WO2020128616A2 - Methods and compositions for controlling cell migration and proliferation - Google Patents

Abstract

Compositions and methods of treating epithelial damages with an effective amount of a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants thereof are provided. The presently disclosed subject matter provides compositions and methods for treating epithelial damages and accelerating the healing of a damaged eye or skin by activating a P2X7 purinergic receptor.

Description

METHODS AND COMPOSITIONS FOR CONTROLLING CELL MIGRATION

AND PROLIFERATION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119 to U.S. Application Serial No. 62/781,362, filed December 18, 2018, which is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The presently disclosed subject matter relates to methods and compositions for treating epithelial damages. Specifically, the presently disclosed subject matter relates to methods and compositions for controlling cell migration and proliferation.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing (submitted electronically as a .txt file named“085089.0110_Sequence_Listing_12.18.2019_ST25” on December 18, 2019). The 085089.0110_Sequence_Listing_12.18.2019_ST25.txt file was generated on December 18, 2019, and is 605 bytes in size. The entire contents of the Sequence Listing are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Thymosin Tb4 (Tb4) has biological effects in restoring tissue function and wound healing. See Yu et al., Cell Motil Cytoskeleton 1994 27: 13-25; Conte et al., Ann N Y Acad Sci 2012 1269:69-73; Bao et al., Front Pharmacol 2013 4: 149; Yuan et al., Invest Ophthalmol Vis Sci 2010 51 : 2424-2435; and Zhu et al., Int J Mol Med 2014 34: 816- 821. For example, exogenous Tb4 can support angiogenesis and hair follicle

development. See Philp et al., Mech Ageing Dev 2004 125: 113-115. Tb4 can also promote corneal wound repair by regulating re-epithelialization after injuries and reduces the inflammatory response by suppression of NF-kB activation. See Sosne et al., Invest Ophthalmol Vis Sci 2005 46:2388-2395; Sosne et al., Exp Eye Res 2002 74: 293-299; and Sosne et al., Exp Eye Res 2007 84: 663-669. Moreover, Tb4 can inhibit cell apoptosis. See Sosne et al., Invest Ophthalmol Vis Sci 2004; 45: 1095-1100. Due to such therapeutic benefits, Tb4 has been used in various clinical trials of eye disease as well as skin disease. Although the biological effects of Tb4 on eye and skin disease have been observed, these effects are focused on a macro level, for example, upon review of any impact on targeted tissue. A further understanding of the effects of Tb4 on a cellular level is desired to extend possible therapeutic effects to potential diseases.

Accordingly, there is an ongoing need for further findings on the cellular mechanism of Tb4 and methods for controlling cellular behaviors. Described herein are such methods using compositions containing Tb4.

SUMMARY OF THE INVENTION

The presently disclosed subject matter provides a method of accelerating the healing of a damaged eye or skin in a subject by activating a P2X7 purinergic receptor of a cell of an affected eye or skin of the subject. The P2X7 purinergic receptor can be activated by contacting the cell with a composition containing a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants ( e.g ., a conservative variant) thereof. The disclosed method can further include increasing a calcium influx in the cell by about two-fold and/or increasing the expression of phosphorylated ERK protein of the cell by at least about 15%. In non-limiting embodiments, the disclosed method can further include delivering a P2X7 purinergic receptor activator, a phosphorylated ERK protein activator, a calcium enhancer, or combinations thereof for synergistically improving a cell migration or a cell

proliferation. Cell migration can increase by at least 30% and cell proliferation can increase by at least about 20%.

The presently disclosed subject matter also provides a method of treating epithelial damages by delivering a peptide agent including amino acid sequence

LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g., a

conservative variant) thereof to a damaged eye or skin of the subject; and delivering a P2X7 purinergic receptor activator to the eye or skin of the subject. In non-limiting embodiments, the P2X7 purinergic receptor activator can be delivered simultaneously with the peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof. In some embodiments, the P2X7 purinergic receptor activator and the peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof can be delivered sequentially. In certain embodiments, delivering the P2X7 purinergic receptor activator and the peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants ( e.g ., a conservative variant) thereof can elicit a synergistic enhancement of a calcium influx or a phosphorylated ERK protein expression on a cell of the affected tissue. The cell can be an epithelial cell, and the P2X7 purinergic receptor activator includes adenosine triphosphate.

The presently disclosed subject matter provides a composition for treating epithelial damages and accelerating the healing of the damaged tissue. The disclosed composition can include an effective amount of a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g., a conservative variant) thereof to increase proliferation and migration of an epithelial cell through P2X7 purinergic receptor. The composition can include about 0.001% to about 1% by weight the peptide agent including amino acid sequence LKKTET (SEQ ID NO:

1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof. In non-limiting embodiments, the composition can further include an additive. The additive can include a P2X7 purinergic receptor activator (e.g., adenosine triphosphate). In some embodiments, the composition can be formulated as a solution, suspension, semi-liquid, semi-solid gel, gel, ointment, spray, or cream. In certain embodiments, the composition can include a pharmaceutically or topically acceptable carrier such as alkanols, vegetable oils, mineral oils, or combinations thereof. The composition can be adapted to be delivered to a damaged eye or skin of the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 A provides a graph showing human corneal epithelial cell (HCEC) proliferation at each time point in the presence or absence of a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof. Figure IB provides exemplary images for HCEC migration in the scratch gap closure assay in the presence or absence of various amounts of the peptide agent at the indicated time points. Figure 1C provides a graph illustrating the gap area closed by HCECs at the indicated time points.

Figure 2A provides a graph showing extracellular ATP levels at different time points with various concentration of a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof. Figure 2B provides exemplary images of HCEC intracellular calcium influx after the peptide treatment. Figure 2C provides a graph showing the level of Fluo-8 fluorescence in HCECs.

Figure 3 A provides a graph showing the expression of P2X7 receptors on HCECs. Figure 3B provides exemplary images illustrating a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g., a conservative variant) thereof induced HCEC migration in the scratch gap closure assay in the presence or absence of P2X7 inhibitors, oxATP, or A438079, at the indicated time points. Figure 3C is a graph showing the gap area closed by HCEC migration.

Figure 4A provides exemplary western blot images showing the expression of phospho-ERKl/2 from HCECs. Figure 4B provides a graph illustrating phospho- ERKl/2 expression level quantified by ImageJ software. Figure 4C provides a schematic diagram representing the peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof mediated signals.

Figure 5 A provides representative images showing the morphology of epithelial cell clusters. Figure 5B provides a graph illustrating an expression level of hEpCAM in epithelial cells.

DETAILED DESCRIPTION OF THE DISCLOSURE

Provided herein are methods of controlling cellular behaviors, wherein the method is directed to the use of a composition that contains a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants ( e.g ., a conservative variant) thereof. These and other aspects of the presently disclosed subject matter are discussed more in the detailed description and examples.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: The Cambridge Dictionary of Science and

Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Certain terms are defined below to provide additional guidance in describing the compositions and methods of the disclosed subject matter and how to use them.

As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. Abbreviations used herein have their conventional meaning within the chemical and biological arts.

Unless specifically stated or obvious from context, as used herein, the term“or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms“a”,“an”, and“the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term “about” or“approximately” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. About with respect to a concentration range of the compositions of the current disclosure also refers to any variation of a stated amount or range which would be an effective amount or range.

As used herein,“additive” can include any additional components that can be added to the composition as described herein. One or more additives can be added to the composition. Exemplary additives can include active agents, preservatives, viscosity agents, buffering agents, hypertonic agents, isotonic agents, stabilizers, and pH adjustment agents. Additives in the current disclosure can be used in any suitable amount.

As used herein, the term“administering” or“delivering” can mean any suitable route, i.e., via oral administration, via topical administration (e.g., eye drops or a spray), or intraocular administration.

As used herein, the term“co-administer” is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of additional therapies. The composition of the disclosure can be administered alone or can be co-administered with a second composition/therapeutic agent to a subject. Co administration is meant to include simultaneous or sequential administration of the composition individually or in combination with a second composition/therapeutic agent. Additionally, the first and second agents can be formulated separately or together in one or more compositions.

As used herein,“comprises,”“comprising,”“containing” and“having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean“includes,” “including,” and the like;“consisting essentially of’ or“consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

As used herein,“concurrent administration” includes overlapping in duration at least in part. For example, when two agents ( e.g ., any of the compositions described herein) are administered concurrently, their administration occurs within a certain desired time. The compositions’ administration can begin and end on the same day. The administration of one composition can also precede the administration of a second composition by day(s) as long as both compositions are taken on the same day at least once. Similarly, the administration of one composition can extend beyond the administration of a second composition as long as both compositions are taken on the same day at least once. The compositions do not have to be taken at the same time each day to include concurrent administration.

As used herein,“conservative variant” or grammatical variations thereof can denote the replacement of an amino acid residue by another biologically similar residue. Examples of conservative variations include the replacement of a hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, the replacement of a polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like.

As used herein, the term“cream” can refer to a thick (high viscosity) liquid or semi-liquid that can be used for therapeutic treatment of a disease, syndrome, or condition.

As used herein, the term“dispersion” can refer to a multi-phase liquid medium. For example, a first phase (e.g., particles) can be dispersed throughout a second phase which can be a continuous phase. The dispersion can be formed when at least two non- mixable phases are compounded. The term“dosage” is intended to encompass a formulation expressed in terms of total amounts for a given timeframe, for example as pg/kg/hr, pg/kg/day, mg/kg/day, or mg/kg/hr. The dosage is the amount of an ingredient administered in accordance with a particular dosage regimen. A“dose” is an amount of an agent administered to a mammal in a unit volume or mass, e.g ., an absolute unit dose expressed in mg of the agent. The dose depends on the concentration of the agent in the formulation, e.g. , in moles per liter (M), mass per volume (m/v), or mass per mass (m/m). The two terms are closely related, as a particular dosage results from the regimen of administration of a dose or doses of the formulation. The particular meaning in any case will be apparent from the context.

As used herein,“dry eye” or“dry eye syndrome” or“DES” can refer to an ophthalmic syndrome or ocular surface condition. The Dry Eye Workshop (DEWS) has redefined dry eye as“a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface, accompanied by increased osmolarity of the tear film and inflammation of the ocular surface.” Dry eye and tear film instability can damage the ocular surface. It is accompanied by increased osmolarity of the tear film and

inflammation of the ocular surface. The tear film instability can be initiated by several etiologies such as xerophthalmia, ocular allergy, topical preservative use and contact lens wear. The tear film instability can cause surface hyperosmolarity.

As used herein, an“effective amount” or“therapeutically effective amount” is that amount sufficient to affect a desired biological effect, such as beneficial results, including clinical results. As such, an“effective amount” depends upon the context in which it is being applied. An effective amount can vary according to factors known in the art, such as the disease state, age, sex, and weight of the individual being treated. Several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. In addition, the compositions/formulations of this disclosure can be administered as frequently as necessary to achieve a therapeutic amount.

As used herein, the term“fragment” or“peptide” or“peptide fragment” comprises a portion of a protein (e.g., Tb4 protein) with homology or percent amino acid sequence identity. Peptides can be biologically occurring short chains of amino acid monomers linked by peptide (amide) bonds. As used herein,“gel” can refer to a material which is not a readily flowable liquid and is not a solid, i.e., a semi-solid gel. Gels can be formed from naturally occurring or synthetic materials. The gels can be non-ordered to slightly ordered showing some birefringence, liquid crystal character. A semi-solid gel formulation apparent viscosity can increase with concentration. Gels can be administered topically.

As used herein,“homology” or“percent (%) amino acid sequence identity” is used with respect to a protein (; i.e ., Tb4 or fragment thereof). The homology or percent amino acid sequence identity can be defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be“substantially identical”. This definition also refers to, or can be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2 or ALIGN software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

As used herein,“intermittent administration” includes the administration of a composition for a period of time (which can be considered a“first period of

administration”), followed by a time during which the composition is not taken or is taken at a lower maintenance dose (which can be considered an“off-period”) followed by a period during which the composition is administered again (which can be considered a“second period of administration”). Generally, during the second phase of administration, the dosage level of the composition will match that administered during the first period of administration but can be increased or decreased as medically necessary.

As used herein,“liquid” is a dosage form consisting of a composition in its liquid state. A liquid is pourable; it flows and conforms to its container at room temperature. Liquids display Newtonian or pseudoplastic flow behavior. In certain embodiments, a“semi-liquid” as used herein can have properties of both a liquid and another formulation (z.e., a suspension, an emulsion, a solution, a cream, a gel, a jelly, and the like).

As used herein,“ocular surface” includes the cornea and the conjunctiva. The ocular surface is covered by a thin layer of fluid or tear film. The tear film is not only responsible for the majority of the refractive power of the eye and clear vision; it is also responsible for nourishing the cells on the surface of the eye and preventing infection. The surface of the eye can suffer many kinds of diseases. One of the most common diseases of the surface of the eye is DES.

As used herein,“ocular surface disorder”“ophthalmic disease,”“ophthalmic disorder,” and the like, includes, but is not limited to, dry eyes, epithelial defects, Superior limbic keratoconjunctivitis, keratoconjunctivitis sicca, Neurotrophic keratopathy, Neurotrophic keratitis, Sjogren’s syndrome, Stevens- Johnson syndrome, ocular cicatricial pemphigoid, Medicamentosa, Graft-versus-host disease, and corneal ulcerations and erosions.

As used herein,“ointment” can refer to a highly viscous liquid or semi-liquid formulation that can be used for therapeutic treatment of a disease, syndrome, or condition (z.e., DES).

As used herein,“composition” refers to a composition intended for various applications to a target tissue (e.g., eye and skin) or its related or surrounding tissues. The term also includes compositions intended to therapeutically treat conditions of the target tissue itself or the tissues surrounding the target tissue. The composition can be applied topically or by other techniques, known to persons skilled in the art, such as injection. Examples of suitable topical administration include administration in solution drops, gels, creams, ointments and spray formulations. A further suitable topical administration route is by subconjunctival/subdermal injection. As used herein,“patient,”“patient in need thereof,”“subject,” and“subject in need thereof’ are used interchangeably and refer to an animal or living organism (human or nonhuman) suffering from or prone to a disease or condition that can be treated by administration using the methods and compositions provided herein. Non-limiting examples of subjects include humans, other mammals, bovines, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammalian animals. In certain embodiments, the subject is human.

As used herein,“pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The type of carrier can be selected based upon the intended route of administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, gels, creams and sterile powders for the extemporaneous preparation of sterile topical solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the composition ( e.g ., Tb4 or fragments thereof), use thereof in the compositions for the disclosure is contemplated.

The term“preservative” as used herein can include any agents included in a composition for the purpose of inhibiting the growth of microorganisms (e.g., bacteria, fungi, viruses, and protozoa) in the product, thereby helping to maintain sterility during use. Additionally, the term“anti-microbial agent” can be used herein to denote a specific active agent which provides the anti-microbial efficacy. Exemplary preservatives can include, for example, benzalkonium chloride, thimerosal, chlorobutanol, chi orhexi dine, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium sorbic acid, Onamer M Polyquat, cetyl bromide, cetyl pyridinium chloride, benzyl bromide, EDTA, phenylmercury nitrate, phenylmercury acetate, thimerosal, merthiolate, acetate and phenylmercury borate, polymyxin B sulphate, methyl and propyl parabens, quaternary ammonium chloride, sodium benzoate, sodium propionate, and sodium perborate, and other agents known to those skilled in the art, or a combination thereof.

As used herein, the terms“prevent,”“preventing,” or“prevention,”“prophylactic treatment” and the like, refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition. The prevention can be complete (i.e., no detectable symptoms) or partial, so that fewer symptoms are observed than would likely occur absent treatment. The terms further include a prophylactic benefit. For disease or condition to be prevented, the compositions can be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease cannot have been made.

Ranges can be expressed herein as from“about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent“about,” it is understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein and that each value is also herein disclosed as“about” that particular value in addition to the value itself. It is also understood that throughout the application, data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point“10” and a particular data point“15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 (and values in between) are also disclosed.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9,

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33

34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges,“nested sub ranges” that extend from either endpoint of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 can include 1 to 10, 1 to

20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction. As used herein,“sequential administration” includes that the administration of two agents ( e.g ., compositions described herein) occurs separately on the same day or does not occur on the same day (e.g., occurs on consecutive days).

As used herein, a“solution” is a clear, homogeneous liquid dosage form that contains one or more chemical substances (i.e., Tb4 or fragments thereof) dissolved in a solvent or mixture of mutually miscible solvents. A solution is a liquid preparation that contains one or more dissolved chemical substances in a suitable solvent or mixture of mutually miscible solvents. Because molecules of a drug substance in solution are uniformly dispersed, the use of solutions as dosage forms generally provides assurance of uniform dosage upon administration and good accuracy when the solution is diluted or otherwise mixed. For example and not limitation, a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof can be dissolved in a solution comprised of sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, sodium citrate dihydrate, or combinations thereof , with a pH of approximately 7.0.

The term“solvent,” as used herein, refers to a liquid solvent either aqueous or non-aqueous. The selection of the solvent depends notably on the solubility of the composition on said solvent and on the mode of administration. An aqueous solvent can consist solely of water, or can consist of water plus one or more miscible solvents, and can contain dissolved solutes such as sugars, buffers, salts or other excipients. The more commonly used non-aqueous solvents are the short-chain organic alcohols, such as methanol, ethanol, propanol, short-chain ketones, such as acetone, and polyalcohols, such as glycerol.

“Suspension,” as used herein, is a liquid dosage form that contains solid particles dispersed in a liquid vehicle.

As used herein, the term“syndrome” can refer to a group of symptoms that consistently occur together or a condition characterized by a set of associated symptoms. A syndrome (e.g., DES) can be a set of medical signs and symptoms that are correlated with a specific disease. A disease, on the other hand, can be a health condition that has a clearly defined reason behind it. A syndrome (from the Greek word meaning‘run together’), however, can produce a number of symptoms without an identifiable cause. They can suggest the possibility of an underlying disease or even the chances of developing a disease.

As used herein,“thymosin beta 4” or“Tb4” refers to a human protein. Tb4 encodes for an actin sequestering protein which plays a role in the regulation of actin polymerization. The protein is also involved in cell proliferation, migration, and differentiation. The thymosin beta 4 peptide, if used after a heart attack, has been shown to potentially reactivate cardiac progenitor cells to repair damaged heart tissue. The safety of topical Tb4 formulations has been demonstrated, both in dermal preparations and in a preservative-free formulation used in the eye. Based on its multifunctional activities during tissue regeneration, Tb4 has the potential for clinical application in a wide range of pathological conditions including ocular surface diseases. The NCBI Reference Sequence of human Tb4 is available under accession number NP 066932.1.

The terms“treat,”“treating” or“treatment,” and other grammatical equivalents as used herein, include alleviating, abating, ameliorating, or preventing a disease, condition or symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and are intended to include prophylaxis. The terms further include achieving a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient can still be afflicted with the underlying disorder.

As used herein,“viscosity” refers to a fluid's resistance to flow. Exemplary viscosity agents that can be used include, for example, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propyl cellulose, carbomer, other agents known to those skilled in the art, or a combination thereof.

As used herein, the term“weight percent” or“% (w/w)” refers to a percentage of a component in a solution that is calculated on the basis of weight for the component and the solvent. For example, a 1% (w/w) solution of a component would have 1 g of the component dissolved in 100 g of solvent. The term“volume percent” or“% (v/v)” refers to a percentage of a component in a solution that is calculated on the basis of volume for the component and the solvent. For example, a 1% (v/v) solution of a component would have 1 mL of the component dissolved in 100 mL of solvent. The term“weight/volume percent” or“% (w/v)” refers to a percentage of a component in a solution that is calculated on the basis of weight for the component and on the basis of volume for the solvent. For example, a 1.0% (w/v) solution of a component would have 1 g of the component dissolved in 100 mL of solvent.

Compositions

The present disclosure provides for compositions comprising a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants ( e.g ., a conservative variant) thereof, in an effective amount to treat various symptoms in a subject in need thereof. In certain embodiments, the disclosed

composition can include from about 0.001% to about 1% by weight of a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g., a conservative variant) thereof. The disclosure also provides a composition comprising Tb4, a Tb4 fragment, a Tb4 isoform, or a Tb4 derivative in an effective amount to treat various symptoms in a subject in need thereof. The

composition can include from about 0.001% to about 1% by weight of the Tb4, Tb4 fragments, Tb4 isoform, or Tb4 derivative.

In some embodiments, the composition can include a Tb4 isoform. Tb4 is a polypeptide composed of 43 amino acids having 4.9 kDa, which can be first isolated from thymus and then identified from various tissues. This protein can upregulate the migration and proliferation of epithelial cells. Tb4 isoforms can have about 70%, or about 75%, or about 80% or more homology to the known amino acid sequence of Tb4. Such isoforms can include, for example, Tb4³¹³, Tb9, TbIO, Tbΐ ΐ, Tb12, Tb 13, Tb14 and Tb15. Tb4 of the presently disclosed subject matter can also be an N-terminal variant or C-terminal variant of wild-type Tb4.

In certain embodiments, the composition can include a peptide agent comprising amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or a variant (e.g, a conservative variant) thereof. Amino acid sequence LKKTET (SEQ ID NO: 1) and LKKTNT (SEQ ID NO: 2) appear to be involved in mediating actin sequestration or binding. Tb4 has anti-inflammatory activity, and can also modulate actin polymerization (e.g. b-thymosins appear to depolymerize F-actin by sequestering free G-actin). Tp4's ability to modulate actin polymerization can be due to its ability to bind to or sequester actin via the LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2) sequence. Thus, as with Tb4, other proteins which are anti-inflammatory and/or bind or sequester actin, or modulate actin polymerization, including Tb4 isoforms having the amino acid sequence LKKTET (SEQ ID NO: 1), are likely to be effective, alone or in combination with Tb4, as set forth herein. For example and not limitation, other agents or proteins having anti-inflammatory activity and/or actin sequestering or binding capability, or that can mobilize actin or modulate actin polymerization, as demonstrated in an appropriate sequestering, binding, mobilization or polymerization assay, or identified by the presence of an amino acid sequence that mediates actin binding, such as LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), for example, can similarly be employed in the disclosed subject matter. Such proteins can include gelsolin, vitamin D binding protein (DBP), profilin, cofilin, depactin, Dnasel, vilin, fragmin, severin, capping protein, b-actinin and acumentin.

In certain embodiments, the composition can include oxidized forms of Tb4 including Tb4 sulfoxide or a variant (e.g. , a conservative variant) thereof. Oxidized Tb4 is a form of Tb4 in which a methionine residue, 6 amino acids from the N-terminus (Met6), is oxidized such that the residue is converted to methionine sulfoxide. The oxidized Tb4 can be obtained by reacting native Tb4 under oxidizing conditions, for example, by treating with hydrogen peroxide.

Although the present invention is described primarily hereinafter with respect to Tb4 and Tb4 fragments, it is to be understood that the following description is intended to be equally applicable to amino acid sequence LKKTET (SEQ ID NO: I) or LKKTNT (SEQ ID NO: 2), peptides and fragments comprising or consisting essentially of LKKTET (SEQ ID NO: I) or LKKTNT (SEQ ID NO: 2), conservative variants thereof and/or Tb4 isoforms, analogues or derivatives, including oxidized Tb4, N-terminal variants of Tb4, and C-terminal variants of Tb4.

In certain embodiments, the Tb4, Tb4 fragments, Tb4 isoforms, Tb4 derivatives, peptide agents including amino acid sequence LKKTET (SEQ ID NO: I) or LKKTNT (SEQ ID NO: 2), and variants (e.g., a conservative variant) thereof can be encapsulated in at least one particle. For example, the Tb4, Tb4 fragments, Tb4 isoforms, Tb4 derivatives, peptide agents including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), and variants ( e.g ., a conservative variant) can be

encapsulated within phospholipid membranes, liposomes, biodegradable microspheres, nanoparticles, or biodegradable lactone based polymers that includes polyesters made by polycondensation of L-lactide, glycolide, caprolactone, dioxanone, poly(lactic-acid), poly(glycolic-acid), cyclic carbonates or their derivatives.

In non-limiting embodiments, the Tb4, Tb4 fragments, Tb4 isoforms, Tb4 derivatives, peptide agents including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), and variants (e.g., a conservative variant) thereof can be conjugated to the at least one particle. For example, the Tb4, Tb4 fragments, Tb4 isoforms, Tb4 derivatives, peptide agents including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), and variants (e.g, a conservative variant) thereof can conjugate with synthetic and/or natural polymers such as polyethylene glycol (PEG) and dextran, including the cyclodextrans for an improve pharmacokinetic profile, resulting in a decrease in the disclosed peptide clearance. In some embodiment, the disclosed composition can include empty particles which do not include the disclosed active agents, such as Tb4, Tb4 fragments, Tb4 isoforms, Tb4 derivatives, peptide agents including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), and variants (e.g, a conservative variant) thereof.

In certain embodiments, the disclosed particles can have various sizes. For example, the particles can be less than about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 10, about 5, or about 1 micron in size. The size of the particles can be adjusted to reduce irritation to a subject.

In non-limiting embodiments, the disclosed particles can release and replenish the disclosed active agents. For example, each particle can have different degradation rates. When they are applied to a target tissue, some particles can be dissolved to release the active agents, while other particles can adhere to the target tissue without being dissolved. As the active agent is absorbed by the target tissue, the undissolved particles can be partially degraded to replenish the released active agent. By including particles which can release and replenish the active agents, the disclosed composition can increase the contact time and duration of the composition.

In certain embodiments, the composition can include carriers which can be suitable for topical or intravitreal administration. The carriers can include, for example, and not limitation, water; a mixture of water and water-miscible solvents such as C1-C7 alkanols, vegetable oils or mineral oils such as from about 0.5 to about 5 wt. % of hydroxyethyl cellulose, ethyl oleate, carboxymethyl cellulose, polyvinyl pyrrolidone, and other non-toxic water-soluble polymers for ophthalmic/dermal use, for example, cellulose derivatives such as methyl cellulose, alkali-metal salts of carboxymethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose and hydroxypropyl cellulose, acrylates or methacrylates such as salts of polyacrylate or ethyl acrylate, polyacrylamides; natural products such as gelatin, alginate, pectin, tragacanth, karaya gum, xanthan gum, carrageenan, agar, acacia, starch derivatives such as starch acetate and hydroxylpropyl starch; and other synthetic products, for example, polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl methylether, polyethylene oxide, preferably, cross-linked polyacrylic acid such as neutral carbopol, or mixtures of the above polymers. Preferable carriers can include water, cellulose derivatives, for example, methyl cellulose, alkali-metal salts of carboxymethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose and hydroxypropyl cellulose, neutral carbopol, or mixtures thereof.

In certain embodiments, the composition can include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In non limiting embodiments, the pharmaceutically acceptable carrier can include one or more pharmaceutically acceptable excipients including but not limited to stabilizers, buffers, preservatives, tonicity agents, and viscosity enhancers.

In certain embodiments, the composition can include stabilizers. The stabilizers according to the presently disclosed subject matter can include, for example, and not limitation, tyloxapol, aliphatic glycerol poly-lower alkylene glycol esters, aliphatic poly- lower alkylene glycol esters, polyethylene glycols, glycerol ethers, acetic acid, citric acid, ascorbic acid, EDTA/disodium edetate, glutathione, acetylcysteine or mixtures of these compounds. Acetic acid used herein is a weak acid represented by formula CH₃COOH. In the presently disclosed subject matter, this can be used in the form of acetate. The acetate can include at least one molecule of water. For example and not limitation, mono-, sesqui-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca-, undeca-, or dodeca-hydrate forms of acetate can be added into the composition. In particular, sodium acetate trihydrate can be included in an amount of from about 0.01% (w/v) to about 1.5% (w/v) based on the total volume of the composition. Further, acetic acid or its salt can be included in an amount of from about 0.1% (w/v) to about 0.8% (w/v), and preferably, from about 0.2% (w/v) to about 0.5% (w/v). Citric acid used herein is a compound represented by formula CeHsCb. In the presently disclosed subject matter, citric acid can be used in the form of one or more citrates. The citrate can be a derivative of citric acid. Additionally, the citrate can include at least one molecule of water. For example and not limitation, mono-, sesqui-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca-, undeca-, or dodeca-hydrate forms of citrate can be added into the composition. In particular, the citrate can be sodium citrate and sodium citrate dihydrate. In this case, citric acid or its salt can be included in an amount of from about 0.01% (w/v) to about 0.5% (w/v). Further, citric acid or its salt can be included in an amount of from about 0.05% (w/v) to about 0.25% (w/v), and preferably, from about 0.1% (w/v) to about 0.3% (w/v). They are typically added in an amount sufficient to dissolve active ingredients.

In certain embodiments, the composition can include a buffer. For example, the buffer can include any forms of acetate, ascorbate, borate, hydrocarbonate/carbonate, gluconate, phosphate, propionate, acetic acid, citric acid and/or tromethamine (TRIS) buffers. The buffer can be added, for example, in an amount to ensure and maintain a physiologically acceptable pH range. Such pH can be in the range of from about 3.5 to about 9, from about 4 to about 7, from about 4.5 to about 6.5, or from about 5 to about 6, from about 5 to about 6, from about 5.5 to about 6.0, from about 6 to about 8.2, or from about 6.8 to about 8.1.

In other embodiments, the pH value of the formulations can range from about 3.5 to about 9, from about 3.5 to about 7, from about 4.5 to about 6.5, or from about 5 to about 6, from about 5 to about 6, from about 5.5 to about 6.0, from about 4.5 to about 8, and from about 5.5 to about 7.8, and can be about pH 7.0.

The composition in accordance with the presently disclosed subject matter can further include an acid selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, etc. The composition can further include a base selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate etc., specifically, sodium hydroxide. For example and not limitation, hydrochloric acid or sodium hydroxide can be suitably added to adjust a pH of the composition. As such, the pH of the composition can be from about 3.5 to about 9, from about 4 to about 7, from about 4.5 to about 6.5, or from about 5 to about 6, from about 5 to about 6, from about 5.5 to about 6.0, from about 6 to about 8.2, or from about 6.8 to about 8.1.

In certain embodiments, the composition can include preservatives. The preservatives can include, for example, quaternary ammonium salts such as Cetrimide, benzalkonium chloride or benzoxonium chloride; alkyl-mercury salts of thiosalicylic acid such as thimerosal, phenylmercuric nitrate, phenylmercuric acetate or

phenylmercuric borate, parabens such as phenylparaben or propylparaben, alcohols such as chlorobutanol, benzyl alcohol or phenyl ethanol, guanidine derivatives such as chlorohexidine or polyhexamethylene biguanide or sorbic acid. Preferable preservatives can include cetrimide, benzalkonium chloride, benzoxonium chloride and parabens. The preservative can be added in a sufficient amount to prevent secondary contamination caused by bacteria and fungi during the use.

In certain embodiments, the composition can include a tonicity agent to adjust the composition closer to isotonicity ( e.g ., 0.9% saline). For instance and not limitation, any form of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, dextrose, mannitol, or combinations thereof, can be added to the composition comprising thymosin b4 according to the presently disclosed subject matter. The tonicity agents can include at least one molecule of water. For example and not limitation, mono-, sesqui-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca-, undeca-, or dodeca-hydrate forms of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, or combinations thereof, can be added into the composition. An amount of the tonicity agent depends upon the kind of active agents to be added. In general, particular compositions of the present disclosed subject matter can include a tonicity agent therein to enable the final composition to have an osmolality acceptable for ophthalmic/dermal use, i.e., preferably in a range of from about 150 to about 450 mOsm, and more preferably in a range of from about 250 to about 350 mOsm. Preferable tonicity agents can include, for example, sodium salts and/or potassium salts, in particular, sodium chloride and/or potassium chloride. Most preferably, the tonicity agent can be sodium chloride. Further, a concentration of sodium chloride can range from about 0.1 to about 1.2% (w/v) or from about 0.3 to about 1.0% (w/v). Preferably, it ranges from about 0.5 to about 0.7% (w/v). Further, a concentration of potassium chloride can range from about 0.01 to about 0.15% (w/v) or from about 0.03 to about 0.12% (w/v). Preferably, it ranges from about 0.05 to about 0.09% (w/v). Further, a concentration of calcium chloride dihydrate can range from about 0.01 to about 0.12% (w/v) or from about 0.03 to about 0.09% (w/v). Preferably, it ranges from about 0.03 to about 0.06% (w/v). Further, a concentration of magnesium chloride hexahydrate can range from about 0.01 to about 0.12% (w/v), and preferably, from about 0.01 to about 0.05% (w/v). Although the tonicity agents are described primarily herein with respect to adjusting the tonicity of the composition, the disclosed tonicity agents can also be used as electrolytes.

In certain embodiments, the composition can include a viscosity enhancer.

Suitable viscosity enhancers in formulations and their concentration ranges used in certain inventive compositions can include but are not limited to: (a) Monomeric polyols, such as tyloxapol (from about 0.1 to about 1%), glycerol (from about 0.2 to about 1%), propylene glycol (from about 0.2 to about 1%), ethylene glycol (from about 0.2 to about 1%); (b) Polymeric polyols, such as polyethylene glycol ( e.g ., PEG 300, PEG 400) (from about 0.2 to about 1%); (c) Cellulose derivatives (polymers of the cellulose family), such as hydroxyethylcellulose (from about 0.2 to about 2.5%), hypromellose (from about 0.2 to about 2.5%), hydroxypropylmethyl cellulose (from about 0.2 to about 2.5%), methylcellulose (from about 0.2 to about 2.5%), carboxym ethyl cellulose sodium (from about 0.2 to about 2.5%), hydroxylpropylcellulose (from about 0.2 to about 2.5%); (d) Dextrans, such as dextran 70 (at about 0.1% when used with another polymeric demulcent agent); (e) Water-soluble proteins such as gelatin (at about 0.01%); (f) Vinyl polymers such as polyvinyl alcohol (from about 0.1 to about 4%), polyvinyl pyrollidine (from about 0.1 to about 4%); (g) Other polyols, such as polysorbate 80 (from about 0.2 to about 1%), povidone (from about 0.1 to about 2%); (h) Carbomers, such as carbomer 934P, carbomer 941, carbomer 940, and carbomer 974P, and (i)

Polysaccharides/Glycosaminoglycans, such as hyaluronan (hyaluronic acid/hyaluronate) (from about 0.1 to about 3%), chondroitin sulfate (from about 0.1 to about 3%).

In certain embodiments, the amount and type of excipient(s) added can be varied depending on specific requirements, the excipient(s) is generally used in a range of about 0.0001 to about 90 wt. %, and within the range commonly used in ophthalmic/dermal fields.

In certain embodiments, the composition is formulated as a solution, suspension, semi-liquid, semi-solid gel, gel, ointment, spray or cream. For example, the formulated solution, suspension, semi-liquid, semi-solid gel, gel, ointment, spray or cream can be administered to a target tissue (e.g., dermal tissue) for contacting the composition with the target tissue. According to one embodiment, the topical formulation containing the active compound can also contain a physiologically compatible vehicle, as those skilled in the art can select using conventional criteria.

In certain embodiments, the composition can be formulated into a unit dosage form to provide a total daily dosage and can be suitably filled in a container for dermal use, which can enable the quantitative administration of the composition. The total daily dosage can be various based on the target tissue (e.g., size of damaged tissue). For this purpose, the composition can be formulated to be used once or several times. For example, several divided doses can be administered daily. In non-limiting embodiments, the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

In certain embodiments, the composition can include from about 0.001 to about 1% by weight of Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g., a conservative variant) thereof. The composition can further include stabilizers, a thickener, and/or preservatives for dermal applications. The stabilizers can include disodium-EDTA or EDTA, trehalose, propylene glycol, and the like, with propylene glycol or a mixture of propylene glycol and disodium-EDTA at a concentration of 0.01 to 100 mg/ml. The thickener can include cellulose derivatives such as polyvinyl alcohol, carboxymethyl cellulose, hydroxypropyl methyl cellulose, and carbomer at a concentration of 1 to 100 mg/ml. The preservative can include, for example, alcohols, methylparaben, propyl paraben, chlorobutanol, benzyl alcohol, and/or guanidine derivatives. In non-limiting embodiments, the pH of the composition for a dermal application can be in the range of from about 4 to about 7, from about 4.5 to about 6.5, or from about 5 to about 6, or from, about 5.5 to about 6 in order to minimize the occurrence of Tb4 sulfoxide.

In certain embodiments, the composition is administered in the form of eye drops. The composition can be, where appropriate, adjusted and/or buffered to the desired pH and, where appropriate, a stabilizer, or a tonicity enhancing agent can be added. Where appropriate, preservatives and/or other excipients can be added to a composition. In specific embodiments, the composition can be formulated as a preservative-free, sterile eye-drop solution in a single unit dropper. In certain embodiments, the composition can be formulated into a unit dosage form to provide a total daily dosage of from about 0.08 to about 2.0 ml and can be suitably filled in a container for ophthalmic use, which can enable quantitative administration of the composition. For this purpose, the composition can be formulated into a unit dosage form with a dose of from about 0.01 to about 10 ml that can be used once or several times. Further, in order to suitably provide the pharmaceutical composition in a total daily dosage of from about 0.08 to about 2.0 ml, the composition can be contained in an eye drop container dropping from about 0.01 to about 2.0 ml per droplet.

In certain embodiments, the composition can include from about 0.001 to about 1% by weight of Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants ( e.g ., a conservative variant) thereof. The composition can be used for ophthalmic use and can be in a solution comprised of sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, sodium citrate dihydrate, or combinations thereof. The pH of the composition can be adjusted to about 6.5 to about 7.5 using an acid or a base. The acid can be selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, etc. The base can be selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate, etc.

In certain embodiments, the composition can further include an additive. The additive can include a P2X7 purinergic receptor activator, a calcium influx enhancer, a phosphorylated ERK activator, or combinations thereof.

In certain embodiments, the P2X7 purinergic receptor activator can include adenosine triphosphate (ATP), 2’(3’)-0-(4-benzoylbenzoyl) adenosine 5’ -triphosphate (BzATP), human cathelicidin-derived peptide LL37, 2-methylthioadenosine 5'- triphosphate (2MeSATP), adenosine-5'-(y-thio)-tri phosphate (ATPyS) or combinations thereof. The disclosed composition can include from about 1 nM to about ImM of the P2X7 purinergic receptor activator. The combination of a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g., a conservative variant) thereof and the P2X7 purinergic receptor activator can provide synergistic therapeutic benefits as compared to when the peptide agent or the P2X7 purinergic receptor activator is administered alone. In non-limiting embodiments, the calcium influx enhancer can include ATP, epidermal growth factor (EGF), parathyroid hormone (PTH), protein kinase C (PKC), or combinations thereof. The disclosed composition can include from about 1 nM to about ImM of the P2X7 purinergic receptor activator. The combination of Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g., a conservative variant) thereof and the calcium influx enhancer can provide synergistic therapeutic benefits as compared to when the peptide agent or the calcium influx enhancer is administered alone.

In non-limiting embodiments, the phosphorylated ERK activator can include ATP, EGF, dinucleotides (e.g., diadenosine polyphosphates), or combinations thereof. The disclosed composition can include from about 1 nM to about ImM of the

phosphorylated ERK activator. The combination of Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof and the phosphorylated ERK activator can provide synergistic therapeutic benefits as compared to when the peptide agent or the phosphorylated ERK activator is administered alone.

In certain embodiments, the composition can further include a P2X7 purinergic receptor inhibitor to regulate cell migration and proliferation. The P2X7 purinergic receptor inhibitor can include oxidized ATP, a competitive P2X7 receptor antagonist or a combination thereof. The disclosed composition can include from about 1 nM to about 10 mM, from about 1 mM to 50 mM, or from about 1 pM to 10 pM of the P2X7 purinergic receptor inhibitor. The P2X7 purinergic receptor inhibitor can reduce or slow the cell migration and proliferation induced by the disclosed peptide agent or the P2X7 purinergic receptor activator.

Methods

The present disclosure provides, inter alia , a method of treating epithelial damage or signs or symptoms thereof in a subject in need of such treatment. The method includes administering to damaged tissue (e.g., skin or eye) of the subject the disclosed composition including Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or in an effective amount to treat the epithelial damage and signs and symptoms thereof. In other examples, the method includes administering to damaged tissue (e.g., skin or eye) of the subject the disclosed peptide agents including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants ( e.g ., a conservative variant) thereof, in an effective amount to treat the epithelial damage and signs and symptoms thereof.

The presently disclosed subject matter provides methods for treating epithelial damage and accelerating the healing of the damaged skin or eye. The methods include delivering Tb4, Tb4 fragments, Tb4 isoforms, Tb4 derivatives, peptide agents including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g., a conservative variant) thereof to a target tissue. In non-limiting embodiments, the Tb4, Tb4 fragments, Tb4 isoforms, Tb4 derivatives, peptide agents including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof can be formulated as a solution, suspension, semi-liquid, semi-solid gel, gel, ointment, spray, or cream. In certain embodiments, delivering the composition including a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof to a target tissue can activate a P2X7 purinergic receptor, enhance a calcium influx, and/or increase expression of phosphorylated ERK protein from the target tissue, leading to increased cell migration and proliferation. For example, the cell migration and proliferation can increase by at least about 5%, least about 10%, least about 15%, least about 20%, least about 25%, least about 30%, least about 50%, or least about 100%.

In certain embodiments, the disclosed methods can further include delivering an additive to the target tissue. The additive can include a P2X7 purinergic receptor activator, a calcium influx enhancer, a phosphorylated ERK activator, or combinations thereof. The combination of a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof and additive can provide synergistic therapeutic benefits as compared to when the peptide agent or the additive is administered alone. For example, the combination of Tb4 and additive can synergistically improve cell migration, proliferation, and healing rate by at least about 5% - 300%. In certain embodiments, the additive can be delivered simultaneously or sequentially with the Tb4, Tb4 fragments, Tb4 isoforms, Tb4 derivatives, peptide agents including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof. In non-limiting embodiments, the disclosed methods can be used for various conditions including dry eyes, epithelial defects, Superior limbic keratoconjunctivitis, keratoconjunctivitis sicca, Neurotrophic keratopathy, Neurotrophic keratitis, Sjogren’s syndrome, Stevens- Johnson syndrome, ocular cicatricial pemphigoid, Medicamentosa, Graft-versus-host disease, corneal ulcerations and erosions, skin inflammation, skin ulceration, and skin damages.

Co-administration

In certain embodiments, the methods of treating epithelial damage can be managed as an ongoing condition. In certain embodiments, if there is an underlying disease, that disease is concurrently treated.

In certain embodiments, the composition including Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants ( e.g ., a conservative variant) thereof can be administered at the same time, just prior to, or just after the administration of additional therapies. The composition of the disclosure can be administered alone or can be co-administered with a second composition/therapeutic agent to a subject.

Co-administration can be meant to include simultaneous or sequential

administration of the composition individually or in combination with a second composition/therapeutic agent. In some embodiments, the composition is administered prior to the administration of the second composition/therapeutic agent. In other embodiments, the second composition/therapeutic agent is administered prior to administration of the composition.

In certain embodiments, the method includes treating epithelial damage with a composition of the present disclosure in combination with artificial tears. Artificial tears can include any ocular ointments, drops, or sprays and the like known in the art.

Exemplary artificial tears can include, for example, Celluvisc, Clear Eyes CLR, GenTeal, Hypotears, Isopto Tears, Lacri-Lube S.O.P., Liquitears, Moisture Drops, Oasis Tears, Opti-Free Rewetting Drops, Optive, Refresh, Soothe, Systane, TheraTears, Ultra Fresh, Visine Tears, and the like.

In certain embodiments, the method includes treating epithelial damage with a composition of the present disclosure in combination with treatments of skin ulcers. Treatments of skin ulcers can include any dermal ointments, spray, gel, or cream and the like known in the art. Exemplary treatments of skin ulcers can include, for example, recombinant human platelet-derived growth factor (PDGF), recombinant human epidermal growth factor (EGF), and recombinant human basic fibroblast growth factor (bFGF) treatments.

Dosage Regimens

For example and not limitation, the methods can include contacting a target tissue with an effective amount of a composition including Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants ( e.g ., a conservative variant) thereof as an active ingredient. The administration can be topical or intravitreal administration. An example of topical administration can include direct application of the composition in the form of, for example, a solution, lotion, plaster, gel, cream, paste, spray, suspension, dispersion, hydrogel, ointment, oil or foaming agent to a subject in order to contact same with eye or skin tissues

In certain embodiments, a method of treating epithelial damage in a subject in need thereof includes administering to a target tissue of the subject, a composition including human Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g., a conservative variant) thereof is formulated in the form of a solution, a suspension, a semi-solid gel, a gel, an emulsion, semi-liquid, an ointment, a cream, foam gel, a spray, or a controlled-release/sustain-release vehicle. For example, the composition can be in the form of a solution, wash, drop, gel, ointment, spray, and the like.

The following dosage regimens can be used to treat epithelial damage in general and can be used to treat both inflammatory responses and increase epithelial healing by administering effective amounts of the composition including Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a

conservative variant) thereof. The dosage regimens provided herein can be used to activate a P2X7 purinergic receptor, enhance a calcium influx, and/or increase expression of phosphorylated ERK protein from the target tissue, leading to increased cell migration and proliferation.

In a particular embodiment, the composition can be administered topically to a target tissue in a dosage range from about 5 pg to about 400 pg, from about 5 pg to about 200 pg, from about 5 pg to about 100 pg, from about 5 pg to about 60 pg, or from about 5 pg to about 50 pg. In other embodiments, the composition can be administered topically to a target tissue in a dose range from about 5 pg to about 400 pg, from about 50 pg to about 400 pg, from about 100 pg to about 400 pg, or from about 200 pg to about 400 pg. In certain embodiments, the dosage of the disclosed composition can be altered based on the target tissue.

In certain embodiments, the target tissue can be contacted with the formulated solution, suspension, semi-liquid, semi-solid gel, gel, cream, paste, ointment, linen, lotion, hydrogel or aerosol including the disclosed dosage of the Tb4, Tb4 fragments,

Tb4 isoforms, Tb4 derivatives, or peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants ( e.g ., a conservative variant) thereof.

In non-limiting embodiments, the dosage for the target tissue can be about 1 to about 10 drops of solution. In certain embodiments, the dosage for the target tissue can be 1, 2, 3, 4, or 5 drops of solution. Each drop of composition in the solution can correspond to about 10 pL to about 150 pL of composition. Each drop of the

composition in a solution can correspond to about 20 pL to about 70 pL of the composition.

In certain embodiments, the composition can be administered to target tissue by administering the disclosed dosage of the composition to the target tissue, 1 to 24 times daily. For example, the composition can be applied, 1, 2, 3, 4, 8, 12, 18 or 24 times a day, or more. In certain embodiments, the composition can be applied by administering the disclosed dosage of the composition to the target tissue, once daily or twice daily, or three times daily, or four times daily, or 5 times daily, or 6 times daily. For example and not limitation, the composition can be applied by administering the disclosed dosage of the composition to the target tissue four times daily, including, for example, in the morning, noon, afternoon, and evening. In non-limiting embodiments, the disclosed composition can be administered in appropriate amounts divided into several portions for a specific period of time. For example, the composition can be administered about once a week, once every two days, once a day, or twice a day.

In certain embodiments, the method of treating epithelial damage includes administering a composition including Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants ( e.g ., a conservative variant) thereof to a subject in any suitable or therapeutically effective amount, e.g., from about 0.001 percent by weight to about 90 percent by weight of the composition, from about 0.001 percent by weight to about 1 percent by weight, from about 0.001 percent by weight to about 10 percent by weight, from about 0.001 percent by weight to about 20 percent by weight, from about 0.001 percent by weight to about 30 percent by weight, from about 0.001 percent by weight to about 40 percent by weight, from about 0.001 percent by weight to about 50 percent by weight, from about 0.001 percent by weight to about 60 percent by weight, from about 0.001 percent by weight to about 70 percent by weight, from about 0.001 percent by weight to about 80 percent by weight, from about 0.01 percent by weight to about 90 percent by weight, from about 0.01 percent by weight to about 0.5 percent by weight, from about 0.01 percent by weight to about 1 percent by weight, from about 0.01 percent by weight to about 10 percent by weight, from about 0.01 percent by weight to about 20 percent by weight, from about 0.01 percent by weight to about 30 percent by weight, from about 0.01 percent by weight to about 40 percent by weight, from about 0.01 percent by weight to about 50 percent by weight, from about 0.01 percent by weight to about 60 percent by weight, from about 0.01 percent by weight to about 70 percent by weight, from about 0.01 percent by weight to about 80 percent by weight, from about 0.5 percent by weight to about 90 percent by weight, from about 0.5 percent by weight to about 1 percent by weight, from about 0.5 percent by weight to about 10 percent by weight, from about 0.5 percent by weight to about 20 percent by weight, from about 0.5 percent by weight to about 30 percent by weight, from about 0.5 percent by weight to about 40 percent by weight, from about 0.5 percent by weight to about 50 percent by weight, from about 0.5 percent by weight to about 60 percent by weight, from about 0.5 percent by weight to about 70 percent by weight, from about 0.5 percent by weight to about 80 percent by weight, or any range in between, of the composition. In certain embodiments, the method of treating epithelial damage includes administering a composition including Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof to a subject at about 0.001% by weight. In certain embodiments, the method of treating epithelial damage includes administering a composition including Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g., a

conservative variant) thereof to a subject at about 0.01% by weight. In certain

embodiments, the method of treating epithelial damage includes administering a composition including Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or a peptide agent including amino acid sequence LKKTET(SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g., a conservative variant) thereof to a subject at about 0.5% by weight. In certain embodiments, the method of treating epithelial damage includes administering a composition including Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof to a subject at about 1% by weight.

In certain embodiments, the method of treating epithelial damage includes administering a composition including Tb4, a Tb4 fragment, a Tb4 isoform, a Tb4 derivative, or a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof to a subject in any suitable or therapeutically effective amount, e.g., from about 0.01 to about 900 mg/ml, from about 0.01 to about 800 mg/ml, from about 0.01 to about 700 mg/ml, from about 0.01 to about 600 mg/ml, from about 0.01 to about 500 mg/ml, from about 0.01 to about 400 mg/ml, from about 0.01 to about 300 mg/ml, from about 0.01 to about 200 mg/ml, from about 0.01 to about 100 mg/ml, from about 0.01 to about 50 mg/ml, from about 0.01 to about 10 mg/ml, from about 0.01 to about 5 mg/ml, from about 0.1 to about 900 mg/ml, from about 0.1 to about 800 mg/ml, from about 0.1 to about 700 mg/ml, from about 0.1 to about 600 mg/ml, from about 0.1 to about 500 mg/ml, from about 0.1 to about 400 mg/ml, from about 0.1 to about 300 mg/ml, from about 0.1 to about 200 mg/ml, from about 0.1 to about 100 mg/ml, from about 0.1 to about 50 mg/ml, from about 0.1 to about 10 mg/ml, from about 0.1 to about 5 mg/ml, or any range in between.

In certain embodiments, the composition can further include an additive. The additive can include a P2X7 purinergic receptor activator, a calcium influx enhancer, a phosphorylated ERK activator, or combinations thereof. The method of treating epithelial damage includes administering a composition including the additive with a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a conservative variant) thereof to a subject in any suitable or therapeutically effective amount, e.g., from about 0.001 percent by weight to about 90 percent by weight of the composition, from about 0.001 percent by weight to about 1 percent by weight, from about 0.001 percent by weight to about 10 percent by weight, from about 0.001 percent by weight to about 20 percent by weight, from about 0.001 percent by weight to about 30 percent by weight, from about 0.001 percent by weight to about 40 percent by weight, from about 0.001 percent by weight to about 50 percent by weight, from about 0.001 percent by weight to about 60 percent by weight, from about 0.001 percent by weight to about 70 percent by weight, from about 0.001 percent by weight to about 80 percent by weight, from about 0.01 percent by weight to about 90 percent by weight, from about 0.01 percent by weight to about 1 percent by weight, from about 0.01 percent by weight to about 10 percent by weight, from about 0.01 percent by weight to about 20 percent by weight, from about 0.01 percent by weight to about 30 percent by weight, from about 0.01 percent by weight to about 40 percent by weight, from about 0.01 percent by weight to about 50 percent by weight, from about 0.01 percent by weight to about 60 percent by weight, from about 0.01 percent by weight to about 70 percent by weight, from about 0.01 percent by weight to about 80 percent by weight, from about 0.5 percent by weight to about 90 percent by weight, from about 0.5 percent by weight to about 1 percent by weight, from about 0.5 percent by weight to about 10 percent by weight, from about 0.5 percent by weight to about 20 percent by weight, from about 0.5 percent by weight to about 30 percent by weight, from about 0.5 percent by weight to about 40 percent by weight, from about 0.5 percent by weight to about 50 percent by weight, from about 0.5 percent by weight to about 60 percent by weight, from about 0.5 percent by weight to about 70 percent by weight, from about 0.5 percent by weight to about 80 percent by weight, or any range in between, of the composition.

In certain embodiments, the method of treating epithelial damage includes administering a composition including a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants (e.g, a

conservative variant) thereof from about 0.001% to about 1% by weight and the additive from about 1 nM to 1 mM to a subject.

EXAMPLES The following examples are merely illustrative of the presently disclosed subject matter, and they should not be considered as limiting the scope of the disclosed subject matter in any way.

Example 1: Purinergic signaling regulates thymosin p4-mediated corneal epithelial cell migration

Study objectives. The objective of this study was to determine the effect of thymosin beta 4 (Tb4) on human corneal epithelial cell migration and the downstream signaling pathways.

Materials and methods. Cell culture. Primary human corneal epithelial cells (HECEs) were obtained commercially (ATCC^®PCS-700-010™) and were cultured in the recommended medium (ATCC^®PCS-700-030™) with growth factors (ATCC^®PCS-700- 040™) and antibiotics (ATCC^®PCS-999-002™) in a humidified atmosphere of 5 % C02 at 37°C. After seeding the cells into culture dishes, the medium was replaced every day. HCECs were used between passages 2 and 5.

Flow cytometry. The surface markers on HCECs were identified using 1 x 10⁵ cells suspended in 50 pL of binding buffer (PBS + 2% FBS) and then incubated for 30 min at 4°C in the dark. The following primary antibodies were used: human anti- EpCAM (PE; 1B7; eBiosciences) and P2X7 (Santa Cruz; Cat. No. sc-514962). In the case of P2X7, the cells were additionally incubated for 30 min more with a secondary antibody (anti-mouse IgG-PE; Santa Cruz). The controls included unstained cells incubated with the appropriate concentration of control antibody (BD Biosciences).

Flow cytometry on HCECs at passage 3 employed a FACSCalibur flow cytometer with analysis by Cell Quest Pro software (BD Biosciences).

Cell proliferation assay. The proliferation of primary HCECs was determined using the CCK-8 kit (Dojindo, Japan). Cells (3 x 10³ cells in 100 pL/well) in 96-well plates were exposed to various Tb4 concentrations (0-1000 ng/mL). At various incubation time points (12- 48 h), the CCK-8 reagent (10 pL) was added, and the cells were further incubated for 2 h. An automatic microplate reader was used to measure the optical density (OD) at 450 nm.

Gap closure assay. Primary HCECs (3 x 10⁵ cells/well) in 6-well plates were allowed to form a complete monolayer. Using a sterile 200 pi pipette tip, a scratch was made across the center of each well. After washing the cells with medium to remove debris, various concentrations of Tb4 (0, 1, 10, 100, and 1000 ng/mL) were added to the medium. After 6, 12, and 24 hours, the migrated cells were photographed under an inverted microscope. Gap closure was quantitated by the reduction in the denuded area by the migrated cells. For P2X7 inhibition, the antagonist A438079 (3-[[5-(2,3- dichlorophenyl)-lH-tetrazol-l-yl]methyl]pyridine; Tocris Biosciences) and oxidized ATP (ox ATP; Sigma-Aldrich) were treated at 10 mM for 24 h.

Measurement of extracellular ATP. Primary HCECs (3 x 10⁴ cells/well) in a 96- well plate were incubated overnight. After treatment with different Tb4 concentrations (0, 1, 10, 100, and 1000 ng/mL) for 1, 2, 4, and 6 hours, extracellular ATP (eATP) was determined in the culture supernates using the ATP Lite One-Step kit (Perkin-Elmer, Waltham, MA, USA). Cellular debris was removed by centrifugation of the plates. A luminometer was used to quantitate luminescence, and the ATP concentration was presented as relative luminescence units (RLUs).

Measurement of intracellular Ca²⁺ levels. Primary HCECs cells (5 x 10⁴ cells/well) in black wall-clear bottom 96-well plates were cultured overnight. Two hours after Tb4 treatment (0, 1, 10, 100, and 1000 ng/ml), the Fluo-8 No Wash Calcium Assay kit (Abeam, Cambridge, MA) was used to measure the Tb4-induced changes in intracellular Ca²⁺ concentration. A GloMaxTM Multi+ Microplate Reader chamber (Promega, Seoul, Korea) at 490 mm was used on the excited cells. Then, the Ca²⁺-bound fluo-8 emission was quantitated at 525 nm. Fluorescence was measured using Image-Pro Plus software.

Immunoblot analysis. Standard immunoblot assays were performed using total cellular protein (10 pg) that was separated by SDS-PAGE and then transferred onto polyvinylidene diflouride membranes. After blocking with 5% nonfat dried milk, the membranes were incubated with anti-human phospho-ERKl/2 (#9101), ERK1/2

(#9102), and GAPDH (#5174; Cell Signaling Technology, Danvers, MA) primary antibodies. An enhanced chemiluminescence (ECL) kit (Ab Frontier) was used to visualize the HRP-conjugated secondary antibodies. Then, protein expression levels were quantified by ImageJ software.

Statistical analysis. Graph Pad Prism Software was used for the statistical analyses. After cell migration was analyzed by one-way ANOVA, a pair-wise comparison to the untreated control group was performed using a paired t-test. A p- value £ 0.05 was considered as significant. Tb4 increases the proliferation and migration ofHCECs. Cell growth and migration ofHCECs after 48 hours of Tb4 treatment were quantitated. Tb4 at 1000 ng/ml significantly increased the proliferation ofHCECs over that of untreated HCECs by 48 hours (Fig. 1 A). When the confluent HCECS were scratch wounded, the denuded area of the untreated control group was mostly covered by 24 hours due to cell migration (Fig. IB and 1C), Tb4 dose-dependently significantly increased the rate of gap closure over that seen in the untreated group (Fig. IB and 1C). The most extensive migration was seen in the 100 and 1000 ng/ml Tb4-treated group at 24 hours. Thus, Tb4 has the ability to promote both HCEC proliferation and migration.

Tb4 increases extracellular ATP and then Ca²⁺ influx in HCECs. The HCEC extracellular ATP level and intracellular Ca²⁺ influx were assessed. The ATP level and intracellular Ca²⁺ influx were enhanced by Tb4 treatment. Tb4 caused an active release of extracellular ATP from HCECs, which peaked at 2 hours with the highest dose of Tb4 at 1000 ng/ml (Fig. 2A).

In addition, HCECs were loaded with Fluo-8NW to verify Ca²⁺ influx. Based on fluorescence microscopy, incubation with Tb4 also caused an enhancement of intracellular Ca²⁺ influx into the HCECs by two hours (Fig. 2B and 2C). The highest dose of Tb4 at 1000 ng/ml showed the largest influx. These results suggest that Tb4 can regulate HCECs proliferation and migration via extracellular ATP and Ca²⁺ influx.

P2X7 is an important purinergic receptor for Tb4 migration and signals via ERK phosphorylation. Extracellular ATP acts with a cognate purinergic receptor, such as the P2X receptor on the cell surface. P2X7, one of the P2X receptors, is a known purinergic receptor for signaling in the human corneal epithelium. The presence of the P2X7 receptor (P2X7R) on HCECs was assessed using flow cytometry and a specific P2X7 receptor antibody (Fig. 3 A). The results with the P2X7 inhibitors, oxATP, and A438079 show that P2X7 is a specific mediator for Tb4-induced extracellular ATP signaling using the scratch wound migration assay. Both P2X7 inhibitors blocked the 1000 ng/ml Tb4- induced extracellular ATP signaling/migration with the oxATP being more inhibitory than that of A43079.

As shown in Fig. 4A and 4B, phosphorylation of ERK1/2 protein in HCECs was up-regulated by Tb4 treatment (1000 ng/ml) as demonstrated in Western blot. These results indicate that Tb4 functions in HCECs by initially increasing extracellular ATP levels that lead to an increase in intracellular Ca²⁺ influx and ERK1/2 phosphorylation (Fig. 4C). Therefore, the P2X7 receptor is a mediator in the signaling for Tp4-induced HCEC migration.

Morphology and characteristics ofHCECs. Primary human corneal epithelial cells formed tightly aggregated clusters typical of epithelial cells when cultured in vitro (Fig. 5 A). Almost all HCECs expressed EpCAM, a major epithelial cell-specific marker (Fig. 5B). These cells have normal morphology and the characteristics of epithelial cells.

Tb4 can regulate actin polymerization. Tb4 in mammalian cells can show various biological activities and demonstrate regenerative activities. Although Tb4 has various biological activities, little is known about receptors that can mediate these effects.

Extracellular ATP can be required for many physiological processes, such as cell growth, migration, differentiation, and neuronal signaling. In particular, extracellular ATP can be required for the growth of T lymphocytes and microglial cells. Extracellular ATP can also exert the opposite effect on some target cells. For example, extracellular ATP is toxic to tumor cells, including melanoma and glioma cells. High ATP levels can effectively induce apoptosis in leukemic cells, but not in hematopoietic stem cells. Thus, activation or inhibition of extracellular ATP can be a target cell-specific phenomenon which can have the potential for new therapeutic strategies.

Extracellular ATP can activate P2X and P2Y plasma membrane nucleotide binding receptors. Among these purinergic receptors, P2X receptors are ligand-gated ion channels that can regulate extracellular cations when exposed to ATP. These receptors can behave as a bifunctional molecule and upon ATP binding a small channel opens up to allow passage of cations and then a larger pore form that permits the passage of macromolecules. Activation of P2X7R with the opening of the pore can also have cytotoxic effects on certain cells. Moreover, P2X7, one of the P2X receptors, can be one of the major receptors in HCECs. Migration of mesenchymal stem cell (MSC) can be mediated by ATP-induced Ca²⁺ signaling mechanisms. Therefore, the results show that the elevation of extracellular ATP levels (Fig. 2A) and Ca²⁺ influx (Fig. 2B and 2C) can be induced by Tb4. HCECs migration can be inhibited by specific P2X7 inhibitors (Fig. 3B and 3C). These results show that Tb4 signals in HCECs affect extracellular ATP, P2X7, Ca²⁺ influx, and ERK1/2 signaling. Results also show that Tb4 can promote HCEC migration via the P2X7 receptor showing a new therapeutic potential that may be utilized in regenerative medicine. Tb4 can accelerate the repair of injuries via reduced inflammation, increased cell migration, and enhanced laminin-332 synthesis. However, a Tb4 receptor and the intracellular signaling pathway in the eye have not been investigated. Through the disclosed experimental results, ATP synthase, as a Tb4 receptor, which acts via purinergic signaling in HCECs, was identified. In addition, the disclosed results of corneal epithelial cells further demonstrate increased ATP and Ca²⁺ influx and identify a downstream effector pathway, ERK1/2 phosphorylation. These results also demonstrate the importance of the purinergic signaling in HCEC cell migration based on specific inhibitors that block Tb4-induced migration. The P2X7R can increase tumor cell growth when acting as a Ca²⁺ channel. Cell migration and tumor cell invasion can be enhanced by activation of the ERK-MAPK pathway. The disclosed results show that Tb4 application to the eye can promote corneal epithelial and stem cell proliferation and migration in patients with ophthalmic injury by increasing extracellular ATP through cell surface ATP synthase. In turn, this increase in ATP can activate the P2X7 purinergic receptor to increase Ca²⁺ influx that can subsequently activate the ERK1/2

phosphorylation pathway.

In conclusion, exogenous Tb4 can enhance HCEC proliferation and migration. These effects of Tb4 can be regulated by Tb4 binding to ATP synthase, resulting in elevation of extracellular ATP levels followed by purinergic receptor P2X7-mediated intracellular Ca²⁺ influx and phosphorylation of ERK1/2.

EXAMPLE 2: Combinatorial/synergistic effects of thymosin b4 with an additive on cell migration and proliferation

Cell Migration Experiment. A single cell layer is formed by inoculating primary human corneal epithelial cells (HCEC) onto a 6-well plate with 3 ^c 10⁵ cells per well. Then, using a sterile 200 mΐ pipette tip, a scratch is made across the center of each well. After washing the wells with the culture medium to remove cell debris, the composition comprising thymosin b4 and an additive. The additive can include a P2X7 purinergic receptor activator (e.g., ATP, human cathelicidin-derived peptide LL37, BzATP, 2MeSATP, ATPyS, or combinations thereof), a calcium influx enhancer(e.g., ATP,

EGF, PTH, PKC, or combinations thereof), and/or a phosphorylated ERK activator (ATP, EGF, dinucleotides (e.g., diadenosine polyphosphates), or combinations thereof). After 0 h, 6 h, 12 h, 24 h, and 48 h, each well is photographed with an inverted microscope combined with a digital camera. Gap closure is quantitated by the reduction in the denuded area by the migrated cells.

Tb4 at about 1-1000 ng/ml significantly increases the migration of HCECs over that of untreated HCECs. Tb4 at 1000 ng/ml with the additive synergistically increases the migration of HCECs over that of the Tb4-oh1g treated group or the additive-only treated group. The addition of additive synergistically increases the rate of cell migration compared to the Tb4-oh1g treated group in a dose-dependent manner.

Cell Proliferation Experiment. The effects of Tb4 with/without an additive on the proliferation of primary HCECs are determined using the CCK-8 kit (Dojindo, Japan). After inoculating 3 ^c 10³ cells in 100 pL onto each well of a 96-well plate (Thermo, Ltd.), the compositions comprising thymosin b4 with/without an additive are added on the plate. The additive includes a P2X7 purinergic receptor activator (e.g., ATP, human cathelicidin-derived peptide LL37, BzATP, 2MeSATP, ATPyS, or combinations thereof), a calcium influx enhancer(e.g., ATP, EGF, PTH, PKC, or combinations thereof), and/or a phosphorylated ERK activator (ATP, EGF, dinucleotides (e.g., diadenosine polyphosphates), or combinations thereof). 0 h, 6 h, 12 h, 24 h, and 48 h after administering the compositions comprising thymosin b4 with/without an additive,

10 pi of CCK-8 reagent is added to the plate, followed by incubation at 37° C for 2 hours. Optical density (OD450) at 450 nm is measured by using a microplate reader.

Tb4 at about 1-1000 ng/ml significantly increases the proliferation of HCECs over that of untreated HCECs. The compositions comprising Tb4 and an additive (e.g., a P2X7 purinergic receptor activator, a calcium influx enhancer, and/or a phosphorylated ERK activator) synergistically promote the cell proliferation, compared to the composition comprising thymosin b4 only or the additive only.

Synergistic Effects of the TB4 compositions on extracellular ATP, Ca²⁺ influx, and ERK phosphorylation in HECEs. The HCEC extracellular ATP level, intracellular Ca²⁺ influx, and phosphorylation of ERK1/2 protein are assessed. The ATP level, intracellular Ca²⁺ influx, and expression of phosphorylated ERK protein increase after the Tb4 treatment. 1000 ng/ml of Tb4 causes the largest Ca²⁺ influx into the HCECs, the release of extracellular ATP from HCECs ane phosphorylated ERK expression.

The compositions comprising Tb4 and an additive (e.g., a P2X7 purinergic receptor activator, a calcium influx enhancer, and/or a phosphorylated ERK activator) synergistically promote the Tb4 mediated therapeutic effects. Tb4 at 1, 10, 100, and 1000 ng/ml with the additive synergistically increases the HCEC extracellular ATP level, intracellular Ca²⁺ influx, and phosphorylation of ERK1/2, compared to the Tb4-oh1g treated group or the additive-only treated group. The additive includes a P2X7 purinergic receptor activator (e.g., ATP, human cathelicidin-derived peptide LL37, BzATP, 2MeSATP, ATPyS, or combinations thereof), a calcium influx enhancer (e.g., ATP, EGF, PTH, PKC, or combinations thereof), and/or a phosphorylated ERK activator (ATP, EGF, dinucleotides (e.g., diadenosine polyphosphates), or combinations thereof). * * *

All patents, patent applications, publications, product descriptions, and protocols, cited in this specification are hereby incorporated by reference in their entireties. In case of a conflict in terminology, the present disclosure controls.

While it will become apparent that the subject matter herein described is well calculated to achieve the benefits and advantages set forth above, the presently disclosed subject matter is not to be limited in scope by the specific embodiments described herein. It will be appreciated that the disclosed subject matter is susceptible to modification, variation, and change without departing from the spirit thereof. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for accelerating the healing of a damaged eye or skin in a subject, comprising:

activating a P2X7 purinergic receptor of a cell of an affected eye or skin of the subject by contacting the cell with a composition containing a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants thereof, in an effective amount to accelerate the healing of the damaged eye or skin in the subject.

2. The method of claim 1, wherein the cell is an epithelial cell.

3. The method of claim 1 or 2, wherein the composition comprises about 0.001% to about 1% by weight the peptide agents including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants thereof.

4. The method of any one of claims 1-3, wherein a rate of cell migration increases by at least about 30%.

5. The method of any one of claims 1-4, wherein the method further comprises increasing a calcium influx in the cell by about two-fold.

6. The method of any one of claims 1-5, wherein the method further comprises increasing expression of phosphorylated ERK protein of the cell by at least about 15%.

7. The method of any one of claims 1-6, wherein the method further comprises delivering a P2X7 purinergic receptor activator, a phosphorylated ERK protein activator, a calcium enhancer, or combinations thereof for synergistically improving a cell migration or a cell proliferation.

8. The method of any one of claims 1-7, wherein a proliferation rate of the cell increases by at least about 20%.

9. A method of treating epithelial damage in a subject, comprising:

delivering a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants thereof to a damaged eye or skin of the subject; and

delivering a P2X7 purinergic receptor activator to the eye or skin of the subject,

wherein the peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants thereof and the P2X7 purinergic receptor activator are delivered in an amount effective to treat epithelial damage in the subject.

10. The method of claim 9, wherein the P2X7 purinergic receptor activator includes adenosine triphosphate.

11. The method of claim 9 or 10, wherein the P2X7 purinergic receptor activator and the peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants thereof are formulated as a solution, suspension, semi-liquid, semi-solid gel, gel, ointment, spray, or cream.

12. The method of claim 11, wherein the solution, suspension, semi-liquid, semi solid gel, gel, ointment, spray, or cream comprises about 0.001% to about 1% by weight the peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants thereof.

13. The method of any one of claims 9-12, wherein the P2X7 purinergic receptor activator is delivered simultaneously with the peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants thereof.

14. The method of any one of claims 9-13, wherein the P2X7 purinergic receptor activator and the peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants thereof are delivered sequentially.

15. The method of any one of claims 9-14, wherein the delivering the P2X7

purinergic receptor activator and the peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants thereof elicits a synergistic enhancement of a calcium influx or a phosphorylated ERK protein expression on an epithelial cell of the affected eye or skin.

16. A composition comprising:

an effective amount of a peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants thereof to increase proliferation and migration of an epithelial cell through P2X7 purinergic receptor.

17. The composition of claim 16, further comprising an additive.

18. The composition of claim 16 or 17, further comprising a P2X7 purinergic

receptor activator.

19. The composition of any one of claims 16-18, further comprising a

pharmaceutically or topically acceptable carrier.

20. The composition of claim 19, wherein the carrier is selected from the group consisting of alkanols, vegetable oils, and mineral oils.

21. The composition of claim 18, wherein the P2X7 purinergic receptor activator includes adenosine triphosphate.

22. The composition of any one of claims 16-21, wherein the composition is

formulated as a solution, suspension, semi-liquid, semi-solid gel, gel, ointment, spray, or cream.

23. The composition of any one of claims 16-22, wherein the composition is

delivered to a damaged eye or skin of the subject.

24. The composition of any one of claims 16-23, wherein the composition comprises about 0.001% to about 1% by weight the peptide agent including amino acid sequence LKKTET (SEQ ID NO: 1) or LKKTNT (SEQ ID NO: 2), or variants thereof.