Therapeutic verteporfin drug-biomolecule conjugates

Abstract

Water-soluble conjugates of verteporfin are provided, which find therapeutic use, including by topical administration. Also provided are liposomal verteporfin formulations, which may be admixed with biomolecules such as hyaluronic acid.

Description

THERAPEUTIC VERTEPORFIN DRUG-BIOMOLECULE CONJUGATESGOVERNMENT SUPPORT RESEARCH

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 611 ,602, filed December 18, 2023, the disclosure of which application is herein incorporated by reference.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This invention was made with Government support under contracts EY033363, EY034168, and EY034600 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0003] Corneal scars can develop during corneal regeneration following traumatic injuries, often leading to irreversible moderate-to-severe vision impairment. When the cornea is injured, quiescent corneal stromal keratocytes differentiate into myofibroblasts. This differentiation is triggered by transforming growth factor beta 1 (TGF-(31 ), which is secreted by injured and immune cells. This cytokine induces the translocation of Yes-associated protein (YAP), a transcription coactivator, into the nucleus, where it activates the expression of downstream genes associated with myofibroblast formation. The resulting myofibroblasts secrete abnormal extracellular matrix (ECM) proteins, causing irregular stromal remodeling and corneal haze.

[0004] Currently, there are no FDA-approved drugs available to prevent or treat corneal scars. However, various chemical agents, such as losartan, mitomycin C, steroids, and rosiglitazone, have demonstrated anti-scarring effects on the cornea. More recently, verteporfin (VP) has emerged as a potential antifibrotic agent. By blocking Engrailed-1 activation, VP has shown efficacy in treating fibrosis across various diseases, including pulmonary fibrosis, systemic sclerosis, hepatic fibrosis, glaucoma, and renal fibrosis.

[0005] The topical application of VP for treating corneal defects and preventing scarring presents several challenges. A major limitation is VP’s poor water solubility, making it unsuitable for use as a simple aqueous mixture. To overcome this, VP is encapsulated in liposomes as seen in the commercial product Visudyne®, which was developed to treat age- related macular degeneration with photodynamic therapy. Similarly, for ocular surface applications, VP must be formulated to enhance its apparent solubility. Additionally, the low bioavailability of topical eye drops (~5%) due to the high clearance rate of the ocular surface is a concern. This rapid clearance reduces therapeutic efficacy, making it crucial to develop formulations that can adhere to the corneal surface and increase residence time. Moreover,these formulations should also facilitate corneal regeneration to promote complete wound healing.

[0006] Improved verteporfin compounds are of interest for wound healing, and decreasing the risk of fibrosis or scarring.SUMMARY

[0001] Verteporfin conjugates are provided; and methods of use thereof. The conjugates increase solubility of Verteporfin in aqueous solution relative to the unconjugated drug, allowing topical administration. The conjugate may also be improved relative to the unconjugated drug in bioavailability, retention time, and therapeutic potency.

[0002] In some embodiments, the conjugate has a structure (I):As shown in structure (I), hyaluronic acid (HA) is a polymer of disaccharides composed of D- glucuronic acid and N-acetyl-D-glucosamine, linked via alternating |3-(1— >4) and |3-(1 — >3) glycosidic bonds. Conjugates of the disclosure can be based on an HA polymer of at least 1 , at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5,000, at least 10,000, at least 50,000, at least 100,000 or more disaccharide subunits, having a molecular weight of from about 1000 Da, up to 106Da or more. Shown in structure (I), n indicates the degree of substitution (DoS), that is the percentage of HA disaccharides that are functionalized with verteporfin, where n+m must equal 1. The value of n may range from at least about 0.01 , at least about 0.025, at least about 0.05, at least about 0.075, at least about0.1 , at least about 0.125, at least about 0.15, at least about 0.175, at least about 0.2, or more. A structure (I) where n is 0.2 will have a DoS of 20%. In some embodiments the DoS is from about 1% to about 20%, or from about 2% to about 10%.

[0003] In some embodiments, a formulation is provided comprising a drug conjugate (I) and a pharmaceutically acceptable carrier. In some embodiments the formulation comprises an effective dose of the conjugate in a unit dosage amount. In some embodiments the formulation is suitable for topical administration. In some embodiments the formulation is suitable for topical administration to the eye. In some embodiments the formulation is free of liposomes.

[0004] In another embodiment, an admixed formulation is provided of liposomal verteporfin, e.g. a formulation as disclosed herein, VISUDYNE™, etc., admixed with a gel of high molecular weight HA, where the verteporfin concentration in the hyaluronic acid is from about 0.5 to about 5 mg / ml verteporfin equivalents, and may be from about 0.75 mg / ml. to about 1 .25 mg / ml, or about 1 mg / ml. The HA gel may comprise about 5 mg / ml HA to about 15 mg / ml HA, and may be around 10 mg / ml HA, with an average molecular weight of from about 1 x 106Da to about 5 x 106Da, and may be around 2.5 x 106Da.

[0005] In some embodiments a method is provided for use of a formulation comprising a conjugate (I). In some embodiments the formulation is topically administered to a body surface, e.g. the ocular surface. In other embodiments the body surface is skin, e.g. at a wound site.

[0006] In some embodiments, following topical administration the body surface is subjected to light. In some embodiments the light is in a dose effective to induce cross-linking. The individual for treatment may be suffering from keratoconus and other corneal ectasias, infection, wounds. In some embodiments the conjugate is used to crosslink biomaterials onto the cornea as a defect filling gel.

[0007] In an embodiment, one or more therapeutically effective doses of conjugate (I) or an admixture are administered to a subject before, during, or after an ocular surgical procedure or after an ocular injury to treat or reduce the risk of developing fibrosis in ocular tissues. In certain embodiments, the ocular surgery is glaucoma surgery. In certain embodiments, the ocular injury is caused by physical trauma or a chemical exposure. In certain embodiments, the ocular injury or ocular surgery causes a corneal wound or a conjunctival wound. In certain embodiments, the ocular injury increases risk of corneal or conjunctival fibrosis or scarring.

[0008] In certain embodiments, the conjugate or admixture is administered in one or more doses. In some embodiments, each administration provides a dose ranging from 0.001 mg to 10 mg of conjugate, including any dose within this range such as 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0. 01 mg, 0.02 mg, 0,03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg,6 mg, 7 mg, 8 mg, 9 mg, or 10 mg. In some embodiments, each administration provides a dose ranging from 0.01 mg to 1 mg of verteporfin equivalents. In some embodiments, the conjugate is administered in a volume ranging from 0.1 ml to 1 ml, e.g. with a concentration of verteporfin from around 0.1 mg / ml, 0.25 mg / ml, 0.5 mg / ml, 0.75 mg / ml, 1 mg / ml, 1.25 mg / ml, 1 .5 mg / ml or more.

[0009] In certain embodiments, the conjugate or admixture is administered locally under the conjunctiva or topically on the cornea, e.g. to repair a corneal wound. In certain embodiments, the wound is the result of ocular surgical procedure comprising incision of the conjunctiva. In certain embodiments, the ocular surgical procedure is glaucoma filtration surgery (trabeculectomy), refractive surgery or cross-linking, e.g. LASIK™, PRKTM, etc., minimally invasive glaucoma surgery (MIGS), insertion of a glaucoma drainage implant, insertion of a Xen gel stent, or insertion of other stents or devices in the subconjunctival space, a revision of a trabeculectomy, a revision of MIGS, a revision of a glaucoma drainage implant, a revision of a Xen gel stent, or revision of other stents or devices in the subconjunctival space. In some embodiments, the ocular surgical procedure comprises incisional surgery or bleb needling to revise a previous trabeculectomy, previous tube shunt surgery, or other previous incisional surgery. Wounding may also be the result of a corneal infection or ulcer, e.g. bacterial, viral and fungal infections of the cornea.

[0010] In certain embodiments, multiple cycles of treatment are administered to the subject. In certain embodiments, the conjugate or admixture is administered to the subject intermittently. In certain embodiments, the conjugate or admixture is administered no more frequently than biweekly. In certain embodiments, the conjugate or admixture is administered for at least one month, at least two months, or at least three months.

[0011] In certain embodiments, the conjugate or admixture is administered before, during, or after an ocular surgical procedure or ocular injury; and subsequent administrations of are no more frequent than biweekly up to postoperative month three. In some embodiments, the conjugate or admixture is administered up to five times subsequently. In some embodiments, the method further comprises administering the conjugate or admixture after postoperative month three. In some embodiments, the method further comprises administering the conjugate or admixture one, two, three, four, five, six, seven, eight, or nine additional times after the fifth administration. In some embodiments, the additional doses are administered monthly.

[0012] In certain embodiments, the administration of the verteporfin is performed in conjunction with bleb needling or other manipulation of the conjunctiva, performed in the office or in the operating room.

[0013] In an embodiment, a method of treating fibrosis in ocular tissues is provided, the method comprising: identifying a subject who has fibrosis in ocular tissues or is at risk of developing fibrosis or scarring in ocular tissues after a previous ocular surgery or ocular injury; administering to the subject a first dose of the conjugate or admixture; and administering to the subject subsequent doses of the conjugate no more frequently than biweekly, wherein up to five doses of the conjugate are administered to the subject. Administration may be topical on the cornea or under the conjunctiva.

[0014] In certain embodiments, the ocular surgical procedure is glaucoma filtration surgery (trabeculectomy), refractive surgery or cross-linking, e.g. LASIK™, PRKTM, etc., minimally invasive glaucoma surgery (MIGS), insertion of a glaucoma drainage implant, insertion of a Xen gel stent, or insertion of other stents or devices in the subconjunctival space, a revision of a trabeculectomy, a revision of MIGS, a revision of a glaucoma drainage implant, a revision of a Xen gel stent, or revision of other stents or devices in the subconjunctival space. In some embodiments, the ocular surgical procedure comprises incisional surgery or bleb needling to revise a previous trabeculectomy, previous tube shunt surgery, or other previous incisional surgery. Wounding may also be the result of a corneal infection or ulcer, e.g. bacterial, viral and fungal infections of the cornea. In certain embodiments, the method further comprises administering one, two, three, four, five, six, seven, eight, or nine additional doses of the conjugate or admixture after the fifth dose. In some embodiments, the additional doses are administered monthly. In certain embodiments, each administration of the conjugate or admixture is performed in conjunction with bleb needling or other manipulation of the conjunctiva, performed in the office or in the operating room.

[0015] In certain embodiments, a method of reducing corneal scarring in an eye of a subject after a corneal injury or infection is provided, the method comprising administering a therapeutically effective amount of the conjugate or admixture to the subject. In some embodiments, the fibrosis is conjunctival fibrosis. In some embodiments administration is provided after a corneal injury to reduce corneal scarring. In some embodiments the method comprises: identifying a subject who has fibrosis in ocular tissues or is at risk of developing fibrosis or scarring in ocular tissues after a previous ocular surgery or ocular injury; administering to the subject a first dose of the conjugate. The subject may subsequently be administered subsequent doses of verteporfin no more frequently than biweekly, wherein up to five doses are administered to the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to commonpractice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0017] FIGS. 1 A-11. Schematic illustration of the study design and characterization of the HA- VP NPs. (a) Schematic illustration, (b) Scheme for synthesis of the HA- VP conjugate using sulfo-NHS / EDC coupling reagents with VP and amine-modified HA (HA-NH2). (c)1H NMR spectra (500 MHz) of the VP, HA-NH2, and HA-VPhigh conjugates in DMSO-de. (d) FTI R spectra and (e) absorbance spectra of the VP, HA-NH2, and HA-VPhigh conjugate with four different concentrations, (f) TEM image of the HA-VP NPs; scale bar = 200 nm. (g) Biodegradability study of the HA-VP NPs (n = 3) against hyaluronidase, (h) In vivo fluorescence imaging of the HA-VPiow and the HA-VPhigh in cornea-defected rats, (i) Relative fluorescent intensity of the HA-VP conjugates in the rat eyes (n = 3).

[0018] FIGS. 2A-2D. Inhibitory effect of the HA-VP NPs on YAP localization and a-SMA expression in CSCs. Cells were treated with TGF-(31 to induce changes in YAP localization and a-SMA expression, (a) Confocal microscopy images showing intracellular YAP localization and fluorescence intensity profiles. The profiles were obtained from the orange square areas in the images. Green and blue colors indicate the intensities of YAP and DAPI, respectively. Scale bar = 50 pm. (b) The intensity ratio of YAP in the nuclear and cytosolic fractions, calculated from 25 cells in each group, (c) Confocal microscopy images of a-SMA expression. Red, yellow, and blue colors indicate the fluorescence intensities of a-SMA, VP, and DAPI, respectively. Scale bar = 50 pm. (d) Quantification of ACTA2 gene expression by RT-qPCR. The control group (Con) represents normal cells without TGF-p1 or any treatments.

[0019] FIGS. 3A-3B. Cellular uptake of the HA-VP NPs by CSCs. (a) Confocal microscopy images of CSCs treated with the HA-VP NPs for 60 min. Calcein-AM is used for labeling live cells, (b) Monitoring of VP intensities from 41 to 55 min. Yellow arrows indicate sphere-shaped aggregates around the cell membrane. Scale bar = 50 pm.

[0020] FIGS. 4A-4D. Corneal residence of the HA-VP NPs in rats assessed by fluorescence imaging (n = 4). (a) Representative in vivo fluorescence images of the Visudyne®- and HA-VP NPs-treated eyes, and (b) their EBR values at each time point, (c) Fluorescence images of the Visudyne®- and HA-VP NPs-treated eyes enucleated at 4h, and (d) their corresponding EBR values.

[0021] FIGS. 5A-5G. In vivo evaluation of the antifibrotic effects of HA-VP NPs in rats (n = 4). (a) The rat ALK model and corneal regeneration study design, (b) OCT images of the defected corneas on days 0, 7, and 14 after treatment, (c) Representative slit-lamp images of corneas, (d) Percentage of haze area on corneas measured from c). (e) E:S ratio based on OCT images. For the normal group, data from day 7 was also used for day 14 for plotting purposes.(f) Fluorescein-stained images of corneas under blue light, (g) Measurement of fluorescein- stained area from f).

[0022] FIGS. 6A-6F. Immunohistochemistry of regenerated cornea, (a) Representative images of ALDH3A1 expression, (b) Relative intensities of ALDH3A1 in epithelial layers (n = 4). (c) Representative images of a-SMA expression, (d) Relative intensities of a-SMA in stromal layers (n = 4). (e) Representative images of CK12 expression, (f) Relative intensities of CK12 in epithelial layers (n = 4).

[0023] FIG. 7. Cytotoxicity of HA- VP NPs in CSCs (n = 5). The cells were treated with HA- VP NPs at various concentrations, ranging from 2 to 1000 pg / mL (as VP). Cell viability was measured using a Resazurin assay (Abeam, Cambridge, UK).

[0024] FIG. 8. VP intensity change in the cytosol of CSCs (n = 7), calculated from FIG. 3b. The intensity values were normalized to those at 41 min.

[0025] FIG. 9. Solubility of VP in various solvents. An excess amount of VP powder was incubated in the solvents for two days at 37°C, protected from light. The incubated suspensions were then centrifuged (17,000g, 10 min), and the concentration of VP in the supernatant was analyzed using a microplate reader.

[0026] FIG. 10. Complete OCT images from the in vivo study (n = 4).

[0027] FIG. 11 . OCT images of normal rat corneas.

[0028] FIG. 12. H&E staining of corneas obtained from the in vivo rat study on scarless corneal regeneration (n = 4). Yellow arrows indicate blood vessels. Scale bar = 200 pm.

[0029] FIG. 13. In vitro VP release pattern from Visudyne® (n = 3). Visudyne® was loaded into a dialysis bag (MWCO 3.5 kDa) and immersed in a 0.1% Triton X-100 solution, which was added to maintain sink conditions.

[0030] FIG. 14. Fluorescence images of Visudyne® and unlabeled HA-VP NPs at Ex / Em = 675 / 730 nm (n = 3). (a) A representative image, (b) Normalized intensity.

[0031] FIGS. 15A-15H. A. Mixing liposomal verteporfin with a hyaluronic acid gel results in sustained-release of verteporfin for more than 3 days. The higher concentration of liposomal verteporfin led to more release of drug due to a larger concentration discrepancy-driven force for diffusion. B and C. Cells grown in the presence of transforming growth factor |31 (TGFp-1 ) were induced to differentiate from CSSC to myofibroblast phenotype, evidenced by the upregulation of ACTA2 gene expression, which encodes alpha-smooth muscle actin (aSMA). VP-HA conjugate and lipoVP / HA mixture both inhibited myofibroblast activity with verteporfin concentrations greater than 5 pg / ml. The lipoVP / HA gel treatment group showed the fastest re-epithelialization rate compared to HA alone, or liposomal verteporfin alone. (D) Corneal wound depth justification. The average wound depth was approximately 50% of the central cornea. No significant differences in the wound depth were found among the four groups. Anordinary one-way ANOVA test was used to calculate the p values. (E) Representative fluorescein staining images indicated the corneal epithelial wound closure over time. (F) Quantification of fluorescein staining images showed a non-significantly faster corneal re- epithelialization in the lipoVP / HA gel-treated group compared to the other groups. A repeated measures ANOVA test was used to calculate the p values. The lipoVP / HA gel treatment prevented scar formation during corneal wound healing. (G) Representative clinical photos showed corneal opacity changes over time. (H) Quantified gray values of the corneas and (C) blindly graded corneal haze score showed that the corneas treated with lipoVP / HA gel were more transparent than those treated with the other treatments. A repeated measures ANOVA test was used to calculate the p values.DETAILED DESCRIPTION OF EMBODIMENTS

[0032] Verteporfin conjugates and admixtures are provided; and methods of use thereof. The conjugates increase solubility of verteporfin in aqueous solution relative to the unconjugated drug, allowing topical administration. The conjugate may also be improved relative to the unconjugated drug in bioavailability, retention time, and therapeutic potency. In some embodiments the conjugate comprises verteporfin covalently linked to a hyaluronic acid polymer.

[0033] Before the compositions and methods are described, it is to be understood that this invention is not limited to particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0034] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this inventionbelongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0036] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0037] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a drug" includes a plurality of such drugs and reference to "the drug" includes reference to one or more drugs and equivalents thereof known to those skilled in the art, and so forth.

[0038] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0039] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. The term "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and / or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and / or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and / or symptom(s). Those in need of treatment include those already inflicted (e.g., those with fibrosis in ocular tissues) as well as those in which prevention is desired (e.g., those with increased susceptibility to developing fibrosis or scarring in ocular tissues because of having an ocular surgical procedure or ocular injury).

[0040] By "therapeutically effective dose or amount" is intended an amount that, when administered, as described herein, brings about a positive therapeutic response, such as improved recovery from a wound, an ocular surgical procedure or ocular injury, etc., including reducing or preventing fibrosis and / or scarring. In particular, a "therapeutically effective dose or amount" may reduce or prevent fibrosis in ocular tissues after an ocular surgical procedure or ocular injury. Additionally, treatment may reduce eye pain, redness, and / or scarring. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, mode of administration, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

[0041] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0042] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0043] "Substantially purified" generally refers to isolation of a component such as a substance (e.g., compound, drug) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography, gel filtration, and sedimentation according to density.

[0044] The terms “recipient”, “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and refer to any vertebrate subject for whom diagnosis, treatment, or therapy is desired, particularly humans. By “vertebrate subject” is meant any member of the subphylum Chordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats;laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered.

[0045] Corneal wound refers to any surgical process, infection, or injury that results in damage to the cornea, e.g. causing a disruption or loss of cells in the corneal epithelium, incision or erosion of the cornea, etc.

[0046] Corneal and conjunctival wounds may be the result, for example of an ocular surgical procedure, such as glaucoma filtration surgery (trabeculectomy), refractive surgery or crosslinking, e.g. LASIK™, PRKTM, etc., minimally invasive glaucoma surgery (MIGS), insertion of a glaucoma drainage implant, insertion of a Xen gel stent, or insertion of other stents or devices in the subconjunctival space, a revision of a trabeculectomy, a revision of MIGS, a revision of a glaucoma drainage implant, a revision of a Xen gel stent, or revision of other stents or devices in the subconjunctival space. In some embodiments, an ocular surgical procedure comprises incisional surgery or bleb needling to revise a previous trabeculectomy, previous tube shunt surgery, or other previous incisional surgery.

[0047] Corneal wound may also be the result of an injury, e.g. a traumatic injury. Wounding may be the result of a corneal infection or ulcer, e.g. bacterial, viral and fungal infections of the cornea.Pharmaceutical Compositions

[0048] A verteporfin conjugate or admixture can be formulated into pharmaceutical compositions, optionally comprising one or more pharmaceutically acceptable excipients. In some embodiments the formulation comprises an effective dose of the conjugate in a unit dosage amount. In some embodiments the formulation is suitable for topical administration. In some embodiments the formulation is suitable for topical administration to the eye. In some embodiments the formulation is free of liposomes. In some embodiments the formulation is topically administered to a body surface, e.g. the ocular surface. In other embodiments the body surface is skin, e.g. at a wound site.

[0049] In some embodiments, the conjugate has a structure (I):As shown in structure (I), hyaluronic acid (HA) is a polymer of disaccharides composed of D- glucuronic acid and N-acetyl-D-glucosamine, linked via alternating (3-(1 — >4) and p-(1 — >3) glycosidic bonds. Conjugates of the disclosure can be based on an HA polymer of at least 1 , at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5,000, at least 10,000, at least 50,000, at least 100,000 or more disaccharide subunits, having a molecular weight of from about 1000 Da, up to 106Da or more. Shown in structure (I), n indicates the degree of substitution (DoS), that is the percentage of HA disaccharides that are functionalized with verteporfin, where n+m must equal 1. The value of n may range from at least about 0.01 , at least about 0.025, at least about 0.05, at least about 0.075, at least about 0.1 , at least about 0.125, at least about 0.15, at least about 0.175, at least about 0.2, or more. A structure (I) where n is 0.2 will have a DoS of 20%. In some embodiments the DoS is from about 1% to about 20%, or from about 2% to about 10%.

[0050] In other embodiments an admixture is provided. Verteporfin admixtures comprise liposomal verteporfin. The liposome may be fusogenic or non-fusogenic. In some embodiments the liposomes comprise one or more of 2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG), and 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP). In some embodiments the liposomes comprise each of DMPC, DSPE-PEG, and DOTAP. In some embodiments the liposomes comprise each of DMPC and DSPE-PEG. DMPC may be present at a concentration of from about 75% to about97%. DSPE-PEG may be present at a concentration of from about 3% to about 5%, from about 3.5% to about 4%, and may be from about 3.75% to about 3.85%. Fusogenic liposomal verteporfin may comprise, for example DMPC, DSPE-PEG, and DOTAP at a ratio of about 76.2:3.8:20. Non-fusogenic liposomal verteporfin may comprise, for example, DMP and, DSPE-PEG at a ratio of 96.2:3.8.

[0051] Exemplary excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerine, vegetable oils, phospholipids, and surfactants. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and / or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0052] A composition can also comprise an antimicrobial agent for preventing or deterring microbial growth. Nonlimiting examples of antimicrobial agents include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

[0053] An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the verteporfin, or other components of the preparation. Suitable antioxidants for use include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0054] A surfactant can be present as an excipient. Exemplary surfactants include: polysorbates, such as "Tween 20" and "Tween 80," and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other such suitable cations.

[0055] Acids or bases can be present as an excipient in the composition. Nonlimiting examples of acids that can be used include those acids selected from the group consisting ofhydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.

[0056] The amount of the conjugate (e.g., when contained in a drug delivery system) in the composition will vary depending on a number of factors, but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., a vial). A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition in order to determine which amount produces a clinically desired endpoint.

[0057] The amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1 % to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred. These foregoing pharmaceutical excipients along with other excipients are described in "Remington: The Science & Practice of Pharmacy", 19th ed., Williams & Williams, (1995), the "Physician’s Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000. Examples of such excipients may include but are not limited to hyaluronic acid, collagen, carboxymethylcellulose, alginate, hypromellose, polyvinyl alcohol, polyethylene glycol, chondroitin sulfate, as well as combinations and / or derivatives thereof.

[0058] The compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted with a solvent prior to use, as well as ready for injection solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate bufferedsaline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. With respect to liquid pharmaceutical compositions, solutions and suspensions are envisioned. Additional compositions include those for oral, topical, ocular, or localized delivery. Formulations suitable for oral, topical, ocular, or localized administration may be prepared through use of appropriate suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Such formulations may be utilized as liquid drops or with a means to provide continuous administration, for example, incorporation into slow-release pellets or controlled- release patches.

[0059] The pharmaceutical preparations herein can also be housed in a syringe, an implantation device, or the like, depending upon the intended mode of delivery and use. Preferably, the compositions comprising the verteporfin are in unit dosage form, meaning an amount of a composition appropriate for a single dose, in a premeasured or pre-packaged form.

[0060] The compositions herein may optionally include one or more additional agents, such as analgesics, anti-inflammatory agents, anti-thrombotic / anti-fibrotic agents, antimetabolites, antibodies, and / or other medications used to treat a subject for fibrosis in ocular tissues. Compounded preparations may include the verteporfin and one or more other agents, such as, but not limited to, analgesics, including acetaminophen, non-steroidal anti-inflammatory agents (NSAIDs) such as aspirin, ibuprofen and naproxen, COX-2 inhibitors such as rofecoxib, celecoxib, and etoricoxib, and opioids such as morphine, codeine, oxycodone, hydrocodone, dihydromorphine, and pethidine; anti-inflammatory agents, including steroids such as prednisolone, betamethasone, dexamethasone, fluoromethoIone, hydrocortisone, and medrysone; anti-thrombotic / anti-fibrotic agents, including tissue plasminogen activator, urokinase, pirfenidone, and saratin; antimetabolites, including mitomycin C and 5-fluorouracil; and antibodies, including anti-VEGF monoclonal antibodies such as bevacizumab, and anti- TGF-|32 monoclonal antibodies such as lerdelimumab; and the like. Alternatively, such agents can be contained in a separate composition from the composition comprising the verteporfin and co-administered concurrently, before, or after the composition comprising the verteporfin.Administration

[0061] At least one therapeutically effective dose of the conjugate or admixture is administered. By "therapeutically effective dose or amount" is intended an amount that, when administered, as described herein, brings about a positive therapeutic response, such as improved recovery from a wound, an ocular surgical procedure or ocular injury, including reducing or preventing fibrosis. In particular, a "therapeutically effective dose or amount" mayreduce or prevent fibrosis in ocular tissues after an ocular surgical procedure or ocular injury. Additionally, treatment may reduce eye pain, redness, and / or scarring.

[0062] The subject methods can be used to treat fibrosis and / or reduce or prevent fibrosis and / or scarring in ocular tissue including, without limitation, surface ocular tissue such as the cornea, conjunctiva, and sclera; and deeper ocular tissue such as tissue of the uvea, including the choroid, ciliary body, pigmented epithelium, and iris; and retina tissue. In some embodiments, verteporfin is used to treat a corneal wound or a conjunctival wound resulting from an ocular surgical procedure or ocular injury.

[0063] In some embodiments, a step of exposing the tissue to light is performed following administration. Generally the far-red peak at 689 nm is utilized in clinical practice, which allows good penetration through melanin, blood and fibrotic tissue enabling effective treatment of pigmented or haemorrhagic lesions. A laser delivery system provides localized light irradiation of the target lesion. For example, the minimum effective light dose may be greater than about 25 J / cm2and the maximum tolerated light dose less than about 150 J / cm2. Tissue selectivity can also be influenced by the time interval between administration and light irradiation. In some embodiments irradiation is performed with about 1 hour, within about 30 minutes, within about 10 minutes following topical administration.

[0064] In some embodiments additional cross-linking materials are included in the formulation, e.g. to fill defects in a corneal surface.

[0065] In some embodiments, one or more therapeutically effective doses of the conjugate or admixture are administered to a subject before, during, or after an ocular surgical procedure or injury to prevent or reduce the risk of developing fibrosis. The ocular surgical procedure may comprise incision of the conjunctiva. In some embodiments, the ocular surgical procedure is glaucoma filtration surgery (trabeculectomy), minimally invasive glaucoma surgery (MIGS), insertion of a glaucoma drainage implant, insertion of a Xen gel stent, or insertion of other stents or devices in the subconjunctival space, a revision of a trabeculectomy, a revision of MIGS, a revision of a glaucoma drainage implant, a revision of a Xen gel stent, or revision of other stents or devices in the subconjunctival space. In some embodiments, the surgical procedure is incisional surgery or bleb needling to revise a previous trabeculectomy, previous glaucoma drainage implant surgery, previous minimally invasive glaucoma surgery, or other previous incisional surgery.

[0066] In some embodiments, one or more therapeutically effective doses of the conjugate or admixture are administered to a subject after an ocular injury to prevent or reduce the risk of developing fibrosis or scarring. Verteporfin can be used to treat any type of eye injury that increases the risk of developing fibrosis or scarring in ocular tissue, including eye injuriesresulting from physical trauma or a chemical exposure. In some embodiments, at least one therapeutically effective dose of the conjugate is administered to reduce corneal scarring in an eye of a subject after a corneal injury.

[0067] In certain embodiments, multiple therapeutically effective doses of the conjugate or admixture are administered according to a daily dosing regimen, or intermittently. For example, a therapeutically effective dose can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. By “intermittent” administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days, every three days, and so forth. For example, in some embodiments, the verteporfin will be administered twice-weekly or thrice-weekly for an extended period of time, such as for 1 , 2, 3, 4, 5, 6, 7, 8, 10, 15, 24 weeks, and so forth. By “twice-weekly” or “two times per week” is intended that two therapeutically effective doses of the conjugate are administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By “thrice weekly” or “three times per week” is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present invention, this type of dosing is referred to as “intermittent” therapy. In accordance with the methods of the present invention, a subject can receive intermittent therapy (i.e., twice-weekly or thrice-weekly administration of a therapeutically effective dose) for one or more weekly cycles until the desired therapeutic response is achieved. In certain embodiments, the conjugate is administered no more frequently than biweekly. In certain embodiments, the conjugate is administered for at least one month, at least two months, or at least three months. The conjugate can be administered by any acceptable route of administration as noted herein below.

[0068] In other embodiments, the pharmaceutical composition comprising the conjugate or admixture is a sustained-release formulation, or a formulation that is administered using a sustained-release device. Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for drug delivery over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.

[0069] Verteporfin conjugate or admixture can be administered alone or in combination with one or more other therapeutic agents, such as analgesics, anti-inflammatory agents, anti- thrombotic / anti-fibrotic agents, antimetabolites, antibodies, and / or other medications used to treat a subject for fibrosis, including, but not limited to, analgesics, including acetaminophen, non-steroidal anti-inflammatory agents (NSAIDs) such as aspirin, ibuprofen and naproxen,COX-2 inhibitors such as rofecoxib, celecoxib, and etoricoxib, and opioids such as morphine, codeine, oxycodone, hydrocodone, dihydromorphine, and pethidine; anti-inflammatory agents, including steroids such as prednisolone, betamethasone, dexamethasone, fluoromethoIone, hydrocortisone, and medrysone; anti-thrombotic / anti-fibrotic agents, including tissue plasminogen activator, urokinase, pirfenidone, and saratin; antimetabolites, including mitomycin C and 5-fluorouracil; and antibodies, including anti-VEGF monoclonal antibodies such as bevacizumab, and anti-TGF-|32 monoclonal antibodies such as lerdelimumab; and the like, and other medications used to treat a particular condition or disease according to a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Preferred compositions are those requiring dosing no more than once a day.

[0070] The conjugate or admixture can be administered prior to, concurrent with, or subsequent to other agents. If provided at the same time as other agents, the conjugate can be provided in the same or in a different composition. Thus, the conjugate and one or more other agents can be presented to the individual by way of concurrent therapy. By “concurrent therapy” is intended administration to a subject such that the therapeutic effect of the combination of the substances is caused in the subject undergoing therapy. For example, concurrent therapy may be achieved by administering a dose of a pharmaceutical composition comprising the conjugate and a dose of a pharmaceutical composition comprising at least one other agent, such as another drug for treating ocular fibrosis, which in combination comprise a therapeutically effective dose, according to a particular dosing regimen. Similarly, the conjugate and one or more other therapeutic agents can be administered in at least one therapeutic dose. Administration of the separate pharmaceutical compositions can be performed simultaneously or at different times (i.e., sequentially, in either order, on the same day, or on different days), as long as the therapeutic effect of the combination of these substances is caused in the subject undergoing therapy.

[0071] Toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50(the dose lethal to 50% of the population) or the LD o (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in further optimizing and / or defining a therapeutic dosage range and / or a sub-therapeutic dosage range (e.g., for use in humans). The exact formulation,route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

[0072] The pharmaceutical compositions comprising the conjugate or admixture and optionally other agents may be administered using the same or different routes of administration in accordance with any medically acceptable method known in the art. Suitable routes of administration include parenteral administration, such as intraocular, subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), intravenous (IV), or infusion, and oral, pulmonary, topical, and transdermal. In some embodiments, the pharmaceutical composition comprising the conjugate is administered topically. In some embodiments, the conjugate is administered locally under the conjunctiva. In some embodiments, the conjugate is administered by subconjunctival injection. In some embodiments, the conjugate is administered topically as eye drops, on a patch, or in a gel. For example, the conjugate may be administered topically on the cornea.

[0073] In certain embodiments, the conjugate or admixture is administered in one or more doses (e.g., topically on the cornea or by subconjunctival injection). In some embodiments, a dose is 0.001 mg to 10 mg of verteporfin equivalent, including any dose within this range such as 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0. 01 mg, 0.02 mg, 0,03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg. In some embodiments, a dose ranges from 0.01 mg to 1 mg of verteporfin equivalent. In some embodiments, the conjugate or admixture is administered in a volume ranging from 0.1 ml to 1 ml.

[0074] In certain embodiments, a first dose of the conjugate or admixture is administered before, during, or after an ocular surgical procedure; and subsequent doses of verteporfin are administered no more frequently than biweekly up to postoperative month three. In some embodiments, up to five subsequent doses of the conjugate are administered. In some embodiments, the method further comprises administering additional doses of the conjugate after postoperative month three. In some embodiments, the method further comprises administering one, two, three, four, five, six, seven, eight, or nine additional doses of the conjugate after the fifth dose of the conjugate. In some embodiments, the additional doses of the conjugate or admixture are administered monthly. In certain embodiments, the administration of the verteporfin is performed in conjunction with bleb needling or other manipulation of the conjunctiva, which may be performed in an office or in an operating room.

[0075] Factors influencing the respective amount of the various compositions to be administered include, but are not limited to, the mode of administration, the frequency of administration (i.e., daily, or intermittent administration, such as twice- or thrice-weekly), the type of ocular surgery, the particular location of ocular injury, the severity of the ocular injury,the degree of risk of ocular fibrosis or scarring, the history of ocular disease, whether the individual is undergoing concurrent therapy with another therapeutic agent, and the age, height, weight, health, and physical condition of the individual undergoing therapy. Generally, a higher dosage of an agent is preferred with increasing weight of the subject undergoing therapy.Kits

[0076] Kits may comprise one or more containers of the pharmaceutical compositions, described herein, comprising verteporfin, for example conjugated verteporfin, liposomal verteporfin, HA, etc. Compositions can be in liquid form or can be lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kit can further comprise a container comprising a pharmaceutically-acceptable buffer, such as phosphate- buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device. The kit may also provide a delivery device pre-filled with a pharmaceutical composition.

[0077] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods (i.e., instructions for treating or reducing risk of developing fibrosis and / or scarring in ocular tissue with verteporfin, as described herein). These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, Blu-ray, flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.

[0078] It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.EXPERIMENTAL

[0079] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, andare not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0080] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0081] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. All such modifications are intended to be included within the scope of the appended claims.Example 1

[0082] Materials. Verteporfin (VP), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), and 2-mercaptoethanol were purchased from Sigma-Aldrich. Hyaluronate amine (HA-amine) with a molecular weight (MW) of 250 kDa and a 50% degree of substitution (DoS) was acquired from Creative PEGWorks. Spectra / Por 3 RC dialysis membrane tubing with a molecular weight cut-off (MWCO) of 3.5 kDa was obtained from Spectrum. Alpha modification of minimum essential medium (MEM) (1 X) with Earle's salts, without ribonucleosides, deoxyribonucleosides, and L- glutamine, was purchased from Corning. GlutaMAX supplement (100X) and MEM non- essential amino acids (NEAA) Solution (100X) were acquired from Gibco. The resazurin assay solution was purchased from Abeam. Milli-Q type 1 ultrapure water was used for the preparation of all aqueous solutions. Syringe filters with a pore size of 0.2 pm were used for the filtration of all aqueous and cell media solutions.

[0083] Synthesis of HA-VP conjugate. Table 1 lists the reagents used to prepare HA-VP at a 10% DoS. The molar ratio between VP and HA-amine was adjusted accordingly to prepare HA-VP variants with DoS ranging from 2% to 10%. The entire reaction process was conducted under light protection. Initially, VP (5.0 mg, 7.0 pmol, 1 eq.) was dissolved in 0.5 mL of DMSO within a 20 mL amber glass vial and stirred for 10 min. Subsequently, 0.1 M of MES buffer (4.5 mL; pH 5.1 ) was added and stirred for an additional 10 min. The addition of sulfo-NHS(30.2 mg, 139.1 nmol, 20 eq.) was followed, and the mixture was stirred for 10 min. EDC (13.3 mg, 69.6 pmol, 10 eq.) was subsequently added and stirred for 2 hr, after which 2- mercaptoethanol (97.6 pL, 1391.2 pmol, 200 eq.) was introduced and stirred for 15 min. The pH was adjusted to 7.1 by adding 1 M K2PO (1 mL; pH 9.7). The addition of HA-amine (26.4 mg, 34.8 pmol, 5 eq.) was followed, and the mixture was stirred overnight. The resulting reaction mixture was dialyzed against ultrapure water using dialysis membrane tubing with a 3.5 kDa MWCO for a duration of 3-5 days. Finally, the dialyzed mixture was lyophilized and stored at -20 °C until further use.Table 1. Reagents used in the preparation of HA- VP component eq. MW # mol Mass volume(pmol) (mg) (pL)VP 1 718.79 7.0 5.0 sulfo-NHS 20 217.13 139.1 30.2EDC 10 191 .7 69.6 13.32-mercaptoethanol 200 78.13 1391 .2 97.6HA-amine (50% DoS) 5 250000 34.8 26.4

[0084] Characterization techniques, (i) Nuclear magnetic resonance (NMR) spectroscopy.1H NMR spectrum of HA-VP (2% DoS) was recorded on a Bruker AVANCE NEO 500 MHz spectrometer. Approximately 5 mg of HA-VP was dissolved in 0.5 mL of deuterium oxide. The chemical shifts (0) were reported relative to the residual H2O signal, which was observed at 5 = 4.79 ppm. (ii) Microplate reader. Absorbance and fluorescence measurements were performed using a TECAN Spark microplate reader.

[0085] Resazurin cell viability assay. Human corneal stromal stem cells (CSSCs) were used for the in vitro cell viability assay. In brief, 4 to 5 x 105cells at passage 6 were seeded into 75- cm2T-flasks containing MEM Alpha supplemented with 1 % GlutaMAX, 1% NEAA, 1 % penicillin / streptomycin, and 10% fetal bovine serum. The cells were cultured at 37°C in a 5% CO2atmosphere. The medium was refreshed every 2-3 days, and after a subsequent 2-3 days of incubation, the cells were trypsinized and sub-cultured. Cells at passages 7 and 8 were utilized for the subsequent assays.

[0086] In 96-well plates, 5,000 or 10,000 CSSCs were seeded into each well and incubated for 24 hr with 100 pL of cell culture medium per well. Subsequently, the cells were treated with another 100 pL of cell culture medium per well, which contained varying concentrations of HA- VP (10% DoS) or VP. After an additional 24 hours of incubation, 110 pL of the resazurin assay solution (comprising 100 pL of cell culture medium and 10 pL of stock resazurin assay solution) was added to each well. Following a 2-hr incubation period, the assay results were assessedby measuring absorbance at 570 nm or by measuring fluorescence with excitation at 545 nm and emission at 590 nm wavelengths.Results and discussion

[0087] Synthesis of HA-VP conjugate. The HA-VP conjugate was prepared by using the EDC coupling reagent in conjunction with amine-modified HA, as shown in FIG. 1 B. Initially, the carboxylic acid group in VP was activated with EDC and sulfo-NHS at pH 5.1 . The incorporation of sulfo-NHS served to stabilize the O-acylisourea intermediate by forming an ester, thereby preventing the undesirable formation of N-acylurea by-products. Notably, the use of sulfo-NHS enhanced the solubility of the intermediate species in comparison to NHS. Subsequent to the activation step, 2-mercaptoethnol was added to quench any excess EDC, which might otherwise react with residual carboxylic acid groups in the HA-amine. Finally, HA- amine was added at pH 7.1 . The manipulation of the molar ratio between HA-amine and VP enabled the synthesis of HA-VP with DoS ranging from 2% to 10%.

[0088] NMR spectroscopy of HA-VP. The covalent conjugation between HA and VP was confirmed by1H NMR spectroscopy. The alkene protons of VP were discernible at 5 = ~6.3 and 8.2 ppm, indicating the covalent attachment of VP to HA-amine. The alkyl protons of VP were overlapped with the predominant HA signals.

[0089] Determination of wt% and DoS of VP in the conjugate. FIG. 1 E illustrates the absorbance spectra of VP and HA-VP across the wavelength range of 300 to 800 nm. Notably, it was observed that the maximum absorbance wavelengths of VP, with the exception of the one at 698 nm, exhibited slight shifts following the conjugation with HA. Consequently, the absorbance signal at 698 nm, remaining unaffected by the conjugation process, was used to construct a calibration curve for VP.

[0090] Subsequently, this calibration curve was used to measure the weight percentage (wt%) of VP within the HA-VP conjugate. Depending on the feed molar ratio between HA and VP employed during synthesis, the wt% of VP ranged from 4% to 16%, indicative of the near- quantitative progress of the conjugation reaction. Furthermore, the measured wt% of VP in the conjugate was used to calculate the DoS, which ranged from 2% to 10%. The DoS provides insight into the relative number of repeat units within the HA strands that underwent conjugation with VP.

[0091] Resazurin cell viability assay. The cytotoxicity of the HA-VP conjugate (10% DoS) on CSSCs was evaluated using the resazurin cell viability assay. Initially, to assess the potential toxicity arising from the amine modification of HA, CSSCs were exposed to varying concentrations of HA-amine. The results demonstrated that the amine modification of HA strands did not induce any cytotoxic effects on CSSCs up to a concentration of 4 mg / mL,which is approximately equivalent to the amount of HA-amine present in the highest concentration of HA-VP tested in the following assay.

[0092] FIG. 7 illustrates the viability of CSSCs following exposure to different concentrations of HA-VP (10% DoS). Notably, the cells maintained an approximately 90% viability when treated with HA-VP concentrations up to 6 mg / mL (equivalent to 1 mg / mL VP within the conjugate). The results show that the HA conjugation significantly improved the solubility of VP, enabling the preparation of high VP concentrations in aqueous solutions. This modification eliminated the need for the initial solubilization of the highly hydrophobic VP in DMSO, which could otherwise introduce unintended cytotoxicity due to the use of the organic solvent.Example 2

[0093] Verteporfin liposomes were made with 1 ,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, 850345C), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG, 880120C), and 1 ,2- dioleoyl-3-trimethylammonium-propane (DOTAP, 890890C). The liposomes are prepared from DMPC, DSPE-PEG, and DOTAP at the molar ratio of 76.2:3.8:20 (fusogenic) or 96.2:3.8:0 (non-fusogenic). All lipids are dissolved in chloroform at 10 mg / mL. Verteporfin is dissolved in DMSO at 50 mg / mL. The lipid films were prepared by evaporating the organic solvents, with 72.55 pL of DMPC, 15.16 pL of DSPE-PEG, 19.63 pL of DOTAP, and 0.2-20 pL verteporfin. Next, the above lipid was hydrated with 1 mL PBS buffer by pipetting and stirring at 40 °C in the dark for 10 minutes. The mixtures were then extruded through (50-200 nm) polycarbonate membrane 20 times. The formed liposomes were stored at 4°C in dark.Example 3 Liposomal Admixture

[0094] Liposomal verteporfin, either commercially available or using the formulation disclosed above, is admixed with 1% high molecular weight sodium hyaluronic acid such that the verteporfin concentration in the hyaluronic acid is 1 mg / ml. As shown in FIG. 15A-C, mixing liposomal verteporfin with a hyaluronic acid gel results in sustained-release of verteporfin for more than 3 days. The admixture shows inhibitory activity at concentrations greater than 5 p.g / ml.

[0095] Shown in FIG. 15D-E, in an in vivo model, the admixture group had a faster re- epithelialization rate compared to HA alone, or liposomal verteporfin alone. Representative fluorescein staining images indicated the corneal epithelial wound closure over time, with a non-significantly faster corneal re-epithelialization in the lipoVP / HA gel-treated group compared to the other groups.

[0096] Shown in FIG. 15F-G, the lipoVP / HA admixture treatment prevented scar formation during corneal wound healing. Quantified gray values of the corneas and blindly graded corneal haze score showed that the corneas treated with lipoVP / HA gel were more transparent than those treated with the other treatments.Example 4Antifibrotic Nanoparticle for Corneal Regeneration Following Traumatic Injuries

[0097] Corneal scars often develop during the regenerative process following traumatic injuries, leading to irreversible vision impairment. Verteporfin (VP), a Yes-associated protein (YAP) inhibitor, has shown promise as an antifibrotic agent, but its application to ocular surface is limited by unfavorable physicochemical properties. Here, we develop hyaluronic acid- verteporfin nanoparticles (HA-VP NPs) to increase the solubility of VP and prolong time on the eye, in order to promote corneal regeneration and prevent scar formation following injuries. The HA-VP NPs effectively prevent the transformation of human corneal stromal cells (CSCs) into myofibroblasts by inhibiting the translocation of YAP into the nucleus. The HA-VP NPs interact with CD44 receptors, facilitating endocytosis in CSCs and contributing to prolonged residence time in corneal defects. In a rat anterior lamellar keratoplasty model, the HA-VP NPs are retained on corneal defects for at least 4 hours, resulting in excellent corneal regeneration and significant reduction in scarring. These findings demonstrate that the HA-VP NPs provide a therapeutic approach for preventing vision impairment caused by corneal scarring.

[0098] In this study, we developed self-assembling nanoparticles by conjugating VP to hyaluronic acid (HA-VP NPs) aimed at promoting corneal regeneration. Hyaluronic acid (HA) was selected as a structural backbone for its excellent corneal wound healing efficacy, bioadhesiveness, and biocompatibility, making it an ideal candidate for ocular applications. The HA-VP NPs were designed to not only improve VP’s solubility and ocular residence but also to facilitate the wound healing processes. The HA-VP NPs were administered topically to eyes with corneal defects, to provide a patient-friendly treatment method. We assessed the physicochemical properties and antifibrotic effects of the HA-VP NPs in both in vitro and in vivo models, comparing their efficacy with that of the liposome-based formulation (Visudyne®).Results

[0099] Synthesis and characterization of HA-VP NPs. The HA-VP conjugate was prepared using the / V-(3-dimethylaminopropyl)- / V-ethylcarbodiimide (EDC) coupling reagent in conjunction with amine-modified HA, as shown in FIG. 1 b. First, the carboxylic acid group inVP was activated with EDC and / hydroxy-sulfosuccinimide (sulfo-NHS) at pH 5.1 . The incorporation of sulfo-NHS served to stabilize the O-acylisourea intermediate by forming an ester, thereby preventing the undesirable formation of A / -acylurea by-products. Importantly, the use of sulfo-NHS enhanced the solubility of the intermediate species in comparison to NHS. After the activation step, 2-mercaptoethnol was added to quench any excess EDC, which might otherwise react with residual carboxylic acid groups in the HA-NH2. Finally, HA- NH2 was added at pH 7.1 . By manipulating the molar ratio between HA- NH2 and VP (Table S1 ), we synthesized the HA-VP conjugates with different degrees of substitution (DoS): (i) the HA-VPiow with 2.2 ± 0.1% DoS and 17.8 ± 0.1 mg / g drug content, and (ii) the VPhigh with 10.4 ± 0.8% DoS and 83.0 ± 4.2 mg / g drug content.

[0100] The conjugation of VP to HA-NH2was confirmed using various spectroscopic techniques. First, as shown in the nuclear magnetic resonance (NMR) spectra (FIG. 1c), HA- NH2lacked signals above 7 ppm; however, new peaks appeared after conjugation, indicating the presence of attached VP. In the Fourier-transform infrared (FTIR) spectra (FIG. 1 d), VP showed a unique absorption peak at approximately 1211 cm1, likely originating from ester (C- O stretching). This peak was present in the HA-VPhigh but absent in the HA-NH2, confirming the successful conjugation of VP to the HA-NH2. The HA-VP conjugates also exhibited unique peaks from HA-NH2, including 1639 cm1(N-H bending), 1377 cm1(C-H bending), and 1041 cm1(C-N stretching). The HA-VP conjugation was also corroborated by absorbance spectroscopy (FIG. 1 e). The VP solution displayed characteristic peaks at 356, 434, 576, and 688 nm, while the HA-NH2solution exhibited negligible absorption peaks within this range. Compared to the VP solution, the HA-VP conjugates generally exhibited broader peaks, with absorbances proportional to the conjugate concentrations.

[0101] The physical structure of the HA-VP conjugate was observed using transmission electron microscopy (TEM) (FIG. 1 f). Due to the amphiphilic nature of the HA-VP conjugates, they self-assembled into sphere-shaped, nano-sized particles (HA-VP NPs). In this text, we refer to the material interchangeably as HA-VP conjugate and HA-VP NPs.

[0102] The biodegradability of the HA-VP NPs was evaluated using hyaluronidase over 4 days (FIG. 1g). In the absence of the enzyme, the HA-VP NPs exhibited minimal structural degradation (2.2 ± 2.2%). However, the degradation rate increased with higher enzyme concentrations (6.5 ± 5.2% at 5 unit / mL 7.6 ± 8.0% at 10 unit / mL; 10.9 ± 7.4% at 20 unit / mL; 13.7 ± 1 1.0% at 50 unit / mL on day 4).

[0103] To assess the impact of the different DoS on corneal residence time, we utilized an in vivo fluorescence imaging technique in a rat lamellar keratectomy (ALK) model (FIG. 1 h and 1 i). After performing ALK to remove half of the corneal thickness, the HA-VP|OWor HA-VPhigh was topically applied to the defected eyes. For this test, the surfaces of these conjugates werefunctionalized with fluorescent dyes (Alexa Fluor 647) to enable visualization upon excitation. Notably, most of the HA-VP|OWwas not retained on the eyes 1.5 h after administration. In contrast, the HA-VPhigh-treated eyes exhibited strong fluorescent signals in the center of the eyes, where the corneal defects were located. The signals persisted for 3 h, indicating superior corneal residence by the HA-VPhigh. To compare the signal quantitatively, the relative fluorescence intensity in the eyes was significantly higher in the HA-VPhigh (2.54-fold) than in the HA-VPiow. Consequently, the HA-VPhigh was selected for further evaluation.

[0104] Inhibitory effect of the HA-VP NPs on YAP localization in CSCs. Human corneal stromal cells (CSCs) can differentiate into myofibroblasts when the cornea is injured, leading to the secretion of irregular ECM proteins and the formation of corneal scars. YAP, a transcription coactivator, plays a key role in this transformation. To assess the pharmacological effects of the HA-VP NPs on YAP, we examined YAP localization in CSCs (FIG. 2a). In this study, TGF-[31 was added to all groups to induce CSC differentiation into myofibroblasts and mimic injured conditions. In the no treatment group, YAP intensities were highly concentrated in the nucleus, with a nucleus-to-cytosol intensity ratio (NC ratio) of 4.16 ± 1.71 (FIG. 2b). This localization did not change with HA-NH2 treatment, whereas the VP solution and Visudyne® (VP-loaded liposomal formulation) groups showed reduced nuclear intensity and increased cytosolic intensity, with NC ratios of 1 .44 ± 0.29 and 2.09 ± 0.63, respectively. Most importantly, the HA-VP NPs group had an NC ratio of 1.17 ± 0.20, the lowest among the groups containing VP, indicating its superior inhibitory effect on YAP activation.

[0105] Inhibitory effect of the HA-VP NPs on a-SMA expression in CSCs. In addition to YAP localization, we investigated the expression of alpha smooth muscle actin (a-SMA) to evaluate the transformation of CSCs into myofibroblasts (FIG. 2c). When TGF-|31 was added, CSCs expressed a-SMA, and HA-NH2alone did not alter this expression pattern. However, treatment with VP-containing formulations (VP solution, Visudyne®, and HA-VP NPs) significantly reduced a-SMA expression. These groups also showed clear VP signals in the cytosol.

[0106] The pharmacological activity of the HA-VP NPs was further analyzed by quantifying the expression of the ACTA2 gene, which encodes a-SMA, using reverse transcription quantitative polymerase chain reaction (RT-qPCR) (FIG. 2d). The HA-VP NPs group exhibited concentration-dependent inhibitory effects against ACTA2, with an IC50 value of 2.90 ± 0.20 pg / ml. This value was not significantly different from that of Visudyne® (2.81 ± 0.21 pg / mL). Additionally, we observed negligible cytotoxicity of the HA-VP NPs up to 1000 pg / mL.

[0107] Cellular uptake of the HA-VP NPs. Hyaluronic acid acts as a ligand for the CD44 receptor on the cell membrane, facilitating endocytosis. This receptor is highly expressed when quiescent stromal cells transform into fibroblasts. To verify the cellular uptake of the HA-VP NPs by CSCs, we monitored the process using confocal microscopy (FIG. 3a). After treatment with the HA-VP NPs, VP intensity in CSCs increased in a time-dependent manner. Notably, sphere-shaped aggregates were observed around the cell membrane (yellow arrows). To track the movement of these aggregates, we captured images every minute from 41 to 55 min (FIG. 3b). The aggregates eventually disappeared as the cellular VP intensity increased, with the intensity at 55 min being 2.60-fold higher than at 41 min.

[0108] In vivo corneal residence of the HA-VP NPs in rats. Prolonged residence time of drugs on the ocular surface is critical for improving efficacy, considering the inherently low bioavailability of eye drop formulations (~ 5%). To investigate the residence of the HA-VP NPs in corneal defects, we applied the Alexa Fluor 647-labeled HA-VP NPs to the artificially created corneal defects in rats (FIG. 4a). The fluorescent dye (Alexa Fluor 647) was covalently attached to the surface of the HA-VP NPs for visualization purposes. In this study, Visudyne®, an FDA-approved liposomal formulation of VP, was used for comparison. Immediately after administration (0 h), most of the Visudyne® was not retained on the cornea, and no significant signal was observed on the corneal defects. In contrast, the HA-VP NPs group exhibited a strong signal on the corneal defect, and the intensity remained for 4 h. The intensity was quantitatively compared using the eye-to-background intensity ratio (EBR) (FIG. 4b). The EBR values of Visudyne® were calculated to be 1.52 ± 0.32 at 0.5 h, decreasing to 1.07 ± 0.12 at 1 h, 0.98 ± 0.18 at 2 h, and 0.96 ± 0.10 at 4 h. Meanwhile, the HA-VP NPs exhibited significantly higher EBR values than Visudyne® (17.63 ± 4.23 at 0.5 h; 8.50 ± 3.81 at 1 h; 7.18 ± 3.01 at 2 h; and 5.34 ± 2.32 at 4 h). To further compare ocular residence, the treated eyes were enucleated and imaged separately at 4 h (FIG. 4c and 4d). Strong fluorescence was clearly observed in the HA-VP NPs-treated eyes, and their EBR values (3.52 ± 0.80) were significantly higher than those of Visudyne® (1 .00 ± 0.17).

[0109] In vivo corneal regeneration and antifibrotic effects in rats. The therapeutic antiscarring efficacy of the HA-VP NPs was evaluated using a rat ALK model, in which an artificial corneal defect with a 2.5-mm diameter and a 40-50% depth was created (FIG. 5a). Slit-lamp and fluorescein-stained images were obtained on days 0 ( / .e., after ALK), 1 , 2, 3, 7, and 14 to monitor the wound healing process, while optical coherence tomography (OCT) images were obtained less frequently on days 0, 7, and 14 to minimize NIR exposure from the instrument to the light-sensitive HA-VP NPs. The concentration of treatment solutions (1 mg / mL as VP, much higher than the IC50 values) was selected considering the inherently low bioavailability of eye drop solutions. We could not include a VP solution group for comparison due to its very poor water solubility. The treatment solutions were applied to the defected eyes once a day until day 3.

[0110] FIG. 5b shows the OCT images used to monitor the topographic changes in the defected corneas. The sham group (treated with PBS) exhibited cone-shaped thick corneas in half of the rats on day 14 (FIG. S4), which was not typically observed in normal corneas (FIG. S5). In contrast, the HA- VP NPs-treated eyes fully restored corneal thickness by day 14, with no abnormal shapes observed in any of the rats. The corneal opacity was evaluated with slit-lamp images (FIG. 5c and 5d). In the sham group, the corneas began to become opaque two days after the defect formation, and distinct scars were observed on day 14. Similarly, the HA solution group also did not show significant improvement. Although VP-containing Visudyne® eye drops showed a trend towards scar reduction at day 2, the reduction was not significant. Meanwhile, the HA-VP NPs treatment resulted in fewer scars throughout the evaluation period compared to other groups, and a significantly smaller scar area was observed on day 14. Additionally, the HA-VP treatment tended to alleviate the corneal neovascularization and inflammation on day 14.

[0111] As an indicator of the degree of corneal scar formation, changes in corneal reflectivity were calculated by measuring the epithelial-to-stromal reflectivity ratio (E:S ratio) (FIG. 5e). The E:S ratios for the sham and HA groups were 0.90 ± 0.02 and 0.92 ± 0.06 on day 14, respectively, both significantly lower than that of the normal, un-defected cornea (1 .30 ± 0.10). The Visudyne® group (0.91 ± 0.14) also showed no significant differences from the sham group. Notably, the HA-VP NPs group showed a higher value (1 .15 ± 0.07) than the other groups, which was not significantly different from that of the normal cornea. Re- epithelialization was also evaluated by staining the corneas with fluorescein (FIG. 5f and 5g). By day 7, corneas in all groups were no longer stained with fluorescein, with no significant differences between groups.

[0112] Immunohistochemistry of regenerated corneas. The eyes were enucleated on day 14 and sliced for immunohistochemical analysis. We first analyzed aldehyde dehydrogenase 3A1 (ALDH3A1 ), an enzyme contributing to transparency in corneal transparency (FIG. 6a and 6b). In the HA solution group, ALDH3A1 expression was polarized in the superficial cells of epithelium. In contrast, the Visudyne® group exhibited expression across the entire epithelial layers, but the overall intensity was not significantly different from the sham and HA groups. Notably, the HA-VP NPs group exhibited significantly stronger intensities throughout the entire epithelium layer than other groups.

[0113] We also investigated a-SMA expression to evaluate myofibroblast formation (FIG. 6c and 6d). While the sham and HA groups exhibited strong intensities in the anterior stromal layers, the HA-VP NPs group displayed significantly lower intensities. The Visudyne® group also tended to show lower intensity, though not significantly.

[0114] We also measured the expression of CK12, a cornea-specific epithelial marker, to evaluate the epithelial regeneration (FIG. 6e and 6f). While the sham group displayed an irregular and low expression pattern, the Visudyne® group showed higher intensity than the sham group, although it was polarized in the superficial layers. The HA and HA-VP NPs groups exhibited significantly higher and more homogeneous intensities compared to both the Visudyne® and sham groups.

[0115] Our research aimed to develop the HA-VP NPs to increase the apparent water solubility of VP and its residence time on corneal defects. In vitro cell studies employed VP solution and Visudyne® as control groups since the concentration used was low enough to be within the solubility limits of VP. However, due to solubility issues, the VP solution group was not feasible for use in in vivo animal experiments.

[0116] Visudyne®, the liposomal formulation of VP, was originally used in photodynamic therapy to treat age-related macular degeneration by inhibiting choroidal neovascularization. This drug is administered via intravenous injection, and to the best of our knowledge, no research has explored its topical application ( / .e., eye drops) for preventing corneal scarring. In this study, we primarily focused on evaluating the efficacy of the HA-VP NPs, while also exploring the potential of this liposomal formulation as an eye drop treatment.

[0117] The HA-VP conjugates are amphiphilic polymers consisting of hydrophilic HA and hydrophobic VP. This amphiphilicity drives the self-assembly of the polymers into sphereshaped nanoparticles. Nanoparticles can increase the solubility and stability of hydrophobic molecules, and their hydrophilic surface facilitates interactions between the nanoparticles and biomolecules. In the absorbance spectra, the HA-VP NPs exhibited a spectrum similar to that of the VP solution, although their peaks are as sharp. Anchoring VP to the polymer structure appears to influence light absorption. Despite the broader peaks, the absorbance intensities are proportional to the HA-VP NP concentration, suggesting successful conjugation of VP to HA structures.

[0118] Myofibroblasts lead to irregular corneal remodeling and haze, induced by TGF-|31 secreted from injured and immune cells. During corneal injuries, fibroblasts stimulated by TGF- |31 undergo YAP translocation into the nucleus, upregulating multiple genes related to differentiation, with a-SMA serving as a biomarker for myofibroblast formation. In cellular studies, the VP solution significantly inhibited YAP translocation and a-SMA expression, confirming its pharmacological activities in CSCs. Notably, both the Visudyne® and HA-VP NPs exhibited similar inhibitory activities to the VP solution, with the HA-VP NPs being more effective than Visudyne®. This suggests that the therapeutic efficacy of VP is well preserved after conjugation, with increased water solubility.

[0119] The VP solution, Visudyne®, and HA-VP NPs have distinct mechanisms of cellular uptake. VP solution can diffuse through cell membranes, while Visudyne® (liposomes) may require cellular engulfment and incorporation into vesicles. Another possible mechanism for Visudyne® could involve VP release from the liposome followed by diffusion into cells; however, this possibility is unlikely given the negligible VP release observed. The HA-VP NPs can be internalized by CD44 receptor-mediated endocytosis. The sphere-shaped aggregates observed in FIG. 3 may result from interactions between surface receptors (CD44) and NPs. Following endocytosis, the HA-VP NPs would be biodegraded by intracellular enzymes, releasing free VP molecules to enable their pharmacological effects. Collectively, the cellular studies indicate that the HA-VP NPs are sufficiently biodegraded to show efficacy comparable to VP solution, and they are more effective than the liposome formulation, Visudyne®.

[0120] Prolonged residence time of VP in corneal defects is crucial for enhanced bioavailability and therapeutic outcomes. We hypothesized that the hyaluronic acid-based NPs would enhance residence on corneal defects through interactions with surface CD44 receptors on fibroblasts and through their innate bioadhesiveness derived from hyaluronic acid. As expected, the HA-VP NPs exhibited prolonged corneal residence, remaining on the defects for at least 4 h. The in vivo and ex vivo fluorescence images indicate that the HA-VP NPs are more effective in corneal residence than Visudyne®. For the in vivo imaging, we functionalized the HA-VP NPs with a fluorescent dye because the intrinsic fluorescence of VP markedly decreased after conjugation with HA. Although the dye’s excitation / emission wavelengths differ from those of Visudyne® ( / .e., the intrinsic fluorescence of VP), their kinetics can be compared by calculating the EBR.

[0121] For in vivo evaluation of therapeutic efficacy, we used a rat ALK model mimicking traumatically injured corneas. In OCT images, all rats exhibited mild-to-moderate inflammation after the corneal defect formation, leading to corneal swelling and edema for 7 days (FIG. 5b). The sudden increase in haze areas in the HA-VP NPs group during this timeframe might be attributed to these inflammatory symptoms (FIG. 5d). By day 14, the sham group showed relieved symptoms, but two rats displayed abnormal stromal regeneration, resulting in cone- shaped corneas. Additionally, inflammation and swelling were not fully resolved in one sham rat. The corneal neovascularization observed in the corneas on day 14 may be attributed to severe inflammation. Similarly, one rat in the Visudyne® group showed uncontrollable inflammation and swelling on day 14. Interestingly, these symptoms were absent in the HA and HA-VP NPs groups, possibly due to the anti-inflammatory and wound healing effects of hyaluronic acid. It is noteworthy that while corneal structures regenerated in both the HA solution and HA-VP NPs groups, the HA solution did not reduce haze areas. Only the HA-VPNPs significantly improved corneal opacity, showing their anti-scarring effects derived from VP.

[0122] Immunohistochemical analysis confirmed the anti-scarring and regenerative effects of the HA-VP NPs. The sham group exhibited high a-SMA expression in the stroma and low ALDH3A1 expression in the epithelium, indicating increased corneal scar formation and reduced transparency. In addition, low expression of CK12 suggests incomplete epithelial healing in the sham group. HA solution eye drops improved epithelial regeneration, likely due to the corneal wound healing effects of HA molecules. However, they did not prevent corneal scar formation. Visudyne® eye drops tended to reduce myofibroblast transformation, but their anti-scarring effect was not significantly greater than that of the sham and HA groups, as shown in the OCT and slit-lamp images in FIG. 5. Thus, Visudyne® eye drops are not effective in preventing corneal scar formation. In contrast, the HA-VP NPs significantly decreased a- SMA expression and increased ALDH3A1 and CK12 expression, confirming their anti-scarring effects after corneal injury.

[0123] We developed verteporfin-conjugated hyaluronic acid nanoparticles (HA-VP NPs) as a therapeutic for scarless corneal regeneration following traumatic injuries. The amphiphilic HA-VP conjugates were biodegradable and self-assembled into nanoparticles, significantly increasing the water solubility of VP and enabling its use in vivo. The HA-VP NPs effectively prevented stromal keratocytes from transforming into myofibroblasts by decreasing YAP localization to the nucleus. In a rat ALK model, the HA-VP NPs exhibited significantly longer residence time on corneal defects compared to Visudyne®, leading to improved stromal remodeling and transparency. These findings show the utility of HA-VP NPs as a therapeutic approach for achieving scarless corneal regeneration.Materials and Methods

[0124] Materials. HA-NH2 (molecular weight (MW) 250k; DoS 45%) was obtained from Creative PEGWorks (Durham, NC, USA). VP, EDC, sulfo-NHS, 2-mercaptoethanol, and hyaluronidase were obtained from Sigma-Aldrich (Burlington, MA, United States). Spectra / Por 3 RC dialysis membrane tubing with a molecular weight cut-off (MWCO) of 3.5 kDa was obtained from Spectrum. Alpha modification of minimum essential medium (MEM) (1X) with Earle's salts, without ribonucleosides, deoxyribonucleosides, and L-glutamine, was purchased from Corning (NY, USA). GlutaMAX supplement (100X) and MEM non-essential amino acids (NEAA) Solution (100X) were acquired from Gibco (Waltham, MA, USA). The resazurin assay solution was purchased from Abeam (Cambridge, UK). Milli-Q type 1 ultrapure water was used for the preparation of all aqueous solutions. Syringe filters with a pore size of 0.2 pm were used for the filtration of all aqueous and cell media solutions.

[0125] Synthesis of the HA-VP NPs. Table 2 lists the reagents used to prepare the HA- VP at approximately 10% DoS. The molar ratio between VP and HA-amine was adjusted accordingly to prepare the HA-VP variant with approximately 2% DoS. All reactions were conducted under light-protected conditions. Initially, VP (5.0 mg, 7.0 pmol, 1 eq.) was dissolved in 0.5 mL of dimethyl sulfoxide (DMSO) in a 20 mL amber glass vial and stirred for 10 min. Subsequently, 0.1 M of 2-(N-morpholino)ethanesulfonic acid buffer (4.5 mL; pH 5.1 ) was added, and the mixture was stirred for an additional 10 min. The addition of sulfo-NHS (30.2 mg, 139.1 pmol, 20 eq.) was followed, and the mixture was stirred for 10 min. EDC (13.3 mg, 69.6 pmol, 10 eq.) was subsequently added, and the reaction mixture was stirred for 2 h, after which 2- mercaptoethanol (97.6 pL, 1391.2 pmol, 200 eq.) was introduced and stirred for 15 min. The pH was adjusted to 7.1 by adding 1 M K2PO4(1 mL; pH 9.7). The addition of HA-NH2(26.4 mg, 34.8 pmol, 5 eq.) was followed, and the mixture was stirred overnight. The resulting reaction mixture was dialyzed against ultrapure water using dialysis membrane tubing with a 3.5 kDa MWCO for a duration of 3-5 days. Finally, the dialyzed mixture was lyophilized and stored at -20 °C until further use.Table 2.Reagents used in the preparation of HA-VPhigh NPs# mol mass volume component eq. MW(pmol) (mg) (pL)VP 1 718.79 7.0 5.0 sulfo-NHS 20 217.13 139.1 30.2EDC 10 191 .7 69.6 13.32-mercaptoethanol 200 78.13 1391.2 97.6HA-NH2(45% DoS) 5 250000 34.8 26.4Table 3.DoS and drug content of HA-VPhigh and HA-VP|OW(n > 3).The values were calculated by measuring the absorbance of (i) VP and (ii) primary amines of HA-NH2using the Primary amine kit (ProFoldin, MA, US).HA-VPiow HA-VPhighDoS (%)* 2.2 ± 0.1 10.4 ± 0.8Content (mg / g)** 17.8 ± 0.1 83.0 ± 4.2* DoS (%) = (mole of conjugated VP / mole of primary amine of HA-NH2) x 100 ** Content (mg / g) = (weight of conjugated VP) / (weight of HA-VP)

[0126] Characterization of the HA-VP NPs. The HA- VP conjugates were analyzed using FTIR spectroscopy with a range of 400 - 4000 cm1, and the absorbance spectra of the HA-VP NPs were measured from 200 to 1000 nm. To investigate concentration-dependent absorbance patterns, four different concentrations of the HA-VP NPs were prepared. The VP solution group was prepared by dissolving VP powder in DMSO. Absorbance was measured using a microplate reader (TECAN, Mannedorf, Switzerland).1H NMR spectra were recorded on a Bruker AVANCE NEO 500 MHz spectrometer. The chemical shifts (0) were reported relative to the residual DMSO signal, which was observed at 0 = 2.50 ppm. The shape of the HA-VP NPs was observed using TEM. Samples were negatively stained with uranyl acetate before imaging. Biodegradability was evaluated by incubating the HA-VP NPs with hyaluronidase (0, 5, 10, 20, and 50 unit / mL). The mixtures of NPs and hyaluronidase were placed into dialysis bags (MWCO: 3.5 kDa), which were then submerged into 0.1% Triton X-100 solution in PBS. The release medium was collected at 7, 24, 48, 72, and 96 h. The collected samples were analyzed using the microplate reader.

[0127] Cell culture. Primary human CSCs were harvested from human donor corneas provided by Lions Eye Institute as previously described. The harvested CSCs were positive for CD74 and CD90, and negative for CD45. The cells were cultured in MEM, alpha 1 X containing 10% fetal bovine serum, 1 % penicillin-streptomycin solution, 1% non-essential amino acid solution, and 1 % Glutamax. The cells were incubated at 37SC in a 5% CO2atmosphere, and the proliferated cells were passaged at 75% confluency using trypsin-EDTA solution.

[0128] YAP localization and a-SMA expression in CSCs. To investigate the intracellular localization of YAP when treated with the HA-VP NPs, CSCs (3 x 105cells) were seeded on cell culture dishes (density: 1 x 103cells / mm2) and incubated for one day. Then, the cells were incubated with fresh media containing recombinant human TGF-p1 protein (10 ng / mL; Abeam, Cambridge, UK) and the HA-VP NPs (10 pg / mL, based on VP concentration) for 24 h. Three different control groups were prepared as follows: (i) liposome-encapsulated VP (Visudyne®; Bausch and Lomb, Laval, Canada), (ii) VP solution in 1 % DMSO, and (iii) HA-NH2(equivalent to the amount of HA-NH2in the HA-VP conjugate). After treatment, the cells were washed twice with PBS and fixed with 4% paraformaldehyde solution for 15 min. The fixed cells were then washed, and their membranes were permeabilized using a permeabilizing solution (0.5% Triton X-100 and 5% goat serum) for 30 min at room temperature. For YAP localization analysis, intracellular YAPs were stained with Alexa Fluor 488-conjugated anti-YAP (1 :200; Cell Signaling Technology, Danvers, Massachusetts, US) for 2 h at room temperature. For a- SMA expression analysis, the cells were stained using Alexa Fluor 488-conjugated anti-a- SMA (1 :500; Abeam). The stained cells were washed two times and further stained with 4', 6-diamidino-2-phenylindole (DAPI; 1 :1000, Thermo Fisher Scientific, Waltham, MA, US). After two additional washes, the cells were analyzed using a confocal microscope (Carl Zeiss, Oberkochen, Germany).

[0129] RT-qPCR. CSCs (1 x105cells) were seeded on 12-well cell culture plates and incubated for one day. The cells were then incubated with fresh media containing TGF- 1 (10 ng / mL) and VP formulations (Visudyne® or HA- P) at various concentrations (2, 10, 20, 100, and 200 pg / mL, based on VP concentration). Control groups included a normal control (without TGF- 1 or any formulations) and a no-treatment control (represented as 0 pg / mL; with TGF-pi but without formulations). After incubation, RNAs were extracted from the treated cells using Trizol and chloroform. The extracted RNAs were purified using RNeasy Plus Mini Kit (Qiagen, Germantown, MD, US). RNA concentrations were measured using a Nanodrop One (Thermo Scientific). After adjusting the RNA concentrations to the same level, the RNAs were transcribed to cDNAs using iScript™ Reverse Transcription Supermix for RT-qPCR (Biorad, Hercules, CA, US). Then, TaqMan gene expression master mix (Bio-Rad) and primers for ACTA2 (Thermo Scientific) were added to the cDNAs. RT-qPCR was performed on an Applied Biosystems QuantStudio 7 Flex Real-Time PCR System with 40 amplification cycles. ACTA2 expression levels were compared to the normal-negative control (i.e. TGF- 1 - untreated cells), and GAPDH was used to normalize endogenous RNA expression within the samples. Comparative fold changes in gene expression levels were analyzed by calculating the 2AACt.

[0130] Animal model. Brown rats (Rattus norvegicus; 150 - 200 g) provided by Charles River Laboratories (San Francisco, CA, US) were used for in vivo experiments. The animal experiments were designed to conform to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were reviewed and approved by the Stanford University Institutional Animal Care and Use Committee (protocol #: APLAC-32765). To perform ALK, a mixture of ketamine (100 mg / mL), xylazine (20 mg / mL), and water (2:1 :3, v / v / v) was administered via intramuscular injection at a dose of 1.68 mL / kg. For follow-up studies, rats received a half-dose of the mixture. During all surgical and follow-up studies, proparacaine hydrochloride ophthalmic solution (0.5%; Bausch and Lomb) was applied to the eyes prior to examination. Corneal defects were created by performing ALK on one eye of each rat using a 2.5-mm trephine to make a deep circular cut (40-60% depth of the corneas) and a spatula to remove the collagen layers of the stroma.

[0131] In vivo fluorescence imaging for corneal residence assessment. To compare corneal residence between the HA-VP|OWand HA-VPhigh, the conjugates were labeled with Alexa Fluor 647 NHS ester (Lumiprobe; Maryland, USA). The fluorophore-labeled conjugates were topically administered to the defected eyes of the rats. Fluorescent signals (Ex / Em = 640 / 689nm) were detected using an Optical Imaging Lago System (Spectral Instruments Imaging, AZ, US). The comparison between the Alexa 647-labeled HA-VP NPs and Visudyne® was conducted under the same experimental conditions, although Visudyne® was not labeled with fluorescent dyes. The intrinsic fluorescence of Visudyne® was detected at different wavelengths (Ex / Em = 675 / 730 nm). EBR was calculated by comparing the intensity of the eye to that of the body. The treated eyes were enucleated at 4 h, and their intensities were analyzed to confirm ocular residence.

[0132] In vivo evaluation of corneal scarring. Corneal defects were assessed using OCT (Heidelberg Engineering, Heidelberg, Germany) and digital photography. To evaluate stromal exposure, fluorescein solution (1 .5%; one droplet) was applied to the eyes, followed by gentle rinsing with balanced salt solution (BSS). Then, the fluorescein-stained areas were visualized under blue light, and the percentage of the stained area was calculated using Imaged software. As part of post-operative care, ofloxacin ophthalmic solution (0.3%, Bausch and Lomb) was administered to prevent bacterial infections. To compare therapeutic efficacy, four groups were prepared: sham, HA, Visudyne®, and HA-VP NPs, with the VP concentration set at 1 mg / mL. The sham and HA groups were treated with PBS and HA-NH2 solution (equivalent to the amount of HA in the HA-VP), respectively. The treatment solutions (10 pL) were applied to the defected eyes. Slit-lamp and fluorescein-stained images of the defects were captured on days 0, 1 , 2, 3, 7, and 14. Meanwhile, OCT images were taken on days 0 (before treatment), 7, and 14 to minimize light exposure of VP from the instrument. The rats were euthanized on day 14, and their eyes were enucleated and cryo-sectioned for immunohistochemical analysis. The thickness of the epithelial layers and entire corneas on day 14 was measured using confocal microscopy.

[0133] Immunohistochemical analysis. Expression of corneal biomarkers was evaluated in regenerated corneas harvested on day 14. The cryo-sectioned corneal slices were stained with one of three markers: Alexa Fluor 488-conjugated anti-a-SMA (1 :500, Abeam), anti- ALDH3A1 (1 :500, Abeam), or Alexa Fluor 488-conjugated anti-CK-12 (1 :100, Abeam). The stained slices were also counterstained with DAPI (1 :1000, Thermo Fisher Scientific) and Alexa Fluor 555-conjugated anti-phalloidin (1 :400, Thermo Fisher Scientific). For slices stained with anti-ALDH3A1 , Alexa Fluor 488-conjugated anti-rabbit IgG (1 :200, Life Technologies Corporation) was used as a secondary antibody. Fluorescent signals were detected using a confocal microscope (Zeiss, Jena, Germany).

[0134] Statistical analysis. All statistical analyses were conducted using GraphPad Prism software (version 8.0.2.263). One-way ANOVA followed by Tukey’s post-hoc analysis was used for multiple comparisons. For pairwise comparisons, a two-tailed unpaired t-test wasused to determine significance between groups. The significance threshold was set at p < 0.05; otherwise, differences were labeled as “not significant (n.s.).”References

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Claims

What is claimed is:

1. A water soluble conjugate of verteporfin and a pharmaceutically acceptable biopolymer.

2. The conjugate of claim 1 , with a structure (I):where n is from about 0.1 to about 0.25, and n+m=1 .

3. A pharmaceutical formulation of the conjugate of claim 1 or claim 2.

4. The formulation of claim 3, in a unit dose.

5. The formulation of claim 3 or claim 4, in a formulation suitable for topical administration.

6. The formulation of any of claims 3-5, where the formulation is free of liposomes.

7. An admixture formulation comprising dose of from about 0.5% to 2.0% liposomal verteporfin and from about 5% to 15% high molecular weight hyaluronic acid gel.

8. The admixture of claim 7, wherein the liposomal verteporfin comprises one or more of 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE- PEG), and 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

9. The admixture of claim 7 or claim 8, wherein the liposomal verteporfin comprises DMPC, DSPE-PEG, and DOTAP at a ratio of about 76.2:3.8:20.

10. The admixture of claim 7 or claim 8, wherein the liposomal verteporfin comprises DMP and DSPE-PEG at a ratio of about 96.2:3.8.1 1 . A method of treating an individual, comprising topical administration of a formulation of any of claims 3-10 to a body surface.

12. The method of claim 1 1 , wherein the body surface is an ocular surface.

13. The method of claim 1 1 , wherein the body surface is skin.

14. The method of any of claims 1 1 -13, wherein the body surface is exposed to a dose of light sufficient to induce verteporfin cross-linking.

15. A method of treating fibrosis or reducing risk of developing fibrosis or scarring in an eye of a subject after an ocular injury or ocular surgery, the method comprising administering a therapeutically effective amount of the formulation of any of claims 3-10 to the subject.

16. The method of claim 15, wherein each administration of provides a dose ranging from 0.001 mg to 10 mg of verteporfin equivalent.

17. The method of claim 15 or 16, wherein the formulation is administered topically on the cornea or locally under the conjunctiva.

18. The method of any one of claims 15-17, wherein the formulation is administered before, during, or after an ocular surgical procedure that increases the risk of developing fibrosis in ocular tissues.

19. The method of any one of claims 15-18, wherein administration of the verteporfin is performed in conjunction with bleb needling or other manipulation of the conjunctiva, performed in the office or in the operating room.

20. The method of any one of claims 15-19, wherein the formulation is administered after an ocular injury that increases the risk of developing fibrosis or scarring of ocular tissues.21 . A method of treating fibrosis in ocular tissues, the method comprising: identifying a subject who has fibrosis in ocular tissues or is at risk of developing fibrosis or scarring in ocular tissues after a previous ocular surgery or ocular injury; administering to the subject a first dose of a formulation of any of claims 3-10; and administering to the subject subsequent doses no more frequently than biweekly, wherein up to five doses of verteporfin are administered to the subject.

22. A method of reducing corneal scarring in an eye of a subject after a corneal injury, the method comprising administering a formulation according to any of claims 3-10 to the subject.

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