Pentapeptides and methods of use thereof

Abstract

The present invention describes synthetic peptides comprising the sequence SHXGY (SEQ ID NO: 2) and methods of using the peptides to promote wound healing and epithelial cell migration.

Description

[0001] introduction

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 62 / 941,226, filed on November 27, 2019, the contents of which are incorporated herein by reference in their entirety.

[0003] This invention was made with government support under Grant Nos. 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 Office of Research and Development. The U.S. Government has certain rights in this invention. Background Art

[0004] Histamine (HTN) is a small histidine-rich cationic peptide found in saliva and 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). Histamines range in size from 7 to 38 amino acid residues in length and represent a group of antimicrobial peptides with antibacterial and significant antifungal properties. In addition, histamine is associated with 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 that contribute to the 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 1545:86-95). In this regard, histamine and its fragments, multimers and combinations have been suggested for use in the treatment of a variety of different 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).

[0005] Cyclic analogs of histamine have also been described. For example, US 6,555,650 describes a cyclic analog of HTN5 with a disulfide bridge that creates a cyclic portion of positions 5-16 of the amino acid unit. In addition, head-to-tail cyclization of HTN5 has been shown to increase the amphipathicity of the peptide without affecting its antimicrobial efficacy (Sikorska & Kamysz (2014) J. Pept. Sci. 20: 952-7). In addition, cyclization of histamine-1 has been shown to enhance molar activity by approximately 1000 times (Oudhoff et al., (2009) FASEB J. 23: 3928-35) and improve wound closure activity (Bolscher et al., (2011) FASEB J. 25: 2650-8). Furthermore, cyclized analogs of histamine with enhanced potency have been suggested for the treatment of microbial infections (US 2010 / 0173833; Brewer & Lajoie (2002) Biochemistry 41:5526-5536). Summary of the invention

[0006] The present invention provides a synthetic peptide or a pharmaceutically acceptable salt thereof, wherein the peptide has a structure of Formula I:

[0007] ZR 1 -[LR 2 ] n (I)

[0008] Where R 1 or R 2 At least one of the peptides is a peptide of 5 to 10 amino acid residues comprising the amino acid sequence SHXGY (SEQ ID NO: 1), wherein X is R, K, H, D or E, and R 1 or R 2 The other of the peptides is a metal binding peptide, a wound healing peptide or an antimicrobial peptide; Z is present or absent and, when present, is an exogenous peptide; L is a linker; and n is 0 or ≥ 1, provided that when n is 0, R 1It is a peptide of 5 to 10 amino acid residues comprising the amino acid sequence SHXGY (SEQ ID NO: 1). In some cases, L may include the same or different linkers at each occurrence; the synthetic peptide may be linear or cyclized; and / or the peptide may include a modification selected from glycosylation, acetylation, amidation, formylation, hydroxylation, methylation, myristoylation, phosphorylation, sulfonation, PEGylation or lipidation. In other cases, the metal binding peptide may have the amino acid sequence HEXXH (SEQ ID NO: 14), wherein X is K, R or H; the wound healing peptide may have the amino acid sequence SNYLYDN (SEQ ID NO: 26) or SHXGY (SEQ ID NO: 1), wherein X is R, K, H, D or E; and the antimicrobial peptide may have the amino acid sequence RKFHEKHHSHRGYR (SEQ ID NO: 28) or AKRHHGYKRKFH (SEQ ID NO: 29). A pharmaceutical composition is also provided, comprising one or more of the synthetic peptides or pharmaceutically acceptable salts thereof mixed with a pharmaceutically acceptable carrier or excipient, and a kit and method for using the synthetic peptides or pharmaceutically acceptable salts thereof or pharmaceutical compositions containing them to promote wound healing and / or epithelial cell migration and increase activation of extracellular signal-regulated protein kinase (ERK), wherein the synthetic peptide or pharmaceutically acceptable salt thereof is administered in an amount ranging from 1 nanomolar to 500 micromolar. In some cases, the kit and method may also include the use of antimicrobial agents, antiviral agents, antiparasitic agents, immunomodulators, anti-scarring agents, collagen, gelatin, analgesics, anesthetics, or combinations thereof. In addition, the pentapeptides prepared according to the principles of the present disclosure may be manipulated to amplify them, orient them in a linear arrangement or other configuration, and maintain their function. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 It was shown that peptides containing SHRGY (SEQ ID NO: 2) are necessary and sufficient to accelerate the rate of scratch closure. The efficacy of the peptides in accelerating the rate of scratch closure was tested. Negative controls included untreated and various scrambled peptide (SP) controls. All peptides were tested at a concentration of 80 μM. All experiments were performed in triplicate, using three technical replicates per experiment. Statistical significance was determined by one-way ANOYA and Dunnett's post hoc test. *p<0.05; **p<0.01; ***p<0.001. Relative closure = (% closure of treated samples) / (% closure of untreated controls).

[0010] Figure 2It was shown that administration of histamine-5 or SHRGY (SEQ ID NO: 2) peptide accelerated the rate of corneal wound closure in a murine corneal epithelial injury model. The wound area was treated with Hst5, SHRGY (SEQ ID NO: 2) or SP1 peptide (n = 7 per group), and each compound was tested at a concentration of 80 μM. The wound area at multiple time points was measured using Image J software. Measurement of the % of the remaining corneal wound area at 18 hours and 24 hours compared to baseline showed statistically significant improvements in the Hst5 and SHRGY (SEQ ID NO: 2) groups compared to SP1. Statistical significance was determined by two-way ANOVA and Bonferroni post hoc test. **P<0.01, ***P<0.001. % of wound area = (wound area at X / wound area at 0) x 100.

[0011] Figure 3 The scratch closure rate of different salt forms of the SHRGY (SEQ ID NO: 2) peptide is shown. Scratches were made on human corneal epithelial cells using a standard scratch assay and the enhancement of wound healing rate after application of different salt forms of the pentapeptide was tested. AA, acetic acid; HCL, hydrochloride; TFA, trifluoroacetate. DETAILED DESCRIPTION

[0012] Epithelial migration, adhesion and proliferation are essential for wound healing in all areas of the body. Without adequate epithelialization, abnormal wound healing can be seen, with consequences such as scarring, inflammation, pain, contracture, loss of vision, impaired function, infection and abnormal vascular proliferation. Several cell types and wound models are often used to test the suitability of agents to enhance wound healing. An exemplary system is the anterior segment and ocular surface of the eye. The cornea and other elements of the anterior surface of the eye are used as wound healing models because abnormal epithelialization can lead to devastating loss of vision or even loss of the eye. It has now been found that the pentapeptide SHXGY (SEQ ID NO: 1) is able to enhance epithelial wound healing. Specifically, peptides containing the sequence SHXGY (SEQ ID NO: 1), such as SHRGY (SEQ ID NO: 2) and SHDGY (SEQ ID NO: 3), have been shown to significantly enhance epithelial migration in several cell types (immortalized human corneal epithelial cells, immortalized human limbal epithelial cells, HeLa human cells) and in a mouse corneal epithelial wound model. When topically applied to mice that have been wounded using standard wounding methods, peptides containing SHXGY (SEQ ID NO: 1) or multimers thereof (e.g., SHRGY-(CH2)6-SHRGY-(CH2)6-SHRGY-(CH2)6-SHRGY; SEQ ID NO: 51) can significantly improve corneal healing. In addition, synthetic peptides containing the SHRGY (SEQ ID NO: 2) sequence have been shown to stimulate the immunolocalization of phosphorylated extracellular signal-regulated protein kinase 1 / 2 (pERK1 / 2) to wound healing sites, indicating that in addition to the function of enhancing wound healing, the SHRGY (SEQ ID NO: 2) peptide also has immunomodulatory activity. Therefore, this core pentapeptide sequence is particularly useful in promoting epithelialization and cell migration, which are important for wound healing, inflammation, cancer, response to infection or injury, and other phenomena.

[0013] Therefore, the present invention provides a synthetic peptide or a pharmaceutically acceptable salt thereof and a method of using them to promote wound healing and / or epithelial cell migration. The synthetic peptide of the present invention has the general structure of Formula I:

[0014] ZR 1 -[LR 2 ] n (I)

[0015] in

[0016] (i)R 1 or R 2 At least one of the peptides is a peptide of 5 to 10 amino acid residues having an amino acid sequence of SHXGY (SEQ ID NO: 1), wherein X is R, K, H, D or E, and R 1 or R2 The other of which is a metal binding peptide, a wound healing peptide, or an antimicrobial peptide;

[0017] (ii) Z is present or absent, and when present is an exogenous peptide;

[0018] (iii) L is a linker; and

[0019] (iv) n is 0 or ≥ 1

[0020] The premise is that when n is 0, R 1 It is a peptide of 5 to 10 amino acid residues having the amino acid sequence SHXGY (SEQ ID NO: 1).

[0021] As indicated, R 1 and R 2 At least one of the peptides is a peptide of 5 to 10 amino acid residues comprising the amino acid sequence SHXGY (SEQ ID NO: 1), wherein X is R (Arg), K (Lys), H (His), D (Asp) or E (Glu). 1 and R 2 At least one of the peptides is a peptide comprising 5, 6, 7, 8, 9 or 10 amino acid residues of 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). 1 or R 2 At least one of the sequences comprises the sequence SHXGY (SEQ ID NO: 1), which may have 1 to 5 additional amino acid residues at the C-terminus and / or N-terminus. In some cases, the 1 to 5 additional amino acid residues are endogenous or native amino acid residues. A "native" or "endogenous" amino acid residue is an amino acid residue that is present at the recited position in a naturally occurring protein. For example, the sequence SHRGY (SEQ ID NO: 2) is present in histamine 3 as described below: DSHAKRHHGYKRKFHEKHH SHRGY RSNYLYDN (SEQ ID NO: 7). 1 and / or R 2 When derived from histamine, 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), KHHSHRGY (SEQ ID NO: 11), HH SHRGY (SEQ ID NO: 12) or H SHRGY (SEQ ID NO: 13).

[0022] In some cases, the synthetic peptide consists only of R 1 According to this case, the synthetic peptide is a peptide of 5, 6, 7, 8, 9 or 10 amino acid residues comprising or consisting of the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0023] In other cases, the synthetic peptide comprises one or more R 2 peptide (i.e. n ≥ 1). In this case, the synthetic peptide may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more peptides connected by a linker. In one embodiment, the R of the synthetic peptide of the present invention is 1 and R 2 In other cases, R of the synthetic peptides of the present invention is 1 and R 2 are different. In other cases, each R 2 They may be the same or different. Ideally, the total length of the synthetic peptide is in the range of 20 to 100 amino acid residues.

[0024] When R 1 or R 2 When at least one of the two is a peptide having 5 to 10 amino acid residues of the amino acid sequence SHXGY (SEQ ID NO: 1), R 1 or R 2 The other of can be metal binding peptide, wound healing peptide or antimicrobial peptide.In this case, synthetic peptide of the present invention can be made up of the combination of peptide with 5 to 10 amino acid residues of amino acid sequence SHXGY and (i) metal binding peptide, (ii) wound healing peptide, (iii) antimicrobial peptide or (iv) any combination of (i)-(iii).In some cases, synthetic peptide of the present invention is made up of the combination of peptide with 5 to 10 amino acid residues of amino acid sequence SHXGY and the second wound healing peptide.

[0025] As used herein, the term "metal binding peptide" refers to an amino acid motif that binds to or forms a complex with a metal. Structural and functional characterization of histamine revealed the presence of two metal binding motifs: an amino-terminal Cu(II) / Ni(II) binding (ATCUN) motif with a histidine residue in the third position (NH2-X1X2H, wherein X1 is Asp or Glu, and X2 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), wherein X represents a basic amino acid residue such as K (Lys), R (Arg), or H (His). Thus, in certain embodiments, the metal binding peptide comprises the sequence DSH, ESH, DAH, EAH, DTH, ETH, DMH or EMH. In other embodiments, the metal binding peptide 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 metal binding peptide may comprise the specific sequence of the metal binding peptides mentioned above, or may comprise 1 to 6 additional native histamine amino acid residues at the C- and / or N-terminus of the metal binding peptide. For example, the metal binding peptide may have the sequence GYKRKF HEKHH SHR (SEQ ID NO: 24) or HEKRH H (SEQ ID NO: 25).

[0026] In certain embodiments, the synthetic peptides of the present invention include one metal binding peptide. In other embodiments, the synthetic peptides include two metal binding peptides. In other embodiments, the synthetic peptides include three metal binding peptides. In certain embodiments, the metal binding peptide has the sequence HEXXH (SEQ ID NO: 14), wherein each X is a basic amino acid residue. As will be readily appreciated by those skilled in the art, the inclusion of one or more metal binding peptides in a synthetic peptide imparts metal ion chelating, anti-inflammatory, matrix metalloproteinase inhibition and / or anti-angiogenic activity to the synthetic peptide. In view of its anti-angiogenic activity, such synthetic peptides may be used to treat age-related macular degeneration, diabetic retinopathy, cancer, and chronic or acute severe uveitis. In view of its metal ion chelating activity, such synthetic peptides may also be used to inhibit tissue destruction mediated by matrix metalloproteinases and other metal-dependent enzymes in inflammatory and infectious diseases such as infectious keratitis, intraocular uveitis, endophthalmitis, inflammatory keratitis, dry eye, and ocular surface or intraocular diseases.

[0027] As used herein, "wound healing peptide" refers to an amino acid motif that promotes or facilitates wound healing. In some cases, the wound healing peptide is derived from histamine. An example of a wound healing peptide derived from histamine is a peptide comprising the sequence SNYLYDN (SEQ ID NO: 26). In another case, the wound healing peptide comprises the amino acid sequence SHXGY (SEQ ID NO: 1), wherein X is R, K, H, D or E. In particular, when included in the synthetic peptides of the present invention, the SHXGY (SEQ ID NO: 1) sequence has the additional advantage of providing immunomodulatory activity to the synthetic peptide. The wound healing peptide may include the specific sequence of the wound healing peptide mentioned above, or may include 1 to 6 additional amino acid residues at the C- and / or N-terminus of the wound healing peptide. By way of example, a wound healing peptide derived from histamine may have the sequence YGDYG SNYLYDN (SEQ ID NO: 27) or any one of SEQ ID NO: 2 or 8-13.

[0028] In certain embodiments, the synthetic peptides of the present invention include a second wound healing peptide in addition to the wound healing peptide of SEQ ID NO: 1. In other embodiments, the synthetic peptides include two additional wound healing peptides in addition to the wound healing peptide of SEQ ID NO: 1. In other embodiments, the synthetic peptides include three additional wound healing peptides in addition to the wound healing peptide of SEQ ID NO: 1. As will be readily appreciated by those skilled in the art, the inclusion of one or more wound healing peptides in the synthetic peptides imparts epithelial cell migration and spreading activity to the synthetic peptides. Thus, such synthetic peptides can be used for wound healing as well as retinal pigment epithelial healing, dry age-related macular degeneration, ocular surface diseases and ocular surface inflammatory disorders, ocular neovascularization including the cornea and intraocular, retinal or choroidal, and treatment of dry eye.

[0029] For the purposes of the present invention, "antimicrobial agents" include both antibacterial agents and antifungal agents. Thus, when used herein, the term "antimicrobial" peptide refers to an amino acid motif that exhibits cytostatic or cytocidal activity against bacterial and / or fungal cells. Characterization of histamines indicates that the positive net charge and amino terminal moiety of HTN mediate antimicrobial activity. Specifically, the amino acid sequence RKFHEKHHSHRGYR (SEQ ID NO: 28) of HTN3 has been shown to exhibit fungicidal activity (Oppenheim et al., (2012) PLoS ONE 7 (12): e51479). Similarly, the sequence AKRHHGYKRKFH (SEQ ID NO: 29), also known as P-113, exhibits fungicidal activity against Candida albicans (Jang et al., (2008) Antimicrob. Agents Chemother. 5292: 497-504). Therefore, the antimicrobial peptide may include the specific sequence of the antimicrobial peptide mentioned above, or may include 1 to 6 additional amino acid residues at the C- and / or N-terminus of the antimicrobial peptide.

[0030] In some embodiments, the synthetic peptide comprises an antimicrobial peptide. In other embodiments, the synthetic peptide comprises two antimicrobial peptides. In other embodiments, the synthetic peptide comprises three antimicrobial peptides. In some embodiments, the antimicrobial peptide has the sequence RKFHEKHHSHRGYR (SEQ ID NO: 28). In other embodiments, the antimicrobial domain has the sequence AKRHHGYKRKFH (SEQ ID NO: 29). As those skilled in the art will readily appreciate, including one or more antimicrobial peptides in the synthetic peptide confers antifungal and / or antibacterial activity to the synthetic peptide. Therefore, this synthetic peptide can be used to treat microbial infections such as Candida eye infections and prevent infections associated with surgical implants.

[0031] Examples of synthetic peptides containing identical or different repeating units are presented in Table 1.

[0032] Table 1

[0033]

[0034] In certain aspects of the invention, an exogenous or heterologous molecule is included in the synthetic peptide. Specifically, in certain aspects, the synthetic peptide includes a 1 and R 2 One or both of the "Z" and / or "L" components, wherein both the "Z" and "L" components are for R 1 and R 2 The term "heterologous molecule" or "foreign molecule" refers to a foreign or heterologous molecule that is not normally present in the peptide or that is not normally associated with R in nature. 1 and / or R 2 A molecule that is accompanied by an amino acid sequence.

[0035] In some cases, the synthetic peptide includes a "Z" component. In other cases, the "Z" component 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 a peptide of 1 to 30 amino acid residues, or more preferably a peptide of 1 to 20 amino acid residues, wherein the exogenous peptide may or may not have a function. For example, the exogenous peptide may exhibit metal binding, wound healing, immunomodulatory and / or antimicrobial activity, or may be a random peptide sequence, such as in the SP2 peptide HSHKEGHHYKRFKRKHHAD SHRGY In some cases, Z is a random peptide sequence of 1-50, 1-30, or 1-20 amino acid residues.

[0036] As used herein, the term "L" or "linker" or "spacer" refers to a 1 Connect, link or join to R 2 and connect, link or join the various R 2 As used herein, the terms "connection", "linking" or "coupling" generally refer to a functional connection between two adjacent or neighboring amino acid sequences to produce a molecule that does not exist in nature. Typically, the amino acid sequences being connected are adjacent or neighboring to each other and retain their respective operability and function when connected. The connector can provide the desired flexibility to allow the desired expression, activity and / or conformational positioning of the synthetic peptide.

[0037] In certain 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 certain cases, the linker (L) may comprise the same or different linkers at each occurrence.

[0038] The linker used in the synthetic peptide of Formula I can be flexible, rigid, cleavable in vivo, or a combination thereof. In addition, the linker can be composed of amino acid residues (i.e., peptide linkers) or composed of hydrocarbon chains (i.e., hydrocarbon linkers). The peptide linker can have any suitable length to connect R 1 and R 2 or each R 2 components, and are preferably designed to allow R 1 and R 2 The correct folding and / or function and / or activity of the connector peptide. Therefore, the connector peptide may have a length of no more than 3, no more than 5, no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 55 or no more than 60 amino acids. In certain embodiments, the connector peptide may 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 certain embodiments, the linker comprises at least 10 and no more than 60 amino acids, at least 10 and no more than 55 amino acids, at least 10 and no more than 50 amino acids, at least 10 and no more than 45 amino acids, at least 10 and no more than 40 amino acids, at least 10 and no more than 35 amino acids, at least 10 and no more than 30 amino acids, at least 10 and no more than 25 amino acids, at least 10 and no more than 20 amino acids, or at least 10 and no more than 15 amino acids.

[0039] "Flexible" connector refers to a hydrocarbon or peptide connector that does not have a fixed structure (secondary or tertiary structure) in solution. Therefore, this flexible connector is free to adopt a variety of different conformations. The flexible connector used in this article includes a hydrocarbon connector and a peptide connector composed of small non-polar (e.g., Gly) and / or polar (e.g., Ser or Thr) amino acid residues. Simple amino acids (e.g., amino acids with simple side chains (e.g., H, CH3 or CH2OH)) are advantageous for use in peptide connectors because these amino acids lack branched side chains, thereby providing greater flexibility (e.g., two-dimensional or three-dimensional flexibility) in the connector and therefore in the polypeptide composition. The flexible connector may contain additional amino acids such as Thr and Ala to maintain flexibility, and polar amino acids such as Lys and Glu to improve solubility. The amino acids may alternate / repeat in any manner consistent with the connector retaining function (e.g., producing expressed and / or active polypeptides). Flexible linkers are described, for example, in Chen et al. (2013) Adv. Drug Deliv. Rev. 65(10): 1357-1369; US 2012 / 0232021; US ​​2014 / 0079701; WO 1999 / 045132; WO 1994 / 012520 and WO 2001 / 1053480.

[0040] In certain cases, the flexible linker is a hydrocarbon linker. 1 and R 2 or each R 2 The hydrocarbon component should be of sufficient length and flexibility to allow the synthetic peptide to achieve the desired conformation. In certain embodiments, the hydrocarbon is composed of one or more methylene groups (-CH2-). In certain embodiments, the hydrocarbon includes 3 to 25 methylene groups, i.e., -(CH2) n -, wherein n is 3 to 25. In certain embodiments, the hydrocarbon linker has the structure -(CH2)6-. Other carbon-based linkers such as diol linkers can also be used in the synthetic peptides of the present invention.

[0041] In other embodiments, the connector is a rigid connector. A "rigid" connector refers to a molecule that adopts a relatively clear conformation when in solution. Therefore, a rigid connector is a connector with a specific secondary and / or tertiary structure in solution. A rigid connector generally has a size sufficient to provide a secondary or tertiary structure to the connector. Such connectors include aromatic molecules (see, for example, US 6,096,875 or US 5,948,648), proline-rich peptide connectors, or peptide connectors with a non-flexible helical structure. Rigid connectors are described, for example, in Chen et al., (2013) Adv. Drug Deliv. Rev. 65 (10): 1357-1369, US 2010 / 0158823, and US 2009 / 10221477.

[0042] In other embodiments, the linker is an in vivo cleavable linker. An in vivo cleavable linker may include a cleavable disulfide bond formed between two cysteine ​​residues or a linker having a protease recognition sequence, such as that recognized by matrix metalloproteinases (MMPs).

[0043] Examples of peptide linkers suitable for use with the synthetic peptides are provided in Table 2.

[0044] Table 2

[0045]

[0046]

[0047] n is 1 to 5. X may be any amino acid residue, but is preferably Ala, Lys or Glu.

[0048] Each individual connector of the synthetic peptide of the present invention can be the same or different. In certain embodiments, the synthetic peptide includes at least one flexible connector. In certain embodiments, at least one flexible connector is a hydrocarbon connector. In other embodiments, at least one flexible connector is a peptide connector. In specific embodiments, each connector of the synthetic peptide is a hydrocarbon connector. In certain embodiments, each connector of the synthetic peptide has the structure -(CH2)6-.

[0049] Examples of synthetic peptides containing combinations of repeat units and flexible linkers are presented in Table 3.

[0050] Table 3

[0051]

[0052] In some cases, the synthetic peptides of the present invention are prepared into pharmaceutically acceptable salts. When used in this article, the term "pharmaceutically acceptable salt" refers to the synthetic peptides that are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., within the scope of reasonable medical judgment, and salts 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. Salts can be prepared in situ during the final separation and purification of the peptides of the present invention, or prepared separately by reacting a free alkali with a suitable organic acid. Examples of pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts formed by amino groups with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or formed by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Other pharmaceutically acceptable salts include nontoxic ammonium, quaternary ammonium and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates and arylsulfonates, where appropriate.

[0053] The synthetic peptides described herein are generally referred to as fusions or chimeric peptides. Such molecules can be synthesized by conventional methods including recombinant protein expression, chemical synthesis or a combination thereof. In certain embodiments, the synthetic peptides of the present invention are recombinantly synthesized using recombinant DNA technology. Therefore, the present invention provides polynucleotides encoding the synthetic peptides of the present invention. In relevant cases, the present invention provides vectors, particularly expression vectors, with the polynucleotides encoding the synthetic peptides of the present invention. In certain embodiments, the vector provides replication, transcription and / or translation regulatory sequences to facilitate the recombinant synthesis of the desired synthetic histamine in eukaryotic or prokaryotic cells. Therefore, the present invention also provides host cells for the recombinant expression of the synthetic peptides, as well as methods for harvesting 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 method can be used.

[0054] In another embodiment, the synthetic peptide is synthesized by any chemical synthesis technique known in the art, particularly solid phase synthesis technique, for example using a commercially available automatic peptide synthesizer. See, for example, Stewart & Young (1984) Solid Phase Peptide Synthesis, 2nd Edition, 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.

[0055] The synthetic peptides can be separated and / or purified by any suitable method known in the art, including but not limited to gel filtration and affinity purification. In certain embodiments, the synthetic peptides are produced with tags, such as epitope tags, to facilitate separation of the synthetic peptides. In one case, when measured by SDS-PAGE, the synthetic peptides are at least 1% pure, such as at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, and at least 90% pure. After separation and / or purification, the properties of the synthetic peptides can be easily verified by techniques known to those skilled in the art.

[0056] Derivatives and analogs of the synthetic peptides described herein are all contemplated and can be prepared by altering their amino acid sequences with substitutions, additions and / or deletions / truncations or by introducing chemical modifications that produce functionally equivalent molecules. It will be appreciated by those of ordinary skill in the art that certain amino acids in the sequence of any polypeptide can be replaced with other amino acids without adversely affecting the activity of the polypeptide.

[0057] In certain embodiments, the synthetic peptides of the present invention comprise one or more modifications, including but not limited to phosphorylation, glycosylation, hydroxylation, sulfonation, amidation, acetylation, carboxylation, palmitoylation, PEGylation, the introduction of non-hydrolyzable bonds and disulfide bond formation. The modifications can improve the stability and / or activity of the synthetic peptides.

[0058] For example, the C-terminus can be modified by amidation, addition of peptide alcohols and aldehydes, addition of esters, addition of p-nitroanilide and thioesters. The N-terminus and side chains can be modified by PEGylation, acetylation, formylation, addition of fatty acids, addition of benzoyl, addition of bromoacetyl, addition of pyroglutamyl, succinylation, addition of tetrabutoxycarbonyl and addition of 3-mercaptopropyl, acylation (e.g., lipopeptides), biotinylation, phosphorylation, sulfation, glycosylation, addition of maleimido groups, chelating moieties, chromophores or fluorophores.

[0059] In one embodiment, the synthetic peptide is coupled to a fatty acid, for example, the synthetic peptide is myristoylated. For example, a fatty acid can be coupled to the N-terminus of the synthetic peptide. Such fatty acids include caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, etc. In addition, the cysteine ​​in the synthetic peptide can be palmitoylated. In one embodiment, the synthetic peptide is myristoylated, stearoylated, or palmitoylated at the N-terminal amino acid.

[0060] In addition to or as an alternative to post-translational modification, the synthetic peptide can be coupled or linked to another peptide, such as a carrier peptide. The carrier peptide can promote cell penetration and can include peptides such as antennapedia, penetratin, TAT, transportan, or polyarginine. In one embodiment, the synthetic peptide is coupled or linked to antennapedia peptide RQIKIWFQNRRMKWKK (SEQ ID NO: 56).

[0061] The synthetic peptides of the present invention can also be cyclized. When used in this article, the term "cyclization" or "cyclic" refers to an analog of a linear peptide that incorporates at least one bridging group (e.g., amide, thioether, thioester, disulfide, urea, carbamate, hydrocarbon or sulfonamide) between two amino acid residues to form a cyclic structure. The bridging group can be present on the side chain of the amino acid residue or the terminal amino acid residue, thereby providing side chain cyclization (e.g., lactam bridge, thioester), head-to-tail cyclization or hydrocarbon-stapling peptides.

[0062] In certain embodiments, the cyclic synthetic peptide has a disulfide bridge between the two terminal cysteine ​​residues. Representative amino acid sequences used to prepare cyclic synthetic peptides are provided in Table 4.

[0063] Table 4

[0064]

[0065] In other embodiments, the cyclic synthetic peptide is prepared from a linear peptide by cyclization with a sortase. "Cyclization with a sortase" or "cyclization with a sortase" refers to a method of cyclizing a linear peptide using a sortase. Sorting enzyme-based cyclization is known in the art for making large cyclic peptides. See Bolscher et al., (2011) FASEB J. 25(8): 2650-2658 and references cited therein.

[0066] Butelase cyclization has also been used to cyclize peptides. Addition of the tripeptide Asn–His–Val at the C-terminus provides a substrate for Butelase to cyclize synthetic peptides at a significantly faster rate than sortase A. See Nguyen et al. (2016) Nat. Protocols 11: 1977-88; Tam et al. (June 2015) Peptides 2015: Proc. 24 th Am. Pept. Symp., Orlando, FL, p. 27.

[0067] Those skilled in the art will recognize that the synthetic peptides of the present invention will be beneficial for treating diseases. Therefore, for ease of administration, the present invention also provides compositions containing one or more endogenous and / or synthetic peptides and pharmaceutically acceptable carriers or excipients. The pharmaceutical compositions provided herein can be formulated for oral, ocular, intravenous, intravitreal, subconjunctival, subcutaneous, intramuscular, intraperitoneal, intracerebral, intraarterial, intraportal, intralesional, intrathecal or intranasal administration or topical administration. Suitable pharmaceutical compositions can be determined by those skilled in the art according to, for example, planned routes of administration, delivery forms and desired doses. See, for example, Remington's Pharmaceutical Sciences (19th edition, 1995).

[0068] The synthetic peptide can be incorporated into conventional dosage forms, such as gels, lotions, creams, tablets, capsules, pills, solutions, eye drops, sprays, bandages, contact lenses, depot preparations, injections, implants or sustained release preparations. The dosage form can also include necessary physiologically acceptable carrier materials, excipients, lubricants, buffers, surfactants, antibacterial agents, bulking agents (such as mannitol), antioxidants (ascorbic acid or sodium bisulfite), etc.

[0069] Acceptable formulation materials are preferably nontoxic to recipients at the dosages and concentrations employed. The pharmaceutical composition may contain formulation materials used to alter, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight The invention relates to a pharmaceutical composition comprising a polypeptide, a salt-forming counterion (such as sodium), a preservative (such as benzalkonium chloride, benzoic acid, salicylic acid, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide), a solvent (such as glycerol, propylene glycol or polyethylene glycol), a sugar alcohol (such as mannitol or sorbitol), a suspending agent, a surfactant or a wetting agent (such as PLURONICS, PEG, sorbitan esters, polysorbates such as polysorbate 20 and polysorbate 80, TRITON, tromethamine, lecithin, cholesterol or tyloxapol), a stability enhancer (such as sucrose or sorbitol), an osmotic enhancer (such as an alkali metal halide, preferably sodium chloride or potassium chloride, mannitol or sorbitol), a delivery vehicle, a diluent, an excipient and / or a pharmaceutical adjuvant. See, for example, Remington's Pharmaceutical Sciences, supra.

[0070] The main carrier or excipient in the pharmaceutical composition can be aqueous or non-aqueous in nature. For example, suitable carriers or excipients can be water for injection, physiological saline solution or artificial cerebrospinal fluid, and may also be supplemented with other materials common in the composition of parenteral administration. Neutral buffered saline or saline mixed with serum albumin is a further exemplary excipient. The pharmaceutical composition may include a Tris buffer of about pH 7.0-8.5 or an acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute. The pharmaceutical composition of the present invention for storage can be prepared by mixing the selected composition with the desired purity with the optional formulation (Remington's Pharmacy, the same) in the form of a lyophilized cake or an aqueous solution. In addition, the synthetic peptide of the present invention can be prepared by mixing a suitable excipient such as sucrose into a lyophilized product.

[0071] The route of administration of pharmaceutical composition of the present invention comprises oral route, by intravenous, intraperitoneal, intracerebral (in substance), intraventricular, intramuscular, intraocular, intraarterial, intraportal vein or intralesional approach injection, or by sustained release system or by implant device.Described pharmaceutical composition can be administered by bolus injection, or by infusion or implant device continuous administration.Described pharmaceutical composition also can be administered by implantation of film, sponge or another kind of material that has been adsorbed or encapsulated with synthetic histamine on it.When using implant device, described device can be implanted in any suitable tissue or organ, and the sending of endogenous or synthetic histamine can be carried out by diffusion, timed release bolus or continuous administration.

[0072] When considering parenteral administration, the composition used in the present invention can take the form of pyrogen-free, parenteral acceptable aqueous solution, which contains endogenous or synthetic histamine of the present invention in a pharmaceutically acceptable vehicle. The particularly suitable vehicle for parenteral injection is sterile distilled water, in which the synthetic peptide is formulated into a sterile isotonic solution, which is suitably preserved. Preparation can include the preparation of the synthetic peptide with a controlled or sustained release reagent such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), pearls or liposomes that can provide the synthetic peptide, which can then be delivered by reservoir injection. Specifically, the preparation with hyaluronic acid has the effect of promoting the circulation duration.

[0073] The composition can also be formulated for inhalation. In these embodiments, the synthetic peptides of the invention are formulated as dry powders for inhalation, or can also be formulated as inhalation solutions with propellants for aerosol delivery, such as by nebulization. Pulmonary administration is further described, for example, in WO 1994 / 020069.

[0074] The pharmaceutical composition of the present invention can be delivered by the digestive tract, for example, orally. The preparation of such pharmaceutically acceptable compositions is within the technical scope of the art. The synthetic peptides of the present invention administered in this manner can be formulated with or without the carriers conventionally used for solid dosage forms, such as tablets and capsules. The capsule can be designed to release the active part of the preparation at a point where bioavailability is maximized and pre-systemic degradation is minimized in the gastrointestinal tract. Other agents can be included to promote the absorption of the synthetic peptides. Diluents, flavoring agents, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants and adhesives can also be used.

[0075] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. By including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc., it is possible to ensure that the action of microorganisms is prevented. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. By including agents that delay absorption such as aluminum monostearate and gelatin, extended absorption of injectable drug forms can be achieved.

[0076] In certain embodiments, the synthetic peptides are formulated for treating ocular diseases or conditions on and within the eye. In specific embodiments, the synthetic peptides of the invention can be formulated and administered to the eye in the form of drops; in the form of a topical gel; as a solid formulation (e.g., similar to LACRISERT, a hydroxypropylcellulose ophthalmic insert); by injection into the anterior chamber of the eye, by injection into the posterior chamber of the eye, for inhibiting angiogenesis, inhibiting destructive MMP activity, or enhancing epithelial wound healing; by coating surgical devices (intraocular lenses, glaucoma devices, corneal prostheses, tear duct cannulas, tear duct bypass tubes); by coating contact lenses; or by coating microbeads, nanobeads, or other similar structures.

[0077] 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; can be used as adjunctive therapy with antimicrobial agents (e.g., histamine, cystatin, lachrymogen, lactoferrin LL-37), antiviral agents, antiparasitic agents, immunomodulators (e.g., glucocorticoids, cyclosporine, NSAIDs), anti-scarring agents (e.g., mitomycin C or similar antimetabolites), collagen, gelatin, analgesics, anesthetics, or combinations thereof; and / or can be used in a plan involving rotation between any or all of these agents, thereby reducing long-term exposure to any one agent (and the side effects thus produced).

[0078] The present invention also provides a kit containing one or more of the synthetic peptides or a pharmaceutical composition containing them, and one or more of an optional antimicrobial, antiviral, antiparasitic, immunomodulator, anti-scarring agent, collagen, gelatin, analgesic or anesthetic. The kit is usually provided in a suitable container (e.g., foil, plastic or cardboard packaging). In certain embodiments, the kit may include one or more pharmaceutical excipients or carriers, pharmaceutical additives, etc. described herein. In other embodiments, the kit may include tools for correct administration, such as graduated cups, syringes, needles, cleaning aids, intraocular lenses, glaucoma devices, orbital implants, corneal prostheses, tear duct cannulas, tear duct bypass tubes, contact lenses, etc. In certain embodiments, the kit may include instructions for correct administration and / or preparations for correct administration.

[0079] In view of the wound healing and epithelial cell migration promoting 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 one or more synthetic peptides of the present invention to a subject in need of such treatment in an amount effective to promote wound healing and / or epithelial cell migration. When used herein, "subject" is intended to include humans and non-human animals, particularly those suffering from diseases or disorders, and may benefit from the promotion of wound healing and / or epithelial cell migration, such as wounds or other body surface injuries such as surgical wounds, excision wounds, blisters, ulcers, other injuries, scratches, avulsions, cuts, dirty wounds, furuncles, and heat or corrosive burns as treatments for wounds associated with epithelial defects and recurrent epithelial erosions. Such wounds may be caused by both mechanical damage and other diseases, such as diabetes, corneal dystrophy, uremia, malnutrition, vitamin deficiency, obesity, infection, immunodeficiency, or complications associated with systemic use of steroids, radiotherapy, nonsteroidal anti-inflammatory drugs, and anticancer drugs.

[0080] Notably, synthetic peptides comprising the SHRGY (SEQ ID NO: 2) sequence have also been shown to increase ERK1 / 2 activation. Thus, the present invention also provides methods for increasing ERK activation by administering one or more synthetic peptides of the present invention to a subject in need of treatment in an amount effective to increase ERK activation. It is well known 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, administration of Hst5 increased the signal intensity of phosphorylated ERK1 / 2 throughout the epithelial monolayer, indicating that Hst5 increases ERK1 / 2 activation of injured epithelium. Furthermore, although untreated and SP1 peptide (which lacks SHRGY (SEQ ID NO: 2)) treated samples had similar pERK1 / 2 localization, Hst5 and SP2 treated samples had elevated immunolocalization of pERK1 / 2 at wound healing sites. This unexpected result suggests that the SHRGY (SEQ ID NO: 2) peptide, which heretofore has no known ability to modulate ERK and would not be predicted to modulate ERK based on understanding of the functional domains, can confer immunomodulatory activity to the synthetic peptides disclosed herein.

[0081] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a synthetic peptide of the present invention or a pharmaceutical composition containing the synthetic peptide sufficient to achieve the desired result stated. In some cases, the effective amount provides a measurable improvement in epithelial cell migration rate, wound closure rate or time, and / or ERK increase and survival pathway regulation compared to subjects who have not yet received such treatment. The amount of the peptide that constitutes an "effective amount" or "therapeutically effective amount" may vary with the severity of the disease, the condition of the patient to be treated, the weight or age, the frequency of administration, or the route of administration, but can be routinely determined by one of ordinary skill in the art. Depending on the site and condition to be treated, the synthetic peptide can be used in a dose ranging from 1 nanomolar to 500 micromolar or higher. The clinician can adjust the dose or route of administration to obtain the best therapeutic effect. Depending on the above factors, a typical dose ranges from about 0.1 μg / kg to up to about 100 mg / kg or higher. In certain embodiments, the dosage may be in the range of 0.1 μg / kg up to about 100 mg / kg, or 1 μg / kg up to about 100 mg / kg, or 5 μg / kg up to about 100 mg / kg.

[0082] "Treating" a subject means achieving one or more of the following: (a) reducing the severity of a disease or condition; (b) arresting the development of a disease or condition; (c) inhibiting the worsening of a disease or condition; (d) limiting or preventing the recurrence of the disease or condition in patients who have previously suffered from the disease or condition; (e) causing regression of the disease or condition; (f) ameliorating or eliminating the symptoms of the disease or condition; and / or (g) improving survival.

[0083] According to the present invention, the synthetic peptides are specifically used to treat ocular diseases or conditions, including but not limited to ocular surface inflammatory disorders such as corneal inflammation (e.g., Moren's or inflammatory and infected ulcers), necrotizing scleritis, ocular surface diseases mediated by inflammation, 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.

[0084] In addition to treating eye diseases and conditions, the synthetic peptides can also be tailored to promote wound healing and / or epithelial migration in other tissues or organs of interest, particularly to treat wounds, inflammation, cancer, infection, or injury. In one embodiment, laminar tissue, neural tissue, connective tissue, vascular tissue, muscle tissue, bone tissue, or blood components are treated. In another embodiment, organs such as the skin, liver, lungs, kidneys, heart, or intestines are treated.

[0085] The following non-limiting examples are provided to further illustrate the present invention.

[0086] Example 1: Materials and Methods

[0087] Peptide Synthesis. Histamine-5 peptides were synthesized using standard Fmoc-based solid phase synthesis chemistry on a Symphony peptide synthesizer (Protein Technologies, Tucson, AZ) with the first amino acid (Fmoc-Tyr-OH) covalently attached to Wang resin. The peptides were synthesized in cycles that began with removal of the Fmoc group in 20% piperidine in N,N-dimethylformamide (DMF). The next amino acid was coupled using 0.1 M HBTU in DMF containing 0.4 M 4-methylmorpholine for 30 min x 2 and this process was continued to complete the synthesis. The resin-bound peptide was deprotected and cleaved from the resin using trifluoroacetic acid (TFA). Ether was added to precipitate the peptide from the TFA solution. The precipitated peptide was then dissolved in 50% acetonitrile in water and freeze-dried. BioCad SPRINT TM The crude peptides were HPLC-ELISA (Applied Biosystems, Foster City, CA) on a Kinetex TM Purification was performed on a reverse phase C18 column 150 x 21.1 mm (Phenomenex, CA). Pure peptide fractions were identified by electrospray ionization mass spectrometry (ESI MS) and freeze-dried as appropriate. The cyclized form of SHRGY (SEQ ID NO: 2) was purified using a 1:1 PCR reaction mixture consisting of 6-(Fmoc-amino)hexanoic acid, 6-(Fmoc-amino)hexanoic acid (C 21 H 23 NO4) composed of linker / spacer preparation.

[0088] The peptides were dissolved in cell culture grade water to obtain a 10 mM stock solution and stored at -20°C. Scrambled peptides (SP1, SP2, SP3, SP4 and SP5) were used as controls. SP1 is a scrambled peptide of 24 amino acid residues based on the full-length native Hst5 peptide. SP2 includes 19 scrambled N-terminal amino acid residues (based on the N-terminal 19 amino acid residues of the native Hst5 peptide) and a SHRGY (SEQ ID NO: 2) sequence at the C-terminus. SP3 and SP4 are scrambled forms of the SHRGY (SEQ ID NO: 2) peptide, and SP5 is a random pentapeptide with charge characteristics and molecular weight similar to SHRGY (SEQ ID NO: 2). Table 5 shows the sequences of the peptides used in this study.

[0089] Table 5

[0090] Name Sequence SEQ ID NO: Histamine - 5 (Hst5) DSHAKRHHGYKRKFHEKHHSHRGY 62 Hst5(1 - 14) DSHAKRHHGYKRKF 63 Hst5(1 - 19) DSHAKRHHGYKRKFHEKHH 64 Hst5(1 - 21) DSHAKRHHGYKRKFHEKHHSH 65 Hst5(1 - 22) DSHAKRHHGYKRKFHEKHHSHR 66 Hst5(1 - 23) DSHAKRHHGYKRKFHEKHHSHRG 67 Hst5(5 - 24) RHHGYKRKFHEKHHSHRGY 68 SHRGY SHRGY 2 SP1 YHGHRHFRKHKKHKEAHSYDRGSH 69 SP2 HSHKEGHHYKRFKRKHHADSHRGY 70 SP3 RYSGH 71 SP4 RYHGS 72 SP5 TNQQK 73

[0091] Cell culture. Human corneal limbal epithelial (HCLE) cells were cultured in keratinocyte serum-free medium (K-SFM; ThermoScientific, Waltham, MA) supplemented with 0.2 ng / mL rhEGF (ThermoScientific, Waltham, MA), bovine pituitary extract (ThermoScientific, Waltham, MA), and 1% amphotericin B (ThermoScientific, Waltham, MA). Standard cell culture conditions (37° C., 5% CO 2 ,>95% humidity) were used during routine passage. The culture medium was changed every 48 hours after inoculation.

[0092] Human corneal epithelial (HCE) cells were cultured in minimum essential medium (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). MCF-7 cell lines were cultured 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) at 37°C in a humidified atmosphere of 5% CO2 and 95% air.

[0093] Short tandem repeat (STR) analysis was performed on each cell line to confirm the authenticity of each cell line used.

[0094] In vitro scratch test. HCE, HCLE, HELA and MCF-7 cells were cultured at 2.5×10 5The inoculation density of (cells / well) was cultivated in 24-well plates and grown to confluence on 24-well plates. Subsequently, a sterile P200 pipette tip was used to make a straight scratch. The cells were then washed twice with phosphate buffered saline (PBS) to remove cell debris. The wound area was then treated with various concentrations in standard culture medium (0.5% FBS (HCE) in MEM culture medium, 1% FBS (HeLa) in growth factor-free K-SFM culture medium (HCLE), 0.5% FBS (MCF-7) in RPMI 1640 culture medium) under low serum conditions with peptides provided in Table 5. During the experiment, the scratches were photographed under a microscope (ImageExpress Micro, Molecular Devices, San Jose, CA) at 4X magnification per hour. 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 the closure of the untreated wound. For all experiments involving truncated histamine-5, a final concentration of 80 μM was used. PD98059 (Calbiochem, San Diego, CA) was used as a specific inhibitor of MEK at a final concentration of 50 μM. PD98059 was added to the cell culture simultaneously with the histamine peptides.

[0095] 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. C57BL16J (Jackson Laboratory, Bar Harbor, ME) mice aged 12-19 weeks were anesthetized by intraperitoneal injection of ketamine (100 mg / kg) and xylazine (5 mg / kg). After applying 2 drops of topical 0.5% proparacaine, a 2.0-mm area of ​​the central epithelium was delineated using a 2-mm disposable biopsy punch and removed by Alger Brush II (The Alger Company, Lago Vista, TX). In the treatment (n=7) or control (n=7) groups, histamine-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 cornea was treated with fluorescein ( Fluorescein sodium ophthalmic strips were used (Akorn, Lake forest, IL) and photographed using a NIKON FS-2 light slit lamp and a NIKON D200 camera (Melville, NY). For each mouse, wound size was compared to baseline and the percentage of wound closure was measured using Image J software.

[0096] Cell budding assay. The cell budding assay used HCE cells, which were produced under the trade name The HCE cells were plated at 5×10 5 The density of cells / 10 μL spots was embedded in dissolved basement membrane. The cell spots were then exposed to low serum medium (0.5% FBS) (untreated negative control), Hst5 (50 μM) or 10% FBS (positive control). HCE cell migration was then tracked using time-lapse microscopy and imaged at 4X (Image Express Micro; Molecular Devices, CA). Image J was used to measure the cell coverage area at a given time point.

[0097] Western blotting. Western blotting was performed according to standard methods. Protein lysate (20 μg) was plated on NuPAGE TM The samples were boiled in LDS sample buffer (Invitrogen, Carlsbad, CA) for 10 min and then subjected to 12% NuPAGE. TM Electrophoresis was performed on a Bis-Tris gel (Invitrogen, Carlsbad, CA) and then transferred to a nitrocellulose membrane (Amersham Protran, GE Healthcare, Pittsburgh, PA). The membrane was then blocked with Tris-buffered saline containing 3% skim milk powder for 1 hour and incubated with a primary antibody against pERK1 / 2 (Cell Signaling, Danver, MA) (1:1000) at 4°C overnight. After washing in 0.05% Tris-buffered saline containing 0.05% polysorbate 20, the membrane was incubated with goat anti-rabbit antibody-HRP (BD Biosciences, San Jose, CA) (1:2000) as a secondary antibody for 1 hour. The membrane was developed using X-ray film and ECL Pro solution (PerkinElmer, Waltham, MA). β-actin was used as an internal control.

[0098] Immunofluorescence imaging. HCE cells were plated at 9x10 4The inoculation density of (cells / well) was inoculated in 8-well cell culture slides and allowed to incubate to form a confluent monolayer. A sterile P10 pipette tip was then used to make a straight scratch. The cells were then washed with culture medium to remove cell debris. The wound area was then kept untreated, or treated with Hst5, SP1 or SP2 at a concentration of 80 μM in MEM culture medium containing 0.5% FBS for 45 minutes. The cells were then fixed with 4% polyformaldehyde 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 then incubated with the first antibody (1:200) (Cell Signaling, Danver, MA) for p-ERK1 / 2 diluted in 1% BSA at 4°C for 16 hours. After washing three times with PBS, the cells were incubated with goat anti-rabbit IgG antibody (BD Biosciences, San Jose, CA) (1:500) diluted in 1% BSA at room temperature for 60 minutes. After being fully washed with PBS, the cells were stained with 4',6-diamidino-2-phenylindole (DAPI; Roche, Mannheim, GE) for 2 minutes for nuclear staining. The cells were fixed in Fluoro gel (Electron Microscopy Sciences, Hattfield, PA) containing Tris buffer and observed under a confocal microscope (Zeiss LSM710 confocal microscope, Oberkochen, Germany) using a 10X objective.

[0099] For immunofluorescence, the whole mouse eyes cut off were quickly frozen in the optimal cutting temperature compound (OCT; Fisher Healthcare, Galderma, CA). The frozen tissue was cut into 10 μm frozen sections (Thermo Scientific NX50 cryostat, Waltham, MA), and then sealed 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 then fixed in Fluoro gel (Electron Microscopy Sciences, Hattfield, PA) containing Tris buffer and observed using a 10X objective lens under a confocal microscope (Zeiss LSM 710 confocal microscope, Oberkochen, Germany).

[0100] Statistical analysis. Experiments were analyzed using two-way or one-way ANOYA followed by Bonferroni or Dunnett post hoc tests or Student t-tests as appropriate. p values ​​< 0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism software 7.0 (GraphPad Software, La Jolla, CA).

[0101] Power analysis to determine sample size for the murine corneal wound experiment was performed using G-Power to compare between treatment (SHRGY (SEQ ID NO: 2) or Hst5) and control (SP1) groups. Parameters used for calculations included a β of 0.8, an α of 0.05, and an effect size of 25%, resulting in a sample size of n=6 per group.

[0102] Example 2: Histamine-5 promotes cell migration

[0103] Used as a spot embedding in The cell budding assay of HCE cells in the low growth factor dissolved basement membrane (Corning Life Sciences, Tewksbury, MA) sold was used to determine whether HST5 can promote epithelial cell migration. At 72 hours, there was a statistically significant increase in cell migration in the Hst5 (50 μM) treatment condition compared to the vehicle-only control. The effect of Hst5 on cell migration was tested using a standardized scratch assay of the HCLE cell line. HCLE cells were grown to confluence and mechanically scratched using a pipette tip. Cells were treated with Hst5 at different concentrations (20, 50, 80 and 100 μM) or kept untreated as a control. Time-lapse microscopy was performed and wound area was analyzed at different time points. This analysis confirmed that the closure rate of the in vitro scratch assay had a dose-dependent increase when Hst5 was applied compared to the untreated control. The most significant increase in the scratch closure rate was observed at 50 μM. These findings were confirmed 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.

[0104] Example 3: The C-terminal SHR domain of Hst5 is required for promoting epithelial cell migration

[0105] To identify the residues of Hst5 required for epithelial cell migration, a series of truncation experiments were performed with progressive deletion of residues in Hst5. This analysis showed that the C-terminal SHRGY (SEQ ID NO: 2) residue of Hst5 is necessary and sufficient to promote migration ( Figure 1All peptides were tested at 80 μM. Truncated forms of Hst5 excluding the C-terminal SHRGY (SEQ ID NO: 2) sequence, i.e., Hst5(1-14), Hst5(1-19), Hst5(1-21), Hst5(1-22), and Hst5(1-23), did not show a significant increase in the wound closure rate ( Figure 1 However, constructs containing the complete SHRGY sequence, i.e., Hst5, Hst5(5-24), SP2, SHRGY (SEQ ID NO: 2), a polymer of said sequence consisting of four repeating units of SHRGY (SEQ ID NO: 2) (i.e., SHRGY-(CH2)6-SHRGY-(CH2)6-SHRGY-(CH2)6-SHRGY) (SEQ ID NO: 51), and a cyclized SHRGY (SEQ ID NO: 2) peptide (c-SHRGY), showed a significant increase in the scratch closure rate ( Figure 1 ). Scrambled peptides SP3 and SP4 did not significantly increase the wound closure rate. Similarly, random pentapeptide SP5, which has similar molecular weight and charge characteristics to SHRGY (SEQ ID NO: 2), did not show a significant increase in the wound closure rate. Therefore, the SHRGY (SEQ ID NO: 2) sequence is necessary and sufficient to promote epithelial migration rate in the in vitro scratch assay.

[0106] Example 4: ERK activation is essential for the pro-migratory effect of Hst5

[0107] The activation / phosphorylation levels of ERK were studied with and without trauma and with and without Hst5 administration to determine whether the cell signaling pathways underlying the pro-migratory effects of Hst5 are similar to Hst1 in other epithelial cell types. Trauma results in an increase in the phosphorylated form of ERK1 / 2 (p-ERK1 / 2). Immunolocalization was used to determine whether the application of Hst5 to the scratched epithelial layer would affect pERK1 / 2 levels. Western blot analysis confirmed that trauma alone increases pERK1 / 2 levels, and these levels are further increased by the administration of Hst5. The administration of the scrambled peptide SP2 containing SHRGY (SEQ ID NO: 2) increased pERK1 / 2 levels to an intensity comparable to that of Hst5. SP1 without SHRGY (SEQ ID NO: 2) did not induce the same increase in pERK1 / 2 immunolocalization as the peptide containing SHRGY (SEQ ID NO: 2). Co-treatment of Hst5 and the MEK-specific inhibitor PD98059 abolished the effect of Hst5 on promoting wound closure, indicating that the action of Hst5 requires ERK activation.

[0108] Example 5: Hst5 administration promotes wound healing in a murine corneal injury model

[0109] Using a standard corneal injury mouse model, it was observed that topical administration of Hst5 or SHRGY (SEQ ID NO: 2) peptides provided a significant improvement in corneal wound closure rate, at levels superior to that of the scrambled peptide control (SP1) ( Figure 2 ). Histological analysis of wounded corneas (DAPI staining on corneal cross sections) demonstrated pathological evidence of reduced corneal wound size under Hst5-treated conditions compared to SP1-treated controls. Thus, Hst5 and peptides containing SHRGY (SEQ ID NO: 2) can enhance wound healing in a well-examined murine model of corneal epithelial injury.

[0110] Example 6: Toxicity and Salt Forms

[0111] Exposure of human corneal epithelial cells to increasing concentrations of SHRGY (SEQ ID NO: 2) pentapeptide, i.e., 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1000 μM, 2000 μM, 4000 μM and 8000 μM, showed minimal reduction in cell viability after 24 hours at concentrations at or below 4000 μM, as determined using a conventional WST1 assay or in the LDH assay up to cell death induced at concentrations exceeding 4000 μM at 24 hours.

[0112] The cytotoxicity of different salt forms of the SHRGY (SEQ ID NO: 2) pentapeptide was also studied. Three different salt forms of SHRGY (SEQ ID NO: 2), acetate, hydrochloride and trifluoroacetate, were prepared and analyzed for toxicity determined using a conventional WST1 assay at 15.625 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM and 500 μM. All three salt forms of SHRGY (SEQ ID NO: 2) showed no significant toxicity compared to untreated samples at any peptide concentration tested.

[0113] The wound closure rates of the three salt forms were also analyzed according to the methods disclosed herein. Specifically, scratches were made on human corneal epithelial cells using a standard scratch assay and the wound healing rates were measured after application of the acetate, hydrochloride, and trifluoroacetate salt forms of SHRGY (SEQ ID NO: 2). All forms tested showed an enhancement in wound closure rate ( Figure 3 ). Sequence Listing <110> The Board of Trustees of the University of Illinois Aakalu, Vinay K. <120> Pentapeptides and methods of use thereof <130> UIC0087WO <150> US 62 / 941,226 <151> 2019-11-27 <160> 73 <170> PatentIn Version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> Miscellaneous Features <222> (3) <223> X represents Arg, Lys, His, Asp or Glu <400> 1 Ser His Xaa Gly Tyr 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 2 Ser His Arg Gly Tyr 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 3 Ser His Asp Gly Tyr 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 4 Ser His Lys Gly Tyr 1 5 <210> 5 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 5 Ser His Gly Tyr 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 6 Ser His Glu Gly Tyr 1 5 <210> 7 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 7 Asp Ser His Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His Glu 1 5 10 15 Lys His His Ser His Arg Gly Tyr Arg Ser Asn Tyr Leu Tyr Asp Asn 20 25 30 <210> 8 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 8 His His Ser His Arg Gly Tyr Arg Ser Asn 1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 9 His Glu Lys His His Ser His Arg Gly Tyr 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 10 Glu Lys His His Ser His Arg Gly Tyr Arg 1 5 10 <210> 11 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 11 Lys His His Ser His Arg Gly Tyr 1 5 <210> 12 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 12 His His Ser His Arg Gly Tyr 1 5 <210> 13 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 13 His Ser His Arg Gly Tyr 1 5 <210> 14 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> Miscellaneous Features <222> (3)..(4) <223> X represents Lys, Arg or His <400> 14 His Glu Xaa Xaa His 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 15 His Glu Lys Lys His 1 5 <210> 16 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 16 His Glu Lys Arg His 1 5 <210> 17 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 17 His Glu Lys His His 1 5 <210> 18 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 18 His Glu Arg Lys His 1 5 <210> 19 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 19 His Glu Arg Arg His 1 5 <210> 20 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 20 His Glu Arg His His 1 5 <210> twenty one <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty one His Glu His Lys His 1 5 <210> twenty two <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty two His Glu His Arg His 1 5 <210> twenty three <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty three His Glu His His His 1 5 <210> twenty four <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty four Gly Tyr Lys Arg Lys Phe His Glu Lys His His Ser His Arg 1 5 10 <210> 25 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 25 His Glu Lys Arg His His 1 5 <210> 26 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 26 Ser Asn Tyr Leu Tyr Asp Asn 1 5 <210> 27 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 27 Tyr Gly Asp Tyr Gly Ser Asn Tyr Leu Tyr Asp Asn 1 5 10 <210> 28 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 28 Arg Lys Phe His Glu Lys His His Ser His Arg Gly Tyr Arg 1 5 10 <210> 29 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 29 Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His 1 5 10 <210> 30 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> Miscellaneous Features <222> (6)..(9) <223> Xaa is either present or absent, and when present represents Gly <220> <221> Miscellaneous Features <222> (10)..(10) <223> Xaa is either present or absent, and when present indicates Ser <220> <221> Miscellaneous Features <222> (11)..(14) <223> Xaa is either present or absent, and when present represents Gly <220> <221> Miscellaneous Features <222> (15) <223> Xaa is either present or absent, and when present indicates Ser <220> <221> Miscellaneous Features <222> (16)..(19) <223> Xaa is either present or absent, and when present represents Gly <220> <221> Miscellaneous Features <222> (20)..(20) <223> Xaa is either present or absent, and when present indicates Ser <220> <221> Miscellaneous Features <222> (21)..(24) <223> Xaa is either present or absent, and when present represents Gly <220> <221> Miscellaneous Features <222> (25)..(25) <223> Xaa is either present or absent, and when present indicates Ser <400> 30 Gly Gly Gly Gly Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 <210> 31 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 31 Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> 32 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 32 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 1 5 10 <210> 33 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 33 Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 34 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 34 Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe 1 5 10 <210> 35 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> Miscellaneous Features <222> (5)..(6) <223> Xaa is either present or absent, and when present represents Gly <220> <221> Miscellaneous Features <222> (7) <223> Xaa is either present or absent, and when present indicates Ser <220> <221> Miscellaneous Features <222> (8)..(10) <223> Xaa is either present or absent, and when present represents Gly <220> <221> Miscellaneous Features <222> (11)..(11) <223> Xaa is either present or absent, and when present indicates Ser <220> <221> Miscellaneous Features <222> (12)..(14) <223> Xaa is either present or absent, and when present represents Gly <220> <221> Miscellaneous Features <222> (15) <223> Xaa is either present or absent, and when present indicates Ser <220> <221> Miscellaneous Features <222> (16)..(18) <223> Xaa is either present or absent, and when present represents Gly <220> <221> Miscellaneous Features <222> (19)..(19) <223> Xaa is either present or absent, and when present indicates Ser <220> <221> Miscellaneous Features <222> (20)..(20) <223> Xaa is either present or absent, and when present represents Gly <400> 35 Gly Gly Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa 20 <210> 36 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> Miscellaneous Features <222> (3) <223> Xaa is either present or absent, and when present represents Gly <220> <221> Miscellaneous Features <222> (4) <223> Xaa is either present or absent, and when present indicates Ser <220> <221> Miscellaneous Features <222> (5) <223> Xaa is either present or absent, and when present represents Gly <220> <221> Miscellaneous Features <222> (6) <223> Xaa is either present or absent, and when present indicates Ser <220> <221> Miscellaneous Features <222> (7) <223> Xaa is either present or absent, and when present represents Gly <220> <221> Miscellaneous Features <222> (8) <223> Xaa is either present or absent, and when present indicates Ser <220> <221> Miscellaneous Features <222> (9)..(9) <223> Xaa is either present or absent, and when present represents Gly <220> <221> Miscellaneous Features <222> (10)..(10) <223> Xaa is either present or absent, and when present indicates Ser <400> 36 Gly Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 <210> 37 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> Miscellaneous Features <222> (6) <223> Xaa is present or absent, and when present indicates Glu <220> <221> Miscellaneous Features <222> (7)..(9) <223> Xaa is either present or absent, and when present represents Ala <220> <221> Miscellaneous Features <222> (10)..(10) <223> Xaa is present or absent, and when present indicates Lys <220> <221> Miscellaneous Features <222> (11)..(11) <223> Xaa is present or absent, and when present indicates Glu <220> <221> Miscellaneous Features <222> (12)..(14) <223> Xaa is either present or absent, and when present represents Ala <220> <221> Miscellaneous Features <222> (15) <223> Xaa is present or absent, and when present indicates Lys <220> <221> Miscellaneous Features <222> (16) <223> Xaa is present or absent, and when present indicates Glu <220> <221> Miscellaneous Features <222> (17)..(19) <223> Xaa is either present or absent, and when present represents Ala <220> <221> Miscellaneous Features <222> (20)..(20) <223> Xaa is present or absent, and when present indicates Lys <220> <221> Miscellaneous Features <222> (21)..(21) <223> Xaa is present or absent, and when present indicates Glu <220> <221> Miscellaneous Features <222> (22)..(24) <223> Xaa is either present or absent, and when present represents Ala <220> <221> Miscellaneous Features <222> (25)..(25) <223> Xaa is present or absent, and when present indicates Lys <400> 37 Glu Ala Ala Ala Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 <210> 38 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> Miscellaneous Features <222> (7) <223> Xaa is present or absent, and when present indicates Glu <220> <221> Miscellaneous Features <222> (8)..(10) <223> Xaa is either present or absent, and when present represents Ala <220> <221> Miscellaneous Features <222> (11)..(11) <223> Xaa is either present or absent, and when present indicates Lys <220> <221> Miscellaneous Features <222> (12)..(12) <223> Xaa is present or absent, and when present indicates Glu <220> <221> Miscellaneous Features <222> (13)..(15) <223> Xaa is either present or absent, and when present represents Ala <220> <221> Miscellaneous Features <222> (16) <223> Xaa is either present or absent, and when present indicates Lys <220> <221> Miscellaneous Features <222> (17) <223> Xaa is present or absent, and when present indicates Glu <220> <221> Miscellaneous Features <222> (18)..(20) <223> Xaa is either present or absent, and when present represents Ala <220> <221> Miscellaneous Features <222> (21)..(21) <223> Xaa is present or absent, and when present indicates Lys <220> <221> Miscellaneous Features <222> (22)..(22) <223> Xaa is present or absent, and when present indicates Glu <220> <221> Miscellaneous Features <222> (23)..(25) <223> Xaa is either present or absent, and when present represents Ala <220> <221> Miscellaneous Features <222> (26)..(26) <223> Xaa is present or absent, and when present indicates Lys <400> 38 Ala Glu Ala Ala Ala Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala 20 25 <210> 39 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 39 Pro Ala Pro Ala Pro 1 5 <210> 40 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> Miscellaneous Features <222> (1) <223> Xaa represents any amino acid residue <220> <221> Miscellaneous Features <222> (3) <223> Xaa is either present or absent, and when present represents any amino acid residue <220> <221> Miscellaneous Features <222> (4) <223> Xaa is either present or absent, and when present indicates Pro <220> <221> Miscellaneous Features <222> (5) <223> Xaa is either present or absent, and when present represents any amino acid residue <220> <221> Miscellaneous Features <222> (6) <223> Xaa is either present or absent, and when present indicates Pro <220> <221> Miscellaneous Features <222> (7) <223> Xaa is either present or absent, and when present represents any amino acid residue <220> <221> Miscellaneous Features <222> (8) <223> Xaa is either present or absent, and when present indicates Pro <220> <221> Miscellaneous Features <222> (9)..(9) <223> Xaa is either present or absent, and when present represents any amino acid residue <220> <221> Miscellaneous Features <222> (10)..(10) <223> Xaa is either present or absent, and when present indicates Pro <400> 40 Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 <210> 41 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 41 Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp 1 5 10 15 Val <210> 42 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 42 Pro Leu Gly Leu Trp Ala 1 5 <210> 43 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 43 Arg Val Leu Ala Glu Ala 1 5 <210> 44 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 44 Glu Asp Val Val Cys Cys Ser Met Ser Tyr 1 5 10 <210> 45 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 45 Gly Gly Ile Glu Gly Arg Gly Ser 1 5 <210> 46 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 46 Thr Arg His Arg Gln Pro Arg Gly Trp Glu 1 5 10 <210> 47 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 47 Ala Gly Asn Arg Val Arg Arg Ser Val Gly 1 5 10 <210> 48 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 48 Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 49 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 49 Gly Phe Leu Gly 1 <210> 50 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 50 Cys Arg Arg Arg Arg Arg Arg Glu Ala Glu Ala Cys 1 5 10 <210> 51 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> Non-consecutive residues <222> (5)..(6) <223> The residues are linked via -(CH2)6-hydrocarbon <220> <221> Non-consecutive residues <222> (10)..(11) <223> The residues are linked via -(CH2)6-hydrocarbon <220> <221> Non-consecutive residues <222> (15)..(16) <223> The residues are linked via -(CH2)6-hydrocarbon <400> 51 Ser His Arg Gly Tyr Ser His Arg Gly Tyr Ser His Arg Gly Tyr Ser 1 5 10 15 His Arg Gly Tyr 20 <210> 52 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> Non-consecutive residues <222> (5)..(6) <223> The residues are linked via -(CH2)6-hydrocarbon <220> <221> Non-consecutive residues <222> (10)..(11) <223> The residues are linked via -(CH2)6-hydrocarbon <220> <221> Non-consecutive residues <222> (15)..(16) <223> The residues are linked via -(CH2)6-hydrocarbon <400> 52 Ser His Arg Gly Tyr Ser His Asp Gly Tyr Ser His Arg Gly Tyr Ser 1 5 10 15 His Asp Gly Tyr 20 <210> 53 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 53 Ser His Arg Gly Tyr Gly Gly Gly Gly Gly Gly Gly Gly Ser His Arg 1 5 10 15 Gly Tyr Gly Gly Gly Gly Ser Ser His Arg Gly Tyr Gly Gly Gly Gly 20 25 30 Gly Gly Gly Gly Ser His Arg Gly Tyr 35 40 <210> 54 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 54 Ser His Arg Gly Tyr Gly Gly Gly Gly Ser Ser His Arg Gly Tyr Gly 1 5 10 15 Gly Gly Gly Ser Ser His Arg Gly Tyr Gly Gly Gly Gly Ser Ser His 20 25 30 Arg Gly Tyr 35 <210> 55 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 55 Ser His Arg Gly Tyr Gly Gly Gly Gly Gly Gly Gly Gly Ser His Arg 1 5 10 15 Gly Tyr Pro Ala Pro Ala Pro Ser His Arg Gly Tyr Gly Gly Gly Gly 20 25 30 Gly Gly Gly Gly Ser His Arg Gly Tyr 35 40 <210> 56 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 56 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> 57 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> Non-consecutive residues <222> (6)..(7) <223> The residues are linked via -(CH2)6-hydrocarbon <220> <221> Non-consecutive residues <222> (11)..(12) <223> The residues are linked via -(CH2)6-hydrocarbon <220> <221> Non-consecutive residues <222> (16)..(17) <223> The residues are linked via -(CH2)6-hydrocarbon <400> 57 Cys Ser His Arg Gly Tyr Ser His Arg Gly Tyr Ser His Arg Gly Tyr 1 5 10 15 Ser His Arg Gly Tyr Cys 20 <210> 58 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> Non-consecutive residues <222> (6)..(7) <223> The residues are linked via -(CH2)6-hydrocarbon <220> <221> Non-consecutive residues <222> (11)..(12) <223> The residues are linked via -(CH2)6-hydrocarbon <220> <221> Non-consecutive residues <222> (16)..(17) <223> The residues are linked via -(CH2)6-hydrocarbon <400> 58 Cys Ser His Arg Gly Tyr Ser His Asp Gly Tyr Ser His Arg Gly Tyr 1 5 10 15 Ser His Asp Gly Tyr Cys 20 <210> 59 <211> 43 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 59 Cys Ser His Arg Gly Tyr Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser His 1 5 10 15 Arg Gly Tyr Gly Gly Gly Gly Ser Ser His Arg Gly Tyr Gly Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ser His Arg Gly Tyr Cys 35 40 <210> 60 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 60 Cys Ser His Arg Gly Tyr Gly Gly Gly Gly Ser Ser His Arg Gly Tyr 1 5 10 15 Gly Gly Gly Gly Ser Ser His Arg Gly Tyr Gly Gly Gly Gly Ser Ser 20 25 30 His Arg Gly Tyr Cys 35 <210> 61 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 61 Cys Ser His Arg Gly Tyr Gly Gly Gly Gly Gly Gly Gly Gly Ser His 1 5 10 15 Arg Gly Tyr Pro Ala Pro Ala Pro Ser His Arg Gly Tyr Gly Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ser His Arg Gly Tyr Cys 35 40 <210> 62 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 62 Asp Ser His Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His Glu 1 5 10 15 Lys His His Ser His Arg Gly Tyr 20 <210> 63 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 63 Asp Ser His Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe 1 5 10 <210> 64 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 64 Asp Ser His Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His Glu 1 5 10 15 Lys His His <210> 65 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 65 Asp Ser His Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His Glu 1 5 10 15 Lys His Ser His 20 <210> 66 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 66 Asp Ser His Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His Glu 1 5 10 15 Lys His His Ser His Arg 20 <210> 67 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 67 Asp Ser His Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His Glu 1 5 10 15 Lys His His Ser His Arg Gly 20 <210> 68 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 68 Arg His His Gly Tyr Lys Arg Lys Phe His Glu Lys His His Ser His 1 5 10 15 Arg Gly Tyr <210> 69 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 69 Tyr His Gly His Arg His Phe Arg Lys His Lys Lys His Lys Glu Ala 1 5 10 15 His Ser Tyr Asp Arg Gly Ser His 20 <210> 70 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 70 His Ser His Lys Glu Gly His His Tyr Lys Arg Phe Lys Arg Lys His 1 5 10 15 His Ala Asp Ser His Arg Gly Tyr 20 <210> 71 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 71 Arg Tyr Ser Gly His 1 5 <210> 72 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 72 Arg Tyr His Gly Ser 1 5 <210> 73 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 73 Thr Asn Gln Gln Lys 1 5

Claims

1. A synthetic peptide or a pharmaceutically acceptable salt thereof, wherein the synthetic peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 51, SEQ ID NO: 68, SEQ ID NO: 70 and cyclized SHRGY.

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

3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is formulated for topical, oral, ocular, intravenous, intravitreal, subconjunctival, subcutaneous, intramuscular, intraperitoneal, intracerebral, intraarterial, intraportal, intralesional, intrathecal or intranasal administration.

4. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is in the form of a gel, lotion, cream, tablet, capsule, pill, solution, eye drops, spray, bandage, contact lens, depot formulation, injection, implant or sustained release formulation.

5. Use of the synthetic peptide or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for promoting wound healing or epithelial cell migration.

6. The use according to claim 5, wherein the amount of the synthetic peptide or a pharmaceutically acceptable salt thereof is in the range of 1 nanomolar to 500 micromolar.

7. The use according to claim 5, wherein the drug further comprises an antimicrobial agent, an antiviral agent, an antiparasitic agent, an anti-scarring agent, an immunomodulator, collagen, gelatin, an analgesic, an anesthetic or a combination thereof.

8. A pharmaceutical kit comprising one or more synthetic peptides according to claim 1 or pharmaceutically acceptable salts thereof.

9. The kit of claim 8, further comprising an antimicrobial agent, an antiviral agent, an antiparasitic agent, an antifungal agent, an anti-scarring agent, an immunomodulator, collagen, gelatin, an analgesic agent, an anesthetic agent, or a combination thereof.

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