Pentapeptide and method for using the same

Abstract

A synthetic peptide containing the sequence SHXGY (SEQ ID NO: 2) has been described, as has its use to promote wound healing and epithelial cell migration.

Description

Technical Field

[0001] Introduction This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 941,226, filed on November 27, 2019, the content of which is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under Grant Numbers EY024339 and EY029409 awarded by the National Institutes of Health; W81XWH-17-1-0122 awarded by the Department of Defense; and I01BX004080 awarded by the Department of Veterans Affairs. The government has certain rights in the invention.

Background Art

[0003] Background Histatins (HTNs) are histidine-rich cationic small peptides found in saliva as well as in human lacrimal gland epithelium (Aakalu, et al. (2014) Invest. Ophthalmol. Vis. Sci. 55:3115; Ubels, et al. (2012) Invest. Ophthalmol. Vis. Sci. 53(11):6738-47; Steele, et al. (2002) Invest. Ophthalmol. Vis. Sci. 43:98). Histatins range in size from 7 to 38 amino acid residues and represent a group of antimicrobial peptides with antibacterial and pronounced antifungal properties. In addition, histatins have been implicated in wound healing, metal ion chelation, anti-inflammatory effects, and angiogenesis (Melino, et al. (2014) FEBS J. 281:657-72; Oudhoff, et al. (2008) FASEB J. 22(12):3805-12); Oudhoff, et al. (2009) J. Dent. Res. 88(9):846-50; WO 2007 / 142381). Structure-function studies have identified distinct N-terminal and C-terminal domains in both HTN1 and HTN3, which contribute to antimicrobial and wound healing properties, respectively (Melino, et al. (1999) Biochemistry 38:9626-33; Brewer, et al. (1998) Biochem. Cell Biol. 76:247-56; Gusman, et al. (2001) Biochim. Biophys. Acta 1545:86-9). In this regard, histatins, as well as fragments, multimers, and combinations thereof, have been suggested for use in the treatment of various conditions including ocular surface diseases (US 2013 / 0310327;2013 / 0310326;WO 2016 / 060916;WO 2016 / 060917;WO 2016 / 060918;WO 2016 / 060921;US 2016 / 0279194) and wounds (US 2013 / 0288964;US 2011 / 0178010).

[0004] Cyclic analogs of histatin have also been described. For example, US 6,555,650 describes cyclic analogs of HTN5 with disulfide bridges that construct a cyclic moiety of 5 to 16 of the amino acid units. In addition, head-to-tail cyclization of HTN5 has been shown to increase the amphiphilicity of the peptide without affecting its antibacterial ability (Sikorska & Kamysz (2014) J. Pept. Sci. 20:952-7). Furthermore, cyclization of histatin-1 has been shown to enhance the molar activity approximately 1000-fold (Oudhoff, et al. (2009) FASEB J. 23:3928-35) and increase the wound closure activity (Bolscher, et al. (2011) FASEB J. 25:2650-8). Moreover, cyclic analogs of histatin have been proposed for use in the treatment of microbial infections due to their enhanced ability (US 2010 / 0173833; Brewer & Lajoie (2002) Biochemistry 41:5526-5536). SUMMARY OF THE INVENTION

[0005] The present invention provides a synthetic peptide, or a pharmaceutically acceptable salt thereof, wherein the peptide has a structure of formula I:

Number

Brief Description of the Drawings

[0006]

Figure 1

[0007]

Figure 2

[0008]

Figure 3

Best Mode for Carrying Out the Invention

[0009] Detailed Description of the Invention Epithelial migration, adhesion, and proliferation are extremely important for wound healing in all areas of the body. Without sufficient epithelialization, ectopic wound healing can be seen as a result of such things as wounds, inflammation, pain, contracture, vision loss, dysfunction, infection, and abnormal angiogenesis. Several cell types and wound models are generally used to test the applicability of agents for enhancing wound healing. Exemplary systems are the anterior part of the eye and the ocular surface. The cornea and other elements of the front of the eye are used as models of wound healing because abnormal epithelialization can lead to tragic blindness or even loss of the eye. Here, it has been found that the pentapeptide SHXGY (SEQ ID NO: 1) can enhance epithelial wound healing. In particular, peptides containing the sequence SHXGY (SEQ ID NO: 1), for example, SHRGY (SEQ ID NO: 2) and SHDGY (SEQ ID NO: 3), can significantly enhance epithelial migration in several cell types (immortalized human corneal epithelial cells, immortalized human corneal limbal epithelial cells, HeLa human cells), and in a model of mouse corneal epithelial wounds. When topically applied to mice wounded using a standardized wounding method, SHXGY (SEQ ID NO: 1)-containing peptides or their multimers (for example, SHRGY-(CH 2 ) 6 -SHRGY-(CH 2 ) 6 -SHRGY-(CH 2 ) 6-SHRGY (SEQ ID NO: 51) can significantly improve corneal healing. In addition, a synthetic peptide containing the SHRGY (SEQ ID NO: 2) sequence has been shown to stimulate the immunoreactivity of phosphorylated extracellular signal-regulated protein kinases 1 / 2 (pERK1 / 2) against the site of wound healing, suggesting that in addition to its function in enhancing wound healing, the SHRGY (SEQ ID NO: 2) peptide also has immunomodulatory activity. Therefore, this core pentapeptide sequence is particularly used in promoting epithelialization and cell migration, which is important for the response to infection or impairment among wound healing, inflammation, cancer, and other phenomena.

[0010] Accordingly, the present invention provides a synthetic peptide, or a pharmaceutically acceptable salt thereof, and a method for using the same in promoting wound healing and / or epithelial cell migration. The synthetic peptide of the present invention has a general structure of Formula I:

Number

[0011] As indicated, R 1 and R 2At least one of them is a peptide of 5 to 10 amino acid residues containing the amino acid sequence SHXGY (SEQ ID NO: 1), where X is R (Arg), K (Lys), H (His), D (Asp), or E (Glu). Thus, R 1 and R 2 At least one of them is a peptide of 5, 6, 7, 8, 9, or 10 amino acid residues containing the amino acid sequence SHRGY (SEQ ID NO: 2), SHDGY (SEQ ID NO: 3), SHKGY (SEQ ID NO: 4), SHHGY (SEQ ID NO: 5), or SHEGY (SEQ ID NO: 6). R 1 or R 2 At least one of them contains the sequence SHXGY (SEQ ID NO: 1), which may have 1 to 5 additional amino acid residues on the C-terminus and / or N-terminus. In some aspects, the 1 to 5 additional amino acid residues are endogenous or natural amino acid residues. A "natural" or "endogenous" amino acid residue is an amino acid residue that is present at the specified position in a naturally occurring protein. By way of illustration, the sequence SHRGY (SEQ ID NO: 2) is present in histatin 3 as follows: DSHAKRHHGYKRKFHEKHH SHRGY RSNYLYDN (SEQ ID NO: 7). Thus, R 1 and / or R 2 When derived from histatin, R 1 and / or R 2 can have the sequence HH SHRGY RSN (SEQ ID NO: 8), HEKHH SHRGY (SEQ ID NO: 9), EKHH SHRGY R (SEQ ID NO: 10), KHH SHRGY (SEQ ID NO: 11), HH SHRGY (SEQ ID NO: 12), or H SHRGY (SEQ ID NO: 13).

[0012] In some aspects, the synthetic peptide consists of only R 1 (i.e., n = 0). According to this aspect, the synthetic peptide is a peptide of 5, 6, 7, 8, 9, or 10 amino acid residues containing or consisting of the sequences defined in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0013] On the other hand, the synthetic peptide contains one or more R 2 peptides (i.e., n ≧ 1). In this regard, the synthetic peptide can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more peptides linked by a linker. In one aspect, the R 1 and R 2 of the synthetic peptide of the present invention are the same. In another aspect, the R 1 and R 2 of the synthetic peptide of the present invention are different. In a further aspect, each R 2 can be the same or different. Ideally, the total length of the synthetic peptide is in the range of 20 to 100 amino acid residues.

[0014] R 1 or R 2 At least one of them is a 5- to 10-amino acid residue peptide having the amino acid sequence SHXGY (SEQ ID NO: 1), while the other of R 1 or R 2 may be a peptide to which a metal is bound, a wound healing peptide, or an antibacterial peptide. In this regard, the synthetic peptide of the present invention may be composed of (i) a peptide to which a metal is bound, (ii) a wound healing peptide, (iii) an antibacterial peptide, or (iv) a 5- to 10-amino acid residue peptide having the amino acid sequence SHXGY combined with any combination of (i)-(iii). In one aspect, the synthetic peptide of the present invention is composed of a 5- to 10-amino acid residue peptide having the amino acid sequence SHXGY combined with a second wound healing peptide.

[0015] As used herein, the term "peptide that binds to a metal" refers to a motif of amino acids that binds to a metal or forms a complex. Evaluation of the structural and functional properties of histatin has revealed the presence of the following two metal-binding motifs: an amino-terminal Cu(II) / Ni(II)-binding (ATCUN) motif (NH 2 -X 1 X 2 H, where X 1 is Asp or Glu, and X 2 is Ala, Thr, Met, or Ser) (Grogan, et al. (2001) FEBS Lett. 491:76-80; Melino, et al. (2006) Biochemistry 45:15373-83; Melino, et al. (1999) Biochemistry 38:9626-33; Gusman, et al. (2001) Biochim. Biophys. Acta 1545:86-95); and a Zn(II)-binding motif HEXXH (SEQ ID NO: 14), where X represents a basic amino acid residue such as K (Lys), R (Arg), or H (His). Thus, in some embodiments, the peptide to which the metal is bound comprises the sequence DSH, ESH, DAH, EAH, DTH, ETH, DMH, or EMH. In other embodiments, the peptide to which the metal is bound comprises the sequence HEKKH (SEQ ID NO: 15), HEKRH (SEQ ID NO: 16), HEKHH (SEQ ID NO: 17), HERKH (SEQ ID NO: 18), HERRH (SEQ ID NO: 19), HERHH (SEQ ID NO: 20), HEHKH (SEQ ID NO: 21), HEHRH (SEQ ID NO: 22), or HEHHH (SEQ ID NO: 23). The peptide to which the metal is bound can comprise a specific sequence of the peptide to which the metal is bound as described above or can comprise 1 to 6 additional native histatin amino acid residues at the C-terminus and / or the N-terminus of the peptide to which the metal is bound. By way of illustration, the peptide to which the metal is bound can have the sequence GYKRKF HEKHH SHR (SEQ ID NO: 24) or HEKRH H (SEQ ID NO: 25).

[0016] In some embodiments, the synthetic peptides of the invention include peptides to which one metal is bound. In other embodiments, the synthetic peptides include peptides to which two metals are bound. In further embodiments, the synthetic peptides include peptides to which three metals are bound. In one embodiment, the peptide to which a metal is bound has the sequence HEXXH (SEQ ID NO: 14), where each X is a basic amino acid residue. As will be readily recognized by those skilled in the art, the inclusion of peptides to which one or more metals are bound in the synthetic peptides confers metal ion chelation, anti-inflammatory, matrix metalloproteinase inhibition, and / or anti-angiogenic activity to the synthetic peptides. In light of its anti-angiogenic activity, such synthetic peptides would be used in the treatment of age-related macular degeneration, diabetic retinopathy, cancer, and chronic or acute severe uveitis. In light of its metal ion chelating activity, such synthetic peptides would also be used in inhibiting tissue destruction mediated by matrix metalloproteinases and other metal-dependent enzymes in inflammatory and infectious diseases such as infectious keratitis, endophthalmitis, intraocular uveitis, inflammatory keratitis, dry eye disease, and ocular surface or intraocular disease conditions.

[0017] As used herein, "wound healing peptide" refers to an amino acid motif that promotes or accelerates wound healing. In some aspects, the wound healing peptide is derived from histatin. An example of a wound healing peptide derived from histatin is a peptide comprising the sequence SNYLYDN (SEQ ID NO: 26). In another aspect, the wound healing peptide comprises the amino acid sequence SHXGY (SEQ ID NO: 1), where X is R, K, H, D, or E. Notably, when included in the synthetic peptides of the present invention, the SHXGY (SEQ ID NO: 1) sequence has the additional benefit of conferring immunomodulatory activity to the synthetic peptide. The wound healing peptide can comprise the specific sequences of the wound healing peptides described above, or can comprise additional amino acid residues between 1 and 6 on the C and / or N termini of the wound healing peptide. By way of illustration, a wound healing peptide derived from histatin can have the sequence YGDYG SNYLYDN (SEQ ID NO: 27) or any one of SEQ ID NO: 2 or 8 - 13.

[0018] In some embodiments, in addition to the wound healing peptide of SEQ ID NO: 1, the synthetic peptides of the present invention comprise a second wound healing peptide. In other embodiments, in addition to the wound healing peptide of SEQ ID NO: 1, the synthetic peptide comprises two additional wound healing peptides. In further embodiments, in addition to the wound healing peptide of SEQ ID NO: 1, the synthetic peptide comprises three additional wound healing peptides. As will be readily appreciated by those skilled in the art, the inclusion of one or more wound healing peptides in the synthetic peptide results in the synthetic peptide having enhanced epithelial cell migration and spreading. Such synthetic peptides are therefore used in the treatment of wound healing as well as retinal pigment epithelial healing, dry age-related macular degeneration, ocular surface diseases and ocular surface inflammatory disorders, neovascularization of the eye including the cornea and intraocular, retinal or choroidal, and dry eye disease.

[0019] With respect to the object of the present invention, "antibacterial" includes antibacterial and antifungal agents. Thus, as used herein, the term "antibacterial peptide" refers to a motif of amino acids that exhibits cell growth inhibitory or cytotoxic activity against bacterial and / or fungal cells. Characterization of histatin indicates that the positive net charge and the amino-terminal portion of HTN mediate antibacterial activity. In particular, the amino acid sequence RKFHEKHHSHRGYR (SEQ ID NO: 28) of HTN3 has been shown to exhibit antifungal activity (Oppenheim, et al. (2012) PLoS ONE 7(12):e51479). Similarly, the sequence AKRHHGYKRKFH (SEQ ID NO: 29), also known as P-113, exhibits antifungal activity against Candida albicans (Jang, et al. (2008) Antimicrob. Agents Chemother. 5292):497-504). Thus, an antibacterial peptide can comprise a specific sequence of the antibacterial peptides described above, or can comprise additional amino acid residues between 1 and 6 on the C and / or N terminus of the antibacterial peptide.

[0020] In some embodiments, the synthetic peptide comprises one antibacterial peptide. In other embodiments, the synthetic peptide comprises two antibacterial peptides. In further embodiments, the synthetic peptide comprises three antibacterial peptides. In one embodiment, the antibacterial peptide has the sequence RKFHEKHHSHRGYR (SEQ ID NO: 28). In other embodiments, the antibacterial domain has the sequence AKRHHGYKRKFH (SEQ ID NO: 29). As will be readily appreciated by those skilled in the art, the inclusion of one or more antibacterial peptides in a synthetic peptide confers antifungal and / or antibacterial activity to the synthetic peptide. Such synthetic peptides would therefore be used in the treatment of microbial infections such as Candida eye infections, as well as in the defense against infections associated with surgical grafts.

[0021] Examples of synthetic peptides containing identical or different repeating units are presented in Table 1.

Table 1

[0022] In one aspect of the present invention, exogenous or heterologous tissue molecules are included in the synthetic peptide. Specifically, in some aspects, the synthetic peptide directly attaches to one or both of R 1 and R 2 and includes the "Z" and / or "L" moieties associated with one or both of them, where both the "Z" and "L" moieties are exogenous or heterologous tissue molecules with respect to R 1 and R 2 . The terms "heterologous tissue molecule" or "exogenous molecule" refer to molecules that are not normally found in a peptide or that are not typically associated with the R 1 and / or R 2 amino acid sequence in nature.

[0023] In some aspects, the synthetic peptide includes the "Z" moiety. In other aspects, the "Z" moiety is absent. When present, Z is an exogenous peptide as defined herein. According to this aspect, Z is a peptide of 1 to 50 amino acid residues, or preferably 1 to 30 amino acid residues, or more preferably 1 to 20 amino acid residues, where the exogenous peptide may or may not have a function. By way of illustration, the exogenous peptide may exhibit metal binding, wound healing, immunomodulatory, and / or antibacterial activity, or, by way of example, a random peptide sequence as in the SP2 peptide HSHKEGHHYKRFKRKHHAD SHRGY (SEQ ID NO: 70). In one aspect, Z is a random peptide sequence of 1 to 50, 1 to 30, or 1 to 20 amino acid residues.

[0024] As used herein, the term "L" or "linker" or "spacer" connects, joins, or binds R 1 to R 2 , and individual R 2Refers to a molecule of a heterologous tissue or an exogenous molecule used to connect, link, or join moieties. As used herein, the terms "linked," "joined," or "connected" generally refer to a functional linkage between two contiguous or adjacent amino acid sequences to produce a molecule that does not occur naturally. Generally, the linked amino acid sequences are contiguous or adjacent to each other and retain their respective operability and function when joined. A linker may provide desired mobility to enable positioning of the desired expression, activity, and / or conformation of the synthetic peptide.

[0025] In some embodiments, the synthetic peptide comprises one linker, i.e., n = 1. In other embodiments, the synthetic peptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 linkers, i.e., n = 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In one aspect, each occurrence of the linker (L) may comprise the same or a different linker.

[0026] The linker for use in the synthetic peptide of Formula I can be flexible, rigid, cleavable in vivo, or a combination thereof. Additionally, the linker can be composed of amino acid residues (i.e., a peptide linker) or a hydrocarbon chain (i.e., a hydrocarbon linker). The peptide linker can be of any suitable length to connect the 1 and R 2 or individual R 2 moieties and is preferably 1 and R 2is designed to allow for proper folding and / or function and / or activity. Thus, the linker peptide can have a length of 3 or less, 5 or less, 10 or less, 15 or less, 20 or less, 25 or less, 30 or less, 35 or less, 40 or less, 45 or less, 50 or less, 55 or less, or 60 or less amino acids. In some embodiments, the linker peptide can have a length of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids. In some embodiments, the linker comprises at least 10 and 60 or less amino acids, at least 10 and 55 or less amino acids, at least 10 and 50 or less amino acids, at least 10 and 45 or less amino acids, at least 10 and 40 or less amino acids, at least 10 and 35 or less amino acids, at least 10 and 30 or less amino acids, at least 10 and 25 or less amino acids, at least 10 and 20 or less amino acids, or at least 10 and 15 or less amino acids.

[0027] A "flexible" linker refers to a hydrocarbon or peptide linker that has no fixed structure (secondary or tertiary structure) in solution. Such a flexible linker can thus freely adopt various conformations. Flexible linkers used herein include hydrocarbon linkers and peptide linkers composed of small, non-polar (e.g., Gly) and / or polar (e.g., Ser or Thr) amino acid residues. Simple amino acids (e.g., simple side chains (e.g., H, CH 3 , or CH 2Amino acids having (OH) are suitable for use as peptide linkers because the lack of branched side chains on these amino acids provides greater mobility (e.g., two-dimensional or three-dimensional mobility) in the linker and, thus, in the polypeptide composition. The mobile linker may contain additional amino acids such as Thr and Ala to maintain mobility and polar amino acids such as Lys and Glu to improve solubility. The amino acids can be substituted / repeated in any consistent manner that maintains the function (e.g., results in an expressed and / or active polypeptide(s)). Mobile linkers are described, for example, in Chen, et al. (2013) Adv. Drug Deliv. Rev. 65(10):1357-1369; US 2012 / 0232021; US 2014 / 0079701; W0 1999 / 045132; WO 1994 / 012520 and WO 2001 / 1053480.

[0028] In certain aspects, the mobile linker is a hydrocarbon linker. R 1 and R 2 or individual R 2 The hydrocarbon that links the moieties should have sufficient length and mobility so that the synthetic peptide can achieve the desired conformation. In one embodiment, the hydrocarbon is composed of one or more methylene (-CH 2 2 2 ) groups. In one embodiment, the hydrocarbon contains between 3 and 25 methylene groups, i.e., -(CH n 2 2 ) 6 n-, where n is from 3 to 25. In one embodiment, the hydrocarbon linker has the structure -(CH 2 ) 6 n-. Additional carbon-based linkers such as glycol linkers can also be used in the synthetic peptides of the invention.

[0029] In other embodiments, the linker is a fixed linker. A "fixed" linker refers to a molecule that adopts a relatively well-defined conformation when in solution. A fixed linker is thus one that has a specific secondary and / or tertiary structure in solution. Fixed linkers typically have a size sufficient to confer a secondary or tertiary structure on the linker. Such linkers include aromatic molecules (see, e.g., US 6,096,875 or US 5,948,648), proline-rich peptide linkers, or peptide linkers with immobile helix structures. Fixed linkers are described, for example, in Chen, et al. (2013) Adv. Drug Deliv. Rev. 65(10):1357-1369; US 2010 / 0158823, and US 2009 / 10221477.

[0030] In other embodiments, the linker is an in vivo cleavable linker. An in vivo cleavable linker can include a cleavable disulfide bond formed between two cysteine residues or, for example, a linker having a protease recognition sequence recognized by a matrix metalloprotease (MMP).

[0031] Examples of suitable peptide linkers for use in synthetic peptides are provided in Table 2.

Table 2

[0032] Each of the individual linkers of the synthetic peptides of the present invention can be the same or different. In some embodiments, the synthetic peptide comprises at least one flexible linker. In some embodiments, the at least one flexible linker is a hydrocarbon linker. In other embodiments, the at least one flexible linker is a peptide linker. In certain embodiments, each linker of the synthetic peptide is a hydrocarbon linker. In one embodiment, each linker of the synthetic peptide has the structure -(CH 2 ) 6 -.

[0033] Examples of synthetic peptides containing combinations of repeat units and flexible linkers are presented in Table 3. [Table 3]

[0034] In some aspects, the synthetic peptides of the present invention are prepared as pharmaceutically acceptable salts. As used herein, the term "pharmaceutically acceptable salts" refers to those salts of the synthetic peptides that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. See, for example, Berge, et al. (1977) J. Pharmaceutical Sciences 66:1-19. The salts can be prepared in situ during the final isolation and purification of the peptides of the present invention, or by separately reacting the free base with a suitable organic acid. Examples of pharmaceutically acceptable salts include those formed with an amino group and an inorganic acid such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or an organic acid such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or non-toxic acid addition salts by using other methods used in the art such as ion exchange, but are not limited thereto. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfonate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, peroxydisulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc., but are not limited thereto.Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts, where appropriate, include non-toxic ammonium, quaternary ammonium, and amine cations formed with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl having from 1 to 6 carbon atoms, sulfonates, and arylsulfonates.

[0035] The synthetic peptides described herein are generally referred to as fusion or chimeric peptides. Such molecules can be synthesized by routine methods including recombinant protein expression, chemical synthesis, or combinations thereof. In some embodiments, the synthetic peptides of the invention are synthesized recombinantly using recombinant DNA technology. Accordingly, the invention provides polynucleotides encoding the synthetic peptides of the invention. In a related aspect, the invention provides vectors, particularly expression vectors containing polynucleotides encoding the synthetic peptides of the invention. In one embodiment, the vector provides replication, transcription, and / or translation control sequences that facilitate the recombinant synthesis of the desired synthetic histatin in eukaryotic or prokaryotic cells. Thus, the invention also provides host cells for the recombinant expression of synthetic peptides, and methods for recovering and purifying the synthetic peptides produced by the host cells. The production and purification of recombinant peptides are routine practices for those skilled in the art, and any suitable methodology can be used.

[0036] In another aspect, the synthetic peptide is synthesized by any of the chemical substance synthesis techniques known in the art, particularly by solid-phase synthesis techniques, for example, using a commercially available automated peptide synthesizer. See, for example, Stewart & Young (1984) Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co.; Tarn, et al. (1983) J. Am. Chem. Soc. 105:6442-55; Merrifield (1986) Science 232:341-347; and Barany et al. (1987) Int. J. Peptide Protein Res. 30:705-739.

[0037] The synthetic peptide can be isolated and / or purified by any suitable method known in the art, including but not limited to gel filtration and affinity purification. In some aspects, the synthetic peptide is generated with a tag, such as an epitope tag, to facilitate isolation of the synthetic peptide. In one aspect, the synthetic peptide is at least 1% pure as determined by SDS-PAGE, for example at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure. Once isolated and / or purified, the properties of the synthetic peptide can be readily confirmed by techniques well known to those skilled in the art.

[0038] Derivatives and analogs of the synthetic peptides described herein can all be predicted and created by changing their amino acid sequences by substitution, addition, and / or deletion / cleavage, or by introducing chemical modifications that result in functionally equivalent molecules. It will be well understood by those skilled in the art that an amino acid in the sequence of any polypeptide may be substituted with another amino acid without adversely affecting the activity of the polypeptide.

[0039] In one aspect, the synthetic peptides of the present invention include, but are not limited to, one or more modifications including phosphorylation, glycosylation, hydroxylation, sulfonation, amidation, acetylation, carboxylation, palmitoylation, PEGylation, introduction of non-hydrolyzable bonds, and disulfide formation. The modifications may improve the stability and / or activity of the synthetic peptides.

[0040] For example, the C-terminus may be modified by amidation, addition of peptide alcohols and aldehydes, addition of esters, or addition of p-nitroaniline and thioesters. The N-terminus and side chains may be modified by PEGylation, acetylation, formylation, addition of fatty acids, addition of benzoyl, addition of bromoacetyl, addition of pyroglutamyl, succinylation, addition of tetrabutyoxycarbonyl, and addition of 3-mercaptopropyl, acylation (as an example, lipopeptide), biotinylation, phosphorylation, sulfation, glycosylation, introduction of maleimide groups, chelation of moieties, chromophores, or fluorophores.

[0041] In one aspect, the synthetic peptide is conjugated to a fatty acid. For example, the synthetic peptide is myristoylated. For example, the fatty acid may be conjugated to the N-terminus of the synthetic peptide. Such fatty acids include caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, and the like. Furthermore, the cysteine of the synthetic peptide can be palmitoylated. In one aspect, the synthetic peptide is myristoylated, stearylated, or palmitoylated with the N-terminal amino acid.

[0042] In addition, or as a variant, for post-translational modification, the synthetic peptide can be conjugated or linked to another peptide, such as a carrier peptide. The carrier peptide may promote cell-penetration and can include peptides such as antennapedia peptide, penetratin peptide, TAT, transportan, or polyarginine. In one embodiment, the synthetic peptide is conjugated or linked to the antennapedia peptide, RQIKIWFQNRRMKWKK (SEQ ID NO: 56).

[0043] The synthetic peptide of the present invention may be cyclized. As used herein, the terms "cyclized" or "cyclic" refer to analogs of linear peptides that incorporate at least one cross-linking group (e.g., amide, thioether, thioester, disulfide, urea, carbamate, hydrocarbon, or sulfonamide) between amino acid residues to form a cyclic structure. The cross-linking group can be present on a side-chain amino acid residue or a terminal amino acid residue, thereby providing side-chain cyclization (e.g., lactam cross-link, thioester), head-to-tail cyclization, or hydrocarbon stapled peptides.

[0044] In one embodiment, the cyclic synthetic peptide has a disulfide bridge between two terminal cysteine residues. Representative amino acid sequences for preparing the cyclized synthetic peptide are provided in Table 4.

Table 4

[0045] In other aspects, the cyclic synthetic peptides are prepared from linear peptides by cyclization with sortase. "Cyclization with sortase" or "cyclized with sortase" refers to a method of cyclizing a linear peptide using the enzyme sortase. Sortase-based cyclization is known in the art for producing large cyclic peptides. See Bolscher, et al. (2011) FASEB J. 25(8):2650-2658, and the references cited therein.

[0046] Butelase cyclization has also been used to cyclize peptides. The addition of a C-terminal tripeptide Asn-his-Val motif provides a substrate for butelase to cyclize the synthetic peptide at a rate significantly faster than that of sortase A. See Nguyen, et al. (2016) Nat. Protocols 11:1977-88; Tam, et al. (June 2015) Peptides 2015: Proc. 24th Am. Pept. Symp., Orlando, FL, pg. 27.

[0047] One of ordinary skill in the art will recognize that the synthetic peptides of the present invention are beneficial for the treatment of diseases. Accordingly, to facilitate administration, the present invention also provides a composition containing one or more endogenous and / or synthetic peptides, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical compositions provided herein can be formulated for oral, ocular, intravenous, intravitreal, subconjunctival, subcutaneous, intramuscular, intraperitoneal, intracerebral, intraarterial, intraportal, intralesional, intrathecal, intranasal administration or topical administration. Suitable pharmaceutical compositions can be determined by one of ordinary skill in the art, depending, for example, on the intended route of administration, delivery format, and desired dosage. See, for example, Remington's Pharmaceutical Sciences (19th edition, 1995).

[0048] The synthetic peptide(s) can be incorporated in conventional dosage forms such as gels, diluents, creams, tablets, capsules, pills, solutions, eye drops, sprays, bandages, contact lenses, reservoirs, injectables, implants, or sustained release formulations. The dosage forms may contain the necessary physiologically acceptable carrier materials, excipients, lubricants, buffers, surfactants, antibacterial agents, fillers (such as mannitol), antioxidants (ascorbic acid or sodium bisulfite) or other such kind.

[0049] Acceptable pharmaceutical materials are preferably non-toxic to the recipient at the dosages and concentrations employed. The pharmaceutical compositions may contain, for example, pharmaceutical materials for modifying, maintaining, or preserving pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or permeation of the composition. Suitable pharmaceutical materials include, but are not limited to: amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antibacterial substances; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); coloring agents, flavoring agents, and diluents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as polysorbates such as PLURONICS, PEG, sorbitan esters, polysorbate 20 and polysorbate 80, TRITON, trimethamine, lecithin, cholesterol, or tyloxapol); stability enhancers (such as sucrose or sorbitol); isotonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium, mannitol, or sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. See, e.g., Remington's Pharmaceutical Sciences, Id.

[0050] The first carrier or excipient of the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, suitable carriers or excipients may be distilled water for injection, physiological saline solution, or artificial cerebrospinal fluid, supplemented with other materials, which are probably common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary excipients. The pharmaceutical composition can contain a Tris buffer at about pH 7.0 - 8.5, or an acetate buffer at about pH 4.0 - 5.5, which may further contain sorbitol or a suitable substitute. The pharmaceutical composition of the present invention may be prepared for storage by mixing a selected composition having the desired degree of purity with any formulation (Remington's Pharmaceutical Sciences, Id.) in the form of a lyophilized cake or an aqueous solution. Further, the synthetic peptide of the present invention may be formulated as a lyophilized product using a suitable excipient such as sucrose.

[0051] The routes of administration for the pharmaceutical composition of the present invention include the oral route; injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intra-arterial, intraportal, or intralesional routes; or via a sustained release system or by means of an implant device. The pharmaceutical composition may be administered by bolus injection, or continuously by infusion, or by an implant device. The pharmaceutical composition can also be administered locally via the implantation of a membrane, sponge, or another suitable material that has absorbed or encapsulated the synthetic histamine(s). Where an implant device is used, the device may be implanted in any suitable tissue or organ, and the delivery of the endogenous or synthetic histamine(s) may be via diffusion, sustained release bolus, or continuous administration.

[0052] When parenteral administration is intended, the compositions used for the purposes of the present invention may be in the form of a parenterally acceptable aqueous solution containing the endogenous or synthetic histatin(s) of the present invention in a pyrogen-free, pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral infusion is sterile distilled water in which the synthetic peptide(s) is formulated as a sterile isotonic solution and appropriately preserved. Preparation may involve the formulation of the synthetic peptide(s) with an agent that may provide for the controlled or sustained release of the synthetic peptide(s), such as injectable microspheres, bio-erodible particles, high molecular weight compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which may then be delivered via depot injection. In particular, formulations with hyaluronic acid have the effect of promoting the duration in the bloodstream.

[0053] The compositions may be formulated for inhalation. In these embodiments, the synthetic peptide(s) of the present invention may be formulated as a dry powder for inhalation, or the inhalation solution may be formulated with a propellant for aerosol delivery, such as by nebulization therapy. Pulmonary administration is further described, for example, in WO1994 / 020069.

[0054] The pharmaceutical compositions of the present invention can be delivered through the gastrointestinal tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. The synthetic peptide(s) of the present invention administered in this manner may be formulated with or without carriers customarily used in the formulation of solid dosage forms such as tablets and capsules. Capsules may be designed to release the active portion of the formulation at a location in the gastrointestinal tract when bioavailability is maximized and presystemic degradation is minimized. Additional agents can be included to facilitate the absorption of the synthetic peptide(s). Diluents, flavorings, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0055] These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. The inhibition of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenolsorbic acid, and others of the same kind. It may be desirable to include isotonic agents such as sugars, sodium chloride, and others of the same kind. The long-term absorption of an injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0056] In certain embodiments, the synthetic peptide(s) is formulated to treat eye diseases or conditions both on and inside the ocular surface. In particular embodiments, the synthetic peptide(s) of the present invention may be formulated and administered to the eye as follows: in the form of drops; in the form of a topical gel; as a solid formulation (by way of example, similar to LACRISERT, an ocular insert of hydroxypropylcellulose); by injection into the anterior chamber; by injection into the posterior chamber for the inhibition of angiogenesis, the inhibition of destructive MMP activity, or the enhancement of epithelial wound healing; by coating a surgical device (intraocular lens, glaucoma device, corneal patch, lacrimal duct tube, lacrimal bypass tube); by coating a contact lens; or by coating microbeads, nanobeads, or other similar constructs.

[0057] As will be appreciated by those skilled in the art, the compositions described herein can be formulated to minimize adverse side effects. The compositions described herein can be suitable for long-term use alone; useful as adjuvant therapy in combination with antibacterial agents (e.g., histatin, cystatin, lactoferrin, lactoferricin, LL-37), antiviral agents, antiparasitic agents, immunomodulatory agents (e.g., glucocorticoids, cyclosporine, NSAIDs), anti-wound agents (e.g., mitomycin C or similar antimetabolites), collagen, gelatin, analgesics, anesthetics, or combinations thereof; and / or involved in the circulation between any or all of these agents, thereby being useful in a program to reduce long-term exposure to any of the agents (and, therefore, resulting side effects).

[0058] The present invention also provides a pharmaceutical composition containing one or more synthetic peptides, or the same, and optionally a kit containing one or more antibacterial agents, antiviral agents, antiparasitic agents, immunomodulatory agents, anti-wound agents, collagen, gelatin, analgesics, or anesthetics. The kit is typically provided in a suitable container (e.g., a foil, plastic, or cardboard package). In certain embodiments, the kit may include one or more pharmaceutical excipients or carriers, pharmaceutical excipients, or the like, as described herein. In other embodiments, the kit may include means for appropriate administration, such as, for example, a graduated cup, syringe, needle, irrigation aid, intraocular lens, glaucoma device, orbital implant, corneal patch, lacrimal duct tube, lacrimal bypass tube, contact lens, and other like items. In certain embodiments, the kit may include instructions for appropriate administration and / or preparations for appropriate administration.

[0059] In view of promoting wound healing and epithelial cell migration, which is the activity of the synthetic peptides disclosed herein, the present invention also provides a method for promoting wound healing and / or epithelial cell migration by administering to a subject in need of such treatment an effective amount of one or more of the synthetic peptides of the present invention. As used herein, "subject" includes humans, as well as non-human animals, especially those having a disease or condition, which, by way of example, may benefit from the promotion of wound healing and / or epithelial cell migration as a treatment in the treatment of wounds or other body surface disorders related to epithelial defects and recurrent epithelial erosions, such as surgical wounds, excisional wounds, herpes, ulcers, other disorders, scratches, abrasion wounds, cuts, dirty wounds, scalds and thermal or corrosive burns. Such wounds can be caused by mechanical injury and other diseases such as diabetes, corneal dystrophy, uremia, malnutrition, vitamin deficiency, obesity, infection, immune deficiency, or complications associated with the systemic use of steroids, radiation therapy, non-steroidal anti-inflammatory drugs, and anti-cancer drugs.

[0060] Notably, it has been shown that synthetic peptides containing the SHRGY (SEQ ID NO: 2) sequence also increase ERK1 / 2 activation. Thus, the present invention also provides a method for increasing ERK activation by administering to a subject in need of such treatment an effective amount of one or more synthetic peptides of the present invention. It is well established that ERK regulation is important in both the innate and adaptive immune systems (Zhang & Dong (2005) Cell. Mol. Immunol. 2(1):20-27). As demonstrated herein, application of Hst5 increased the signal intensity of phosphorylated ERK1 / 2, indicating increased ERK1 / 2 activation by Hst5 in the wounded epithelium. In addition, untreated and SP1 peptide (lacking SHRGY (SEQ ID NO: 2))-treated samples had a similar localization pattern to pERK1 / 2, and Hst5- and SP2-treated samples increased the immunolocalization of pERK1 / 2 at the site of wound healing. This unexpected result indicates that the SHRGY (SEQ ID NO: 2) peptide, which has no known ability to regulate ERK to date and would not have been predicted to do so based on an understanding of functional domains, can confer immunomodulatory activity to the synthetic peptides disclosed herein.

[0061] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of the synthetic peptides of the invention, or a pharmaceutical composition containing the synthetic peptides, sufficient to achieve the desired result as specified. In some aspects, the effective amount provides a measurable improvement in epithelial cell migration rate, wound closure rate or time, and / or increase in ERK and survival pathway modulation, as compared to a subject not receiving such treatment. The amount of peptide that constitutes an "effective amount" or "therapeutically effective amount" may vary depending on the severity, condition, weight, or age of the disease of the patient being treated, the frequency of administration, or the route of administration, but can be determined routinely by those skilled in the art. Depending on the location and condition being treated, dosages in the range of 1 nanomole to 500 micromoles or more of the synthetic peptide may be used. The clinician may titer the dosage or route of administration to obtain an optimal therapeutic effect. Typical dosages are in the range of about 0.1 μg / kg to up to about 100 mg / kg, or more, depending on the above factors. In certain embodiments, the dosage may range from 0.1 μg / kg to up to about 100 mg / kg, or from 1 μg / kg to up to about 100 mg / kg, or from 5 μg / kg to up to about 100 mg / kg.

[0062] "Treating" a subject means achieving one or more of the following: (a) reducing the severity of a disease or condition; (b) arresting the progression of a disease or condition; (c) inhibiting the worsening of a disease or condition; (d) limiting or preventing recurrence of a disease or condition in a patient who has previously had the disease or condition; (e) causing regression of a disease or condition; (f) ameliorating or eliminating the symptoms of a disease or condition; and / or (g) improving survival.

[0063] According to the present invention, the synthetic peptide is used particularly in the treatment of eye diseases or conditions, including but not limited to corneal inflammation (such as Mooren's or inflammatory and infectious ulcers), necrotizing scleritis, inflammation-mediated ocular surface diseases, alkali burns, and chronic atopic diseases such as atopic or allergic conjunctivitis or eczematous diseases, fungal and bacterial infections, and corneal and conjunctival wounds, especially wounds associated with neurotrophic / diabetic neuropathy.

[0064] In addition to the treatment of eye diseases or conditions, the synthetic peptide can be engineered to promote wound healing and / or epithelial migration of other associated tissues or organs, particularly in the treatment of wounds, inflammation, cancer, infection, or disorders. In one aspect, lamellar tissue, nerve tissue, connective tissue, vascular tissue, muscle tissue, skeletal tissue, or blood components are treated. In another aspect, organs such as the skin, liver, lung, kidney, heart, or intestine are treated.

[0065] The following non-limiting examples are provided to further illustrate the present invention.

[0066] Example 1: Materials and Methods Peptide Synthesis. The histatin-5 peptide was synthesized using standard Fmoc-based solid-phase synthesis chemistry on a Symphony Peptide Synthesizer (Protein Technologies, Tucson, AZ). The first amino acid (Fmoc-Tyr-OH) was covalently attached to Wang resin. Peptide synthesis was initiated by removal of the Fmoc group in 20% piperidine in N,N-dimethylformamide (DMF) and synthesized in cycles. The next amino acid was added for 30 minutes X 2It was coupled using 0.1 M HBTU in DMF containing 0.4 M 4-methylmorpholine, and this process was continued until the synthesis was completed. The resin-bound peptide was deprotected and cleaved from the resin using trifluoroacetic acid (TFA). Ethyl ether was added to precipitate the peptide from the TFA solution. The precipitated peptide was then dissolved in 50% acetonitrile in water and lyophilized. The crude peptide was purified using a BioCad SPRINT™ (Applied Biosystems, Foster City, CA) HPLC system on a Kinetex™ reverse-phase C18 column, 150 × 21.1 mm (Phenomenex, CA). The pure peptide fractions were identified by electrospray ionization mass spectrometry (ESI MS) and appropriately lyophilized. A cyclized version of SHRGY (SEQ ID NO: 2) was prepared using a linker / spacer composed of 6-(Fmoc-amino)caproic acid, (6-(Fmoc-amino)hexanoic acid (C21H23NO4)).

[0067] The peptide was dissolved in cell culture grade water to obtain a 10 mM stock concentration and stored at -20 °C. Scrambled peptides (SP1, SP2, SP3, SP4 and SP5) were used as controls. SP1 was a 24 amino acid residue scrambled peptide based on the full-length native Hst5 peptide. SP2 contained 19 scrambled N-terminal amino acid residues (based on the N-terminal 19 amino acid residues of the native Hst5 peptide) and the SHRGY (SEQ ID NO: 2) sequence at the C-terminus. SP3 and SP4 were scrambled versions of the SHRGY (SEQ ID NO: 2) peptide and SP5 was a random pentapeptide with similar charge characteristics and molecular weight to SHRGY (SEQ ID NO: 2). Table 5 shows the sequences of the peptides used in this study.

Table 5

[0068] Cell culture. Human corneal limbal epithelial (HCLE) cells were cultured in keratinocyte - serum - free medium (K - SFM; Thermo Scientific, Waltham, MA) supplemented with 0.2 ng / mL of rhEGF (Thermo Scientific, Waltham, MA), bovine pituitary extract (Thermo Scientific, Waltham, MA), and 1% amphotericin B (Thermo Scientific, Waltham, MA). Standard cell culture conditions (37 °C, 5% CO 2 , >95% humidity) were used during routine sub - culturing. The culture medium was replaced every 48 hours after seeding.

[0069] Human corneal epithelial (HCE) cells were cultured in Medium Essential Media (MEM; Gibco, Life Technologies, Carlsbad, CA). HeLa cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Life Technologies, Grand Island, NY). Both media were supplemented with 10% fetal bovine serum (Gibco, Life Technologies, Carlsbad, CA) and 1% penicillin - streptomycin (Gibco, Life Technologies, Carlsbad, CA). The MCF - 7 cell line was cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air in Roswell Park Memorial Institute 1640 (RPMI 1640; Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies, Carlsbad, CA) and 1% penicillin - streptomycin (Gibco, Life Technologies, Carlsbad, CA).

[0070] Short tandem repeat (STR) analysis was performed on each cell line to confirm the authenticity of each cell line used.

[0071] In Vitro Scratch Assay. HCE, HCLE, HeLa, and MCF-7 cells were cultured in 24-well plates at a seeding density of 2.5x10 5 (cells / well) and grown to confluence on the 24-well plates. Subsequently, a linear scratch was created with the sterile P200 pipette tip member. The cells were then washed twice with phosphate buffered saline (PBS) to remove cell debris. The wound area was then treated with various concentrations of standard medium with reduced serum conditions (0.5% FBS in MEM medium (HCE), growth factor-free K-SFM medium (HCLE), 1% FBS in DMEM medium (HeLa), and 0.5% FBS in RPMI 1640 medium (MCF-7)) with the peptides provided in Table 5. The scratch was photographed hourly over the course of the experiment at 4x magnification (Image Express Micro, Molecular Devices, San Jose, CA) by microscopy. The wound area at each time point was measured using Image J software (Image J 1.47v, NIH, Thornwood, Bethesda, MD). Relative wound closure was calculated by dividing the closure of the treated wound by that of the untreated wound. For all experiments containing truncated histatin-5, a final concentration of 80 μM was used. At a final concentration of 50 μM, PD98059 (Calbiochem, San Diego, CA)) was used as a specific inhibitor of MEK. PD98059 was added as a histatin peptide simultaneously with cell culture.

[0072] Wound healing assay. Corneal wound experiments in mice were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. The protocol was approved by the Animal Care & Use Committee of the University of Illinois at Chicago. C57BLl6J (Jackson Laboratory, Bar Harbor, ME) mice, 12 - 19 weeks old, were anesthetized by intraperitoneal injection of ketamine (100 mg / kg) and xylazine (5 mg / kg). After application of two drops of topical 0.5% proparacaine, a 2.0 - mm area of the central epithelium was demarcated using a 2 - mm disposable biopsy punch and removed by an AlgerBrush II (The Alger Company, Lago Vista, TX). In the treatment (n = 7) or control (n = 7) groups, Histatin = 5 (80 μM), SHRGY peptide (SEQ ID NO: 2; 80 μM), or SP1 (80 μM) was applied to the cornea three times a day. At 0, 18, and 24 hours, the corneas were stained with fluorescein (FUL - GLO® Fluorescein Sodium ophthalmic strips, Akorn, Lake forest, IL) and photographed using a NIKON FS - 2 photo - slit lamp with a NIKON D200 camera (Melville, NY). The wound size was compared to the baseline for each mouse, and the percentage of wound closure was measured using Image J software.

[0073] Cell sprouting assay. The cell sprouting assay was performed using HCE cells in a soluble basement membrane sold under the trade name MATRIGEL®. (Reduced and diluted in MEM 1:1; Corning Life Sciences, Tewksbury, MA). HCE cells were seeded onto the soluble basement membrane at 5x10 5Cells were seeded at 10 μL spots. The cell spot plates were then exposed to reduced serum medium (0.5% FBS) (untreated negative control), Hst5 (50 μM), or 10% FBS (positive control). HCE cell migration was then tracked by time-lapse microscopy and imaged at 4x (Image Express Micro; Molecular Devices, CA). The cell-covered area at a given time point was measured using Image J.

[0074] Western blot. Western blot was performed following standard methods. Protein lysates (20 μg) were boiled for 10 minutes in NuPAGE™ LDS sample buffer (Invitrogen, Carlsbad, CA) and subjected to electrophoresis on a 12% NuPAGE™ Bis-Tris gel (Invitrogen, Carlsbad, CA), followed by transfer to a nitrocellulose membrane (Amersham Protran, GE Healthcare, Pittsburgh, PA). The membrane was then blocked with Tris-buffered saline containing 3% non-fat dry milk for 1 hour and incubated overnight at 4 °C with the first antibody against pERK1 / 2 (Cell Signaling, Danver, MA) (1:1000). After washing with 0.05% Tris-buffered saline containing 0.05% polysorbate 20, the membrane was then incubated for 1 hour with goat anti-rabbit-HRP (BD Biosciences, San Jose, CA) (1:2000) as the secondary antibody. The membrane was developed using X-ray film and ECL Pro solution (PerkinElmer, Waltham, MA). Beta-actin was used as an internal standard.

[0075] Immunofluorescence imaging. HCE cells were 4(Cell / Well) seeding density, seeded into an 8-well chamber slide, and incubated to form a confluent monolayer. Subsequently, a linear scratch was created with a sterile P10 pipette tip member. Next, the cells were washed with medium to remove cell debris. Subsequently, the wound area was left untreated or treated with Hst5, SP1, or SP2 at a concentration of 80 μM in MEM medium with 0.5% FBS for 45 minutes. The cells were then fixed with 4% paraformaldehyde for 30 minutes and permeabilized with 0.1% Triton X-100 for 5 minutes. After washing with PBS, the cells were incubated with 5% bovine serum albumin (BSA) and 5% normal goat serum in PBS at room temperature for 30 minutes. The cells were subsequently incubated with a first antibody (Cell Signaling, Danver, MA) against p-ERK1 / 2 (1:200) diluted in 1% BSA at 4°C for 16 hours. After washing three times with PBS, the cells were incubated with a fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG antibody (BD Biosciences, San Jose, CA) (1:500) diluted in 1% BSA at room temperature for 60 minutes. After extensive washing with PBS, the cells were then stained with 4',6-diamidino-2-phenylindole for 2 minutes for nuclear staining (DAPI; (Roche, Mannheim, GE). The cells were mounted in fluorogel with Tris buffer (Electron Microscopy Sciences, Hattfield, PA) and observed under a confocal microscope (Zeiss LSM 710 Confocal Microscope, Oberkochen, Germany) using a 10x objective.

[0076] For immunofluorescence, the entire excised mouse eyeball was snap frozen in optimal cutting temperature compound (OCT; (Fisher Healthcare, Galderma, CA)). The frozen tissue was sectioned into 10 μm frozen sections (ThermoScientific NX50 Cryomicrotome, Waltham, MA) and subsequently mounted on Superfrost plus slides (Thermofisher, Waltham, MA). The slides were fixed in methanol for 20 minutes, washed several times with PBS, stained with DAPI for 2 minutes, and further washed with PBS and deionized water. The slides were mounted in fluorogel with tris buffer (Electron Microscopy Sciences, Hattfield, PA) and observed under a confocal microscope (Zeiss LSM 710 Confocal Microscope, Oberkochen, Germany) using a 10X objective.

[0077] Statistical analysis. The experiments were appropriately analyzed using two-way or one-way ANOVA followed by Bonferroni or Dunnett's post hoc test or Student's t-test. A p-value < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism software 7.0 (GraphPad Software, La Jolla, CA).

[0078] Power analysis to determine the sample size for the mouse corneal wound experiment was performed using G-Power to compare between treatment (SHRGY (SEQ ID NO: 2) or Hst5) and control (SP1) groups. The parameters for the calculation included a beta of 0.8, an alpha of 0.05, and an effect size of 25%, and yielded a sample size of n = 6 per group.

[0079] Example 2: Histatin-5 promotes cell migration The cell sprouting assay using HCE cells seeded as spots in growth factor-reduced soluble basement membrane sold under the trade name MATRIGEL® (Corning Life Sciences, Tewksbury, MA) was used to determine whether HST5 can promote epithelial cell migration. At 72 hours, a statistically significant increase was seen in cell migration under the Hst5 (50 μM) treatment condition compared to the vehicle-only control. The effect of Hst5 on cell migration was tested in a standardized scratch assay using the HCLE cell line. HCLE cells were grown to confluence and mechanically wounded with the pipette tip member. Cells were treated with various concentrations of Hst5 (20, 50, 80, and 100 μM) or left untreated as a control. Time-lapse microscopy was performed and the wound area was analyzed at various time points. This analysis demonstrated a dose-dependent increase in the in vitro scratch assay closure rate by the application of Hst5 compared to the untreated control. The most significant increase in scratch closure rate was noted at 50 μM. These findings were reinforced in the HCE corneal cell line with a statistically significant peak effect at 80 μM compared to the scrambled peptide control (SP1). Statistically significant effects were also observed in the HeLa cell line and the MCF-7 breast cancer cell line.

[0080] Example 3: The C-terminal SHR domain of Hst5 is required to promote epithelial cell migration To identify the residues of Hst5 required for epithelial cell migration, serial cleavage experiments were performed, gradually removing residues in Hst5. This analysis showed that the C-terminal SHRGY (SEQ ID NO: 2) residues of Hst5 were necessary and sufficient to drive migration (Figure 1). All peptides were tested at a concentration of 80 μM. Truncated versions of Hst5 that did not contain the C-terminal SHRGY (SEQ ID NO: 2) sequence, namely, Hst5(1-14), Hst5(1-19), Hst5(1-21), Hst5(1-22), and Hst5(1-23)), did not show a significant increase in wound closure rate (Figure 1). However, constructs containing the full SHRGY sequence, namely, Hst5, Hst5(5-24), SP2, SHRGY (SEQ ID NO: 2), a multimer of SHRGY (SEQ ID NO: 2) composed of four repeating units of the above sequence (i.e., SHRGY-(CH 2 ) 6 -SHRGY-(CH 2 ) 6 -SHRGY-(CH 2 ) 6 -SHRGY (SEQ ID NO: 51)), and the cyclized SHRGY (SEQ ID NO: 2) peptide (c-SHRGY) showed a significant increase in scratch closure rate (Figure 1). Scrambled peptides SP3 and SP4 did not significantly increase the wound closure rate. Similarly, the random pentapeptide SP5 with similar molecular weight and charge characteristics to SHRGY (SEQ ID NO: 2) did not show a significant improvement in wound closure rate. Thus, the SHRGY (SEQ ID NO: 2) sequence is necessary and sufficient to drive epithelial migration rate in an in vitro scratch assay.

[0081] Example 4: ERK activation is required for the pro-migratory effect of Hst5 To determine whether the cell signaling pathway that supports the pro-migratory effect of Hst5, in terms of the levels of ERK activation / phosphorylation depending on the presence or absence of trauma and the application of Hst5, was the same as that of Hst1 in other epithelial cell types, it was investigated. Trauma causes an increased phosphorylated form of ERK1 / 2 (p-ERK1 / 2). Immunolocalization was used to determine whether the application of Hst5 to the epithelial scratch sheet affected the pERK1 / 2 levels. Western blot analysis confirmed that trauma alone increased the pERK1 / 2 levels and that these levels were further increased by the application of Hst5. The application of the SHRGY (SEQ ID NO: 2)-containing scrambled peptide SP2 increased the pERK1 / 2 levels relative to the relative intensity of Hst5. SP1, which does not contain SHRGY (SEQ ID NO: 2), did not elicit the same increase in pERK1 / 2 immunolocalization as the SHRGY (SEQ ID NO: 2)-containing peptide. The co-treatment of Hst5 and the MEK-specific inhibitor PD98059 removed the effect of Hst5 in promoting wound closure, indicating that the effect of Hst5 requires ERK activation.

[0082] Example 5: Application of Hst5 promotes wound healing in a mouse corneal injury model Using a standard mouse model of corneal injury, it was observed that the topical administration of Hst5 or the SHRGY (SEQ ID NO: 2) peptide provided a significant improvement in the corneal wound closure rate at levels superior to the scrambled peptide control (SP1) (Figure 2). Histological analysis of the wounded cornea (DAPI staining on cross-sections of the cornea) demonstrated pathological evidence of a decrease in corneal wound size in the Hst5-treated state compared to the SP1-treated control. Thus, Hst5 and the SHRGY (SEQ ID NO: 2)-containing peptide can enhance wound healing in a well-investigated model of mouse corneal epithelial injury.

[0083] Example 6: Toxicity and salt form Human corneal epithelial cells exposed to increasing concentrations of the SHRGY (SEQ ID NO:2) pentapeptide, namely 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1000 μM, 2000 μM, 4000 μM, and 8000 μM, showed a minimal decrease in cell viability at concentrations of 4000 μM and below after 24 hours as determined using the conventional WST1 assay, or induced cell death in the LDH assay at 24 hours up to concentrations exceeding 4000 μM.

[0084] The cytotoxicity of various salt forms of the SHRGY (SEQ ID NO:2) pentapeptide was also investigated. Three different salt forms of SHRGY (SEQ ID NO:2), namely acetate, hydrochloride, and trifluoroacetate, were prepared and their toxicity was analyzed at 15.625 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM as determined using the conventional WST1 assay. All three salt forms of SHRGY (SEQ ID NO:2) did not show significant toxicity at any of the peptide concentrations tested compared to the untreated samples.

[0085] The wound closure rate of the three salt forms was also analyzed according to the method disclosed herein. In particular, human corneal epithelial cells were wounded using a standardized scratch assay and the rate of wound healing was measured after application of the acetate, hydrochloride, and trifluoroacetate salt forms of SHRGY (SEQ ID NO:2). All tested forms presented an increased rate of wound closure (Figure 3).

Claims

1. A synthetic peptide having the structure of Formula I, or a pharmaceutically acceptable salt thereof: 【Chemical 1】 Wherein, R １ or R ２ at least one of which is the amino acid sequence SHRGY (SEQ ID NO: 2), and R １ or R ２ the other is a peptide to which a metal is bound, a wound healing peptide, or an antibacterial peptide; Z is present or absent, and when present, is a peptide of 1 to 20 amino acid residues; L is a hydrocarbon linker; and, n is 0 or ≧1, However, when n is 0, R １ is the amino acid sequence SHRGY (SEQ ID NO: 2), and Z does not exist.

2. The synthetic peptide according to claim 1, wherein each L may be the same or different linker.

3. The synthetic peptide according to claim 1, which is linear or cyclized.

4. The synthetic peptide according to claim 1, comprising a modification selected from glycosylation, acetylation, amidation, formylation, hydroxylation, methylation, myristoylation, phosphorylation, sulfonation, PEGylation, or lipidation.

5. The synthetic peptide according to claim 1, wherein the peptide to which a metal is bound comprises the amino acid sequence HEXXH (SEQ ID NO: 14), where X is K, R, or H.

6. The synthetic peptide according to claim 1, wherein the wound healing peptide comprises the amino acid sequence SNYLYDN (SEQ ID NO: 26) or SHXGY (SEQ ID NO: 1), where X is R, K, H, D, or E.

7. The synthetic peptide according to claim 1, wherein the antibacterial peptide comprises the amino acid sequence RKFHEKHHSHRGYR (SEQ ID NO: 28) or AKRHHGYKRKFH (SEQ ID NO: 29).

8. A pharmaceutical composition comprising one or more synthetic peptides according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

9. The pharmaceutical composition according to claim 8, formulated for topical, oral, ocular, intravenous, intravitreal, subconjunctival, subcutaneous, intramuscular, intraperitoneal, intracerebral, intraarterial, intraportal, intralesional, intrathecal, or intranasal administration.

10. The pharmaceutical composition according to claim 8, which is in the form of a gel, diluent, cream, tablet, capsule, pill, solution, eye drop, spray, bandage, contact lens, reservoir, injectable drug, implantable article, or sustained release formulation.

11. Use of the pharmaceutical composition according to claim 8 in a method for promoting wound healing or epithelial cell migration, comprising administering to a subject in need of treatment an effective amount of the synthetic peptide according to claim 1, or a pharmaceutically acceptable salt thereof, thereby promoting wound healing or epithelial cell migration.

12. The pharmaceutical composition according to claim 11, wherein the amount of the synthetic peptide or a pharmaceutically acceptable salt thereof is in the range of 1 nanomole to 500 micromoles.

13. The pharmaceutical composition according to claim 11, wherein the method further comprises administering an antibacterial agent, an antiviral agent, an antiparasitic agent, an anti-wound agent, an immunomodulatory agent, collagen, gelatin, an analgesic, an anesthetic, or a combination thereof.

14. Use of the pharmaceutical composition according to claim 8 in a method for promoting wound healing or epithelial cell migration, comprising administering an effective amount of the pharmaceutical composition according to claim 8 to a subject in need of treatment, thereby promoting wound healing or epithelial cell migration.

15. The pharmaceutical composition according to claim 14, wherein the amount of the synthetic peptide or a pharmaceutically acceptable salt thereof is in the range of 1 nanomole to 500 micromoles.

16. The pharmaceutical composition according to claim 14, wherein the method further comprises administering an antibacterial agent, an antiviral agent, an antiparasitic agent, an anti-wound agent, an immunomodulatory agent, collagen, gelatin, an analgesic, an anesthetic, or a combination thereof.

17. Use of the pharmaceutical composition according to claim 8 in a method for increasing extracellular signal-regulated protein kinase (ERK) activation, comprising administering an effective amount of the synthetic peptide according to claim 1 or a pharmaceutically acceptable salt thereof to a subject in need of treatment, thereby increasing extracellular signal-regulated protein kinase (ERK) activation.

18. A kit comprising one or more synthetic peptides according to claim 1 or a pharmaceutically acceptable salt thereof.

19. The kit according to claim 18, further comprising an antibacterial agent, an antiviral agent, an antiparasitic agent, an antifungal agent, an anti-wound agent, an immunomodulatory agent, collagen, gelatin, an analgesic, an anesthetic, or a combination thereof.

20. A synthetic peptide having the structure of Formula I, or a pharmaceutically acceptable salt thereof: [Chemical Formula 2] Wherein: R1 is a peptide to which a metal is bonded, a wound healing peptide, or an antibacterial peptide, or a peptide of 5 to 10 amino acid residues containing the amino acid sequence SHRGY (SEQ ID NO: 2); R2 is a peptide of 5 to 10 amino acid residues containing the amino acid sequence SHRGY (SEQ ID NO: 2); Z is present or absent, and when present, is a peptide of 1 to 20 amino acid residues; L is a hydrocarbon linker; And n is ≧1.

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