Compositions and methods for promoting wound healing

Abstract

Novel peptides that target CCR10-eNOS binding are provided as cell-permeable constructs and methods of using the constructs to promote or accelerate wound healing, particularly diabetic wound healing.

Description

[Technical field]

[0001] Introduction This application claims the benefit of priority from U.S. Provisional Application No. 62 / 879,717, filed July 29, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] This invention was made with Government support under Grant Nos. HL125356, HL083298, and RR029879 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] background Cardiovascular complications of type 2 diabetes mellitus (T2DM) are now the third leading cause of death in the United States, significantly reducing quality of life and may form the basis of a 25% lifetime risk of developing a non-healing diabetic foot ulcer (DFU), which precedes amputation in up to 90% of diabetic amputees. DFUs are associated with substantial medical costs estimated to be over $10 billion / year in the United States alone.

[0004] Current interventions to treat DFUs include the use of bioengineered skin equivalents, growth factors, granulocyte colony-stimulating factor, hyperbaric oxygen therapy, and negative pressure wound therapy. However, in a meta-analysis of randomized controlled trials, evidence did not support the above interventions, such as growth factors (Marti-Carvajal, et al. (2015) Cochrane Database Syst. Rev. 10:CD008548), as being beneficial for DFU patients, and thus, there remains a huge unmet need. Summary of the Invention

[0005] SUMMARY OF THE PRESENT APPLICATION The invention provides constructs comprised of a 7-20 amino acid peptide having the amino acid sequence Lys-Ile-Ser-Ala-Ser-Leu-Met (SEQ ID NO: 1) operably linked to one or more carrier moieties, such as a cell penetrating peptide, a lipid, or a combination thereof. In some embodiments, the peptide further comprises one or more modifications selected from substitution, carboxylation, glycosylation, sulfonation, amidation, PEGylation, biotinylation, disulfide formation, and addition of a charged amino acid residue. In some embodiments, the peptide is selected from the group of TRKKTFKEVANAVKISASLM (SEQ ID NO: 2), FKEVANAVKISASLM (SEQ ID NO: 3), VANAVKISASLM (SEQ ID NO: 4), and KISASLM (SEQ ID NO: 1). In certain embodiments, the construct is selected from myr-TRKKTFKEVANAVKISASLM (SEQ ID NO: 61), myr-FKEVANAVKISASLM (SEQ ID NO: 62), myr-VANAVKISASLM (SEQ ID NO: 63) and myr-KISASLM (SEQ ID NO: 64). The invention further provides pharmaceutical compositions comprising the constructs, as well as methods for promoting or accelerating wound healing in a subject, including in a diabetic subject. [Brief description of the drawings]

[0006] [Figure 1] FIG. 1 shows the time course of wound healing in WT C57BL / 6 mice treated with Myr-CBD7 constructs. Four 5 mm full-thickness excision wounds were made on the dorsal skin of mice. Wounds were treated with 30 μl (50 μM) of Myr-CBD7 (CBD7) construct or Myr-control construct (Ctl-P) per wound. Of note, Myr-CBD7 construct treatment significantly reduced wound size on days 5 and 7. *P<0.05 (n=8).

[0007] [Diagram 2]Figure 2 shows re-epithelialization of skin wounds in WT mice with Myr-CBD7 treatment compared to control constructs on day 7. Wound closure was measured and quantified in each condition. *P<0.05 (n=5).

[0008] [Diagram 3] Figure 3 shows enhanced eNOS expression by the Myr-CBD7 construct compared to the control construct in mouse wounds at day 7. *P<0.05; **P<0.005 (n=3-4).

[0009] [Figure 4] FIG. 4 shows that in co-IP experiments, CCR10-eNOS interaction is reduced in mouse wounds following treatment with 50 μM Myr-CBD7 construct for 7 days.

[0010] [Diagram 5] FIG. 5 shows that Myr-CBD7 improves wound healing in db / db mice. Four 5 mm full-thickness excision wounds were made on the dorsal skin of db / db mice. Immediately after punch excision, 30 μl (50 μM) of Myr-control construct (Ctl-P) or Myr-CBD7 (CBD7) construct was topically applied to the wounds. Normalized wound size (day 0 set as 100%) values ​​are shown. *P<0.05; **P<0.005; ***P<0.001 vs. Ctl-P (n=10-12). Of note, compared to the control construct, Myr-CBD7 construct treatment significantly reduced wound size from day 4.

[0011] [Figure 6]Figure 6 shows that disruption of eNOS-CCR10 interaction leads to increased eNOS expression in db / db mouse wounds at day 12 after topical application of Myr-CBD7. Normalized values ​​between eNOS and CCR10 are shown. **P<0.005 vs. Ctl-P (n=5). Of note, eNOS-CCR10 was disrupted by Myr-CBD7 compared to Ctl-P in db / db mouse wounds at day 12. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description of the Invention Cardiovascular complications of type 2 diabetes mellitus (T2DM) significantly reduce quality of life and may form the basis of a 25% lifetime risk of developing a non-healing diabetic foot ulcer (DFU), which precedes amputation in up to 90% of cases. In patients with DFU, persistent hyperglycemia leads to endothelial cell (EC) dysfunction, resulting in defective endothelial nitric oxide synthase (eNOS) activity and reduced nitric oxide (NO) production. eNOS is constitutively expressed in vascular endothelial cells and plays a key role in regulating vascular tone and angiogenesis. It has been reported that the deficiency of wound healing response in obese mice can be overcome by increasing NO production via overexpression of eNOS.

[0013] It has now been found that punch biopsies from human subjects with T2DM and obesity-induced diabetes in mice also exhibit increased expression of the chemokine receptor CCR10, eNOS blockade and inactivation by CCR10, and reduced eNOS expression and NO bioavailability. Thus, a cell-permeable construct has now been developed that targets CCR10-eNOS binding, thereby increasing eNOS / NO levels to improve skin wound healing in obese and diabetic mice. A novel construct named Myr-CBD7 (myristoylated CCR10 binding domain) can prevent CCR10-eNOS interaction and also upregulate eNOS expression and activity in dysfunctional endothelial cells. Myr-CBD7 can be provided at concentrations up to 100 μM without adversely affecting cell viability. The construct efficiently enters vascular endothelial cells, resulting in the rescue of normal endothelial cell function and improving skin wound healing in obese and diabetic mice. Thus, the construct finds use as a novel therapeutic approach to improve skin wound healing, particularly in diabetic patients.

[0014] Thus, the present invention provides a construct comprising a peptide having at least the amino acid sequence KISASLM (SEQ ID NO: 1) fused to a carrier moiety, and its use in compositions and methods for modulating CCR10-eNOS interaction to promote wound healing. Ideally, the construct of the present invention does not interfere with or disrupt the binding of calmodulin to eNOS. For the purposes of the present invention, the term "construct" is used herein to refer to a peptide that targets CCR10-eNOS binding and is modified by recombinant, chemical and / or enzymatic techniques to include one or more carrier moieties that improve the uptake of the peptide. In particular, at least one of the one or more carrier moieties is not naturally associated with the peptide. Ideally, the construct of the present invention is a peptide having at least the amino acid sequence depicted in SEQ ID NO: 1 operably linked to one, two, three, four or more carrier moieties.

[0015] The term "peptide" as used herein broadly refers to a sequence of two or more amino acids joined together by peptide bonds. It should be understood that the term does not imply a specific length of the polymer of amino acids, nor is it intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or naturally occurring. The peptide (or, more specifically, the CCR10 binding domain or CBD peptide) of the construct of the present invention is composed of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues, including all ranges derivable therein. Ideally, the CBD peptide provides a minimal binding site for disrupting the CCR10-eNOS interaction. In particular, the peptide comprises or consists of the amino acid sequence Lys-Ile-Ser-Ala-Ser-Leu-Met (SEQ ID NO: 1). Exemplary peptides of the invention include the sequences KISASLM (SEQ ID NO:1), TRKKTFKEVANAVKISASLM (SEQ ID NO:2), FKEVANAVKISASLM (SEQ ID NO:3) and VANAVKISASLM (SEQ ID NO:4).

[0016] As used herein, a "carrier moiety" refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic molecule that facilitates crossing a lipid bilayer, a micelle, a cell membrane, an organelle membrane, or a vesicle membrane. A carrier moiety attached to another molecule facilitates the molecule to cross a membrane, for example, from the extracellular space to the intracellular space, or from the cytosol into an organelle. In some embodiments, the carrier moiety is covalently linked to the amino terminus of the CBD peptide. In other embodiments, the carrier moiety is covalently linked to the carboxyl terminus of the CBD peptide. Ideally, the carrier moiety is a cell-penetrating peptide, a lipid, or a combination thereof.

[0017] Cell-penetrating peptides (CPPs), also known as peptide transduction domains (PTDs), are a diverse class of peptides that have been reported to cross cell membranes. Representative members of this family, such as Trans-Activator of Transcription (TAT) peptides and penetratin, were initially identified as segments within naturally occurring proteins with proposed membrane-permeability. In some cases, the carrier moiety is a cell-penetrating peptide that is covalently linked or fused to the CBD peptide. In some embodiments, the covalent linkage is a peptide bond. For example, the cell-penetrating peptide can be a peptide having a length of about 5 to about 50 amino acids, including, for example, about 5 to about 10 amino acids, about 10 to about 15 amino acids, about 15 to about 20 amino acids, about 20 to about 25 amino acids, about 25 to about 30 amino acids, about 30 to about 40 amino acids, or about 40 to about 50 amino acids.

[0018] Cell-penetrating peptides are well known in the art and are described, for example, by Bechara & Sagan (2013) FEBS Lett. 587:1693-1702; Copolovici, et al. (2014) ACS Nano 8(3):1972-94; and Guidotti, et al. (2017) Trends Pharmacol. Sci. 38(4):406-24. Exemplary cell-penetrating peptides of use in the present invention include, but are not limited to, those listed in Table 1. [Table 1-1] [Table 1-2]

[0019] Alternatively or in addition, the CBD peptides may contain lipids to facilitate cell penetration. As used herein, lipids generally refer to water-insoluble molecules that are soluble in organic solvents. In some embodiments, the lipids are fatty acids that contain an aliphatic hydrocarbon chain with an acyl group, where the aliphatic chain is either saturated or unsaturated alkyl with one or more double bonds. Exemplary fatty acids include, without limitation, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, valeroic acid, octanoic acid, decanoic acid, and linolenic acid. The fatty acids are attached or can be attached to an acyl group carrier, such as glycerol, sphingosine, cholesterol, and others. The lipophilic group may be attached to the peptide directly or via a linking group. For example, 5-aminovaleroic acid, 8-aminooctanoic acid, or 2-aminodecanoic acid may be attached to the N- and / or C-terminus of the peptide.

[0020] Lipids can also be classified into different lipid classes based on their polarity. Lipids can be non-polar or polar lipids. Examples of such non-polar lipids are mono-, di-, or triacylglycerols (glycerides), alkyl esters of fatty acids, and fatty alcohols. Polar lipids have polar head groups and exhibit surface activity, such as fatty amines, phosphatidic acids (e.g., phosphatidylethanolamine, phosphatidylcholine, etc.), phospholipids, glycolipids, glycosylphosphatidylinositol, etc. In some forms, lipids are attached or linked to nucleosides, nucleotides, nucleic acids, amino acids, proteins, or sugars. Exemplary lipids that can be attached to the CBD peptide include N-myristoyl, palmitoyl, and glycophosphatidylinositol (see, for example, Thompson & Okuyama (2000) Prog. Lipid Res. 39:19-39; Bauman & Menon (2002): Biochemistry of lipids, lipoproteins and membranes, 4th ed., pp. 37-54, in Nance & Vance Ed., Elsevier, Amsterdam). Preferably, the CBD peptide is myristoylated, stearylated or palmitoylated at the N-terminal amino acid residue. More preferably, the CBD peptide is myristoylated at the N-terminal amino acid residue. This modification can be added either co- or post-translationally, for example with N-myristoyltransferase (NMT) which catalyzes the addition of myristate. In certain embodiments, the peptide is myristoylated at the N-terminal amino acid residue to facilitate entry of the peptide into cells.

[0021] As used herein, lipids can also be terpenes, fat-soluble vitamins, phytosterols, terpenoids, steroids, tetracyclic ring compounds based on hydrogenated 1,2 cyclopentenophenanthrene with substitutions at the C-10, C-13 and C-17 carbon atoms. Exemplary steroids include, but are not limited to, cholic acid, deoxycholic acid, chenodeoxycholic acid, estrone, progesterone, testosterone, androsterone, norethindrone, cholesterol, digoxin, and the like. Steroids or sterols as described herein can be attached to or modified with nucleosides, nucleotides, nucleic acids, amino acids, proteins, sugars, oligosaccharides, polysaccharides, and other lipids.

[0022] Moreover, lipids may also include isoprenoids, which are composed of isoprene units C5H8. Isoprenoids include a variety of natural and synthetic terpenes, which may be either linear or, more typically, cyclic, including bicyclic, tricyclic and polycyclic. Exemplary isoprenoids include, by way of example, geraniol, citronellal, zingiberene, β-santanol, β-cadinene, matricarin, copaene, camphene, taxol, carotenoids, steroids, and the like. Isoprenoids may be attached to other molecules, including, but not limited to, nucleosides, nucleotides, nucleic acids, amino acids, proteins, sugars, oligosaccharides, and polysaccharides. As an illustrative example, a prenylated peptide may be attached to an isoprenoid lipid unit, farnesyl (C5H8), via a cysteine ​​thioether bond at or near the carboxyl terminus of the CBD peptide. 15 ) or geranylgeranyl (C 20 ) can be prepared by attachment. The use of Reversible Aqueous Lipidization Technology (REAL) is also contemplated. See Mahajan, et al. (2014) Indian J. Pharmaceut. Ed. Res. 48:34-47.

[0023] Alternatively, CBD peptides can also include other modifications to facilitate cellular uptake. For example, cyclizing a given peptide and / or methylating selected amide bond nitrogens can improve its membrane permeability and / or bioavailability. Such modifications, when done carefully, are believed to facilitate the formation of intramolecular hydrogen bonds in response to the low dielectric environment inside the membrane (Bockus, et al. (2013) Curr. Top. Med. Chem. 13:821-836;Rezai, et al. (2006) J. Am. Chem. Soc. 128:14073-14080;White, et al. (2011) Nat. Chem. Biol. 7:810-817). In addition to permeability, cyclization can increase stability. Thus, in some embodiments, the CBD peptides of the invention are cyclized. CBD peptides can be cyclized head-to-tail, head / tail-to-side-chain, or side-chain-to-side-chain. Cyclization is commonly accomplished through lactamization, lactonization, and sulfide-based cross-linking.

[0024] A variety of inorganic materials have also been proposed to transport protein cargo, including silica, carbon nanotubes, quantum dots, and gold nanoparticles (Du, et al. (2012) Curr. Drug Metab. 13:82-92;Malmsten (2013) Curr. Opin. Colloid Interface Sci. 18:468-480). In addition, N-methylation can be used to reduce hydrogen bonding potential.

[0025] In addition to the carrier moiety, the CBD peptide may include one or more other modifications that improve stability (e.g., half-life) and / or solubility, facilitate automatic assembly into nanoparticles, increase shelf life, increase bioavailability, reduce toxicity, facilitate insertion into membranes, and / or reduce proteolysis of the CBD. Specifically, the CBD peptide may include one or more modifications selected from substitution, lipidation, carboxylation, glycosylation, sulfonation, amidation, PEGylation, biotinylation, disulfide formation, and addition of charged amino acid residues.

[0026] Carboxylation refers to the gamma-carboxylation of glutamic acid residues, and glycosylation refers to the attachment of one or more sugars (e.g., N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose, fucose, or xylose) to a peptide via an N- or O-linkage.

[0027] Sulfonation is the process of converting the sulfonic acid group (SO3 -1 Sulfonation can occur via several types of linkages: ester (O-sulfonation), amide (N-sulfonation) and thioester (S-sulfonation).

[0028] Numerous proteolytic enzymes degrade peptide sequences from the N- and / or C-terminus. Post-translational modifications of the N- and / or C-terminus can often improve peptide stability. For example, N-acetylation and C-amidation can increase resistance to proteolysis. For example, it has been reported that N-terminally acetylated somatostatin analogues are much more stable than the native peptide (Adessi & Soto (2002) Curr. Med. Chem. 9(9):963-78). Similarly, the N-acetylated 7-34 form of GLP-1 has been shown to be much more stable than the unprotected peptide (John, et al. (2008) Eur. J. Med. Res. 13(2):73-8). In addition, N-acetylation and C-amidation, when applied together with amino acid substitutions, improve the resistance of EFK17 peptides to endopeptidase digestion (Stroemstedt, et al. (2009) Antimicrob. Agents Chemother. 53(2):593-602). Moreover, tesamorelin, which has a hexenoyl group attached to the N-terminal tyrosine residue, has a much longer half-life than natural growth hormone releasing hormone (Ferdinandi, et al. (2007) Basic Clin. Pharmacol. Toxicol. 100(1):49-58). In some embodiments, the CBD peptide contains N-acetylation and / or C-amidation as a stabilizing moiety.

[0029] Amidation refers to the addition of an amide group to the end of a polypeptide. Several methods have been described for amidating proteins, including the use of α-amidating enzymes (Beaudry, et al. (1990) J. Biol. Chem. 265(29):17694-17699; US 4,708,934); proteases (US 4,709,014; US 5,580,751); carbodiimide compounds, trapping agents and amine sources (US 5,503,989); and recombinant methods (WO1998 / 050563). In certain embodiments, the CBD protein comprises C-terminal amidation.

[0030] Substitution of natural amino acid residues with non-natural residues can increase stability by decreasing substrate recognition and binding affinity for proteases. For example, replacement of L-Arg with D-Arg in vasopressin increased the half-life of this peptide in humans from 10-35 minutes to 3.7 hours in healthy volunteers (Agerso et al. (2004) Br. J. Clin. Pharmacol. 58(4):352-8). Similarly, substitution of L-amino acids with D-amino acids increased the in vivo half-life of somatostatin from a few minutes to 1.5 hours (Harris (1994) Gut 35(3):S1-4). Modification of natural amino acids can also improve peptide stability by introducing steric hindrance. For example, gonadotropin releasing hormone has a very short half-life (minutes), whereas buserelin, in which one Gly is replaced by t-butyl-d-Ser and another by an ethylamide, has a much longer half-life in humans. The pentapeptide ipamorelin has 2'-naphthylalanine and phenylalanine in the D configuration, and the C-terminal L-alanine is replaced by 2-aminoisobutyric acid, resulting in an improved terminal half-life of about 2 hours in humans (Raun, et al. (1998) Eur. J. Endocrinol. 139(5):552-61; Gobburu, et al. (1999) Pharm. Res. 16(9):1412-6).

[0031] Conjugation to polymers (e.g., polyethylene glycol (PEG) or albumin) is an effective strategy to improve peptide stability. For example, covalently attaching an albumin-binding small molecule to a peptide improves proteolytic stability and extends half-life by indirectly interacting with albumin through the highly conjugated small molecule. Liraglutide is a GLP-1 analog linked to a 16-carbon fatty acid residue via a γ-1-glutamyl spacer. The lipopeptide binds to albumin, thereby decreasing proteolysis and increasing half-life from minutes to 8 hours (Hou, et al. (2012) J. Cereb. Blood Flow Metab. 32(12):2201-10; Levy Odile, et al. (2014) PLoS One 9(2):e87704; Lindgren, et al. (2014) Biopolymers 102(3):252-9).

[0032] Conjugation of peptides to large synthetic or natural polymers or carbohydrates can increase their molecular weight and hydrodynamic volume. Common polymers used for peptide conjugation are PEG, polysialic acid (PSA), and hydroxyethyl starch (HES). One example is the PEGylated synthetic peptide peginesatide, which has an elimination half-life of 18.9 hours in healthy volunteers (Bronson, et al. (2013) Annu. Rep. Med. Chem. 48:471-546). As used herein, "polyethylene glycol" or "PEG" refers to a compound of the general formula H-(O-CH2-CH2) nPEG is a polyether compound of -OH. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), and depending on their molecular weight, PEO, PEE, or POG refer to ethylene oxide oligomers or polymers as used herein. The three names are chemically synonymous, but PEG tends to refer to monomers and polymers with molecular weights less than 20,000 Da, PEO to polymers with molecular weights greater than 20,000 Da, and POE to polymers of any molecular weight. The polymer moiety is preferably water-soluble (amphiphilic or hydrophilic), non-toxic, and pharma- ceutical inert. Polymer molecules suitable for use as stabilizing moieties include polyethylene glycol (PEG), homo- or copolymers of PEG, monomethyl-substituted polymers of PEG, or polyoxyethylene glycerol (POG). Also encompassed are PEGs prepared for the purpose of half-life extension, e.g., monoactivated alkoxy-terminated polyalkylene oxides (POAs), such as monomethoxy-terminated polyethylene glycols (mPEGs); bis-activated polyethylene oxides (glycols) or other PEG derivatives are also contemplated. Suitable polymers vary substantially by weight over the range of about 200 Da to about 40,000 Da, or about 200 Da to about 60,000 Da, which are often selected for purposes of the present invention. In some embodiments, PEGs having molecular weights of 200 to 2,000 or 200 to 500 are used.

[0033] PEGs are also available in different geometries: branched PEGs have 3-10 PEG chains emanating from a central core group; star PEGs have 10-100 PEG chains emanating from a central core group; and comb PEGs have multiple PEG chains usually grafted onto the polymer backbone. PEGs can also be linear. The numbers often included in the names of PEGs indicate their average molecular weight (as an example, a PEG with n=9 would have an average molecular weight of approximately 400 daltons and would be labeled PEG 400). As used herein, "PEGylation" is the act of covalently coupling a PEG structure to the CBD peptides of the invention (which are then referred to as "PEGylated CBD peptides"). In some embodiments, the PEGylated side chain PEGs are PEGs of molecular weight from about 200 to about 40,000.

[0034] According to the invention, short linear PEGs are attached to the C- and / or N-terminus of the CBD peptide. In certain embodiments, the PEG component of the CBD peptide is a PEG monomer of 5-50 units, i.e., n is 5-50 (-CH2CH2O-). n In other embodiments, the CBD peptide comprises up to 50, 45, 40, 35, 30, 25, 20, 15, 10 or 5 PEG units. In certain embodiments, the CBD peptide has between 20-30 PEG units. In certain embodiments, the CBD peptide has up to 30 PEG units. PEG may be linked or attached to the C- and / or N-terminal amino acid residues of the CBD peptide via solid phase synthesis methods, for example by employing PEG building blocks such as O-(N-Fmoc-2-aminoethyl)-O'-(2-carboxyethyl)-undecaethylene glycol, available from commercial sources such as EMD Biosciences (La Jolla, Calif.).

[0035] Plasma proteins such as albumin and immunoglobulin (IgG) fragments have a long half-life of 19-21 days in humans (Pollaro & Heinis (2010) Med. Chem. Comm. 1(5):319-24). Due to their high molecular weight (67-150 kDa), these proteins have low renal clearance, and their binding to the neonatal Fc receptor (FcRn) reduces excretion by pinocytosis through the vascular epithelium. Covalent linkage of CBD peptides to albumin or IgG fragments can reduce renal clearance and prolong half-life. As one example, albumin-exendin-4 conjugate (CJC-1134-PC) has a half-life of approximately 8 days in humans, and the FDA-approved drug albiglutide, a DPPIV-resistant GLP-1 dimer fused to human albumin, has a half-life of 6-7 days, thereby allowing once-weekly dosing for the treatment of type 2 diabetes (Pratley, et al. (2014) Lancet Diabetes Endocrinol. 2(4):289-97).

[0036] The peptide may include the addition of between 1 and 10 charged amino acid residues to the C-terminal end. Charged amino acid residues are those that are protonated under biological conditions. + It is intended to include aspartic acid (Asp or D) or glutamic acid (Glu or E) containing an α-amino group of the NH3 type. In certain embodiments, the peptide may contain between 1-7, 1-5, 1-4, or 1-3 charged amino acid residues at the C-terminal end. More specifically, the CBD peptide may contain between 1-7, 1-5, 1-4, or 1-3 aspartic acid residues at the C-terminal end.

[0037] When the carrier moiety and / or stabilizing moiety are peptides, they can be easily attached or conjugated directly to the CBD peptide via a peptide bond. However, when the carrier moiety and / or stabilizing moiety are not peptides, they can be attached to the CBD peptide via other common bonds such as disulfide, amide, oxime, thiazolidine, urea and carbonyl bonds, or by Diels-Alder or Hueisgen 1,3-dipolar cycloaddition reactions (Lu, et al. (2010) Bioconjug. Chem. 21:187-202; Roberts, et al. (2002) Adv. Drug Deliv. Rev. 54:459-76; WO 2008 / 101017).

[0038] Alternatively, the constructs of the invention can include a linker for tethering or linking the carrier moiety and / or stabilizing moiety to the CBD peptide. For purposes of this invention, the linker is a peptide having any of a variety of amino acid sequences. The linker, being a spacer peptide, can be flexible in nature, but does not exclude other chemical bonds. The linker peptide can have a length of about 1 to about 40 amino acids, for example, about 1 to about 5 amino acids, about 5 to about 10 amino acids, about 10 to about 20 amino acids, about 20 to about 30 amino acids, or about 30 to about 10 amino acids. These linkers can be produced using synthetic, linker-encoding oligonucleotides for coupling proteins. Peptide linkers with some degree of flexibility can be used. The linking peptide can have virtually any amino acid sequence, where in some embodiments, the linker peptide has a sequence that generally results in a flexible peptide. The use of small amino acids such as glycine and alanine is used in the production of flexible peptides. The production of such sequences is routine for those skilled in the art. A variety of linkers are commercially available and are deemed suitable for use.

[0039] An exemplary flexible linker that can be used to tether or link the carrier moiety and / or stabilizing moiety to the CBD peptide is a glycine polymer (G n (e.g., n is an integer from 1 to about 20); glycine-serine polymers (e.g., n is an integer of at least 1 (GS) nFlexible linkers include, but are not limited to, GG, GGG, GGS, GGSG (SEQ ID NO: 70), GGSGG (SEQ ID NO: 71), GGSSG (SEQ ID NO: 72), GSGGG (SEQ ID NO: 73), GGGSG (SEQ ID NO: 74), GSSSG (SEQ ID NO: 75), and the like.

[0040] Non-peptide linker moieties can also be used to tether or link carrier and / or stabilizing moieties to the CBD peptide. The linker molecule is generally about 6-50 atoms in length. The linker molecule can also be, for example, an aryl acetylene, an ethylene glycol oligomer containing 2-10 monomer units, a diamine, a diacid, an amino acid, or a combination thereof. Other linker molecules capable of binding to a peptide can be used in light of the present disclosure.

[0041] Without departing from the scope of this invention, the construct also encompasses CBD peptide variants having one or more deletions, additions, and / or conservative substitutions in the CBD peptides of SEQ ID NO: 1, 2, 3, or 4 that retain at least one of the functional properties of the peptide. Conservative substitutions of amino acids, i.e., replacing an amino acid with a different amino acid with similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically involving minor changes. As is understood in the art, these minor changes can be identified, in part, by considering the hydropathic index of an amino acid (Kyte, et al. (1982) J. Mol. Biol. 157:105-132). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and retain protein function. In one aspect, amino acids with a hydropathic index of ±0.2 are substituted. Amino acid hydrophilicity can also be used to identify substitutions that result in a protein retaining biological function. Considering amino acid hydrophilicity in the context of a peptide allows calculation of the maximum local average hydrophilicity of the peptide, a useful tool that has been reported to correlate well with antigenicity and immunogenicity. See US 4,554,101, fully incorporated herein by reference. As is understood in the art, substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity. Substitutions can be made with amino acids that have hydrophilicity values ​​within ±0.2 of each other. Both the hydrophobic index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with that observation, it is understood that amino acid substitutions that are compatible with biological function, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties, depend on the relative similarity of the amino acids, and in particular on the side chains of those amino acids.

[0042] In some embodiments, the skilled artisan may identify suitable areas of the CBD peptide that may be altered without destroying activity by targeting regions that are not considered important for activity. In further embodiments, even amino acids that are important for biological activity or for structure may undergo conservative amino acid substitutions without destroying the biological activity or adversely affecting the peptide structure. For example, Lys may be replaced by Arg and / or Ser may be replaced by Thr in one or more occurrences. In one embodiment, the variant CBD peptide has at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4. In general, the variant CBD peptide exhibits a binding affinity that is substantially the same or greater than that of the CBD peptide of SEQ ID NO: 1, 2, 3 or 4, for example at least 0.75x, 0.8x, 0.9x, 1.0x, 1.25x or 1.5x. In some embodiments, the CBD peptide or variant thereof has a K in the range of about 10 pM to about 1 μM, or more preferably about 10 pM to about 100 nM, or most preferably about 10 pM to about 10 nM. D It has a value.

[0043] In some embodiments, peptides are glycosylated, phosphorylated, sulfated, animated, carboxylated, or acetylated. For example, the C-terminus can be modified by amidation, addition of peptide alcohols or aldehydes, addition of esters, addition of p-nitroaniline and thioesters. The N-terminus and amino acid 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 tetrabutyric acid and addition of 3-mercaptopropyl, acylation, biotinylation, phosphorylation, sulfation, glycosylation, introduction of maleimide groups, chelating moieties, chromophores and fluorophores.

[0044] In one aspect, a construct of the invention is selected from those presented in Table 2. [Table 2]

[0045] In some embodiments, the CBD peptide or construct is fused to a protein or purification tag, such as chitin-binding protein, maltose-binding protein, glutathione-S-transferase, 6xHis, FLAG§, or HA, to facilitate detection and / or purification. As an example, a Cys residue can be incorporated into a CBD protein, where the N-terminal side of the Cys residue is thioesterified and the tag is attached to the C-terminal side. Upon purification, the tag is cleaved off and the peptide thioester is effectively obtained.

[0046] CBD peptides and constructs can be recombinantly synthesized using recombinant DNA techniques. Thus, in another aspect, the invention provides a polynucleotide encoding a CBD peptide or construct of the invention. In a related aspect, the invention provides vectors, particularly expression vectors, harboring a polynucleotide encoding a CBD peptide or construct. In one embodiment, the vector provides replication, transcription, and / or translation regulatory sequences that facilitate recombinant synthesis of the CBD peptide or construct in eukaryotic or prokaryotic cells. Thus, the invention also provides host cells for recombinant expression of CBD peptides or constructs, and methods for harvesting and purifying the CBD peptides or constructs produced by the host cells. The production and purification of recombinant polypeptides is routine for those skilled in the art. The CBD peptides or constructs can be purified by any suitable method known in the art, including, without limitation, gel filtration and affinity purification. When the CBD peptides or constructs of the invention are produced in the form of a fusion protein, the fused portion (or epitope tag) can optionally be cleaved using a protease before further analysis.

[0047] Alternatively, the CBD peptides or constructs of the invention can be advantageously synthesized by any of the chemical synthesis techniques known in the art, in particular by solid phase synthesis using, for example, a commercially available automated peptide synthesizer. See, for example, Stewart and Young, 1984, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.; Tarn, et al. (1983) J. Am. Chem. Soc. 105:6442; Merrifield (1986) Science 232:341-347; and US 5,424,398. Moreover, a combination of recombinant and chemical synthesis techniques is also contemplated.

[0048] The constructs of the invention can be purified by any suitable method known in the art, including, by way of example, affinity chromatography, ion exchange chromatography, filtration, ultrafiltration, gel filtration, electrophoresis, salting out, dialysis, etc. In one embodiment, the constructs are purified by reverse phase chromatography.

[0049] As demonstrated herein, the constructs of the invention disrupt eNOS-CCR10 interaction, increase eNOS / NO levels, increase re-epithelialization of skin wounds, and improve skin wound healing in obese and diabetic mice. Thus, the invention provides a method for modulating CCR10-eNOS interaction and promoting, accelerating or improving wound healing in a subject. The method of the invention involves administering an effective amount of a CBD peptide construct to a subject in need of treatment (e.g., a subject with a chronic or infected wound). There are various ways to measure wound healing. Often, images are taken to calculate linear dimensions, perimeter and area. The NIH has a free program called Image J that allows wound area measurements from images. A prognosis of final healing can be estimated from the initial healing rate based on the migration of the periphery to the center. This is done using a number of formulas, the most common of which is the modified Gilman equation. In addition to visual inspection, measurement of wound healing can also be assisted by spectroscopy or MRI. For example, see Dargaville, et al. (2013) Biosensors Bioelect. 41:30-42; Tan, et al. (2007) Br. J. Radiol. 80:939-48. In cases of slow / poor healing, a wound edge biopsy may be performed to rule out or determine infection and malignancy. In some embodiments, accelerated or improved wound healing can be assessed by comparing wound closure in treated and control wounds. In some embodiments, accelerated or improved wound healing is at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% faster or better than control.

[0050] In one aspect, the present invention provides a method for promoting / accelerating / improving wound healing with or without active infection, microbial contamination or colonization in the wound. The CDB peptide or construct of the present application can be used to treat an infected wound or promote / accelerate / improve healing of an infected wound. In one embodiment, the CDB peptide or construct can be used to treat a wound or promote / accelerate / improve wound healing in the presence of an infection. In some embodiments, the CDB peptide or construct can be used to treat a wound or promote / accelerate / improve wound healing in the presence of microbial contamination or colonization at risk of infection. In further embodiments, the patient in need of wound healing treatment is a diabetic patient. Thus, in some embodiments, the wound is a diabetic wound, such as a diabetic foot ulcer. In some further embodiments, the wound is an infected diabetic wound, such as an infected diabetic foot ulcer.

[0051] To accelerate the healing of chronic wounds, such as for the treatment of diabetic foot ulcers, administration of a CDB peptide or construct can be combined with one or more additional wound healing agents. Suitable additional wound healing agents include, without limitation, growth factors (e.g., EGF, FGF, IGF, PDGF, TGF, and VEGF), nerve growth factor (NGF), angiogenesis factors (e.g., HGF, TNF-α, angiogenin, IL-8, angiopoietin 1 and 2, Tie-2, integrin α5, matrix metalloproteinases, nitric oxide, COX-2), members of the platelet-derived growth factor (PDGF) family (e.g., PDGF-A, PDGF-B, PDGF-C, and PDGF-D), members of the insulin growth factor (IGF) family (e.g., IGF-I, IGF-II), members of the transforming growth factor (TGF) family (e.g., TGF-α, TGF-β), and anabolic oxygen (vacuum therapy). In certain embodiments, the CDB peptide or construct can be co-administered with one or more additional wound healing agents described herein and / or one or more antimicrobial agents or antibiotics suitable for use in topical administration. See WO2006 / 138468. In such embodiments, the antimicrobial agent can be a sulfur antibiotic, including, without limitation, silver sulfadiazine or silvadeen. The one or more additional agents co-administered can be administered simultaneously, alternately, or sequentially with the CDB peptide or construct.

[0052] In another aspect, the disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a CDB peptide or construct of the invention and one or more pharma- ceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, excipients, or carriers. The pharmaceutical composition can be used, for example, to promote wound healing in a mammal in need thereof. The pharmaceutical composition is particularly useful for promoting diabetic wound healing.

[0053] In some embodiments, the present disclosure provides pharmaceutical compositions comprising a compound of the present disclosure together with one or more pharma- ceutically acceptable excipients or vehicles, and optionally other therapeutic and / or prophylactic ingredients. Such excipients include liquids such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, and the like.

[0054] The term "pharmaceutical acceptable vehicle" refers to a diluent, adjuvant, excipient or carrier administered with the compounds of the present disclosure. The term "effective amount" or "pharmacologically effective amount" refers to a non-toxic but sufficient amount to provide a desired biological result. The result may be reduction and / or alleviation of signs, symptoms, or causes of disease, or any other desired change in a biological system. An appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

[0055] "Pharmaceutically acceptable carriers" for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990). For example, sterile saline and phosphate-buffered saline at physiological pH can be used. Preservatives, stabilizers, dyes, and even flavorings can be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid can be added as preservatives. Ibid., at 1449. In addition, antioxidants and suspending agents can be used. Ibid.

[0056] Suitable excipients for non-liquid formulations are also known to those skilled in the art. A thorough discussion of pharma- ceutically acceptable excipients and salts is available in Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990).

[0057] Additionally, auxiliary substances such as wetting or emulsifying agents, biological buffers, surfactants, and the like, may be present in such vehicles. The biological buffer may be any solution that is pharmacologically acceptable and provides the formulation with the desired pH, i.e., a pH that is in the physiologically acceptable range. Examples of buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank's buffered saline, and the like.

[0058] In some embodiments, the peptides of the present invention may be formulated as a topical ointment or cream containing an amount of active ingredient(s) generally ranging from about 0.01 to about 20% by weight, in some embodiments from about 0.1 to about 20% by weight, in some embodiments from about 0.1 to about 10% by weight, and in some embodiments from about 0.5 to about 15% by weight. When formulated as an ointment, the active ingredient is typically combined with either a paraffinic or water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream, for example with an oil-in-water cream base. Such formulations are known in the art and generally include additional ingredients to enhance the skin penetration or stability of the active ingredient or formulation. Such known topical formulations and ingredients are encompassed within the scope of the present invention.

[0059] In various embodiments, the formulation herein can be in the form of aqueous gel, anhydrous gel, water-in-oil emulsion, oil-in-water emulsion or suspension.Examples of gel formation procedures for DHEA can be found in US5,709,878 and US4,978,532, the contents of which are incorporated herein by reference in their entirety.Gels are any semi-solid systems that contain suspended small inorganic particles (two-phase gel) or liquid interpenetrating organic polymers (single-phase gel).Emollients such as petrolatum, paraffin wax, beeswax, cetyl palmitate, and lanolin can be included in the formulation herein.When formulated for presentation as a gel, the composition of the present invention can contain gelling agents such as finely divided solids and / or thickeners at concentrations that produce a loose molecular network that inhibits the free movement of liquid components. Thus, a typical gel composition of the present invention comprises a concentration of peptide ranging from about 0.1 to about 20 grams per 100 grams of composition, and in some embodiments, from about 0.25 to about 5 grams per 100 grams; a concentration of phospholipids ranging from about 2 to about 50 grams per 100 grams of composition, and in some embodiments, from about 3 to about 25 grams per 100 milliliters; a concentration of finely divided solids ranging from about 0 to about 15 grams per 100 grams of composition; and a concentration of thickening agent ranging from about 0 to about 15 grams per 100 grams of composition.

[0060] Gelling agents may also be included in the formulation. These agents are typically non-ionic or cationic polymers, such as hydroxyethylcellulose, methylcellulose, guar gum, xanthan gum, hydroxypropylcellulose and cationic cellulosics. A particular example is SEPIGEL. In one embodiment, gels containing the peptides of the invention can be made by mixing lower alkyl alcohols, polysorbates, water and peptides, and optionally adding and mixing a thickening agent, followed by incubating the components until gel formation. Various temperatures can be used for incubation to allow gel formation.

[0061] Examples of thickeners that can be added to the gel or solution formulations described herein include: cellulosic thickeners, such as cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, and hydroxypropyl methyl cellulose; and acrylic thickeners. An example of an acrylic thickener is a carbomer, such as a nonlinear polymer of acrylic acid crosslinked with a polyalkenyl polyether. Examples of carbomers that can be used in the present invention include carboxypolymethylene, carboxyvinyl polymers, and alkyl acrylates, such as acrylic acid / alkyl methacrylate copolymers. All of the above are available from Noveon, including carboxypolymethylene sold under the trade name CARBOPOL® 980, carboxyvinyl polymer sold as CARBOPOL® 940, and acrylic acid / alkyl methacrylate copolymer sold as PEMULEN® TR-1.

[0062] In some embodiments, the formulation of the present invention can be applied by misting or spraying the formulation onto the skin, either via a metered dose device or from a unit dose container.In this way, the formulation can be distributed evenly over a larger area, thereby providing a quick means for absorption.Alternatively, the formulation can be applied via an applicator such as a roll-on applicator, a metered pump dispenser or a sponge.

[0063] Topical oil-in-water emulsion compositions can be prepared by forming a solution containing the peptide of the invention and adding an immiscible phase (e.g., a biocompatible oil phase) and optionally an emulsifier. 12~15 Irritation reducing agents such as alkyl benzoates, octyl methoxycinnamate, octyl dimethyl PABA, octocrylene, menthyl anthranilate, and homomenthyl salicylate may also be included.

[0064] In some embodiments, foams containing the compounds of the present application can be prepared. An example of a foam-forming procedure can be found in US 7,141,237. For example, a fast-acting foaming agent can be used that includes an active agent in solution as described herein and a mixture of cetyl alcohol and stearyl alcohol dissolved in an ethanol solution. This composition can be packaged in an aluminum can lined with polyamideimide and pressurized with a propane / butane mixture as a propellant. Under the pressure of packaging, the hydrocarbon propellant liquefies and becomes miscible with the water / ethanol solution.

[0065] The formulations herein may include an emulsifier and / or surfactant. A wide variety of such agents can be employed. In one embodiment, the compositions of the present invention include from about 0.05% to about 95%, in some embodiments from about 10% to about 80%, and in some embodiments from about 3.5% to about 60% of at least one surfactant. The surfactant must, at a minimum, be sufficiently hydrophilic to be dispersed in ethanol or other solvent system. Surfactants useful herein can include any of a wide variety of cationic, anionic, zwitterionic, and amphoteric surfactants disclosed in prior patents and other references. The exact surfactant chosen will depend on the pH of the composition and other components present.

[0066] In one embodiment, the composition comprises a hydrophilic emulsifier or surfactant. In some embodiments, the compositions of the present invention comprise from about 0.05% to about 5%, in some embodiments from about 0.05% to about 3.5%, of at least one hydrophilic surfactant. Without intending to be limited by theory, it is believed that the hydrophilic surfactant aids in dispersing hydrophobic materials.

[0067] The hydrophilic surfactant is selected from non-ionic surfactants. Among the non-ionic surfactants useful herein are long chain alcohols, e.g., C 8~30They can be broadly defined as the condensation products of an alcohol with a sugar or starch polymer, i.e., a glycoside. Representative sugars include glucose, fructose, mannose, and galactose. Examples of long chain alcohols from which the alkyl groups can be derived include decyl alcohol, cetyl alcohol, stearyl alcohol, lauryl alcohol, myristyl alcohol, oleyl alcohol, and the like. Commercially available examples of these surfactants include decyl polyglucoside and lauryl polyglucoside.

[0068] Other useful nonionic surfactants include the condensation products of alkylene oxides with fatty acids (i.e., alkylene oxide esters of fatty acids); the condensation products of alkylene oxides with two moles of fatty acids (i.e., alkylene oxide diesters of fatty acids); the condensation products of alkylene oxides with fatty alcohols (i.e., alkylene oxide ethers of fatty alcohols); and the condensation products of alkylene oxides with both fatty acids and fatty alcohols. Non-limiting examples of these alkylene oxide derived nonionic surfactants include ceteth-6, ceteth-10, ceteth-12, ceteareth-6, ceteareth-10, ceteareth-12, steareth-6, steareth-10, steareth-12, PEG-6 stearate, PEG-10 stearate, PEG-100 stearate, PEG-12 stearate, PEG-20 glyceryl stearate, PEG-80 glyceryl tallowate, PEG-10 glyceryl stearate, PEG-30 glyceryl cocoate, PEG-80 glyceryl cocoate, PEG-200 glyceryl tallowate, PEG-8 dilaurate, PEG-10 distearate, and mixtures thereof.

[0069] Other nonionic surfactants suitable for use herein include sugar esters and polyesters, alkoxylated sugar esters and polyesters, C1-C 30 Fatty alcohols C1-C 30 Fatty acid esters, C1-C 30 Fatty alcohols C1-C 30Alkoxylated derivatives of fatty acid esters, C1-C 30 Alkoxylated ethers of fatty alcohols, C1-C 30 Polyglyceryl esters of fatty acids, polyols C1-C 30 Esters and polyols C1 to C 30 ethers, alkyl phosphates, polyoxyalkylene fatty ether phosphates, fatty acid amides, acyl lactates, and mixtures thereof. Non-limiting examples of these non-silicon-containing emulsifiers include: polyethylene glycol 20 sorbitan monolaurate (polysorbate 20), polyethylene glycol 5 soy sterol, steareth-20, ceteareth-20, PPG-20 methyl glucose ether distearate, ceteth-10, polysorbate 80, cetyl phosphate, potassium cetyl phosphate, cetyl diethanoamine phosphate, polysorbate 60, glyceryl stearate, polyoxyethylene 20 sorbitan trioleate (polysorbate 85), sorbitan monolaurate, polyoxyethylene 4 lauryl ether sodium stearate, polyglyceryl-4 isostearate, hexyl laurate, PPG-20 methyl glucose ether distearate, PEG-100 stearate, and mixtures thereof. Commercially available surfactants include polysorbate 80 (sold under the trade name TWEEN® 80), polysorbate 20 (sold under the trade name TWEEN® 20), polysorbate 40 (sold under the trade name TWEEN® 40), and polysorbate 60 (sold under the trade name TWEEN® 60). In some embodiments, the surfactant includes a polysorbate, and in some embodiments, the surfactant is sold under the trade name TWEEN®.

[0070] The CDB peptides or constructs of this invention can also be administered by a device. Thus administration can be accomplished using a patch, either of the reservoir or porous membrane type, or of a solid matrix variety.

[0071] The device of the present invention, or individual dosage units, can be produced in any manner known to those skilled in the art. After the dermatological composition is formed, it can be contacted with the backing layer in any manner known to those skilled in the art. Such techniques include calendar coating, hot melt coating, solution coating, and the like. Backing materials are well known in the art and can include plastic films such as polyethylene, vinyl acetate, ethylene / vinyl acetate copolymer, polyvinyl chloride, polyurethane, metal foils, nonwovens, fabrics, and commercially available laminates. The backing material generally has a thickness ranging from 2 to 1000 micrometers, and the dermatological composition is generally disposed on the backing material at a thickness ranging from about 12 to 250 micrometers.

[0072] Suitable release liners are also well known in the art and include commercially available products from Dow Corning Corporation designated BIO-RELEASE™ liner and Syl-off7 7610 liner. For embodiments in which polysiloxane is part of a multi-polymer adhesive carrier, the release liner must be compatible with the silicone adhesive. An example of a suitable commercially available liner is 3M's 1022 SCOTCH PAK™. The configuration of the transdermal delivery system of the present invention can be of any shape or size necessary or desired. Illustratively, a single dosage unit can be 1-200 cm. 2 In some embodiments, the size ranges from 5 to 60 cm 2 It is.

[0073] In some embodiments, when the carrier is a flexible, finite polymer, one or more polymers are blended with PVP to result in a pressure-sensitive adhesive composition, or with the drug delivery system adhesive system (with the incorporated parent drug) to control the delivery of the incorporated parent drug through the epidermis. In some embodiments of the present invention, a suitable drug delivery system is prepared by mixing soluble PVP, polyacrylate, polysiloxane, parent drug / prodrug, optional enhancer(s), co-solvent(s) and adhesive in a suitable volatile solvent(s), then casting the mixture and removing the solvent(s) by evaporation to form a film. Suitable volatile solvents include, but are not limited to, alcohols such as isopropanol and ethanol; aromatics such as xylene and toluene; aliphatics such as hexane, cyclohexane and heptane; and alkanoic acid esters such as ethyl acetate and butyl acetate.

[0074] Also included are delivery systems for administration by: passive patch, heated passive patch, passive patch applied over RF treated skin, and spray-on-skin systems where the total amount applied is fixed and delivery is improved by a co-formed permeation enhancer.

[0075] In some embodiments, the composition is administered to the recipient by means of a delivery system or patch. Delivery is accomplished by exposing a source of the substance to be administered to the recipient's skin for an extended period of time. Typically, the formulation is incorporated into or adsorbed onto a matrix or reservoir from which it is released onto the recipient's skin. The rate of release can be controlled by a membrane placed between the reservoir and the skin, by direct diffusion from the reservoir, or by the skin itself acting as a rate-controlling barrier. Thus, numerous suitable topical delivery systems and reservoirs are known, ranging in complexity from simple gauze pads impregnated with the substance to be administered and secured to the skin with a bandage, to multi-layered and multi-component structures. Some of the systems are characterized by the use of a shaped article, flexible enough to fit snugly against the recipient's skin, used with the substance to be administered, thereby acting both as a reservoir through which the substance is delivered to the recipient's skin and as a barrier to prevent loss or leakage of the substance from the area of ​​the skin to which the substance is delivered. Topical delivery systems or patches may also contain added substances that aid in the penetration of the active ingredient through the skin, often referred to as skin enhancers or permeability enhancers. The delivery systems may contain ethoxylated oils, such as ethoxylated castor oil, ethoxylated jojoba oil, ethoxylated corn oil, and ethoxylated emu oil. Alcohol mixed with ethoxylated oils may form a permeability enhancer.

[0076] The present invention also provides dosages for CBD peptides or constructs. For localized wound healing, about 0.001 mg / cm 2 ~about 10mg / cm 2 Wound area, approximately 0.05mg / cm 2 ~about 5mg / cm 2 Wound area, approximately 0.01mg / cm 2 ~About 1mg / cm 2 Wound area, approximately 0.05mg / cm 2 ~about 0.5mg / cm 2 Wound area, approximately 0.01mg / cm2 ~about 0.5mg / cm 2 Wound area, approximately 0.05mg / cm 2 ~about 0.2mg / cm 2 wound area, or approximately 0.1 mg / cm 2 ~about 0.5mg / cm 2 One or more doses of about 0.01 mg / cm of wound area (or any combination thereof) may be administered to the patient. 2 , 0.02 mg / cm 2 , 0.03 mg / cm 2 , 0.04 mg / cm 2 , 0.05 mg / cm 2 , 0.06 mg / cm 2 , 0.07 mg / cm 2 , 0.08 mg / cm 2 , 0.09 mg / cm 2 , 0.1 mg / cm 2 , 0.15 mg / cm 2 , 0.2 mg / cm 2 , 0.25 mg / cm 2 , 0.3 mg / cm 2 , 0.4 mg / cm 2 , or 0.5 mg / cm 2 One or more doses of the wound area may be administered to the patient. Such doses may be administered intermittently, for example, once a week or every three weeks (for example, such that the patient receives from about 2 to about 20, or about 6, doses of the CBD peptide or construct). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may be useful. The progress of this treatment is easily monitored by conventional techniques and assays. EXAMPLES

[0077] The following non-limiting examples are provided to further illustrate the present invention.

[0078] Example 1: Materials and Methods Reagents: PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), calcium ionophore A23187, NG-Nitro-L-arginine methyl ester (L-NAME), wortmannin STO-609, U0126, BSA, and RIPA buffer, and F-127 gel sold under the trade name PLURONIC® were from Sigma (St. Louis, MO). n-Octyl-β-D-glucopyranoside (ODG) was from RPI Corp. (Mt Prospect, IL). siRNA against eNOS, CCR10, and transfection reagent sold under the trade name DHARMAFECT®1 were from Dharmacon (Lafayette, CO). Transfection reagent sold under the trade name TRANSIT® was from Mirus Bio (Madison, WI). Control siRNA, mouse anti-CCR10, and mouse eNOS antibodies were purchased from Santa Cruz Biotechnologies (Dallas, TX). 4',6-diamidino-2-phenylindole (DAPI), fluorescently labeled secondary antibodies were purchased from Invitrogen (Carlsbad, CA). Mouse anti-eNOS, mouse anti-β-catenin, rabbit and mouse anti-actin antibodies, human recombinant CCL28, and extracellular matrix sold under the trade name MATRIGEL® were from BD Biosciences (San Diego, CA). Rabbit anti-EEA1, mouse anti-LAMP1 antibody, mouse anti-Bcl-2, rabbit anti-CD31, rabbit anti-phospho-eNOS (pSer1177), rabbit anti-phospho-p85 (pTyr458), rabbit anti-phospho-Src (pTyr416), rabbit anti-phospho-ERK (pT202 / Y204) and corresponding total antibodies, and Griess reagent kit were from Cell Signaling Technology (Danvers, MA). Skin punch biopsy tools were from Acuderm Inc. (Fort Lauderdale, FL). Hematoxylin, eosin, High-Def and Bluing were from StatLab (McKinney, TX).Transfection reagent sold under the trade name LIPOFECTAMINE® 2000, 4',6-diamidino-2-phenylindole (DAPI), fluorescently labeled secondary antibodies, DAF-FM diacetate, nucleic acid isolation reagents sold under the trade name TRIZOL®, and SYBR Green PCR mix were from ThermoFisher Scientific (Waltham, MA). Mouse ELISA kits were purchased from R&D Systems (Minneapolis, MN). Nitrocellulose membranes were from Bio-Rad Laboratories (Hercules, CA). Chemiluminescent substrate sold under the trade name SUPERSIGNAL® West Femto and RESTORE™ Western stripping buffer were from Pierce (Rockford, IL).

[0079] Cell culture and transfection. Human umbilical vein endothelial cells (HUVEC; Lonza, Walkersville, MD) and human dermal microvascular endothelial cells (HDMVEC; Cell Biologics, Chicago, IL) were cultured in EGM-2 (EGM™-2 plus BULLETKIT™; Lonza, Walkersville, MD) supplemented with 10% (v / v) FBS. cDNA and siRNA transfection in endothelial cells was performed according to known methods (Chen, et al. (2018) Mol. Biol. Cell 29(10):1190-1202; Chen, et al. (2020) FASEB J. 34(4):5838-5850). HEK293 cells (ATCC, Rockville, MD) and HEK / eNOS cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin. cDNA transfection in endothelial and HEK cells was performed using transfection reagents sold under the trade names TRANSIT® and LIPOFECTAMINE® 2000, respectively, according to established methods (Chen, et al. (2018) Mol. Biol. Cell 29(10):1190-1202; Chen, et al. (2020) FASEB J. 34(4):5838-5850; Chen, et al. (2015) Ann. Rheum. Dis. 74:1898-1906). Cells transduced with fluorescently tagged proteins were verified by fluorescence microscopy and immunoblot analysis.

[0080] Construction of CCR10-GFP plasmid. Wild-type, C-terminal, GFP-tagged CC motif CCR10, full-length Homo sapiens CCR10 cDNA (Addgene, Cambridge, Mass.) was used as DNA template in PCR reactions with DNA PHUSION® high fidelity polymerase (New England BioLabs, Ipswich, Mass.) using the following primers lacking stop codons: [ka] (SEQ ID NO: 76) and [ka] (SEQ ID NO: 77). The resulting PCR fragment was digested with restriction enzymes 5'-EcoRI and 3'-KpnI and ligated into pEGFP-N1 vector (Clontech, Mountain View, CA) digested with the same restriction enzymes using T4 DNA ligase (New England BioLabs, Ipswich, MA). The ligated reaction was transferred into Subcloning Efficiency DH5α competent cells (ThermoFisher, Waltham, MA).

[0081] Nitric oxide (NO) measurement using DAF-FM diacetate. Confluent HUVEC monolayers on 96-well plates were loaded with 2.5 μM DAF-FM and incubated at 37° C. for 45 minutes. NO concentration was measured using a SpectraMax Microplate Reader (Molecular Devices, San Jose, CA) as described in the art (Chen, et al. (2020) FASEB J. 34(4):5838-5850).

[0082] Nitrite measurement using Griess reagent. Supernatants from confluent endothelial cells on 10 cm dishes with or without stimulation at 37°C were analyzed for nitrite levels using the Griess reagent kit according to the manufacturer's instructions. As described in the art, nitrite levels are normalized to eNOS expression levels in cells (Chen, et al. (2020) FASEB J. 34(4):5838-5850).

[0083] Immunoblots and co-immunoprecipitation (co-IP). After serum starvation, inhibitors were added 45 min before stimulation for the indicated times at 37°C, and cells were then harvested and lysed for Western blot analysis. After probing for p-eNOS (Ser1177), p-p85 (Tyr458), p-Src (Tyr416), and p-ERK (T202 / Y204), the same blots were stripped and reprobed for total proteins as above. For detection of CCL28-induced protein levels of β-catenin and Bcl-2, near-confluent endothelial cells were incubated with 500 ng / ml CCL28 for 24 h in EGM-2 medium containing 1% FBS.

[0084] Co-IP experiment. Cells or mouse wounds were collected after treatment and dissolved in 2% ODG in Tris buffer. Homogenates were further prepared for co-IP experiment as described in the art (Chen, et al. (2018) Mol. Biol. Cell 29(10):1190-1202; Chen, et al. (2020) FASEB J. 34(4):5838-5850; Chen, et al. (2015) Ann. Rheum. Dis. 74:1898-1906).

[0085] Endothelial cell tube formation. HUVECs were transfected with siRNA for eNOS (50 nM), CCR10 (100 nM), or scrambled control (100 nM), and after 48 hours, cells were transferred to 96-well plates preloaded with extracellular matrix sold under the trade name MATRIGEL®. To investigate the CCL28 signaling pathway, HUVECs were preincubated with vehicle or specific inhibitors and then stimulated with or without CCL28 as previously described (Chen, et al. (2015) Ann. Rheum. Dis. 74:1898-1906).

[0086] Confocal microscopy. For cell immunostaining, confluent monolayers of cells grown on coverslips were prepared as previously described (Chen, et al. (2012) Mol. Biol. Cell 23(7):1388-1398). Fluorescent images were obtained using a Zeiss LSM 880 confocal microscope. Colocalization coefficients in identified regions of interest (ROIs) were determined using Zeiss Zen software. Briefly, desired ROIs were drawn along the cell edges using the Overlay tool, and values ​​for the ROIs were tabulated. Data from different treatment groups were collected and analyzed for statistical significance as previously described (Chen, et al. (2020) FASEB J. 34(4):5838-5850).

[0087] Synthesis of myristoylated peptides. Myristoylated peptides were synthesized using a stepwise solid-phase method using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on Wang resin (AnaSpec, Fremont, CA) using a 12-channel multiplex peptide synthesizer (Protein Technologies, Tucson, AZ) as previously described (Chen, et al. (2020) FASEB J. 34(4):5838-5850; Jayathilaka, et al. (2007) J. Biomol. Tech. 18:46).

[0088] Mouse healing model. All animal experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). WT C57BL / 6, eNOS - / - and db / db mice were purchased from Jackson Laboratory (Bar Harbor, ME). - / - Mice have been described in the art, see, for example, Jin, et al. (2011) J. Immunol. 185(10):5723-31. Four 5 mm or 8 mm full-thickness excision wounds were made on the dorsal skin of mice (male, 9 weeks old) using a standard skin biopsy punch under ketamine (100 mg / kg) and xylazine (5 mg / kg) anesthesia. After wounding, scrambled myristoylated (Myr-) control or Myr-CBD7 peptide was applied topically to the wound in F-127 gel sold under the trade name PLURONIC® (Mirza, et al. (2009) Am. J. Pathol. 175:2454-2462). Wound size was determined as previously described (Chen, et al. (2020) FASEB J. 34(4):5838-5850; Zhao, et al. (2016) PLoS ONE. 11:e0146451), and wounds were collected for the indicated measurements at the indicated times.

[0089] Hematoxylin and Eosin (H&E) staining. H&E staining was performed as previously described (Chignalia, et al. (2015) Am. J. Pathol. 185:1251-1263). Slides with 5 μm tissue sections were baked for 30 min at 60°C and stained with an Autostainer XL (Leica Microsystems, Wetzlar, Germany) using a preset protocol. Briefly, slides were deparaffinized in three changes of xylene, rehydrated in 100% and 95% ethanol, incubated for 1.5 min, followed by a brief immersion in High-Def and Bluing to obtain clear nuclear details. Slides were then incubated in eosin for 2 min, dehydrated, and mounted with Micromount media (Leica Biosystems). Whole slide images were acquired using an Aperio AT2 brightfield scanner (Leica Microsystems, Wetzlar, Germany).

[0090] CD31 staining in mouse wound tissue. Formalin-fixed mouse skin samples were processed using standard overnight processing protocols on an ASP300 S automated tissue processor (Leica Biosystems, Buffalo Grove, IL) and embedded into paraffin blocks. Tissues were sectioned at 5 μm and stained with rabbit anti-CD31 antibody. Sections were deparaffinized, rehydrated, and stained on a BOND™ RX automated stainer (Leica Biosystems, Buffalo Grove, IL) using a preset protocol. Briefly, sections were subjected to EDTA-based antigen retrieval in BOND™ ER2 solution (Leica Biosystems, Buffalo Grove, IL) for 20 minutes at 100°C. Endogenous peroxidase activity and nonspecific binding were blocked by treating samples with peroxidase block and protein block for 15 and 30 minutes at room temperature using Background Sniper (Biocare Medical, Pacheco, CA). Sections were then incubated with primary antibodies diluted 1:200 for 30 minutes at room temperature. BOND™ Polymer Refine Detection kit (Leica Biosystems, Buffalo Grove, IL) was used for detection. All sections were then counterstained with hematoxylin for 10 minutes and mounted with Micromount media (Leica Biosystems, Buffalo Grove, IL). Whole slide images were acquired at 20x magnification using an Aperio AT2 digital automated scanner (Leica Biosystems, Buffalo Grove, IL).

[0091] Real-time RT-PCR. Total cellular RNA was extracted from mouse skin wounds using a nucleic acid extraction reagent sold under the trade name TRIZOL®. The mRNA expression of various genes was examined by real-time RT-PCR employing SYBR Green PCR mix. Relative gene expression was calculated as ΔΔC based on GAPDH levels as previously described. t The immunoglobulin levels were determined by the method described above (Chen, et al. (2013) J. Immunol. 190:5256-5266).

[0092] ELISA measurements. Mouse skin wounds were harvested, lysed, and prepared for ELISA measurements of mouse CCL28 and IL-6 according to the manufacturer's instructions.

[0093] Blood perfusion measurements. Immediately after the creation of a dorsal skin wound using an 8 mm punch on db / db mice, 50 μl of 50 μM Myr-scrambled control or Myr-CBD7 peptide in F-127 gel sold under the trade name PLURONIC® was applied topically to the wound of db / db mice. Blood perfusion was analyzed based on the movement detected under the surface of the wound using a near-infrared laser-based PeriCam PSI laser speckle contrast analysis system (LASCA) as previously described (Elgharably, et al. (2013) Wound Repair Regen. 21(3):473-481; Das, et al. (2016) J. Immunol. 196(12):5089-5100).

[0094] Statistical analysis. Data are expressed as the mean ± SEM from at least three independent experiments. Statistical analysis was performed by Student's t test or one-way ANOVA using GraphPad InStat software (San Diego, CA). Statistical significance was defined as P < 0.05.

[0095] Example 2: CCL28 activated eNOS-dependent Src, PI3K and MAPK signaling pathways in human endothelial cells CCR10 levels were found to be highly expressed in human umbilical vein endothelial cells (HUVEC) and human endothelial progenitor cells (EPC). NO production was also observed in HUVEC following treatment with 500 ng / ml human recombinant CCL28, a ligand for CCR10. The signaling pathways activated by CCL28 binding to CCR10 in endothelial cells were further investigated. Phosphorylation of eNOS (Ser1177), p85 (Tyr458; PI3K), Src kinase (Tyr418), and ERK (T202 / Y204; MAPK) were all significantly increased following CCL28 treatment for up to 60 min. However, when eNOS was depleted with siRNA, phosphorylation of p85, Src, and MAPK was abolished, suggesting that CCL28 promotes eNOS-dependent Src, PI3K, and MAPK signaling pathways in endothelial cells.

[0096] The role of the CCL28 / CCR10 signaling pathway in angiogenesis was further investigated. Pretreatment of cells with 10 μM wortmannin (PI3K inhibitor), 5 μM U0126 (MAPK / ERK inhibitor), 1 μM L-NAME (eNOS inhibitor), 10 μM PP2 (Src kinase inhibitor), and 10 μM STO-609 (CaMKII inhibitor) reduced endothelial cell tube formation induced by 500 ng / ml CCL28. These results suggest that eNOS / NO, Src, PI3K, and MAPK mediate, at least in part, CCL28-dependent angiogenesis. In addition, CCL28 stimulation of endothelial cells for 24 h enhanced the expression levels of the cell adhesion protein β-catenin and the antiapoptotic protein B-cell leukemia / lymphoma-2 (Bcl-2), supporting the proliferative capacity of endothelial cells in response to stimulation with CCL28.

[0097] Example 3: CCR10 regulation of eNOS-dependent angiogenesis in HUVECs To further investigate the role of CCR10 and eNOS in endothelial cell tube formation, siRNA targeting CCR10 (100nM) or eNOS (50nM) was used, and tube formation was measured in HUVEC. This analysis suggested that knockdown of either CCR10 or eNOS abolished endothelial cell tube formation, indicating that both CCR10 and eNOS proteins play a crucial role in angiogenesis. Western blot analysis showed that eNOS knockdown did not affect CCR10 expression, however, CCR10 knockdown reduced eNOS expression levels by 60%. The regulation of eNOS by CCR10 in endothelial cells was further demonstrated by confocal microscopy in that eNOS colocalized with CCR10 in cells treated with control siRNA. Consistent with the Western blot results, CCR10 fluorescence intensity was not affected in eNOS-depleted cells, however, eNOS fluorescence intensity was dramatically reduced in CCR10-depleted cells. These results may suggest that eNOS depletion in endothelial cells treated with CCR10 siRNA reflects the stabilization of the pool of eNOS that colocalizes with CCR10. Taken together, these results suggest that CCR10 plays an important role in angiogenesis through the regulation of eNOS expression and activation.

[0098] Example 4: Regulation of eNOS expression and function by CCR10 Since eNOS expression can be regulated by CCR10 in primary cultured human endothelial cells, the effect of CCR10 expression on eNOS activity in human endothelial cells was further investigated. After 72 hours of treatment with 100 nM CCR10 siRNA, endothelial cells were stimulated with 5 μM calcium ionophore A23187, and cells were harvested and prepared for Western blot analysis. Both CCR10 and eNOS expression levels were reduced. However, phosphorylation of eNOS was significantly increased compared to control siRNA even at 0 minutes, suggesting that CCR10 binding essentially suppresses eNOS activity. Nitrite concentration in cell supernatants was also measured, and eNOS activity was calculated relative to total eNOS expression. Consistent with the increased eNOS phosphorylation, NO production per eNOS molecule in endothelial cells after CCR10 knockdown was significantly higher, suggesting that CCR10 negatively regulates eNOS expression and activity in endothelial cells.

[0099] Example 5: Ligand-induced interaction between CCR10 and eNOS To investigate whether CCR10 directly interacts with eNOS, primary cultured HUVECs were harvested and prepared for co-immunoprecipitation (co-IP) to assess CCR10-eNOS interaction following stimulation with 5 μM A23187. Based on co-IP with anti-CCR10 or anti-eNOS antibodies, enhanced binding of CCR10 to eNOS was observed in endothelial cells stimulated with A23187, with maximum binding observed 5 min after addition of A23187. Similar results were obtained in endothelial cells following stimulation with 500 ng / ml human recombinant CCL28. CCR10-eNOS interaction was further supported by confocal microscopy and high-resolution imaging. Upon stimulation with A23187 or CCL28 for 5 min, CCR10 co-localized to a greater extent with eNOS on the plasma membrane. Colocalization coefficients in regions of interest (ROIs) in the plasma membrane were quantified from Zeiss LSM 880 confocal microscope images using Zeiss Zen software. The colocalization coefficients of CCR10 and eNOS were significantly greater following A23187 (81%) and CCL28 (78%) stimulation compared to untreated control cells (43%). These results suggest an increased interaction between CCR10 and eNOS at or near the plasma membrane of activated endothelial cells.

[0100] Example 6: Blockade of eNOS-CCR10 with myristoylated CCR10 binding domain (Myr-CBD) peptide improves eNOS activity The results suggested that CCR10 may directly interact with eNOS in endothelial cells, that CCR10 regulates eNOS expression and angiogenesis, and inhibits eNOS activity. Therefore, the effect of inhibiting CCR10-eNOS interaction was investigated using a panel of cell-penetrating peptides based on the sequence of the predicted interaction domain on eNOS. Similar peptides have been described in determining the solution structure of calmodulin bound to the target peptide of eNOS (Piazza, et al. (2014) Biochemistry 53(8):1241-9; Piazza, et al. (2016) Biochemistry 55(42):5962-71). First, a myristoylated 20 amino acid peptide (491-TRKKTFKEVANAVKISASLM-510(P1); SEQ ID NO:61) was synthesized based on the sequence of human eNOS, which is conserved in mouse eNOS. Co-IP experiments in HUVECs suggested that pretreatment with 50 μM cell-permeable 20 amino acid myristoylated CCR10 binding domain peptide (Myr-CBD20) significantly reduced CCR10-eNOS interaction in endothelial cells stimulated with 5 μM A23187 for 5 min compared to the control peptide. Following this, it was determined whether truncated versions of the CBD-derived peptides would have the same effect. Therefore, the truncated peptides listed in Table 3 were synthesized. [Table 3]

[0101] Pretreatment with 50 μM P1, P2, P3 or P4 completely blocked CCR10-eNOS interaction in endothelial cells following stimulation with 5 μM A23187 for 5 min. The effect of the Myr-CBD7 peptide on eNOS activity was evaluated, as the Myr-CBD7 peptide exhibited significant inhibition. Co-IP experiments showed a significant reduction in CCR10-eNOS interaction in cells treated with Myr-CBD7 compared to the scrambled control peptide (Myr-MSIALKS; SEQ ID NO:5). Consistent with reduced negative regulation by CCR10-dependent binding, eNOS phosphorylation (Ser1177) was significantly enhanced in the presence of Myr-CBD7.

[0102] Calmodulin (CaM) binding to bovine eNOS has been reported to occur at residues 493-TRKKTFKEVANAVKISASLM-512 (SEQ ID NO:2) (Venema, et al. (1996) J. Biol. Chem. 271(11):6435-6440; Venema, et al. (1995) J. Biol. Chem. 270:14705-14711). Therefore, co-IP was used to assess whether Myr-CBD7 affects eNOS-CaM binding. This analysis suggested that pretreatment of endothelial cells with Myr-CBD7 peptide had no effect on CaM-eNOS binding induced by treatment with 500 ng / ml CCL28 for 5 min. Thus, these data suggest that the 504-KISASLM-510 (SEQ ID NO: 1) domain in human eNOS is a specific binding region for CCR10.

[0103] Example 7: Topical administration of Myr-CBD7 peptide improved dermal wound healing in mice CCL28 (also called mucosa-associated epithelial chemokine) is a CC chemokine that signals through CCR10 as well as CCR3 (Pan, et al. (2000) J. Immunol. 165:2943). CCR10 has been reported to have two functional ligands, CCL27 and CCL28, which are involved in epithelial immunity (Xiong, et al. (2012) Protein Cell 3:571-580). It was observed that CCL28 and CCR10 levels were highly expressed compared to CCL27 and CCR3 in the skin of WT C57BL / 6 mice. Similar to IL-6, the protein levels of CCL28 determined by ELISA were significantly elevated in mouse wounds on days 3 and 7. Therefore, we subsequently investigated whether blocking CCR10-eNOS interaction with Myr-CBD7 peptide has an effect on wound healing in WT C57BL / 6 mice. Four 5 mm diameter full-thickness excision wounds were made on the back skin of mice, and 300 μl Myr-CBD7 peptide (50 μM) was administered topically onto the wounds immediately after punch wound creation. Compared to wounds treated with control peptide, Myr-CBD7 peptide significantly reduced wound size by 20% on day 5 and by 35% on day 7 (Figure 1). Wound size reduction with Myr-CBD7 treatment was associated with re-epithelialization of the skin wounds observed in H&E stained wound sections (Figure 2).

[0104] Myr-CBD7 peptide treatment also improved eNOS protein levels in wounds at day 7 compared to control peptide (Figure 3), and co-IP showed that Myr-CBD7 peptide treatment reduced CCR10-eNOS interaction in wounds at day 7 (Figure 4). Besides eNOS, RNA levels of CD31, another endothelial cell marker, were also increased; CCR10 mRNA levels, however, were unchanged. In addition, Myr-CBD7 peptide treatment resulted in a reduction in mRNA levels of the pro-inflammatory cytokine IL-6, while improving anti-inflammatory cytokine IL-4 mRNA in mouse wounds at day 7. Hepatocyte growth factor (HGF) mRNA levels were increased by 35% in Myr-CBD7-treated wounds, although this was non-significant. Taken together, these results indicate that topical treatment of mouse skin wounds with Myr-CBD7 peptide enhances eNOS / NO levels by blocking CCR10-eNOS interaction, thereby facilitating an anti-inflammatory environment that promotes wound angiogenesis and wound healing.

[0105] Example 8: Reduced eNOS expression and elevated CCR10 levels in type 2 diabetes mellitus (T2DM) patients and genetically diabetic mice Plasma nitric oxide (NO) concentrations have been shown to be higher in lean healthy controls (LHC) than in basal T2DM patients (37.4±10.1 μmol / L vs. 21.6±3.9 μmol / L; Mahmoud, et al. (2016) Physiol. Rep. 4:pii:e12895). In this analysis, expression levels of eNOS, an important endothelial marker protein, were further evaluated in human skeletal muscle biopsies. Protein levels of eNOS were significantly lower in T2DM patients compared to LHC. Following this, levels of the chemokine receptor CCR10 in these human biopsies were measured. The mRNA levels of CCR10 were also determined using real-time RT-PCR, and it was observed to be significantly elevated in biopsies from T2DM relative to LHC. Expression levels of eNOS and CCR10 were also measured in diabetic db / db mice and were compared with those of mice with a genetic leptin receptor db mutation (LEPR db ) mice were used as a spontaneous model of type 2 diabetes. Heavier body weight, elevated blood glucose, and increased expression of proinflammatory cytokines in the dorsal skin were observed in db / db mice compared with WT C57BL / 6 mice. In the dorsal skin of db / db mice, eNOS expression was significantly reduced compared with WT mice, and CCR10 mRNA was significantly elevated compared with WT. Taken together, the reduced eNOS and elevated CCR10 levels observed in human T2DM patients and genetically obese and diabetic db / db mice suggest that CCR10 may play an important role in regulating eNOS expression and is related to the pathological pathogenesis of cardiovascular dysfunction in type 2 diabetes.

[0106] Example 9: Downregulation of eNOS by CCR10 overexpression in mouse dorsal skin and human endothelial cells As determined by real-time RT-PCR, the expression of another receptor for CCL28, CCR3, was much lower compared with CCR10 in the dorsal skin of both WT and db / db mice. Therefore, CCR10 was considered to be the primary CCL28 receptor in further studies. - / - , db / db, WT, and CCR10 - / - Dorsal skin from db / db mice was collected and prepared for determination of eNOS and CCR10 expression. Western blot analysis showed reduced eNOS expression in db / db mice. Interestingly, eNOS expression was significantly higher than CCR10. - / - Moreover, CCR10 mRNA levels in mouse skin, as determined by real-time RT-PCR, were elevated in eNOS mice. - / - The eNOS expression was surprisingly elevated in mice. - / - As in db / db and db / db, CCR10 levels increase and CCR10 decreases, - / - As in mice, eNOS protein levels are increased. These data suggest that there is a correlation between eNOS and CCR10 expression.

[0107] Wound healing time in db / db and WT mice was also significantly correlated with eNOS expression. - / - and CCR10 - / - Mice were compared. Wounds were made on the dorsal skin and images were collected and analyzed every 48 hours. eNOS - / - , db / db and CCR10 - / - Delayed wound healing was observed in diabetic db / db mice compared to WT, starting from day 3. Importantly, delayed wound healing in diabetic db / db mice (85% relative to day 0) was due to the upregulation of eNOS at day 7. - / - This was very similar to that observed in mice (70%), whereas CCR10 - / - Mice (26%) exhibited relatively larger wounds than WT mice (16%).

[0108] CCR10 has been shown to be highly expressed in human umbilical vein endothelial cells (HUVEC) and human endothelial progenitor cells (EPC) (Chen, et al. (2020) FASEB J. 34(4):5838-5850). To understand whether the CCL28 / CCR10 signaling axis plays a role in wound healing in dermal capillaries, 500ng / ml of CCL28 was administered to human dermal microvascular endothelial cells (HDMVEC). eNOS-CCR10 interaction was increased, with maximum binding observed at 5 minutes, which was similar to that observed in HUVEC (Chen, et al. (2020) FASEB J. 34(4):5838-5850). Confocal microscopy was used to further demonstrate CCR10-eNOS interaction. Stimulation with CCL28 for 5 min resulted in a higher degree of colocalization of CCR10 with eNOS on the plasma membrane.

[0109] To understand whether eNOS is regulated by CCR10 in dermal endothelial cells, CCR10-GFP cDNA was transfected into primary cultured HDMVEC. With increasing amounts of transfected CCR10-GFP cDNA, eNOS expression levels were significantly decreased, and consistent with the reduction in eNOS expression, overexpression of CCR10 also reduced tube formation in HDMVEC. Reduced eNOS expression and NO production were also observed in stably transfected HEK / eNOS cells that were further transduced with CCR10-GFP cDNA. However, overexpression of eNOS had no effect on CCR10 levels in stable HEK / CCR10-GFP cells. Taken together, these results suggest that overexpression of CCR10 leads to downregulation of eNOS and that this is associated with reduced angiogenesis in vitro.

[0110] Example 10: Internalization of eNOS by CCR10 can be prevented by Myr-CBD7 peptide in HDMVEC stimulated with CCL28 Upon sustained stimulation, GPCRs are phosphorylated by G protein receptor kinases (GRKs), which leads to the recruitment of β-arrestins that mediate receptor desensitization / downregulation. GPCRs are internalized via clathrin- or caveolae-mediated endocytosis and either targeted to lysosomes for degradation or dephosphorylated and recycled to the cell surface to allow a new round of activation. In preliminary analysis, following 90 min treatment with 500 ng / ml CCL28, eNOS showed more colocalization with EEA1 (early endosomal antigen 1) in HDMVEC, suggesting that CCL28-activated CCR10 in endothelial cells leads to eNOS internalization. Following longer treatment times with CCL28 (3 h), co-IP experiments suggested that both eNOS and CCR10 were associated with LAMP1 (lysosome-associated membrane protein 1). These results suggested that both proteins underwent degradation compared to total cellular protein levels, which were unchanged.

[0111] Since the myristoylated 7 amino acid CCR10 binding domain (Myr-CBD7) peptide can block eNOS interaction with CCR10 and increase eNOS activity in HUVECs, we assessed whether Myr-CBD7 can prevent eNOS internalization and degradation in HDMVECs following CCL28 stimulation. Pretreatment with Myr-CBD7 prevented the interaction of eNOS but not CCR10 with LAMP1 compared to a scrambled control peptide, while total protein levels were unchanged. Confocal microscopy further confirmed that following pretreatment with Myr-CBD7 prior to 3 h of stimulation with CCL28, eNOS localization remained on the plasma membrane of endothelial cells, whereas eNOS colocalized with LAMP1 in the perinuclear region in cells pretreated with the control peptide. In addition, pretreatment with Myr-CBD7 significantly increased NO production in HEK / eNOS cells with or without co-expression of CCR10-GFP compared to the control peptide. These data suggest that eNOS undergoes internalization and degradation following activation of endothelial cells with CCL28 due to its interaction with CCR10. However, blocking the eNOS-CCR10 interaction with Myr-CBD7 peptide leads to the maintenance of eNOS on the cell surface and its ability to activate eNOS, and increases NO production.

[0112] Example 11: Local administration of Myr-CBD7 onto dorsal skin wounds in diabetic db / db mice improved wound healing by increasing eNOS / NO levels and microvessel density Topical administration of Myr-CBD7 peptide onto dermal wounds in WT C57BL / 6 mice reduced wound healing time. Furthermore, treatment with Myr-CBD7 not only improved the expression of eNOS, CD31, and IL-4, but it also reduced CCL28 and IL-6 levels in the wounds. Therefore, the effect of Myr-CBD7 peptide on the restoration of eNOS / NO levels and dorsal skin wound healing in obese and hyperglycemic db / db mice was further investigated.

[0113] Two 8 mm full-thickness excision wounds were made on the dorsal skin of diabetic db / db mice, and then 500 μl of 50 μM Myr-CBD7 peptide was applied topically. As shown in FIG. 5, Myr-CBD7 peptide treatment significantly reduced wound healing time by 4 days (control peptide-treated wounds on days 8, 10 and 12 were comparable to Myr-CBD7-treated wounds on days 4, 6 and 8, respectively). Thus, Myr-CBD7 significantly reduced wound size compared to control peptide, starting from day 4 (at 19%) and continuing through day 12 (at 46%) (FIG. 5). eNOS expression and NO production were significantly elevated in wounds on day 12 following treatment with Myr-CBD7 peptide, as determined by Western blot analysis and nitrite measurement, respectively. Reduced CCR10-eNOS interaction was also observed in mouse wounds following treatment with Myr-CBD7 peptide, as determined by co-IP (Figure 6). Furthermore, immunohistochemistry showed improved microvessel density (CD31 staining) in mouse wounds at day 12 following Myr-CBD7 treatment. These data suggested that Myr-CBD7 peptide treatment prevented eNOS inhibition and internalization by CCR10, thereby facilitating NO production and angiogenesis associated with improved wound healing in diabetic db / db mice.

[0114] Example 12: Shift in wound microenvironment from pro-inflammatory (M1) to anti-inflammatory (M2) in db / db mice following topical administration of Myr-CBD7 peptide ELISA measurements showed higher levels of the pro-inflammatory cytokines IL-6, TNF-α, IL-1β, as well as VEGF in the dorsal skin of db / db mice compared to WT mice. We investigated whether topical administration of Myr-CBD7 affected those factors in db / db mice, which are known to play important roles in the healing process. Mouse wounds were harvested on day 3 (inflammatory phase) and day 12 (proliferative phase) and inflammatory factor levels were determined by ELISA and real-time RT-PCR.

[0115] ELISA measurements demonstrated significantly reduced protein levels of CCL28 and the pro-inflammatory cytokines IL-6, TNF-α, IL-1β in db / db mouse wounds on day 3 following treatment with Myr-CBD7. Consistent with the protein levels, IL-6 mRNA levels were also reduced. In addition, levels of CCR10, which were elevated in db / db dorsal skin compared to WT, were also reduced on day 3 following treatment with Myr-CBD compared to the control peptide.

[0116] On day 12 after Myr-CBD7 treatment, elevated eNOS / NO levels were observed in mouse wounds as well as improved angiogenesis. Furthermore, Myr-CBD7 treatment led to elevated levels of VEGF, anti-inflammatory cytokines IL-13 and IL-4 (M2 markers), and EC markers eNOS, CD31, Flk-1 and VE-cadherin in mouse wounds.

[0117] To further investigate whether Myr-CBD7 promotes the healing of dorsal skin wounds on db / db mice by stimulating angiogenesis, blood perfusion was monitored as detected by laser speckle contrast analysis (LASCA). As shown above, 50 μM Myr-CBD7 or control peptide was administered to the wound, and on day 7, blood perfusion in the same wound was observed to be significantly increased when the wound size was significantly reduced. These results suggest that inhibition of CCR10 binding to eNOS in db / db mouse skin wounds stimulates wound perfusion and promotes healing.

[0118] Taken together, these data suggest that blocking CCR10-eNOS interaction with Myr-CBD7 peptide upregulated eNOS / NO bioavailability leading to a switch of the wound microenvironment from pro-inflammatory (M1) to anti-inflammatory (M2) that promoted angiogenesis, blood perfusion, and improved wound healing.

Claims

1. 1. A pharmaceutical composition for promoting or accelerating wound healing in a subject comprising a construct comprising a peptide of 7 to 20 amino acids comprising the amino acid sequence Lys-Ile-Ser-Ala-Ser-Leu-Met (SEQ ID NO: 1), operably linked to one or more carrier moieties.

2. The pharmaceutical composition of claim 1, wherein one or more carrier portions of the construct comprise a cell-penetrating peptide, a lipid, or a combination thereof.

3. The pharmaceutical composition described in claim 1 or 2, wherein the peptide of the construct further comprises one or more modifications selected from substitution, carboxylation, glycosylation, sulfonation, amidation, PEGylation, biotinylation, disulfide formation, and addition of a charged amino acid residue, and has the function of modulating CCR10-eNOS interaction.

4. A pharmaceutical composition described in claim 1 or 2, wherein the peptide of the construct is selected from the group consisting of TRKKTFKEVANAVKISASLM (sequence number 2), FKEVANAVKISASLM (sequence number 3), VANAVKISASLM (sequence number 4) and KISASLM (sequence number 1).

5. 3. The pharmaceutical composition of claim 1 or 2, wherein the construct is selected from myr-TRKKTFKEVANAVKISASLM (SEQ ID NO: 61), myr-FKEVANAVKISASLM (SEQ ID NO: 62), myr-VANAVKISASLM (SEQ ID NO: 63) and myr-KISASLM (SEQ ID NO: 64).

6. The pharmaceutical composition of claim 1, further comprising a pharma- ceutically acceptable vehicle.

7. A pharmaceutical composition described in any one of claims 1 to 6, wherein the wound is a diabetic wound.

8. The pharmaceutical composition of claim 7, wherein the wound is a diabetic foot ulcer.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the subject is co-administered at least one additional therapeutic agent for improving or accelerating wound healing.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition is for topical administration.

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