-002 was an open-label trial of TTHX1114 (manipulated FGF1) administered as an adjuvant to DSO in FECD patients, demonstrating a dose-dependent acceleration of recovery based on both BCVA and corneal edema. Since efficacy was similar in the two high-dose groups (n=50 in accordance with the protocol), these data were pooled and recovery rates in subset populations were analyzed. 45 / 50 (90%) of eyes recovered to BCVA 20 / 40 or better at the data cutoff.

[0383] Results: The time to 20 / 40 (weeks) was faster in subjects with smaller DSO (4mm = 4.5, >4.5~≦4.5mm = 6.7, ≧5mm = 6.6) and slower in subjects with diabetes (7.7 compared to 5.2 in subjects without diabetes). This trend in those with faster recovery was also observed at day 28, as shown in Figures 18 and 19, with lower corneal edema in subjects with smaller DSO (4mm = 637.6, >4.5~≦4.5mm = 743.2, ≧5mm = 808.8) and slightly higher corneal thickness in subjects with diabetes (719.8 μm compared to 702.9 μm in subjects without diabetes).

[0384] In patients undergoing combined cataract surgery, no significant difference in recovery was observed compared to patients receiving DSO alone. This suggests that TTHX1114 prevents further damage during cataract surgery.

[0385] Conclusion: Small-area Descemet's membrane dissection was associated with early recovery, but comorbid diabetes appeared to delay recovery. Visual outcomes in patients undergoing DSO did not appear to be adversely affected by simultaneous cataract surgery. Although these numbers are small, comorbidity and combinations of planned procedures must be considered when designing trials in DSO.

[0386] Example 2: Protection of endothelial cells by TTHX1114 in a patient with Fuchs endothelial corneal dystrophy (FECD) who underwent Descemet's Stripping Only (DSO) in combination with cataract surgery.

[0387] Objective: TTHX1114 is an FGF1 analog with protective, proliferative, and migration-promoting properties for corneal endothelial cells (CEnCs). Cataract surgery is known to damage CEnCs. This study attempted to analyze data from the STORM trial to evaluate whether TTHX1114 protected endothelial cells from cataract surgery damage in FECD patients who underwent DSO.

[0388] Methods: This clinical trial was an open-label, non-randomized, phase 2 dose-range trial of TTHX1114 in FECD patients who received DSO. In STORM, 46% of eyes underwent cataract surgery in combination with DSO. Subgroup analysis was used to determine whether the group that received the cataract surgery combination had more severe edema, worsened BCVA, or any other signs that cataract surgery had the expected effect of endothelial damage.

[0389] Results: Across all patients, TTHX1114 resulted in dose-dependent improvement of corneal edema and BCVA postoperatively. The proportion of patients experiencing complete resolution of corneal edema at day 28 in the high-dose group (100 ng at surgery, with or without further postoperative doses) was similar between DSO-only patients and DSO-plus cataract patients (25.9% vs. 17.4%), as was the mean central corneal thickness (705+ / -174 μm vs. 700+ / -148 μm). The proportion of patients recovering a good BCVA (69 letters or better) was also similar between these groups, with 58% of DSO-only patients recovering to 69 letters or better at day 28, compared to 61% of DSO-plus cataract patients. In the low-dose (50 ng at surgery) group, central corneal thickness at day 14 was numerically inferior in the DSO + cataract group (961 + / -77) compared to the DSO-only group (828 + / -108 μm), but this was not statistically significant, and the two groups were comparable at all subsequent time points.

[0390] Conclusion: Cataract surgery combined with DSO did not worsen postoperative edema or negatively impact BCVA recovery. These data support the potential use of TTHX1114 for controlling postoperative edema in high-risk patients undergoing cataract surgery.

[0391] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Those skilled in the art will be able to conceive of numerous variations, alterations, and substitutions without departing from the present invention. It should be understood that various alternative forms of the embodiments of the present invention described herein may be employed in carrying out the present invention. The following claims define the scope of the present invention, and the methods and structures within these claims, as well as their equivalents, are intended to be encompassed by these claims.

[0392] [Table 11-1]

[0393] Table 11-2

[0394] Table 11-3

[0395] Table 11-4

[0396] Table 11-5

[0397] Table 11-6

[0398] Table 11-7

[0399] Table 11-8

[0400] Table 11-9

[0401] Table 11-10

[0402] Table 11-11

Claims

1. A method for treating or preventing a corneal disease or illness in a subject, comprising the step of intrachorally administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a modified FGF-1 polypeptide having at least 90% sequence identity to the wild-type FGF-1 amino acid sequence of SEQ ID NO: 1 and having Cys16Ser, Ala66Cys, and Cys117Val mutations, wherein the corneal disease or illness comprises Descemet's membrane detachment without endothelial keratoplasty (DWEK) or Descemet's membrane detachment alone (DSO).

2. The method according to claim 1, wherein the modified FGF-1 polypeptide contains methionine upstream of the first residue of SEQ ID NO:

1.

3. The method according to claim 1 or 2, wherein the modified FGF-1 polypeptide comprises an amino acid sequence that is at least about 90% identical to the sequence of SEQ ID NO:

2.

4. The method according to claim 1 or 2, wherein the modified FGF-1 polypeptide comprises an amino acid sequence that is at least about 95% identical to the sequence of SEQ ID NO:

2.

5. The method according to claim 1 or 2, wherein the modified FGF-1 polypeptide comprises an amino acid sequence that is at least about 99% identical to the sequence of SEQ ID NO:

2.

6. The method according to claim 1 or 2, wherein the modified FGF-1 polypeptide comprises the amino acid sequence of SEQ ID NO:

2.

7. The method according to claim 1, wherein the subject undergoes cataract surgery before the administration step, simultaneously with the administration step, or after the administration step.

8. The method according to claim 1, wherein the corneal disease or illness includes corneal endothelial dystrophy or corneal endothelial injury.

9. (i) The subject undergoes cataract surgery before the administration step, simultaneously with the administration step, or after the administration step. (ii) The method according to claim 1, wherein the disease or illness of the cornea includes corneal endothelial dystrophy or corneal endothelial injury.

10. The method according to claim 9, wherein the corneal endothelial dystrophy or corneal endothelial injury includes Fuchs dystrophy, bullous keratopathy, congenital hereditary endothelial dystrophy 1, congenital hereditary endothelial dystrophy 2, posterior polymorphic corneal dystrophy, corneal endothelial injury following ophthalmic surgery, corneal endothelial injury following cataract surgery, or corneal endothelial injury following oxidative stress.

11. The method according to claim 1, wherein the subject has risk factors for corneal endothelial injury induced by ophthalmic surgery.

12. The method according to claim 11, wherein the risk factors include diabetes mellitus, low corneal endothelial cell density, corneal endothelial dystrophy, small pupil, shallow anterior chamber, mature cataract or brown cataract, or a combination thereof.

13. The method according to claim 1, wherein the subject undergoes cataract surgery before the administration step, simultaneously with the administration step, or after the administration step, and the corneal disease or illness includes Fuchs dystrophy.

14. The method according to claim 7, wherein the DWEK or DSO is performed before the cataract surgery, simultaneously with the cataract surgery, or after the cataract surgery.

15. The method according to claim 9, wherein the corneal endothelial dystrophy or corneal endothelial injury is caused by the cataract surgery.

16. The method according to claim 9, wherein the corneal endothelial dystrophy or corneal endothelial injury is not caused by cataract surgery.

17. The method according to claim 1, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient, or diluent.

18. The method according to claim 1, wherein the pharmaceutical composition comprises a liquid formulation.

19. The method according to claim 17, wherein the pharmaceutically acceptable carrier, excipient, or diluent includes physiological saline.

20. The method according to claim 19, wherein the physiological saline comprises phosphate-buffered saline (PBS).

21. The method according to claim 20, wherein the PBS comprises one or more salts of sodium ions or potassium ions.

22. The method according to claim 21, wherein the one or more salts include sodium chloride, potassium chloride, monosodium phosphate, potassium dihydrogen phosphate monobasic, disodium hydrogen phosphate anhydrous, or dipotassium hydrogen phosphate anhydrous.

23. The method according to claim 21, wherein the one or more salts include sodium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate anhydrous.

24. The method according to claim 17, wherein the pharmaceutically acceptable carrier, excipient, or diluent includes a surfactant.

25. The method according to claim 24, wherein the surfactant comprises polysorbate, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil, or equivalents thereof.

26. The method according to claim 25, wherein the polysorbate comprises polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

27. The method according to claim 25, wherein the polysorbate comprises polysorbate 80.

28. The method according to claim 18, wherein the pharmaceutically acceptable carrier, excipient, or diluent comprises albumin.

29. The method according to claim 28, wherein the albumin comprises human serum albumin.

30. The method according to claim 28, wherein the albumin comprises recombinant human serum albumin.

31. The method according to claim 17, wherein the pharmaceutically acceptable carrier, excipient, or diluent comprises (i) PBS containing sodium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate anhydrous, (ii) polysorbate 80, and (iii) recombinant human serum albumin.

32. The method according to claim 25, wherein the concentration of the polysorbate is at least about 0.001% (w / v), 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), or 0.1% (w / v).

33. The method according to claim 25, wherein the concentration of the polysorbate is approximately 0.01% (w / v).

34. The method according to claim 1, wherein the pharmaceutical composition comprises at least about 0.1 ng / μl, at least about 0.5 ng / μl, at least about 1 ng / μl, at least about 5 ng / μl, and at least about 10 ng / μl of the modified FGF-1 polypeptide.

35. The method according to claim 1, wherein the pharmaceutical composition comprises about 1 ng / μl of the modified FGF-1 polypeptide.

36. The method according to claim 1, wherein the pharmaceutical composition has a pH of about 7.2 to about 7.

6.

37. The method according to claim 1, wherein the pharmaceutical composition does not contain heparin.

38. The method according to claim 1, wherein the administration step includes administering the modified FGF-1 polypeptide in a dose of at least about 1 ng, at least about 3 ng, at least about 5 ng, at least about 10 ng, at least about 30 ng, at least about 50 ng, at least about 100 ng, at least about 150 ng, or at least about 200 ng.

39. The method according to claim 1, wherein the administration step includes administering the modified FGF-1 polypeptide in doses of up to approximately 1 ng, up to approximately 3 ng, up to approximately 5 ng, up to approximately 10 ng, up to approximately 30 ng, up to approximately 50 ng, up to approximately 100 ng, up to approximately 150 ng, or up to approximately 200 ng.

40. The method according to claim 1, wherein the administration step includes administering the modified FGF-1 polypeptide in doses of approximately 1 ng, approximately 3 ng, approximately 5 ng, approximately 10 ng, approximately 30 ng, approximately 50 ng, approximately 100 ng, approximately 150 ng, or approximately 200 ng.

41. The method according to claim 1, wherein the administration step comprises administering a first dose and a second dose, the second dose being less than or equal to the first dose, and the first dose or the second dose being at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of the modified FGF-1 polypeptide.

42. The method according to claim 1, wherein the administration step comprises administering a first dose and a second dose, the second dose being equal to or greater than the first dose, and the first dose or the second dose being at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of the modified FGF-1 polypeptide.

43. The method according to claim 1, wherein the administration step comprises administering a first dose, a second dose, and a third dose, the second dose being less than or equal to the first dose, the third dose being less than or equal to the second dose, and the first dose, the second dose, or the third dose being at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of the modified FGF-1 polypeptide.

44. The method according to claim 1, wherein the administration step comprises administering a first dose, a second dose, and a third dose, the second dose being greater than or equal to the first dose, the third dose being greater than or equal to the second dose, and the first dose, the second dose, or the third dose being at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of the modified FGF-1 polypeptide.

45. The method according to claim 1, wherein the administration step comprises administering a first dose, a second dose, a third dose, and a fourth dose, the first dose, the second dose, the third dose, or the fourth dose being at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of the modified FGF-1 polypeptide.

46. The method according to claim 1, wherein the administration step comprises administering a first dose, a second dose, a third dose, a fourth dose, and a fifth dose, wherein the first dose, the second dose, the third dose, the fourth dose, or the fifth dose is at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of the modified FGF-1 polypeptide.

47. The method according to claim 1, wherein the administration step comprises administering at least five doses, each of which is at least about 1 ng, 3 ng, 5 ng, 10 ng, 30 ng, 50 ng, 100 ng, 150 ng, or 200 ng of the modified FGF-1 polypeptide.

48. The method according to claim 1, wherein the administration step comprises administering a certain dose of the modified FGF-1 polypeptide before, simultaneously with, or after the DWEK or DSO treatment.

49. The method according to claim 1, wherein the administration step comprises administering a certain dose of the modified FGF-1 polypeptide at least one, two, three, four, five, six, seven, two, three, or four weeks prior to the DWEK or DSO treatment.

50. The method according to claim 1, wherein the administration step comprises administering a certain dose of the modified FGF-1 polypeptide up to one day, two days, three days, four days, five days, six days, seven days, two weeks, three weeks, or four weeks prior to the DWEK or DSO treatment.

51. The method according to claim 1, wherein the administration step includes administering a certain dose of the modified FGF-1 polypeptide two to three days before the DWEK or DSO treatment.

52. The method according to claim 1, wherein the administration step comprises administering a certain dose of the modified FGF-1 polypeptide on the same day as the DWEK or DSO treatment.

53. The method according to claim 1, wherein the administration step includes administering a certain dose of the modified FGF-1 polypeptide at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the DWEK or DSO treatment.

54. The method according to claim 1, wherein the administration step includes administering a certain dose of the modified FGF-1 polypeptide at a maximum of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the DWEK or DSO treatment.

55. The method according to claim 1, wherein the administration step includes administering a certain dose of the modified FGF-1 polypeptide weekly at least one week, two weeks, three weeks, four weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, three years, four years, or five years after the DWEK or DSO treatment.

56. The method according to claim 1, wherein the administration step includes administering a certain dose of the modified FGF-1 polypeptide weekly at a maximum of one week, two weeks, three weeks, four weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, three years, four years, or five years after the DWEK or DSO treatment.

57. The method according to claim 1, wherein the administration step comprises administering a certain dose of the modified FGF-1 polypeptide weekly, three weeks after the DWEK or DSO treatment.

58. The method according to claim 1, wherein the administration step comprises administering a certain dose of the modified FGF-1 polypeptide on the 7th, 14th, and 21st days following the DWEK or DSO treatment.

59. The method according to claim 1, wherein the administration step includes administering at least about 10 microliters (mcL), 30 mcL, 50 mcL, and 100 mcL of the pharmaceutical composition having the modified FGF-1 polypeptide at a concentration of 1 ng / mcL.

60. The method according to claim 1, wherein the administration step includes administering the drug once to three times a day.

61. The method according to claim 1, wherein the administration step includes administering the drug once a day.

62. The method according to claim 1, wherein the administration step includes administering the drug twice a day.

63. The method according to claim 1, wherein the administration step includes administering the drug three times a day.

64. The method according to claim 1, wherein the administration step includes administering the drug once to three times a week.

65. The method according to claim 1, wherein the pharmaceutical composition is administered by microneedles.

66. The method according to claim 1, wherein the administration step includes administering to one eye.

67. The method according to claim 1, wherein the administration step includes administering to both eyes.

68. The method according to claim 1, wherein the DWEK or DSO is performed on one eye.

69. The method according to claim 1, wherein the DWEK or DSO is performed in both eyes.

70. The method according to claim 7, wherein the cataract surgery is performed on one eye.

71. The method according to claim 7, wherein the cataract surgery is performed on both eyes.

72. The method according to claim 1, wherein the administration step produces one or more therapeutic effects selected from the group including a reduction in corneal endothelial damage, an increase in corneal endothelial cell migration, an increase in corneal endothelial cell proliferation, a reduction in corneal edema, an improvement in best corrected visual acuity (BCVA), a decrease in corneal thickness, a decrease in central corneal thickness, an improvement in visual function and corneal health (V-FUCHS) measurements, a decrease in intraocular pressure, an improvement in slit-lamp examination, an improvement in central corneal endothelial cell count, an improvement in peripheral corneal endothelial cell count, or a reduction in the time to achieve any of these.

73. The method according to claim 72, wherein the reduction in corneal endothelial damage includes a reduction in cell death, a reduction in functional impairment, or both.

74. The method according to claim 1, wherein the administration step brings about a therapeutic effect at a maximum of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the administration step.

75. The method according to claim 1, wherein the administration step provides a therapeutic effect at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the administration step.

76. The method according to claim 72, wherein the BCVA is measured using an Early Treatment Diabetic Retinopathy Scale (ETDRS) scoring system having at least 69 letters and at least 20 / 40 scores.

77. The method according to claim 1, wherein the administration step includes administering to both eyes, and the administration brings about a therapeutic effect in both eyes that is similar or the same between the two eyes.

78. The method according to claim 1, wherein the administration step includes administering to both eyes, and the administration results in an improvement in BCVA that is similar or the same between the two eyes.

79. The method according to claim 1, wherein the administration step provides a dose-dependent therapeutic effect.

80. The method according to claim 1, wherein the therapeutic effect increases with the dose of the modified FGF-1 polypeptide administered.

81. The method according to claim 1, wherein the administration step yields a dose-independent therapeutic effect.

82. The method according to claim 1, wherein the administration step produces a therapeutic effect in subjects with or without cataract surgery, and the therapeutic effect is similar or the same between subjects with and without cataract surgery.

83. The method according to claim 1, wherein the administration step results in an improvement in corneal edema or central corneal thickness in subjects with or without cataract surgery, and the improvement in corneal edema or central corneal thickness is similar or the same in subjects with and without cataract surgery.

84. The method according to claim 1, wherein the administration step results in an improvement in BCVA.

85. The method according to claim 1, wherein the step of administration results in a reduction or elimination of corneal edema.

86. The method according to claim 1, wherein the administration step results in an accelerated reduction or elimination of corneal edema.

87. The method according to claim 1, wherein the method includes a treatment course of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the DWEK or DSO treatment.

88. The method according to claim 1, wherein the method includes a treatment course of up to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years after the DWEK or DSO treatment.

89. The method according to claim 1, wherein the DWEK / DSO includes a central descemetorhexis of approximately 4 mm to 5 mm.

90. The method according to claim 1, wherein the reduction in the size of the DSO results in a reduction in the time required to achieve a therapeutic effect in the subject.

91. The method according to claim 90, wherein the therapeutic effect includes faster improvement to BCVA 20 / 40, accelerated improvement to BCVA 20 / 40, shortening of the time to BCVA 20 / 40, or reduction of corneal edema.

92. The method according to claim 1, wherein the subject has diabetes.

93. The method according to claim 1, wherein the administration step provides a therapeutic effect including a reduction in corneal thickness or a reduction in corneal edema.

94. The method according to claim 1, wherein the corneal disease or illness includes Fuchs endothelial cell dystrophy with central guttata (FECD) as a contributing cause of visual symptoms.

95. The method according to claim 94, wherein the visual symptoms include decreased visual acuity from the cornea, morning blur, or central corneal edema.

96. The method according to claim 1, wherein the subject has a transparent peripheral cornea with appropriate corneal endothelial cell (CEC) reserve capacity.

97. The method according to claim 1, wherein the subject does not have subepithelial turbidity or fibrosis.

98. The method according to claim 1, wherein the subject does not exhibit secondary corneal pathology, refractive surgery of the eye, or extraocular inflammation caused by non-infectious or infectious pathogens of the eye.

99. The method according to claim 1, wherein the subject is a human.

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