WO2023218095A1 - Agents for stimulating tissue regeneration - Google Patents

Agents for stimulating tissue regeneration Download PDF

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Publication number
WO2023218095A1
WO2023218095A1 PCT/EP2023/062965 EP2023062965W WO2023218095A1 WO 2023218095 A1 WO2023218095 A1 WO 2023218095A1 EP 2023062965 W EP2023062965 W EP 2023062965W WO 2023218095 A1 WO2023218095 A1 WO 2023218095A1
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WIPO (PCT)
Prior art keywords
seq
agent
use according
skin
wound
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PCT/EP2023/062965
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French (fr)
Inventor
Anna HULTGÅRDH NILSSON
Jan Alenfall
Jan Nilsson
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Coegin Pharma Ab
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Publication of WO2023218095A1 publication Critical patent/WO2023218095A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism

Definitions

  • the present invention relates to agents for the regeneration of tissue.
  • the regenerated tissue is preferably skin, i.e. the regeneration of tissue is preferably the healing of wounds.
  • Said wounds can be acute or pathological wounds.
  • Advanced wound care is a fast-growing market mainly composed of 4 main categories: dressings, wound cleansers, negative pressure wound therapy devices and biologies. It is estimated that 1 % to 2 % of the population will experience a chronic wound during their lifetime in developed countries (Gottrup) and this rate is increasing year after year. Stalled wounds may persist for months, even years (Kirker et al., Frykberg et al.) before healing. Hence, non-healing/hard-to-heal wounds represent an economic burden for health services and a large medical problem for patients, representing a global cost of $143 bn.
  • Treatment for acute wounds include debridement, decontamination, moist wound healing, and early closure.
  • Treatment for chronic wounds include debridement, anti-inflammation therapy, antibiotic therapy, synthetic substitutes, modulating cytokines and/or matrix metalloproteinases (MMPs), or recruiting stem cells.
  • MMPs matrix metalloproteinases
  • Wounds may arise from a variety of aetiologies such as peripheral arterial disease, venous hypertension, neuropathy (e.g., diabetes mellitus), pressure (in patients with neuropathy or decreased mobility), vasculitis and burns, etc and are very often hard to heal.
  • a large proportion of acute wounds heal with appropriate management, but some wounds fail to heal and proceed to becoming chronic, non-healing wounds.
  • a ‘completely healed wound’ can be defined as one that has returned to its normal anatomic structure, function, and appearance within a reasonable period of time (normally in about four to six weeks). Any wound that has not reached the above state would be considered a chronic, non-healing wound.
  • Wound care has become increasingly important given the rise of chronic wounds and the morbidity associated with them.
  • An important concept in wound care is the role of debridement, or the removal of non-viable tissue material. Disruption at one or more points in the distinct phases of wound healing (haemostasis, inflammation, proliferation, or remodelling) can cause a wound not to heal.
  • An alternation in the balance between various cytokines, growth factors, protease activity, matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) is observed in chronic wounds.
  • alteration in the morphology and proliferation rate of fibroblasts, keratinocyte activity, and accumulation of oxygen derived free radicals, necrotic tissue and underlying disease all play a role in inhibiting wound healing.
  • the present inventors have identified agents with use in healing wounds.
  • One aspect of the disclosure provides for an agent comprising: a) a peptide selected from the group consisting of:
  • X 5 is D or G
  • X14 is E or G
  • X15 is S or T
  • VDTYDGDISVVYGL SEQ ID NO: 34
  • VDTYDGDISVVYG SEQ ID NO: 35
  • VDTYDGDISVVY SEQ ID NO: 36
  • VDTYDGDISVV SEQ ID NO: 37
  • VDTYDGDISV SEQ ID NO: 38
  • VDTYDGDIS SEQ ID NO: 39
  • VDTYDGRGDSVVYGLR SEQ ID NO: 40
  • VDVPNGDISLAYGL SEQ ID NO: 41
  • VDVPNGDISLAYG SEQ ID NO: 42
  • VDVPNGDISLA SEQ ID NO: 43
  • VDVPNGDIS SEQ ID NO: 44
  • GDPNDGRGDSVVYGLR SEQ ID NO: 45
  • LDGLVRAYDNISPVG SEQ ID NO: 46
  • GDPNGDISVYGLR SEQ ID NO: 47
  • GDPNGDISVYGLR SEQ ID NO
  • the present invention relates to a method for treatment and/or regeneration of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent as defined herein to an subject in need thereof.
  • the present invention relates to a method for treating and/or promoting healing of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent as defined herein to an subject in need thereof.
  • the present invention relates to a method for regeneration of skin at the site of a skin defect and/or for reducing scar formation resulting from healing of a skin defect in a subject, said method comprising administering an agent as defined herein.
  • the present invention relates to use of an agent as defined herein for the manufacture of a medicament for the treatment and/or regeneration of a skin defect in a subject.
  • the present invention relates to use of an agent defined herein for the manufacture of a medicament for the treating and/or promoting healing of a tissue defect in a subject.
  • the present invention relates to use of an agent defined herein for the manufacture of a medicament for the treatment of a skin defect in a subject.
  • the present invention relates to use of an agent defined herein for reducing scar formation resulting from healing of a skin defect in a subject.
  • the present invention relates to use of an agent defined herein for promoting the healing of a skin defect in a subject.
  • the present invention relates to use of an agent defined in herein for promoting vascularisation in a healing tissue, such as skin healing from a skin defect, such as a wound.
  • the present invention relates to use of an agent defined herein in combination with a tissue meshing technique, for promoting healing of a skin defect, such as a wound.
  • the present invention relates to use of an agent as defined herein for promoting grafting of skin.
  • the present invention relates to a method for inserting a stent in a subject, said method comprising administering an agent as defined herein.
  • the present invention relates to a medical device comprising the agent as defined herein.
  • FIG. 1 FOL26 stimulates endothelial and vascular smooth muscle cell proliferation.
  • Human umbilical cord endothelial cells (A) and human coronary artery smooth muscle cells (B) were treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and DNA synthesis analyzed by measuring the uptake of BrdU.
  • FIG. 2 FOL26 enhances endothelial cell tube formation and expression of PECAM-1.
  • Human umbilical cord endothelial cells were treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and endothelial tube formation and gene expression of PECAM-1 (CD31) analyzed.
  • FOL26 increased tube formation compared to control.
  • the expression of PECAM-1 was increased for 100 nM and 1000 nM FOL26.
  • FIG. 3 FOL26 increases endothelial cell migration in a cell culture scratch wound assay. Confluent cultures of human umbilical cord endothelial cells were treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and ability of the cells to migrate into an in vitro scratch injury was analyzed with image J software. Migration of cells increased with increasing concentration of FOL26.
  • FIG. 4 FOL26 increases arterial smooth muscle cell migration in a cell culture scratch wound assay.
  • PCR polymerase chain reaction
  • FOL26 increased expression of both COL1 A1 and COL1 A2.
  • the wound closure at 5h was increased for FOL26 at 10 nM and 100 nM, while the 1000 nM also trended towards increased closure compared to control.
  • FIG. 5 FOL26 inhibits endothelial and arterial smooth muscle cell apoptosis.
  • Cultured human umbilical cord endothelial and coronary artery smooth cells were exposed to soluble Fas ligand (sFasL) to induce apoptosis that subsequently was assessed by determining activation of caspase 3 and 7.
  • Cells were also treated with FOL 26 in concentrations of 10 nM, 100 nM, and 1000 nM to determine the effect on apoptosis activated by sFasL.
  • FOL26 was capable of reversing the sFasL-induced apoptosis, to the highest extent at the 100 nM concentration.
  • FOL26 affects the gene expression of VEGF and the VEGF receptor family in endothelial cells.
  • Human umbilical cord endothelial cells were treated with FOL 26 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding c-Met, neuropilin-1 (NRP-1), VEGF-A and VEGF receptor-2 (VEGFR-2) determined by polymerase chain reaction (PCR).
  • FOL026 was reducing the c-Met expression, in the lowest dose reducing however in the 100nM and 1000nM doses were inducing NRP-1 expression.
  • FOL026 had no effect on VEGF-A expression, however in 10nM it reduced and in 1000nM it induced the expression of VEGFR-2 levels.
  • FOL26 affects the gene expression of cytokines and matrix metalloproteinases in endothelial cells.
  • Human umbilical cord endothelial cells were treated with FOL 26 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding TNF-a, IL-6, MMP2 and MMP3 determined by polymerase chain reaction (PCR).
  • TNF-a is strong pro-inflammatory cytokine while MMP2 is required for activation of smooth muscle cell proliferation and migration.
  • FOL026 reduced the TNF-a expression in 10nM and 100nM doses, had no effect on IL-6 or MMP3 expression, however, MMP2 expression was induced with 200nM and 1000nM doses.
  • FOL26 affects the gene expression of VEGF and the VEGF receptor family in arterial smooth muscle cells.
  • Human coronary artery smooth cells were treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding c-Met, neuropilin-1 (NRP-1), VEGF-A and VEGF receptor-2 (VEGFR- 2) determined by polymerase chain reaction (PCR).
  • FOL026 reduced the c-MET expression in 10nM and 1000nM doses, as well as the NRP-1 expression in 10, 100, and 1000nM doses.
  • FOL26 affects the gene expression of cytokines and matrix metalloproteinases in arterial smooth muscle cells.
  • Human coronary artery smooth cells were treated with FOL 26 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding TNF-a, IL-6, MMP2 and MMP3 determined by polymerase chain reaction (PCR).
  • TNF-a is strong pro-inflammatory cytokine while MMP2 is required for activation of smooth muscle cell proliferation and migration.
  • FOL026 100 and 1000nM induced the TNF-a, IL-6 and MMP3 expression, furthermore, FOL026 induced in 10, 100, and 1000nM doses the MMP2 expression.
  • FIG. 10 FOL26 stimulation of endothelial cell proliferation is NRP-1 dependent.
  • Human umbilical cord endothelial cells were first treated with a scramble non-coding siRNA (NC) or siRNA for NRP-1, subsequently they were treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and DNA synthesis analyzed by measuring the uptake of BrdU.
  • NC scramble non-coding siRNA
  • FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM
  • DNA synthesis analyzed by measuring the uptake of BrdU.
  • An increase in O.D. (450 nm) for the FOL 26 treated cell lines compared to the control showed that FOL26 induced angiogenesis may be NRP-1 dependent as a reduction was observed for FOL26 10 and 100nM.
  • Figure 11 Human umbilical cord endothelial cells were treated with a scramble noncoding siRNA (NC) or siRNA for NRP-1 in concentrations of 1nM, and 5nM, and the NRP-1 expression values and Glyceraldehyde-phosphate dehydrogenase (GAPDH) mRNA expression values was evaluated, the latter was used as an internal control to normalize the data.
  • NC scramble noncoding siRNA
  • GPDH Glyceraldehyde-phosphate dehydrogenase
  • Figure 12 FOL26 enhances endothelial cell tube in an NRP-1 specific manner.
  • Human umbilical cord endothelial cells were first treated with a scramble non-coding siRNA (NC) or siRNA for NRP-1, subsequently treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and endothelial tube formation and the total master segemnts were measured using Imaged, gene expression of PECAM-1 (CD31) analyzed.
  • FOL26 increased tube formation compared to control in a NRP-1 dependent way. Scale bar: 150 pm.
  • Figure 13 Overview of acute wound healing and pathological wound healing models, including experimental parameters.
  • A) Acute wound healing - one way ANOVA and Dunnett’s multiple comparison test vs. vehicle n.s. Unpaired t test * ⁇ 0.05.
  • C) Pathological wound healing - One way ANOVA p 0.143, Dunnett’s multiple comparison test vs vehicle #p ⁇ 0.05. Unpaired t test *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 15 Representative images of wound tongue at day 3, acute model (donor 1). PAS stained skin sections. Dashed lines show the newly formed epithelial tongues. Scale bar represents 100 pm.
  • Figure 16 Representative images of wound tongue at day 3, pathological model (donor 2). PAS stained skin sections. Dashed lines show the newly formed epithelial tongues. Scale bar represents 100 pm.
  • Figure 17 Increased proliferation of epidermal keratinocytes adjacent to the wound bed and decreased keratinocyte apoptosis at 300 nM FOL-005 peptide in the ex vivo pathological wound healing model (6 day protocol, 3 topical applications of FOL peptide).
  • FIG. 18 FOL56 stimulates endothelial and vascular smooth muscle cell proliferation.
  • Human umbilical cord endothelial cells (A) and human coronary artery smooth muscle cells (B) were treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and DNA synthesis analyzed by measuring the uptake of BrdU.
  • FOL56 increases proliferation at all concentrations in both HUVECs and HCASMCs compared to the control.
  • FIG. 19 FOL56 enhances endothelial cell tube formation and expression of PECAM- 1.
  • Human umbilical cord endothelial cells were treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and endothelial tube formation and gene expression of PECAM-1 (CD31) analyzed.
  • FOL56 increased tube formation at all concentrations compared to control, and also increased expression of PECAM-1.
  • FIG. 20 FOL56 increases endothelial cell migration in a cell culture wound model. Confluent cultures of human umbilical cord endothelial cells were treated FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and ability of the cells to migrate into an in vitro scratch injury was analyzed with image J software. FOL56 increased cell migration at all concentrations compared to the control.
  • FIG. 21 FOL56 increases arterial smooth muscle cell migration in a cell culture wound model.
  • Confluent cultures of human coronary artery smooth cells were treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and ability of the cells to migrate into an in vitro scratch injury was analyzed with image J software. Expression of the collagen gene a1 and a2 chains were determined by polymerase chain reaction (PCR).
  • FOL56 increased expression of both COL1A1 and COL1A2. The wound closure at 5h was increased for FOL56 at all concentrations compared to the control.
  • FIG. 22 FOL56 inhibits endothelial and arterial smooth muscle cell apoptosis.
  • Cultured human umbilical cord endothelial and coronary artery smooth cells were exposed to soluble Fas ligand (sFasL) to induce apoptosis that subsequently was assessed by determining activation of caspase 3 and 7.
  • Cells were also treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM to determine the effect on apoptosis activated by sFasL.
  • FOL56 was capable of inhibiting, or trended towards inhibiting apoptosis in both HUVECs and HCASMCs.
  • FOL56 affects the gene expression of VEGF and the VEGF receptor family in endothelial cells.
  • Human umbilical cord endothelial cells were treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding c-Met, neuropilin-1 (NRP-1), VEGF-A and VEGF receptor-2 (VEGFR-2) determined by polymerase chain reaction (PCR).
  • FOL56 affects the gene expression of cytokines and matrix metalloproteinases in endothelial cells.
  • Human umbilical cord endothelial cells were treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding TNF-a, IL-6, MMP2 and MMP3 determined by polymerase chain reaction (PCR).
  • TNF-a is strong pro-inflammatory cytokine while MMP2 is required for activation of smooth muscle cell proliferation and migration.
  • FOL56 affects the gene expression of VEGF and the VEGF receptor family in arterial smooth muscle cells.
  • Human coronary artery smooth cells were treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding c-Met, neuropilin-1 (NRP-1), VEGF-A and VEGF receptor-2 (VEGFR- 2) determined by polymerase chain reaction (PCR).
  • FOL56 affects the gene expression of cytokines and matrix metalloproteinases in arterial smooth muscle cells.
  • Human coronary artery smooth cells were treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding TNF-a, IL-6, MMP2 and MMP3 determined by polymerase chain reaction (PCR).
  • TNF-a is strong pro-inflammatory cytokine while MMP2 is required for activation of smooth muscle cell proliferation and migration.
  • FIG. 27 Effect of FOL-026 peptide on endothelial cell function and gene expression.
  • the ROS level (C) activated by 50ug/ml oxidized Low-density Lipoprotein (oxLDL) for 2 hours in cells pre-incubated with FOL-026 peptides was assessed using H2O2 measurement (n 4-8 per group).
  • oxLDL oxidized Low-density Lipoprotein
  • the effect of FOL-026 peptides on gene expression of VEGF-A (F) and its receptors (G and H), as well as HGF receptor c-Met (I) in HUVECs (n 4 for F-l).
  • Scale bar 150um.
  • FIG. 29 Effect of FOL-026 peptide on angiogenesis in vitro and vivo.
  • A Western blot analysis of phosphorylated AKT (pAKT-T308), AKT, phosphorylated ERK1/2 (p-ERK1/2) and ERK1/2 in endothelial cells stimulated with FOL-26 peptides for 48 hours (B).
  • Figure 31 Effect of NRP-1 knock-down on endothelial cell function induced with FOL- 026 peptide.
  • FIG 32 Representative pictures of endothelial cells at 0 and 5 hours after scratch wounding are shown in (A).
  • FIG 33 Effect of NRP-1 knock-down on tube formation induced with FOL-026 peptide.
  • Figure 34 Effect of FOL-026 peptide on smooth muscle cell function.
  • Figure 36 Effect of NRP-1 knock-down on smooth muscle cell function induced with FOL-026 peptide.
  • qRT-PCR analysis of NRP-1 mRNA expression in HCASMCs with small interfering RNA transfection for 48 hours (n 4) (A).
  • FIG 37 Representative images of smooth muscle cells at 0 and 5 hours after scratch wounding are shown in (A).
  • FIG 38 Effect of FOL-005 peptide on endothelial and smooth muscle cell proliferation and wound healing ability.
  • FIG 40 Effect of FOL-005 peptide on tube formation and the role of NRP-1 in that. Representative images of tube formation in human umbilical vascular endothelial cells (HUVECs) treated with FOL-005 peptides are shown in (A and B).
  • HUVECs human umbilical vascular endothelial cells
  • Preventing includes delaying, stopping, reducing the risk of the onset, of disease, disorder, or condition.
  • the term "skin defect" refers to, but is not limited to: injuries or trauma to internal or external tissue, preferably injury or trauma to the epidermis and/or dermis of the skin.
  • the wound may be an acute wound or a chronic wound.
  • an acute wound may be an incision, laceration, abrasion graze or burn, a puncture wound, a penetration wound or a wound due to dermatologic diseases such as psoriasis, acne and eczema.
  • a chronic wound may be a venous ulcer, a diabetic ulcer, a pressure ulcer, corneal ulcer, digestive ulcer or wounds as a result of ischemia and radiation poisoning.
  • treatment and/or regeneration of a skin defect relates to the promotion, the acceleration, and/or the improvement of healing at the wounded site, i.e. the formation of a functional skin at the wounded site.
  • treatment and/or regeneration of a skin defect ideally result in formation or regeneration of dermis and epidermis, including a basal layer, which characterize functional skin.
  • hard-to-heal wound refers to a wound that has not healed. Wounds that do not heal within approximately 4 to 6 weeks, for example, are considered chronic.
  • a "chronic wound” may be a wound that fails to progress through an orderly and timely sequence of repair or a wound that does not respond to treatment and/or the demands of treatment are beyond the patient's physical health, tolerance or stamina. Many wounds that are first considered to be acute wounds ultimately become chronic wounds due to factors still not well understood. One significant factor is the transition of planktonic bacteria within the wound to form a biofilm. For example, a chronic wound may have an epithelial layer that fails to cover the entire surface of the wound and is subject to bacterial colonization, which can result in biofilm formation, which is resistant to treatment with anti-bacterial agents.
  • venous insufficiency venous insufficiency
  • arterial insufficiency venous insufficiency
  • diabetic complications or is a pressure-related.
  • Hard-to-heal wounds due to venous insufficiency account for 70% to 90% of all hard-to-heal wounds and commonly affect the elderly.
  • Venous insufficiency results in venous hypertension, in which blood flow is abrogated resulting in subsequent ischaemia. Venous insufficiency can occur as a result of obstructions to venous outflow or reflux due to valve damage. Following a period of ischaemia, tissue reperfusion can result in reperfusion injury, causing the tissue damage that leads to wound formation.
  • Exemplary chronic wounds can include "burn ulcers", including first degree burn, which may be a superficial, reddened area of skin; second degree burn, which may be a blistered injury site which may heal spontaneously after the blister fluid has been removed; third degree burn, which may be a burn through the entire skin and usually require surgical intervention for wound healing; scalding burns, which may occur from scalding hot water, grease or radiator fluid; thermal burns, which may occur after contact with flames, usually deep burns; chemical burns, which may come from acid and alkali, usually deep burns; electrical burns; and contact burns, which are usually deep and may occur from muffler tail pipes, hot irons and stoves, or other materials.
  • first degree burn which may be a superficial, reddened area of skin
  • second degree burn which may be a blistered injury site which may heal spontaneously after the blister fluid has been removed
  • third degree burn which may be a burn through the entire skin and usually require surgical intervention for wound healing
  • vascular regeneration Angiogenesis and neovascularisation are understood to be important processes of vascular regeneration and may collectively or alternatively be referred to herein as vascular regeneration.
  • vascular regeneration is to be understood in its broadest sense to also include other repair processes including endothelial cell migration, proliferation and mobilisation.
  • wound dressing particularly refers to any material applied to a wound for protection, absorbance, drainage, etc.
  • films e.g., polyurethane films
  • hydrocolloids hydrophilic colloidal particles bound to polyurethane foam
  • hydrogels cross-linked polymers containing about at least 60% water
  • foams hydrophilic or hydrophobic
  • calcium alginates nonwoven composites of fibers from calcium alginate
  • cellophane cellulose with a plasticizer
  • tissue such as in the context of “tissue defect”, is meant epithelial surfaces of epithelial origin.
  • the agent of the present invention may be a peptide; a polynucleotide encoding said peptide; a vector comprising said polynucleotide; or a cell comprising said polynucleotide or vector.
  • the agent is a peptide or a pharmaceutically acceptable salt thereof.
  • amino acid as used herein includes the standard twenty genetically-encoded amino acids and their corresponding stereoisomers in the ‘D’ form (as compared to the natural ‘L’ form), omega-amino acids and other naturally-occurring amino acids, unconventional amino acids (e.g., a,a-disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatized amino acids (see below).
  • amino acid when an amino acid is being specifically enumerated, such as ‘alanine’ or ‘Ala’ or ‘A’, the term refers to both L-alanine and D-alanine unless explicitly stated otherwise.
  • Other unconventional amino acids may also be suitable components for peptides of the present disclosure, as long as the desired functional property is retained by the peptide.
  • each encoded amino acid residue where appropriate, is represented by a single letter designation, corresponding to the trivial name of the conventional amino acid.
  • the peptide is non-naturally occurring, such as a peptide comprising non-proteinogenic amino acid residues.
  • the agent comprises or consists of a tandem repeat comprising two or more repeat units.
  • the repeat unit comprises or consists of the amino acid sequence of any one or more of the sequences as described herein.
  • the peptide is cyclic.
  • the cyclic structure may be achieved by any suitable method of synthesis.
  • heterodetic linkages may include, but are not limited to formation via disulphide, cysteine, alkylene or sulphide bridges.
  • the peptide is capable of forming at least one intramolecular cysteine bridge.
  • the agent comprises or consists of a peptide comprising or consisting of an amino acid sequence of the general formula:
  • Xs is D or G
  • Xe is I or G
  • X7 is V or L; and Xa is V or A.
  • the peptide comprises or consists of an amino acid sequence of the general formula:
  • VDX2X3X4GX5X6SX7X8YGLR (SEQ ID NO: 2) wherein:
  • X 2 is T or V
  • X 3 is Y or P
  • X4 is D or N
  • Xs is D or G
  • Xe is I or G
  • X 7 is V or L
  • X 8 is V or A.
  • the peptide comprises or consists of an amino acid sequence of the general formula:
  • VDTYX 4 GX5X6SX 7 X 8 YGLR (SEQ ID NO: 3) wherein:
  • X 4 is D or N
  • X 5 is D or G
  • X 6 is I or G
  • X 7 is V or L
  • X 8 is V or A.
  • the peptide comprises or consists of an amino acid sequence of the general formula:
  • VDTYDGZ 7 Z 8 SZIOZIIYGLR (SEQ ID NO: 4) wherein:
  • X 5 is D or G
  • X 8 is I or G
  • X 7 is V or L
  • X 8 is V or A.
  • the peptide comprises or consists of an amino acid sequence of the general formula:
  • VDTYDGZ 7 Z 8 SVVYGLR (SEQ ID NO: 5) wherein:
  • Xs is D or G
  • X 8 is I or G;
  • the agent comprises or consists of a peptide comprising or consisting of an amino acid sequence of the general formula:
  • X14 is E or G
  • X15 is S or T.
  • the peptide comprises or consists of an amino acid sequence of the general formula:
  • X 9 is C, P or G
  • X is E or G
  • Xu is C, D or I
  • X12 is D, I, S or G
  • X13 is S, D or G
  • X14 is E or G
  • X15 is S or T.
  • the peptide comprises or consists of an amino acid sequence of the general formula:
  • X 9 is C, P or G
  • X10 is E or G
  • Xu is C, I or absent
  • X12 is D, G or absent
  • X13 is S, G or absent
  • X14 is E or G.
  • the peptide comprises or consists of an amino acid sequence of the general formula:
  • X 9 is C, P or G; Xio is E or G; and
  • X14 is E or G.
  • the peptide comprises or consisted of the amino acid sequence IELSYGIK (SEQ ID NO: 109).
  • the peptide comprises or consists of VDTYDGGISVVYGLR (SEQ ID NO: 6). In one embodiment, the peptide comprises or consists of AEIDSIELSYGIK (SEQ ID NO: 110). In one embodiment, the peptide comprises or consists of VDTYDGDISVVYGLR (SEQ ID NO: 7). In one embodiment, the peptide comprises or consists of DTYDGDISVVYGLR (SEQ ID NO: 8). In one embodiment, the peptide comprises or consists of TYDGDISVVYGLRS (SEQ ID NO: 9). In one embodiment, the peptide comprises or consists of TYDGDISVVYGLR (SEQ ID NO: 10).
  • the peptide comprises or consists of YDGDISWYGLRS (SEQ ID NO: 11). In one embodiment, the peptide comprises or consists of YDGDISVVYGLR (SEQ ID NO: 12). In one embodiment, the peptide comprises or consists of DGDISVVYGLRS (SEQ ID NO: 13). In one embodiment, the peptide comprises or consists of DGDISVVYGLR (SEQ ID NO: 14). In one embodiment, the peptide comprises or consists of GDISVVYGLRS (SEQ ID NO: 15). In one embodiment, the peptide comprises or consists of GDISVVYGLR (SEQ ID NO: 16).
  • the peptide comprises or consists of DISVVYGLRS (SEQ ID NO: 17). In one embodiment, the peptide comprises or consists of DISVVYGLR (SEQ ID NO: 18). In one embodiment, the peptide comprises or consists of VDVPNGDISLAYGLR (SEQ ID NO: 19). In one embodiment, the peptide comprises or consists of DVPNGDISLAYGLRS (SEQ ID NO: 20). In one embodiment, the peptide comprises or consists of DVPNGDISLAYGLR (SEQ ID NO: 21). In one embodiment, the peptide comprises or consists of VPNGDISLAYGLRS (SEQ ID NO: 22).
  • the peptide comprises or consists of VPNGDISLAYGLR (SEQ ID NO: 23). In one embodiment, the peptide comprises or consists of PNGDISLAYGLRS (SEQ ID NO: 24). In one embodiment, the peptide comprises or consists of PNGDISLAYGLR (SEQ ID NO: 25). In one embodiment, the peptide comprises or consists of NGDISLAYGLRS (SEQ ID NO: 26). In one embodiment, the peptide comprises or consists of NGDISLAYGLR (SEQ ID NO: 27). In one embodiment, the peptide comprises or consists of GDISLAYGLRS (SEQ ID NO: 28). In one embodiment, the peptide comprises or consists of GDISLAYGLR (SEQ ID NO: 29).
  • the peptide comprises or consists of DISLAYGLRS (SEQ ID NO: 30). In one embodiment, the peptide comprises or consists of DISLAYGLR (SEQ ID NO: 31). In one embodiment, the peptide comprises or consists of VDTYDGDGSVVYGLR (SEQ ID NO: 32). In one embodiment, the peptide comprises or consists of VDVPEGDISLAYGLR (SEQ ID NO: 33). In one embodiment, the peptide comprises or consists of KPLAEIDSIELSYGIK (SEQ ID NO: 111). In one embodiment, the peptide comprises or consists of KCLAECDSIELSYGIK (Cyclic) (SEQ ID NO: 112).
  • the peptide comprises or consists of KPLAEDISIELSYGIK (SEQ ID NO: 113). In one embodiment, the peptide comprises or consists of KPLAEISDIELSYGIK (SEQ ID NO: 114). In one embodiment, the peptide comprises or consists of KPLAEIGDIELSYGIK (SEQ ID NO: 115). In one embodiment, the peptide comprises or consists of KPLAEGDIELSYGIK (SEQ ID NO: 116). In one embodiment, the peptide comprises or consists of KPLAEIELSYGIK (SEQ ID NO: 117). In one embodiment, the peptide comprises or consists of KPLAEIDSIELTYGIK (SEQ ID NO: 118).
  • the peptide comprises or consists of KPLAEIDGIELSYGIK (SEQ ID NO: 119). In one embodiment, the peptide comprises or consists of KPLAEIDGIELTYGIK (SEQ ID NO: 120). In one embodiment, the peptide comprises or consists of KPLAEIGSIELSYGIK (SEQ ID NO: 121). In one embodiment, the peptide comprises or consists of KGLAEIDSIELSYGIK (SEQ ID NO: 122). In one embodiment, the peptide comprises or consists of KPLAGIDSIGLSYGIK (SEQ ID NO: 123). In one embodiment, the peptide comprises or consists of Cyclic KCLAEIDSCELSYGIK (SEQ ID NO: 124).
  • the peptide comprises or consists of LAEIDSIELSYGIK (SEQ ID NO: 125). In one embodiment, the peptide comprises or consists of EIDSIELSYGIK (SEQ ID NO: 126). In one embodiment, the peptide comprises or consists of IDSIELSYGIK (SEQ ID NO: 127). In one embodiment, the peptide comprises or consists of DSIELSYGIK (SEQ ID NO: 128). In one embodiment, the peptide comprises or consists of SIELSYGIK (SEQ ID NO: 129). In one embodiment, the peptide comprises or consists of IELSYGIK (SEQ ID NO: 109).
  • the peptide comprises or consists of VDTYDGDISVVYGL (SEQ ID NO: 34). In one embodiment, the peptide comprises or consists of VDTYDGDISVVYG (SEQ ID NO: 35). In one embodiment, the peptide comprises or consists of VDTYDGDISVVY (SEQ ID NO: 36). In one embodiment, the peptide comprises or consists of VDTYDGDISVV (SEQ ID NO: 37). In one embodiment, the peptide comprises or consists of VDTYDGDISV (SEQ ID NO: 38). In one embodiment, the peptide comprises or consists of VDTYDGDIS (SEQ ID NO: 39).
  • the peptide comprises or consists of VDTYDGRGDSVVYGLR (SEQ ID NO: 40). In one embodiment, the peptide comprises or consists of VDVPNGDISLAYGL (SEQ ID NO: 41). In one embodiment, the peptide comprises or consists of VDVPNGDISLAYG (SEQ ID NO: 42). In one embodiment, the peptide comprises or consists of VDVPNGDISLA (SEQ ID NO: 43). In one embodiment, the peptide comprises or consists of VDVPNGDIS (SEQ ID NO: 44).
  • the peptide comprises or consists of GDPNDGRGDSVVYGLR (SEQ ID NO: 45 In one embodiment, the peptide comprises or consists of LDGLVRAYDNISPVG (SEQ ID NO: 46). In one embodiment, the peptide comprises or consists of GDPNGDISVVYGLR (SEQ ID NO: 47). In one embodiment, the peptide comprises or consists of VDVPNGDISLAYRLR (SEQ ID NO: 48). In one embodiment, the peptide comprises or consists of VDVPEGDISLAYRLR (SEQ ID NO: 49). In one embodiment, the peptide comprises or consists of V(beta-D)TYDGDISVVYGLR (SEQ ID NO: 50).
  • the peptide comprises or consists of VDTY(beta-D)GDISVVYGLR (SEQ ID NO: 51 In one embodiment, the peptide comprises or consists of VDTYDG(beta- D)ISVVYGLR (SEQ ID NO: 52). In one embodiment, the peptide comprises or consists of CLAEIDSC (Cyclic) (SEQ ID NO: 130). In one embodiment, the peptide comprises or consists of CFKPLAEIDSIECSYGIK (Cyclic) (SEQ ID NO: 131). In one embodiment, the peptide comprises or consists of Cyclic CFKPL EIDSIEC (SEQ ID NO: 132).
  • the peptide comprises or consists of KPLAEIDSIELSYGI (SEQ ID NO: 133). In one embodiment, the peptide comprises or consists of KPLAEIDSIELSYG (SEQ ID NO: 134). In one embodiment, the peptide comprises or consists of KPLAEIDSIELSY (SEQ ID NO: 135), KPLAEIDSIELS (SEQ ID NO: 136). In one embodiment, the peptide comprises or consists of KPLAEIDSIEL (SEQ ID NO: 137). In one embodiment, the peptide comprises or consists of KPLAEIDSIE (SEQ ID NO: 138).
  • the agent as defined herein can be in the form of a pharmaceutically acceptable salt or prodrug of said agent.
  • the agent as defined herein can be formulated as a pharmaceutically acceptable addition salt or hydrate of said agent, such as but not limited to K + , Na + , as well as non-salt e.g. H + .
  • the agent is a chemical derivative of a peptide.
  • Chemical derivatives of one or more amino acids may be achieved by reaction with a functional side group.
  • Such derivatives include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulphonyl groups, carboxybenzoxy groups, f-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters and hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
  • Also included as chemical derivatives are those peptides which contain naturally occurring amino acid derivatives of the twenty standard amino acids.
  • 4-hydroxyproline may be substituted for proline
  • 5- hydroxylysine may be substituted for lysine
  • 3-methylhistidine may be substituted for histidine
  • homoserine may be substituted for serine and ornithine for lysine.
  • Derivatives also include peptides containing one or more additions or deletions as long as the requisite activity is maintained.
  • Other included modifications are amidation, amino terminal acylation (e.g. acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g. with ammonia or methylamine), and the like terminal modifications.
  • the agent is further modified such by glycosylation, PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.
  • the agent is further conjugated to a moiety, which may be selected from the group consisting of polyethylene glycol (PEG), monosaccharides, fluorophores, chromophores, radioactive compounds, and cell-penetrating peptides.
  • PEG polyethylene glycol
  • monosaccharides include fluorophores, chromophores, radioactive compounds, and cell-penetrating peptides.
  • the fluorophore is selected from the group consisting of Lucifer yellow, biotin, 5,6-carboxyltetramethylrhodamine (TAMRA), indodicarbocyanine (C5) Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 647, ATTO 488, ATTO 532, 6- carboxyfluorescein (6-FAM), Alexa Fluor® 350, DY-415, ATTO 425, ATTO 465, Bodipy® FL, fluorescein isothiocyanate, Oregon Green® 488, Oregon Green® 514, Rhodamine GreenTM, 5’-Tetrachloro-Fluorescein, ATTO 520, 6-carboxy-4',5'-dichloro- 2',7'-dimethoxyfluoresceine, Yakima YellowTM dyes, Bodipy® 530/550, hexachlorofluorescein, Alexa Fluor® 555, DY-549
  • the agent further comprises a detectable moiety, such as a moiety that is detectable by an imaging technique such as SPECT, PET, MRI, optical or ultrasound imaging.
  • the detectable moiety comprises or consists of a radioisotope, such as selected from the group consisting of 99m Tc, 111 In, 67 Ga, 58 Ga, 72 As, 89 Zr, 123 l and 201 TI.
  • the peptide comprises or consists of a fusion.
  • the peptide may comprise a fusion of two amino acid sequences as disclosed herein.
  • fusion of a peptide relates to an amino acid sequence fused to another peptide.
  • the said peptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said peptide. Examples of such fusions are well known to those skilled in the art.
  • the said peptide may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well-known Myc tag epitope. Fusions to any variant or derivative of said peptide are also included in the scope of the disclosure.
  • the fused portion may be a lipophilic molecule or peptide domain that is capable of promoting cellular uptake of the polypeptide, as known to those skilled in the art.
  • said peptide is of no more than no more than 85, such as no more than 80, such as no more than 75, such as no more than 70, such as no more than 65, such as no more than 60, such as nor more than 55, such as no more than 50, such as no more than 55, such as no more than 40 amino acids, such as no more than 35, such as no more than 30, such as no more than 28, such as no more than 26, such as no more than 24, such as no more than 22, such as no more than 20, such as no more than 19, such as no more than 18, such as no more than 17, such as no more than 16, such as no more than 15, such as no more than 14, such as no more than 13, such as no more than 12, such as no more than 11, such as no more than 10 amino acids in length.
  • no more than 80 such as no more than 75, such as no more than 70, such as no more than 65, such as no more than 60, such as nor more than 55, such as no more than 50, such as no more than 55, such as no more than 40 amino acids, such as no
  • the peptide comprises at least 2 additional amino acids, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 15 or such as at least 20 amino acids conjugated to the N- or C-terminus of the peptide.
  • said peptide is between 5 and 30 amino acids in length, such as between 5 and 20, such as between 8 and 20, such as between 8 and 18, such as between 10 and 16 amino acids in length.
  • the peptide when is T, then the peptide comprises no more than 25 amino acid residues.
  • the agent is a fragment of a peptide described herein, and the fragment comprises 15 or fewer amino acids in length, such as fewer than 14 amino acids, such as fewer than 13 amino acids, such as fewer than 12 amino acids, such as fewer than 11 amino acids, such as fewer than 10 amino acids, such as fewer than 9 amino acids, such as fewer than 8 amino acids, such as fewer than 7 amino acids, such as fewer than 6 amino acids, such as fewer than 5 amino acids in length.
  • fragment at least 5 contiguous amino acids of the amino acid sequence are included, for example at least 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 contiguous amino acids of the amino acid sequence.
  • the fragment may be 15 or fewer amino acids in length, for example 14, 13, 12, 11 , 10, 9, 8, 7, 6 or 5 amino acids in length.
  • the agent is a variant of a peptide as described herein, wherein the variant comprises or consists of a sequence wherein any one amino acid has been altered for another proteinogenic or non-proteinogenic amino acid, with the proviso that no more than five amino acids are so altered.
  • variant refers to a peptide that does not share 100% amino acid sequence identity with the parent peptide, i.e. one or more amino acids must be mutated.
  • “Mutated” refers to altering an amino acid at a specified position in the parent peptide. For example, an amino acid at a specified position may be deleted, altered, substituted or may be the site of an insertion/addition of one or more amino acids. It will be appreciated by persons skilled in the art that the substitutions may be conservative or non-conservative.
  • said peptide variant comprises or consists of a sequence wherein no more than five amino acids are altered for another proteinogenic or non- proteinogenic amino acid, such as no more than 4 amino acids, such as no more than 3 amino acids, such as no more than 2 amino acids, such as no more than 1 amino acid is altered.
  • one or more amino acids are conservatively substituted. “Conservatively substituted” refers to a substitution of one amino acid with another with similar properties (size, hydrophobicity, etc.), such that the function of the peptide is not significantly altered. Thus, by “conservative substitutions” is intended combinations such as Gly, Ala; Vai, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • the peptide has one additional amino acid.
  • said peptide comprises or consists of one or more additional amino acids, inserted at the N- and/or C-terminus and/or internally within the sequence.
  • at least 2 additional amino acids such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 15 or such as at least 20 additional amino acids are inserted.
  • the agent is a polynucleotide encoding upon expression a peptide as described herein.
  • the agent is a vector comprising a polynucleotide as described herein.
  • the agent is a cell comprising a polynucleotide or a vector as described herein.
  • the agent is in a composition.
  • the composition is a pharmaceutical composition.
  • the composition is a cosmetic composition.
  • the composition is a coating.
  • Coatings on various implants are known in the art. Applications in humans include central venous catheters, coronary stents, ventricular assist devices, extracorporeal blood circuits, blood sampling devices, and vascular grafts. Such coatings can be in a gel or non-gel form.
  • a "coating comprising the agent” includes that the agent adsorbed to the surface, bonded to the surface, and imbedded in the polymer surface.
  • the present invention relates to an implant comprising the agent described herein.
  • the implant is coated with a composition comprising the agent.
  • the agent may for example be adsorbed to the surface, bonded to the surface, and imbedded in the polymer surface of the implant.
  • the implant is of a biomaterial, such as bone.
  • the implant is a medical device, such as a stent.
  • the present invention relates to a medical device comprising the agent as defined herein.
  • the medical device is a stent.
  • the medical device is a wound dressing.
  • the medical device is a mesh.
  • a normal wound healing process typically includes the stages of 1) haemostasis via platelets, fibrin, and proteoglycans; 2) inflammation comprising recruitment of neutrophils, macrophages, and lymphocytes; 3) proliferation comprising formation of fibroblasts, epithelial cells, collagen, and endothelial cells; and 4) remodelling including collagen fibril cross-linking and scar maturation.
  • the mode of action of the disclosed polypeptides as outlined herein is likely to play a favourable role in the inflammation, proliferation, and remodelling stages of wound healing through the interaction with the NRP-1 co-receptors such as VEGFR and several others. An interplay of the agents of the disclosure with several important growth factors can therefore improve the wound healing process.
  • the composition of the disclosure is a pharmaceutical composition.
  • the composition of the disclosure is a cosmetic composition.
  • the composition is a coating composition.
  • Tissue meshes have found use in facilitating wound healing.
  • One embodiment of the present disclosure provides for a mesh comprising an agent of the disclosure.
  • One embodiment of the disclosure provides for a stent comprising an agent of the disclosure.
  • One embodiment of the present disclosure provides for a wound dressing comprising the agent of the disclosure.
  • the agents of the present disclosure were found to increase vascularisation/induce angiogenesis, and to induce healing of wounds.
  • One embodiment of the disclosure provides for the agent of the disclosure for use in the treatment and/or regeneration of a tissue defect in a subject.
  • One embodiment of the disclosure provides for an agent of the disclosure for use in the treatment and/or promoting healing of a tissue defect.
  • the agents of the disclosure induce healing of epithelial tissue.
  • the tissue defect is an epithelial tissue defect.
  • the tissue defect is a skin defect.
  • the tissue is epithelium.
  • the tissue is epithelial cells.
  • the tissue is skin.
  • the skin defect is a wound, such as an open wound.
  • the wound is a hard-to-heal wound, such as a chronic wound.
  • the wound is a surgical wound.
  • the wound is associated with venous insufficiency, arterial insufficiency, diabetic complications, sickle cell anaemia, or is a pressure-related wound.
  • the diabetic complication is due to diabetes mellitus, such as Type 1 diabetes mellitus, such as Type 1 diabetes mellitus with foot ulcer and/or Type 1 diabetes mellitus with other skin ulcer, or Type 2 diabetes mellitus, such as Type 2 diabetes mellitus with foot ulcer and/or Type 2 diabetes mellitus with other skin ulcer.
  • the diabetes mellitus presents with gangrene.
  • the diabetes mellitus is associated with a disease of the connective tissue, such as fibrosis. In one embodiment, the diabetes mellitus presents with fibrosis. In one embodiment, the diabetes mellitus is associated with fibrosis. On embodiment of the disclosure provides for an agent of the disclosure for prevention or treatment of fibrosis. In one embodiment of the disclosure, the fibrosis is associated with diabetes mellitus.
  • the wound is a dermal ulcer, or a skin defect due to vascular insufficiency or diabetic affection of the blood vessels.
  • the tissue defect is a diabetic complication.
  • the diabetic complication is a diabetes mellitus related eye condition.
  • the diabetic complication is retinopathy.
  • the tissue defect is damage to the eye and/or orbit, such as injury (trauma) of the eye and/or orbit.
  • the skin defect is a skin disorder provoked by external factors.
  • the skin disorder provoked by external factors is pressure ulceration, dermatoses provoked by friction or mechanical stress, dermatoses due to foreign bodies, dermatoses provoked or exacerbated by exposure to cold, dermatoses provoked by heat or electricity, dermatoses provoked by light or UV radiation, dermatoses due to ionizing radiation, allergic contact dermatitis, photo-allergic contact dermatitis, irritant contact dermatitis, allergic contact urticarial, protein contact dermatitis, allergic contact sensitisation, phototoxic reactions to skin contact with photoactive agents, cutaneous reactions to venomous or noxious animals.
  • the skin defect is an inflammatory dermatosis.
  • the inflammatory dermatosis is dermatitis, eczema, atopic dermatitis, papulosquamous dermatoses, urticaria, angioedema or other urticarial disorders, inflammatory erythemas and other reactive inflammatory dermatoses, immunobullous diseases of the skin, cutaneous lupus erythematosus, scarring or sclerosing inflammatory dermatoses.
  • the skin defect is sclerosis.
  • the sclerosis is systemic sclerosis (scleroderma), such as circumscribed scleroderma.
  • the skin defect is a postprocedural disorder of the skin.
  • the postprocedural disorder of the skin is an unsatisfactory surgical scar of skin, a cutaneous flap necrosis, a myocutaneous flap necrosis, a skin graft failure, a composite graft failure.
  • the postprocedural disorder of the skin is a surgical incision.
  • the skin defect is a genetic and/or developmental disorder affecting the skin.
  • the genetic and/or developmental disorder affecting the skin is epidermolysis bullosa, or genodermatosis, such as pemphigus genodermatosis.
  • the epidermolysis bullosa is selected from the group consisting of epidermolysis bullosa simplex, junctional epidermolysis bullosa, dystrophic epidermolysis bullosa, recessive dystrophic epidermolysis bullosa, syndromic epidermolysis bullosa and epidermolysis bullosa.
  • the skin defect is an external defect. In one embodiment, the skin defect is an open defect. In one embodiment, the skin defect is an inflamed wound. In one embodiment, the skin defect is infected. Infected wounds can be difficult to heal due to formation of biofilm, e.g. from bacteria.
  • the skin defect is an injury or trauma to internal or external tissue, such as injury or trauma to the epidermis and/or dermis of the skin.
  • the skin defect is an acute defect or a chronic defect.
  • the skin defect is full thickness skin defect.
  • the wound is an acute wound or a chronic wound.
  • the acute wound is an incision, laceration, abrasion graze or burn, a puncture wound, a penetration wound or a wound due to dermatologic diseases such as psoriasis, acne and eczema.
  • the chronic wound is a venous ulcer, a diabetic ulcer, a pressure ulcer, corneal ulcer, digestive ulcer or wounds as a result of ischemia and radiation poisoning.
  • the wound is a non-pressure chronic ulcer, such as a nonpressure chronic ulcer of lower limb.
  • the agent is administered topically, orally, subcutaneously, or systemically. In one embodiment, the administration is by instillation.
  • One embodiment of the disclosure provides for an agent of the disclosure for use in the treatment of a disease of the skin, such as injury of external cause.
  • the skin defect is a disease of the skin, such as injury of external cause.
  • the disease of the skin is selected from the group consisting of: a. a skin disorder involving specific cutaneous structures, such as disorders of cutaneous blood and lymphatic vessels, such as dermatoses resulting from vascular insufficiency, such as ischaemic ulceration of the skin, and b. a skin disorder associated with pregnancy, the neonatal period, and infancy, such as dermatoses of infancy, such as erythrodermas of infancy (Leiner’s disease).
  • the injury of external cause is selected from the group consisting of: a. injury to the head, such as intracranial injury, such as diffuse brain injury, such as cerebral contusion, and b. injury to unspecified part of trunk, limb, or body region, such as other injuries of spine or trunk, such as injury of the spinal cord.
  • the subject is suffering from diabetes mellitus.
  • the subject is suffering from stoma.
  • the wound is generated as part of internal acidic fluids.
  • the subject is a mammal. In one embodiment, the mammal is a human.
  • One embodiment of the disclosure provides for a method for treatment and/or regeneration of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent of the disclosure to an subject in need thereof.
  • One embodiment of the disclosure provides for a method for treating and/or promoting healing of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent of the disclosure to an subject in need thereof.
  • One embodiment of the disclosure provides for a method for regeneration of skin at the site of a skin defect and/or for reducing scar formation resulting from healing of a skin defect in a subject, said method comprising administering an agent of the disclosure.
  • One embodiment of the disclosure provides for a use of an agent of the disclosure for the manufacture of a medicament for the treatment and/or regeneration of a skin defect in a subject.
  • One embodiment of the disclosure provides for a use of an agent of the disclosure for the manufacture of a medicament for the treating and/or promoting healing of a tissue defect in a subject.
  • One embodiment of the disclosure provides for a use of an agent of the disclosure for the manufacture of a medicament for the treatment of a skin defect in a subject.
  • One embodiment provides for a use of an agent of the disclosure for reducing scar formation resulting from healing of a skin defect in a subject.
  • One embodiment provides for a use of an agent of the disclosure for promoting the healing of a skin defect in a subject.
  • One embodiment of the disclosure provides a use of an agent of the disclosure for promoting vascularisation in a healing tissue, such as skin healing from a skin defect, such as a wound.
  • One embodiment provides for a use of an agent of the disclosure in combination with a tissue meshing technique, for promoting healing of a skin defect, such as a wound.
  • One embodiment provides for a use of an agent of the disclosure for promoting grafting of skin.
  • One embodiment of the disclosure provides for a method for inserting a stent in a subject, said method comprising administering an agent of the disclosure.
  • the stent is coated with the agent of the disclosure.
  • the agent of the disclosure is part of a cosmetic composition.
  • the peptide comprises or consists of the amino acid sequence VDTYDGDISVVYGLR (SEQ ID NO: 7), and the tissue defect is a wound.
  • the peptide comprises or consists of the amino acid sequence VDTYDGGISVVYGLR (SEQ ID NO: 6), and the tissue defect is a wound. In one embodiment, the peptide comprises or consists of the amino acid sequence AEIDSIELSYGIK (SEQ ID NO: 110), and the tissue defect is a wound.
  • the peptide comprises or consists of the amino acid sequence KPLAEIDSIELSYGIK (SEQ ID NO: 111), and the tissue defect is a wound.
  • One aspect of the disclosure provides for the agent of the disclosure for use in the prevention of fibrosis of the skin in a subject.
  • One aspect of the disclosure provides for an agent of the disclosure for use in the treatment of a disease of the skin, such as injury of external cause.
  • One aspect of the disclosure provides for a method for treatment and/or regeneration of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent of the disclosure to an subject in need thereof.
  • One aspect of the disclosure provides for a method for treating and/or promoting healing of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent of the disclosure to an subject in need thereof.
  • One aspect provides for a use of an agent of the disclosure for reducing scar formation resulting from healing of a skin defect in a subject.
  • One aspect of the disclosure provides a use of an agent of the disclosure for promoting vascularisation in a healing tissue, such as skin healing from a skin defect, such as a wound.
  • An agent comprising: a) a peptide selected from the group consisting of:
  • X 5 is D or G; Xe is I or G;
  • X? is V or L
  • Xs is V or A
  • X14 is E or G
  • X15 is S or T
  • VDTYDGDISVVYGL (SEQ ID NO: 34),
  • VDTYDGDISVVYG (SEQ ID NO: 35),
  • VDTYDGDISVVY (SEQ ID NO: 36),
  • VDTYDGDISVV (SEQ ID NO: 37),
  • VDTYDGDISV (SEQ ID NO: 38),
  • VDTYDGDIS (SEQ ID NO: 39),
  • VDTYDGRGDSVVYGLR (SEQ ID NO: 40),
  • VDVPNGDISLAYGL (SEQ ID NO: 41),
  • VDVPNGDISLAYG (SEQ ID NO: 42),
  • VDVPNGDISLA (SEQ ID NO: 43),
  • VDVPNGDIS (SEQ ID NO: 44),
  • VDVPNGDISLAYRLR (SEQ ID NO: 48)
  • VDVPEGDISLAYRLR (SEQ ID NO: 49),
  • V(beta-D)TYDGDISVVYGLR (SEQ ID NO: 50)
  • VDTYDG(beta-D)ISVVYGLR (SEQ ID NO: 52);
  • KPLAEIDSIELSYG (SEQ ID NO: 134), KPLAEIDSIELSY (SEQ ID NO: 135), KPLAEIDSIELS (SEQ ID NO: 136), KPLAEIDSIEL (SEQ ID NO: 137), and KPLAEIDSIE (SEQ ID NO: 138); b) a polynucleotide encoding upon expression, the peptide of a); c) a vector comprising the polynucleotide of b); or d) a cell comprising the polynucleotide of b), or the vector of c).
  • the agent according to item 1 wherein the peptide comprises an amino acid sequence of the general formula:
  • VDX 2 X3X4GX5X 6 SX7X 8 YGLR (SEQ ID NO: 2) wherein: X 2 is T or V;
  • X 3 is Y or P
  • X 4 is D or N
  • X 5 is D or G
  • X s is I or G
  • X 7 is V or L; and X 8 is V or A.
  • X 4 is D or N
  • Xs is D or G
  • X 8 is I or G
  • X7 is V or L; and Xs is V or A.
  • Xs is D or G
  • Xs is I or G
  • X7 is V or L
  • Xs is V or A.
  • the agent according to any one of the preceding items, wherein the peptide comprises an amino acid sequence of the general formula: VDTYDGZ 7 Z 8 SVVYGLR (SEQ ID NO: 5) wherein:
  • X 5 is D or G; and X 5 is I or G;
  • the agent according to item 1 wherein the peptide comprises an amino acid sequence of the general formula:
  • X 9 is C, P or G
  • X is E or G
  • Xn is C, D or I
  • X12 is D, I, S or G
  • X13 is S, D or G
  • X14 is E or G; and X15 is S or T.
  • the agent according to item 1 wherein the peptide comprises an amino acid sequence of the general formula:
  • Xg is C, P or G
  • X is E or G
  • X11 is C, I or absent
  • X12 is D, G or absent
  • X13 is S, G or absent
  • X14 is E or G. 8. The agent according to item 1 , wherein the peptide comprises an amino acid sequence of the general formula:
  • X9 is C, P or G
  • X is E or G; and X14 is E or G.
  • the agent according to item 1 wherein the peptide comprises or consists of an amino acid sequence selected from the group consisting of: VDTYDGGISVVYGLR (SEQ ID NO: 6),
  • VDTYDGDISVVYGLR (SEQ ID NO: 7), DTYDGDISVVYGLR (SEQ ID NO: 8), TYDGDISVVYGLRS (SEQ ID NO: 9), TYDGDISVVYGLR (SEQ ID NO: 10), YDGDISVVYGLRS (SEQ ID NO: 11), YDGDISVVYGLR (SEQ ID NO: 12), DGDISVVYGLRS (SEQ ID NO: 13), DGDISVVYGLR (SEQ ID NO: 14), GDISVVYGLRS (SEQ ID NO: 15), GDISVVYGLR (SEQ ID NO: 16), DISVVYGLRS (SEQ ID NO: 17), DISVVYGLR (SEQ ID NO: 18), VDVPNGDISLAYGLR (SEQ ID NO: 19), DVPNGDISLAYGLRS (SEQ ID NO: 20), DVPNGDISLAYGLR (SEQ ID NO: 21), VPNGDISLAYGLRS (SEQ ID NO: 22
  • DISLAYGLRS (SEQ ID NO: 30), DISLAYGLR (SEQ ID NO: 31), VDTYDGDGSVVYGLR (SEQ ID NO: 32), VDVPEGDISLAYGLR (SEQ ID NO: 33).
  • KPLAEGDIELSYGIK (SEQ ID NO: 116), KPLAEIELSYGIK (SEQ ID NO: 117), KPLAEIDSIELTYGIK (SEQ ID NO: 118), KPLAEIDGIELSYGIK (SEQ ID NO: 119), KPLAEIDGIELTYGIK (SEQ ID NO: 120), KPLAEIGSIELSYGIK (SEQ ID NO: 121), KGLAEIDSIELSYGIK (SEQ ID NO: 122), KPLAGIDSIGLSYGIK (SEQ ID NO: 123), Cyclic KCLAEIDSCELSYGIK (SEQ ID NO: 124), LAEIDSIELSYGIK (SEQ ID NO: 125), EIDSIELSYGIK (SEQ ID NO: 126), IDSIELSYGIK (SEQ ID NO: 127), DSIELSYGIK (SEQ ID NO: 128), SIELSYGIK (SEQ ID NO: 129), and IEL
  • the peptide comprises or consists of an amino acid sequence VDTYDGGISVVYGLR (SEQ ID NO: 6). 13. The agent according to item 1 , wherein the peptide comprises or consists of an amino acid sequence VDTYDGDISVVYGLR (SEQ ID NO: 7).
  • the peptide comprises no more than 85, such as no more than 80, such as no more than 75, such as no more than 70, such as no more than 65, such as no more than 60, such as nor more than 55, such as no more than 50, such as no more than 55, such as no more than 40 amino acids, such as no more than 35, such as no more than 30, such as no more than 28, such as no more than 26, such as no more than 24, such as no more than 22, such as no more than 20, such as no more than 19, such as no more than 18, such as no more than 17, such as no more than 16, such as no more than 15, such as no more than 14, such as no more than 13, such as no more than 12, such as no more than 11 , such as no more than 10 amino acids.
  • no more than 80 such as no more than 75, such as no more than 70, such as no more than 65, such as no more than 60, such as nor more than 55, such as no more than 50, such as no more than 55, such as no more than 40 amino acids, such as no more than 35,
  • the peptide comprises at least 2 additional amino acids, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 15 or such as at least 20 amino acids conjugated to the N- or C-terminus of the peptide.
  • agent according to any one of the preceding items, wherein the agent comprises or consists of a tandem repeat comprising two or more repeat units.
  • agent is a variant of the peptide, wherein the variant comprises or consists of a sequence wherein any one amino acid has been altered for another proteinogenic or non- proteinogenic amino acid, with the proviso that no more than five amino acids are so altered.
  • the variant comprises or consists of a sequence wherein no more than five amino acids are altered for another proteinogenic or non-proteinogenic amino acid, such as no more than 4 amino acids, such as no more than 3 amino acids, such as no more than 2 amino acids, such as no more than 1 amino acid is altered.
  • agent according to any of the preceding items, wherein the agent further comprises a detectable moiety.
  • radioisotope is selected from the group consisting of 99m Tc, 111 ln, 67 Ga, 68 Ga, 72 As, 89 Zr, 123 l and 201 TI.
  • composition comprising the agent according to any one of the preceding items.
  • composition according to item 38 wherein the composition is a pharmaceutical composition.
  • composition according to item 38 wherein the composition is a cosmetic composition.
  • 41 The composition according to any one of the preceding items, wherein the composition is a coating composition.
  • a medical device comprising an agent according to any one of the preceding items.
  • a mesh comprising an agent according to any one of the preceding items.
  • a stent comprising an agent according to any one of the preceding items.
  • a wound dressing comprising the agent according to any one of the preceding items.
  • tissue defect is a skin defect.
  • the skin defect is a wound, such as an open wound.
  • the wound is a hard-to-heal wound, such as a chronic wound.
  • the wound is associated with venous insufficiency, arterial insufficiency, diabetic complications, sickle cell anaemia, or is a pressure-related wound.
  • diabetes mellitus such as Type 1 diabetes mellitus, such as Type 1 diabetes mellitus with foot ulcer and/or Type 1 diabetes mellitus with other skin ulcer, or Type 2 diabetes mellitus, such as Type 2 diabetes mellitus with foot ulcer and/or Type 2 diabetes mellitus with other skin ulcer.
  • diabetes mellitus is associated with a disease of the connective tissue, such as fibrosis.
  • the wound is a dermal ulcer, or a skin defect due to vascular insufficiency or diabetic affection of the blood vessels.
  • tissue defect is a diabetic complication.
  • the agent for use according to any one of the preceding items, wherein the diabetic complication is retinopathy.
  • the agent for use according to any one of the preceding items, wherein the tissue defect is damage to the eye and/or orbit, such as injury (trauma) of the eye and/or orbit.
  • the agent for use according to any one of the preceding items, wherein the skin defect is a skin disorder provoked by external factors.
  • the skin disorder provoked by external factors is pressure ulceration, dermatoses provoked by friction or mechanical stress, dermatoses due to foreign bodies, dermatoses provoked or exacerbated by exposure to cold, dermatoses provoked by heat or electricity, dermatoses provoked by light or UV radiation, dermatoses due to ionizing radiation, allergic contact dermatitis, photo-allergic contact dermatitis, irritant contact dermatitis, allergic contact urticarial, protein contact dermatitis, allergic contact sensitisation, phototoxic reactions to skin contact with photoactive agents, cutaneous reactions to venomous or noxious animals.
  • the agent for use according to any one of the preceding items, wherein the skin defect is an inflammatory dermatosis.
  • the agent for use according to any one of the preceding items, wherein the inflammatory dermatosis is dermatitis, eczema, atopic dermatitis, papulosquamous dermatoses, urticaria, angioedema or other urticarial disorders, inflammatory erythemas and other reactive inflammatory dermatoses, immunobullous diseases of the skin, cutaneous lupus erythematosus, scarring or sclerosing inflammatory dermatoses.
  • the agent for use according to any one of the preceding items, wherein the skin defect is sclerosis.
  • sclerosis is systemic sclerosis (scleroderma), such as circumscribed scleroderma.
  • skin defect is a postprocedural disorder of the skin.
  • the postprocedural disorder of the skin is an unsatisfactory surgical scar of skin, a cutaneous flap necrosis, a myocutaneous flap necrosis, a skin graft failure, a composite graft failure.
  • agent for use according to any one of the preceding items, wherein the genetic and/or developmental disorder affecting the skin is epidermolysis bullosa, or genodermatosis, such as pemphigus genodermatosis.
  • the epidermolysis bullosa is selected from the group consisting of epidermolysis bullosa simplex, junctional epidermolysis bullosa, dystrophic epidermolysis bullosa, recessive dystrophic epidermolysis bullosa, syndromic epidermolysis bullosa and epidermolysis bullosa.
  • the skin defect is infected.
  • the skin defect is an injury or trauma to internal or external tissue, such as injury or trauma to the epidermis and/or dermis of the skin.
  • the acute wound is an incision, laceration, abrasion graze or burn, a puncture wound, a penetration wound or a wound due to dermatologic diseases such as psoriasis, acne and eczema.
  • the chronic wound is a venous ulcer, a diabetic ulcer, a pressure ulcer, corneal ulcer, digestive ulcer or wounds as a result of ischemia and radiation poisoning.
  • agent for use according to any one of the preceding items wherein the agent is administered topically, orally, subcutaneously, or systemically.
  • the disease of the skin is selected from the group consisting of: a. a skin disorder involving specific cutaneous structures, such as disorders of cutaneous blood and lymphatic vessels, such as dermatoses resulting from vascular insufficiency, such as ischaemic ulceration of the skin, and b. a skin disorder associated with pregnancy, the neonatal period, and infancy, such as dermatoses of infancy, such as erythrodermas of infancy (Leiner’s disease).
  • the injury of external cause is selected from the group consisting of: a. injury to the head, such as intracranial injury, such as diffuse brain injury, such as cerebral contusion, and b. injury to unspecified part of trunk, limb, or body region, such as other injuries of spine or trunk, such as injury of the spinal cord.
  • agent for use according to item 94 wherein the mammal is a human.
  • a method for treatment and/or regeneration of a tissue defect in a subject comprising administering a therapeutically effective amount of an agent according to any one of the preceding items to an subject in need thereof.
  • a method for treating and/or promoting healing of a tissue defect in a subject comprising administering a therapeutically effective amount of an agent according to any one of the preceding items to an subject in need thereof.
  • a method for regeneration of skin at the site of a skin defect and/or for reducing scar formation resulting from healing of a skin defect in a subject comprising administering an agent according to any one of the preceding items.
  • a method for inserting a stent in a subject comprising administering an agent according to any one of the preceding items.
  • 108. The method according to item 107, wherein the stent is coated with the agent according to any one of the preceding items.
  • Example 1 Polypeptides of the disclosure stimulates endothelial and vascular smooth muscle cell proliferation Methods
  • human umbilical cord endothelial cells were first treated with a scramble non-coding siRNA (NC) or siRNA for NRP-1 , subsequently they were treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and DNA synthesis analyzed by measuring the uptake of BrdU.
  • NC scramble non-coding siRNA
  • FOL26 FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM
  • polypeptides of the disclosure stimulates endothelial and vascular smooth muscle cell proliferation.
  • Cells were transfected for 48 hours or performed with 0.5% supplement starvation for 24 hours on 6-well plates as described above and dissociated by Accutase (Gibco, USA) to obtain cell suspension.
  • Geltrex reduced growth factor basement membrane matrix ((Invitrogen, CA, USA) was thawed at 4°C overnight and added into a 96-well plate (50 pl/well) next. The coated 96-well plate was placed in the incubator more than 30 mins for further use and the cell suspension was seeded into it at a density of 1.5 x 104/well.
  • NRP-1 silencing experiments cells were cultured with complete medium.
  • polypeptide treatment experiments cells were stimulated with complete medium plus the different concentration of polypeptide.
  • NRP-1 rescued experiments cells transfected with siRNA were cultured with complete medium and NRP-1 stimulations. After a 24-hour incubation, images were recorded by an inverted microscope (Nikon, Eclipse TE2000-U, Japan) and the sum length of master segments of vessels in each image were quantified using Angiogenesis Analyzer plug-in ImageJ software program (pixel/unit). Data were analyzed with photographs from 8-14 wells per condition.
  • Human umbilical cord endothelial cells were first treated with a scramble non-coding siRNA (NC) or siRNA for NRP-1 , subsequently treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and endothelial tube formation and the total master segemnts were measured using ImageJ. gene expression of PECAM-1 (CD31) analyzed.
  • NC scramble non-coding siRNA
  • FOL26 FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM
  • test polypeptides significantly increased tube formation in the HUVECs.
  • the polypeptides also increased expression of PECAM-1.
  • Figure 12 shows that FOL26 increased tube formation compared to control in a NRP-1 dependent way.
  • Polypeptides of the disclosure induces tube formation in HUVECs.
  • Example 3 Polypeptides of the disclosure induces wound healing in a scratch wound assay on HUVECs and HCASMCs
  • Ibidi Culture-Insert 2 well (Ibidi, Nanterre, France) was used to assess the ability of wound healing in vitro. Briefly, one silicone culture-insert (cut 2 well inlet) was placed in the one well of 24-well plate and 3.5x104 cells were seeded into each inlet with 70 pl complete medium. For NRP-1 silencing and NRP-1 rescued experiments, cells were transfected with siRNA/siRNA-NC for 48 hours before seeding. For test polypeptide treatment experiments, cells were starved with respective low-serum medium for 24 hours and 48 hours after adhering in the inlet. The cell-free gap between two inlets was created by removing the culture-insert, followed by washing with DPBS gently to remove suspended cells.
  • wound closure % 1 - (wound area at T x I wound area at To)] * 100 (To is the wound area at 0 h, and T x is the wound area at corresponding hours after creating the gap (pixel/unit). Data were quantified with pictures from 8-20 wells per group.
  • Polypeptides of the disclosure induces wound healing in a scratch wound assay on HUVECs and HCASMCs.
  • Example 4 Polypeptides of the disclosure prevents apoptosis in HUVECs and HCASMCs
  • HAVECs Human Umbilical Vein Endothelial Cells
  • HCASMCs Human Coronary Artery Smooth Muscle Cells
  • Test polypeptide and sFasL were dissolved in the respective complete medium.
  • Caspase-Gio 3/7 Assay was performed to evaluate cell apoptosis. HUVECs and HCASMCs were seeded into 96-well plates at a density of 3-5 x 103 per well. For additional NRP-1 on cells transfected siRNA, NRP-1 was added 24 hours after transfection and the mixture of Caspase-Gio 3/7 was added 48 hours after polypeptide stimulations. Subsequent steps were performed as manufacturer’s instructions using the FOL26 and FOL56 as test polypeptides. Data were read by Wallac 1420 Victor 2 (Perkin Elmer, USA).
  • Polypeptides of the disclosure prevents apoptosis in HUVECs and HCASMCs.
  • Example 5 Effect on gene expression of polypeptides of the disclosure in HUVECs and HCASMCs
  • real-time qPCR was performed using by KAPA SYBR FAST One-Step qRT- PCR Master Mix Kit (KAPA Biosystems, USA) on LightCycler 480 system (Roche, Mannheim, Germany).
  • Table 1 Growth Factors and Cytokines Affecting Various Steps in Wound Healing.
  • Polypeptides of the disclosure induces a milieu inducing wound healing by TNF and IL- 6 expression likely to promote fibroblast migration and proliferation, collagen secretion, angiogenesis, and induce correct wound healing structure by inducing cell remodelling through MMP expression.
  • Example 6 Healing of wounds in combination with tissue meshing.
  • tissue meshes having a coating of a polypeptide of the disclosure, and/or having a polypeptide dispersed within it will effect faster and/or more complete healing of a wound, compared to tissue meshes without the polypeptide of the disclosure.
  • Example 7 Effect of test polypeptides in acute and pathological wound healing Methods
  • test polypeptides The pharmacological profile of the test polypeptides has been tested using two models, with full-thickness human abdominal skin cultured ex-vivo under normal conditions (WCM medium only, 37 °C, 5% CO2,) representing “acute wound healing” and under extreme culture conditions (VCM, high glucose, H2O2, 37 °C, 5% CO2, 5% O2) representing “pathological wound healing” (Figure 13) (Monasterium Labs, Prof. Ralf Paus, Munster, Germany).
  • Table 2 Number of wound closure observed in acute wound healing-pooled data at 72 hours timer point*. *No full wound closure was observed in the pathological punches. The wound closure in day 6 time point from the first experiment were excluded from this table.
  • Polypeptides of the disclosure induce full wound closure.
  • the ROS level (figure27b) activated by 50ug/ml oxidized Low-density Lipoprotein (oxLDL) for 2 hours in cells pre-incubated with FOL- 026 peptides was assessed using H2O2 measurement (n 4-8 per group).
  • the FOL-026 peptide has a stimulatory effect on the proliferation of endothelial cells.
  • Figures 30 a-d shows the positive effect FOL-026 has on tube formation. It demonstrates the increased vessel density effect of FOL-026 and how FOL-026 induces upregulation of angiogenesis related genes.
  • the FOL-026 peptide affects angiogenesis both in vitro and in vivo.
  • Example 10 Effect of NR P-1 knock-down on endothelial cell function induced with
  • Figures 31a-c shows that reducing the NRP-1 expression blocks the effect of FOL-026
  • Figures 32a-b show that FOL-026 has a positive effect on wound closure in siRNA-NC/ siNRP-1-transfected HUVECs.
  • FOL-026 stimulates wound closure in HUVECs by binding to NRP-1
  • the FOL-026 peptide induce tube formation in siRNA-NC and siNRP-1.
  • Example 12 Effect of FOL-026 peptide on smooth muscle cell function
  • FOL-026 induces smooth muscle cell proliferation, inhibits apoptosis, reduces ROS formation and accelerates wound closure
  • Example 13 Effect of NRP-1 knock-down on smooth muscle cell function induced with FOL-026 peptide
  • Method qRT-PCR analysis of NRP-1 mRNA expression in HCASMCs with small interfering RNA transfection for 48 hours (n 4) (figure 36a).
  • Representative images of smooth muscle cells at 0 and 5 hours after scratch wounding were shown (figure 37a).
  • FOL-026 has a positive effect on wound closure in siRNA-NC/ si NRP-1 -transfected HUVECs.
  • Example 14 The FOL-005 peptide stimulates endothelial and smooth muscle cell proliferation and wound healing ability
  • the results shown in figure 38a indicates that the proliferation of human umbilical vascular endothelial cells (HUVECs) increases with the addition of the FOL-005 peptide.
  • the scratch wound assay in figure 38b and 38c indicates that the FOL-005 peptide accelerate HUVEC wound closure.
  • the FOL-005 peptide has a positive effect on endothelial and smooth muscle cell proliferation and wound healing ability.
  • Gottrup F. A specialized wound-healing center concept: importance of a multidisciplinary department structure and surgical treatment facilities in the treatment of chronic wounds.

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Abstract

The present disclosure relates to agents comprising polypeptides for treatment of tissue defects, in particular for promoting healing of wounds.

Description

Agents for stimulating tissue regeneration
Technical field
The present invention relates to agents for the regeneration of tissue. The regenerated tissue is preferably skin, i.e. the regeneration of tissue is preferably the healing of wounds. Said wounds can be acute or pathological wounds.
Background
Advanced wound care is a fast-growing market mainly composed of 4 main categories: dressings, wound cleansers, negative pressure wound therapy devices and biologies. It is estimated that 1 % to 2 % of the population will experience a chronic wound during their lifetime in developed countries (Gottrup) and this rate is increasing year after year. Stalled wounds may persist for months, even years (Kirker et al., Frykberg et al.) before healing. Hence, non-healing/hard-to-heal wounds represent an economic burden for health services and a large medical problem for patients, representing a global cost of $143 bn.
Most of the clinical wound care practises today are underserved due to increasing costs and limitations of resources. The management of a standard of care for the wounds cannot therefore be followed optimally. There are limited pharmacological treatments available whereas there are many advanced dressings of diverse quality with different claims of improving wound healing. Treatment for acute wounds include debridement, decontamination, moist wound healing, and early closure. Treatment for chronic wounds include debridement, anti-inflammation therapy, antibiotic therapy, synthetic substitutes, modulating cytokines and/or matrix metalloproteinases (MMPs), or recruiting stem cells.
Wounds may arise from a variety of aetiologies such as peripheral arterial disease, venous hypertension, neuropathy (e.g., diabetes mellitus), pressure (in patients with neuropathy or decreased mobility), vasculitis and burns, etc and are very often hard to heal. A large proportion of acute wounds heal with appropriate management, but some wounds fail to heal and proceed to becoming chronic, non-healing wounds. A ‘completely healed wound’ can be defined as one that has returned to its normal anatomic structure, function, and appearance within a reasonable period of time (normally in about four to six weeks). Any wound that has not reached the above state would be considered a chronic, non-healing wound. Wound care has become increasingly important given the rise of chronic wounds and the morbidity associated with them. An important concept in wound care is the role of debridement, or the removal of non-viable tissue material. Disruption at one or more points in the distinct phases of wound healing (haemostasis, inflammation, proliferation, or remodelling) can cause a wound not to heal. An alternation in the balance between various cytokines, growth factors, protease activity, matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) is observed in chronic wounds. In addition, alteration in the morphology and proliferation rate of fibroblasts, keratinocyte activity, and accumulation of oxygen derived free radicals, necrotic tissue and underlying disease all play a role in inhibiting wound healing.
Summary
The present inventors have identified agents with use in healing wounds.
One aspect of the disclosure provides for an agent comprising: a) a peptide selected from the group consisting of:
(i) a peptide comprising or consisting of an amino acid sequence of the general formula:
XsXeSXyXsYGLR (SEQ ID NO: 1) wherein:
X5 is D or G;
X6 is I or G; X7 is V or L; Xs is V or A;
(ii) a peptide comprising or consisting of an amino acid sequence of the general formula:
X14LX15YGIK (SEQ ID NO: 105) wherein:
X14 is E or G;
X15 is S or T;
(iii) a peptide comprising or consisting of an amino acid sequence selected from the group consisting of: VDTYDGDISVVYGL (SEQ ID NO: 34), VDTYDGDISVVYG (SEQ ID NO: 35), VDTYDGDISVVY (SEQ ID NO: 36), VDTYDGDISVV (SEQ ID NO: 37), VDTYDGDISV (SEQ ID NO: 38), VDTYDGDIS (SEQ ID NO: 39), VDTYDGRGDSVVYGLR (SEQ ID NO: 40), VDVPNGDISLAYGL (SEQ ID NO: 41), VDVPNGDISLAYG (SEQ ID NO: 42), VDVPNGDISLA (SEQ ID NO: 43), VDVPNGDIS (SEQ ID NO: 44), GDPNDGRGDSVVYGLR (SEQ ID NO: 45), LDGLVRAYDNISPVG (SEQ ID NO: 46), GDPNGDISVVYGLR (SEQ ID NO: 47) VDVPNGDISLAYRLR (SEQ ID NO: 48), VDVPEGDISLAYRLR (SEQ ID NO: 49), V(beta-D)TYDGDISVVYGLR (SEQ ID NO: 50), VDTY(beta-D)GDISVVYGLR (SEQ ID NO: 51), VDTYDG(beta-D)ISVVYGLR (SEQ ID NO: 52); CLAEIDSC (Cyclic) (SEQ ID NO: 130), CFKPLAEIDSIECSYGIK (Cyclic) (SEQ ID NO: 131), Cyclic CFKPLAEIDSIEC (SEQ ID NO: 132), KPLAEIDSIELSYGI (SEQ ID NO: 133), KPLAEIDSIELSYG (SEQ ID NO: 134), KPLAEIDSIELSY (SEQ ID NO: 135), KPLAEIDSIELS (SEQ ID NO: 136), KPLAEIDSIEL (SEQ ID NO: 137), and KPLAEIDSIE (SEQ ID NO: 138); b) a polynucleotide encoding upon expression, the peptide of a); c) a vector comprising the polynucleotide of b); or d) a cell comprising the polynucleotide of b), or the vector of c) for use in the treatment and/or regeneration of a tissue defect in a subject, such as skin defect, such as a wound.
In one aspect, the present invention relates to a method for treatment and/or regeneration of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent as defined herein to an subject in need thereof.
In one aspect, the present invention relates to a method for treating and/or promoting healing of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent as defined herein to an subject in need thereof.
In one aspect, the present invention relates to a method for regeneration of skin at the site of a skin defect and/or for reducing scar formation resulting from healing of a skin defect in a subject, said method comprising administering an agent as defined herein.
In one aspect, the present invention relates to use of an agent as defined herein for the manufacture of a medicament for the treatment and/or regeneration of a skin defect in a subject.
In one aspect, the present invention relates to use of an agent defined herein for the manufacture of a medicament for the treating and/or promoting healing of a tissue defect in a subject.
In one aspect, the present invention relates to use of an agent defined herein for the manufacture of a medicament for the treatment of a skin defect in a subject.
In one aspect, the present invention relates to use of an agent defined herein for reducing scar formation resulting from healing of a skin defect in a subject.
In one aspect, the present invention relates to use of an agent defined herein for promoting the healing of a skin defect in a subject.
In one aspect, the present invention relates to use of an agent defined in herein for promoting vascularisation in a healing tissue, such as skin healing from a skin defect, such as a wound. In one aspect, the present invention relates to use of an agent defined herein in combination with a tissue meshing technique, for promoting healing of a skin defect, such as a wound.
In one aspect, the present invention relates to use of an agent as defined herein for promoting grafting of skin.
In one aspect, the present invention relates to a method for inserting a stent in a subject, said method comprising administering an agent as defined herein.
In one aspect, the present invention relates to a medical device comprising the agent as defined herein.
Description of Drawings
Figure 1 : FOL26 stimulates endothelial and vascular smooth muscle cell proliferation. Human umbilical cord endothelial cells (A) and human coronary artery smooth muscle cells (B) were treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and DNA synthesis analyzed by measuring the uptake of BrdU. An increase in O.D. (450 nm) for the FOL 26 treated cell lines compared to the control.
Figure 2: FOL26 enhances endothelial cell tube formation and expression of PECAM-1. Human umbilical cord endothelial cells were treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and endothelial tube formation and gene expression of PECAM-1 (CD31) analyzed. FOL26 increased tube formation compared to control. The expression of PECAM-1 was increased for 100 nM and 1000 nM FOL26.
Figure 3: FOL26 increases endothelial cell migration in a cell culture scratch wound assay. Confluent cultures of human umbilical cord endothelial cells were treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and ability of the cells to migrate into an in vitro scratch injury was analyzed with image J software. Migration of cells increased with increasing concentration of FOL26.
Figure 4: FOL26 increases arterial smooth muscle cell migration in a cell culture scratch wound assay. Confluent cultures of human coronary artery smooth cells subjected to a scratch and were following treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and ability of the cells to migrate into an in vitro scratch injury was analyzed with image J software. Expression of the collagen gene a1 and a2 chains were determined by polymerase chain reaction (PCR). FOL26 increased expression of both COL1 A1 and COL1 A2. The wound closure at 5h was increased for FOL26 at 10 nM and 100 nM, while the 1000 nM also trended towards increased closure compared to control.
Figure 5: FOL26 inhibits endothelial and arterial smooth muscle cell apoptosis. Cultured human umbilical cord endothelial and coronary artery smooth cells were exposed to soluble Fas ligand (sFasL) to induce apoptosis that subsequently was assessed by determining activation of caspase 3 and 7. Cells were also treated with FOL 26 in concentrations of 10 nM, 100 nM, and 1000 nM to determine the effect on apoptosis activated by sFasL. FOL26 was capable of reversing the sFasL-induced apoptosis, to the highest extent at the 100 nM concentration.
Figure 6: FOL26 affects the gene expression of VEGF and the VEGF receptor family in endothelial cells. Human umbilical cord endothelial cells were treated with FOL 26 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding c-Met, neuropilin-1 (NRP-1), VEGF-A and VEGF receptor-2 (VEGFR-2) determined by polymerase chain reaction (PCR). FOL026 was reducing the c-Met expression, in the lowest dose reducing however in the 100nM and 1000nM doses were inducing NRP-1 expression. FOL026 had no effect on VEGF-A expression, however in 10nM it reduced and in 1000nM it induced the expression of VEGFR-2 levels.
Figure 7: FOL26 affects the gene expression of cytokines and matrix metalloproteinases in endothelial cells. Human umbilical cord endothelial cells were treated with FOL 26 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding TNF-a, IL-6, MMP2 and MMP3 determined by polymerase chain reaction (PCR). TNF-a is strong pro-inflammatory cytokine while MMP2 is required for activation of smooth muscle cell proliferation and migration. FOL026 reduced the TNF-a expression in 10nM and 100nM doses, had no effect on IL-6 or MMP3 expression, however, MMP2 expression was induced with 200nM and 1000nM doses. Figure 8: FOL26 affects the gene expression of VEGF and the VEGF receptor family in arterial smooth muscle cells. Human coronary artery smooth cells were treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding c-Met, neuropilin-1 (NRP-1), VEGF-A and VEGF receptor-2 (VEGFR- 2) determined by polymerase chain reaction (PCR). FOL026 reduced the c-MET expression in 10nM and 1000nM doses, as well as the NRP-1 expression in 10, 100, and 1000nM doses. FOL026 induced in 10, 100, and 1000nM doses the VEGF-A expression, as well as for FOL026 100nM dose only.
Figure 9: FOL26 affects the gene expression of cytokines and matrix metalloproteinases in arterial smooth muscle cells. Human coronary artery smooth cells were treated with FOL 26 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding TNF-a, IL-6, MMP2 and MMP3 determined by polymerase chain reaction (PCR). TNF-a is strong pro-inflammatory cytokine while MMP2 is required for activation of smooth muscle cell proliferation and migration. FOL026 100 and 1000nM induced the TNF-a, IL-6 and MMP3 expression, furthermore, FOL026 induced in 10, 100, and 1000nM doses the MMP2 expression.
Figure 10: FOL26 stimulation of endothelial cell proliferation is NRP-1 dependent. Human umbilical cord endothelial cells were first treated with a scramble non-coding siRNA (NC) or siRNA for NRP-1, subsequently they were treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and DNA synthesis analyzed by measuring the uptake of BrdU. An increase in O.D. (450 nm) for the FOL 26 treated cell lines compared to the control showed that FOL26 induced angiogenesis may be NRP-1 dependent as a reduction was observed for FOL26 10 and 100nM.
Figure 11: Human umbilical cord endothelial cells were treated with a scramble noncoding siRNA (NC) or siRNA for NRP-1 in concentrations of 1nM, and 5nM, and the NRP-1 expression values and Glyceraldehyde-phosphate dehydrogenase (GAPDH) mRNA expression values was evaluated, the latter was used as an internal control to normalize the data.
Figure 12: FOL26 enhances endothelial cell tube in an NRP-1 specific manner. Human umbilical cord endothelial cells were first treated with a scramble non-coding siRNA (NC) or siRNA for NRP-1, subsequently treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and endothelial tube formation and the total master segemnts were measured using Imaged, gene expression of PECAM-1 (CD31) analyzed. FOL26 increased tube formation compared to control in a NRP-1 dependent way. Scale bar: 150 pm.
Figure 13: Overview of acute wound healing and pathological wound healing models, including experimental parameters.
Figure 14: Results from wound healing models. Mean +/- SEM. N=10-11 punches from 4 independent donors (each dot represents the average value of 3 sections. A) Acute wound healing - one way ANOVA and Dunnett’s multiple comparison test vs. vehicle n.s. Unpaired t test *<0.05. B) Acute wound healing - Kruskal-Wallis test p=0.016, Dunn’s multiple comparison test #p<0.05, ##p<0.01. Mann-Whitney ore Unpaired t test *p<0.05. C) Pathological wound healing - One way ANOVA p=0.143, Dunnett’s multiple comparison test vs vehicle #p<0.05. Unpaired t test *p<0.05, **p<0.01. D) Pathological wound healing - One way ANOVA, Dunnett’s multiple comparison test vs vehicle #p<0.05, ##p<0.01. Unpaired t test *p<0.05, **p>0.01 , ***p>0.001.
Figure 15: Representative images of wound tongue at day 3, acute model (donor 1). PAS stained skin sections. Dashed lines show the newly formed epithelial tongues. Scale bar represents 100 pm.
Figure 16: Representative images of wound tongue at day 3, pathological model (donor 2). PAS stained skin sections. Dashed lines show the newly formed epithelial tongues. Scale bar represents 100 pm.
Figure 17: Increased proliferation of epidermal keratinocytes adjacent to the wound bed and decreased keratinocyte apoptosis at 300 nM FOL-005 peptide in the ex vivo pathological wound healing model (6 day protocol, 3 topical applications of FOL peptide). Mean+/-SEM N=14-16 microscopic images/ experimental group from N=2 punches/ experimental group from 1 donor, one way ANOVA *p<0.05 **p<0.01 , Kruskal Wallis #p<0.05 ##p<0.01.
Figure 18: FOL56 stimulates endothelial and vascular smooth muscle cell proliferation.
Human umbilical cord endothelial cells (A) and human coronary artery smooth muscle cells (B) were treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and DNA synthesis analyzed by measuring the uptake of BrdU. FOL56 increases proliferation at all concentrations in both HUVECs and HCASMCs compared to the control.
Figure 19: FOL56 enhances endothelial cell tube formation and expression of PECAM- 1. Human umbilical cord endothelial cells were treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and endothelial tube formation and gene expression of PECAM-1 (CD31) analyzed. FOL56 increased tube formation at all concentrations compared to control, and also increased expression of PECAM-1.
Figure 20: FOL56 increases endothelial cell migration in a cell culture wound model. Confluent cultures of human umbilical cord endothelial cells were treated FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and ability of the cells to migrate into an in vitro scratch injury was analyzed with image J software. FOL56 increased cell migration at all concentrations compared to the control.
Figure 21 : FOL56 increases arterial smooth muscle cell migration in a cell culture wound model. Confluent cultures of human coronary artery smooth cells were treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and ability of the cells to migrate into an in vitro scratch injury was analyzed with image J software. Expression of the collagen gene a1 and a2 chains were determined by polymerase chain reaction (PCR). FOL56 increased expression of both COL1A1 and COL1A2. The wound closure at 5h was increased for FOL56 at all concentrations compared to the control.
Figure 22: FOL56 inhibits endothelial and arterial smooth muscle cell apoptosis. Cultured human umbilical cord endothelial and coronary artery smooth cells were exposed to soluble Fas ligand (sFasL) to induce apoptosis that subsequently was assessed by determining activation of caspase 3 and 7. Cells were also treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM to determine the effect on apoptosis activated by sFasL. FOL56 was capable of inhibiting, or trended towards inhibiting apoptosis in both HUVECs and HCASMCs.
Figure 23: FOL56 affects the gene expression of VEGF and the VEGF receptor family in endothelial cells. Human umbilical cord endothelial cells were treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding c-Met, neuropilin-1 (NRP-1), VEGF-A and VEGF receptor-2 (VEGFR-2) determined by polymerase chain reaction (PCR).
Figure 24: FOL56 affects the gene expression of cytokines and matrix metalloproteinases in endothelial cells. Human umbilical cord endothelial cells were treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding TNF-a, IL-6, MMP2 and MMP3 determined by polymerase chain reaction (PCR). TNF-a is strong pro-inflammatory cytokine while MMP2 is required for activation of smooth muscle cell proliferation and migration.
Figure 25: FOL56 affects the gene expression of VEGF and the VEGF receptor family in arterial smooth muscle cells. Human coronary artery smooth cells were treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding c-Met, neuropilin-1 (NRP-1), VEGF-A and VEGF receptor-2 (VEGFR- 2) determined by polymerase chain reaction (PCR).
Figure 26: FOL56 affects the gene expression of cytokines and matrix metalloproteinases in arterial smooth muscle cells. Human coronary artery smooth cells were treated with FOL 56 in concentrations of 10 nM, 100 nM, and 1000 nM and the expression of the genes encoding TNF-a, IL-6, MMP2 and MMP3 determined by polymerase chain reaction (PCR). TNF-a is strong pro-inflammatory cytokine while MMP2 is required for activation of smooth muscle cell proliferation and migration.
Figure. 27. Effect of FOL-026 peptide on endothelial cell function and gene expression. The impact of FOL-026 peptide on human umbilical vascular endothelial cells (HUVECs) proliferation (A) and apoptosis (B) induced with low-oxygen condition was determined by BrdU uptake (n=8-9 per group) and active caspase-3/-7 (n=7-10 per group) separately. The ROS level (C) activated by 50ug/ml oxidized Low-density Lipoprotein (oxLDL) for 2 hours in cells pre-incubated with FOL-026 peptides was assessed using H2O2 measurement (n=4-8 per group).
Figure 28. Representative pictures of endothelial migration into an in vitro scratch injury are shown (A) and the wound closure rate (B) in HUVECs stimulated with increasing concentration of FOL-026 peptide was quantified by image J software (n=8-9 per group). The effect of FOL-026 peptides on gene expression of VEGF-A (F) and its receptors (G and H), as well as HGF receptor c-Met (I) in HUVECs (n=4 for F-l). Scale bar =150um.
Figure 29. Effect of FOL-026 peptide on angiogenesis in vitro and vivo. Representative images of tube formation in HUVECs treated with FOL-026 peptides (A). Quantified measurement of total length (B), total master segments length (C), total branching length (D) and total segments length (E) were performed to evaluate angiogenesis in vitro (n=7 per group for B-E).
Figure 30. The quantification of vessel density was assessed with CD31+ positive area (A) (n=13 per group). Western blot analysis of phosphorylated AKT (pAKT-T308), AKT, phosphorylated ERK1/2 (p-ERK1/2) and ERK1/2 in endothelial cells stimulated with FOL-26 peptides for 48 hours (B). Quantification of phosphorylation intensities normalized to respective total expressions (C and D, n=3 per group).
Figure 31. Effect of NRP-1 knock-down on endothelial cell function induced with FOL- 026 peptide. The efficiency of small interfering RNA transfection for 48 hours in HUVECs was confirmed by qRT-PCR (n=4) (A). Effect of FOL-026 peptide stimulation on cell proliferation (B) and cell apoptosis at 0.1% oxygen (C) in HUVECs transfected with siRNA-NC or siNRP-1 was evaluated by BrdU incorporation (n=4-7 per group) and active caspase-3/-7 (n=4-7 per group).
Figure 32. Representative pictures of endothelial cells at 0 and 5 hours after scratch wounding are shown in (A). The wound closure rate (B) of siRNA-NC/ siNRP-1- transfected HUVECs stimulated with increasing concentration of FOL-026 peptide was quantified by image J software (n=9-12 per group). Scale bar =150um.
Figure 33. Effect of NRP-1 knock-down on tube formation induced with FOL-026 peptide. Tube formation with rising dose of FOL-026 peptide treatment in siRNA-NC or siNRP-1 group (A), total length (B), total master segments length (C), total branching length (D) and total segments length (E) was calculated using Angiogenesis Analyzer plug-in Imaged (n=7-12 per group). Scale bar =150um. Figure 34. Effect of FOL-026 peptide on smooth muscle cell function. The effect of FOL-026 peptide treatment on smooth muscle cell proliferation (A) and apoptosis (B) induced with low-oxygen condition was evaluated with the uptake of BrdU (n=10 per group) and active caspase-3/-7 (n=6-8 per group) separately. H2O2 measurement (C) of smooth muscle cells pre-incubated with FOL-26 peptides after 50ug/ml oxidized Low-density Lipoprotein (oxLDL) stimulation for 2 hours (n=6-7 per group).
Figure 35. Representative images of scratch wound assay in vitro are shown in (A) and the ability of wound healing (B) in human coronary smooth muscle cells (HCASMCs) treated with FOL-026 peptides was measured by image J software (n=10 per group). Scale bar =150um.
Figure 36. Effect of NRP-1 knock-down on smooth muscle cell function induced with FOL-026 peptide. qRT-PCR analysis of NRP-1 mRNA expression in HCASMCs with small interfering RNA transfection for 48 hours (n=4) (A). The assessment of BrdU incorporation (B) (n=10-11) and caspase-3/-7 activation (C), (n=3-10). in siRNA-NC/ siNRP-1-transfected HUVECs treated with indicated concentration of FOL-026 peptide.
Figure 37. Representative images of smooth muscle cells at 0 and 5 hours after scratch wounding are shown in (A). The wound closure rate (B) of HCASMCs in siRNA-NC and siNRP-1 group stimulated with increasing concentration of FOL-026 peptide was quantified by image J software (n=9-12 per group). Scale bar =150um.
Figure 38. Effect of FOL-005 peptide on endothelial and smooth muscle cell proliferation and wound healing ability. The endothelial cell proliferation (A) was assessed with BrdU uptake in human umbilical vascular endothelial cells (HUVECs) stimulated with increasing concentration of FOL-005 peptide for 48 hours (n=7-10 per group). The wound closure rate (B) in HUVECs with indicated dose of FOL-005 treatment was quantified by image J software (n=12-14 per group) and representative images of at 0 and 6 hours after scratch wounding are shown in (C).
Figure 39. The effect of FOL-005 peptide on smooth muscle cell proliferation (A) was determined with BrdU incorporation in peptide-treated human coronary smooth muscle cells (HCASMCs). Scratch wound assay (B) of HCASMCs incubated with FOL-005 peptides for 5 hours was measured by image J software (n=10-12 per group) and representative pictures of smooth muscle cell migration into an in vitro scratch injury are shown in (C). Scale bar =150um.
Figure 40. Effect of FOL-005 peptide on tube formation and the role of NRP-1 in that. Representative images of tube formation in human umbilical vascular endothelial cells (HUVECs) treated with FOL-005 peptides are shown in (A and B).
Scale bar =150um.
Detailed description
Definitions
As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly states otherwise.
The term “some embodiments” can include one, or more than one embodiment.
The use of the word “a” or “an” when used throughout the text or in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
“Preventing” or “Prevention” as used herein, includes delaying, stopping, reducing the risk of the onset, of disease, disorder, or condition.
As used herein, the term "skin defect" refers to, but is not limited to: injuries or trauma to internal or external tissue, preferably injury or trauma to the epidermis and/or dermis of the skin. The wound may be an acute wound or a chronic wound. By way of example, an acute wound may be an incision, laceration, abrasion graze or burn, a puncture wound, a penetration wound or a wound due to dermatologic diseases such as psoriasis, acne and eczema. By way of example, a chronic wound may be a venous ulcer, a diabetic ulcer, a pressure ulcer, corneal ulcer, digestive ulcer or wounds as a result of ischemia and radiation poisoning. As used herein "treatment and/or regeneration of a skin defect" relates to the promotion, the acceleration, and/or the improvement of healing at the wounded site, i.e. the formation of a functional skin at the wounded site. As such "treatment and/or regeneration of a skin defect" ideally result in formation or regeneration of dermis and epidermis, including a basal layer, which characterize functional skin.
The terms “hard-to-heal wound”, “difficult-to-heal wound” and “chronic wound” are used herein interchangeably. The terms “hard-to-heal wound” and “chronic wound” as used herein refer to a wound that has not healed. Wounds that do not heal within approximately 4 to 6 weeks, for example, are considered chronic. A "chronic wound" may be a wound that fails to progress through an orderly and timely sequence of repair or a wound that does not respond to treatment and/or the demands of treatment are beyond the patient's physical health, tolerance or stamina. Many wounds that are first considered to be acute wounds ultimately become chronic wounds due to factors still not well understood. One significant factor is the transition of planktonic bacteria within the wound to form a biofilm. For example, a chronic wound may have an epithelial layer that fails to cover the entire surface of the wound and is subject to bacterial colonization, which can result in biofilm formation, which is resistant to treatment with anti-bacterial agents.
Commonly, chronic wounds are classified into three broad categories based on their main cause: venous insufficiency, arterial insufficiency and diabetic complications, or is a pressure-related. Hard-to-heal wounds due to venous insufficiency account for 70% to 90% of all hard-to-heal wounds and commonly affect the elderly. Venous insufficiency results in venous hypertension, in which blood flow is abrogated resulting in subsequent ischaemia. Venous insufficiency can occur as a result of obstructions to venous outflow or reflux due to valve damage. Following a period of ischaemia, tissue reperfusion can result in reperfusion injury, causing the tissue damage that leads to wound formation.
Exemplary chronic wounds can include "burn ulcers", including first degree burn, which may be a superficial, reddened area of skin; second degree burn, which may be a blistered injury site which may heal spontaneously after the blister fluid has been removed; third degree burn, which may be a burn through the entire skin and usually require surgical intervention for wound healing; scalding burns, which may occur from scalding hot water, grease or radiator fluid; thermal burns, which may occur after contact with flames, usually deep burns; chemical burns, which may come from acid and alkali, usually deep burns; electrical burns; and contact burns, which are usually deep and may occur from muffler tail pipes, hot irons and stoves, or other materials.
Angiogenesis and neovascularisation are understood to be important processes of vascular regeneration and may collectively or alternatively be referred to herein as vascular regeneration. As used herein vascular regeneration is to be understood in its broadest sense to also include other repair processes including endothelial cell migration, proliferation and mobilisation.
The term "wound dressing", as used herein, particularly refers to any material applied to a wound for protection, absorbance, drainage, etc. Numerous types of dressings are commercially available, including films (e.g., polyurethane films), hydrocolloids (hydrophilic colloidal particles bound to polyurethane foam), hydrogels (cross-linked polymers containing about at least 60% water), foams (hydrophilic or hydrophobic), calcium alginates (nonwoven composites of fibers from calcium alginate), and cellophane (cellulose with a plasticizer).
By “tissue”, such as in the context of “tissue defect”, is meant epithelial surfaces of epithelial origin.
Agent
The agent of the present invention may be a peptide; a polynucleotide encoding said peptide; a vector comprising said polynucleotide; or a cell comprising said polynucleotide or vector.
Peptides
In one embodiment, the agent is a peptide or a pharmaceutically acceptable salt thereof.
The term ‘amino acid’ as used herein includes the standard twenty genetically-encoded amino acids and their corresponding stereoisomers in the ‘D’ form (as compared to the natural ‘L’ form), omega-amino acids and other naturally-occurring amino acids, unconventional amino acids (e.g., a,a-disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatized amino acids (see below).
When an amino acid is being specifically enumerated, such as ‘alanine’ or ‘Ala’ or ‘A’, the term refers to both L-alanine and D-alanine unless explicitly stated otherwise. Other unconventional amino acids may also be suitable components for peptides of the present disclosure, as long as the desired functional property is retained by the peptide. For the peptides shown, each encoded amino acid residue, where appropriate, is represented by a single letter designation, corresponding to the trivial name of the conventional amino acid.
In one embodiment, the peptide is non-naturally occurring, such as a peptide comprising non-proteinogenic amino acid residues.
In one embodiment, the agent comprises or consists of a tandem repeat comprising two or more repeat units. In one embodiment, the repeat unit comprises or consists of the amino acid sequence of any one or more of the sequences as described herein.
In one embodiment, the peptide is cyclic. The cyclic structure may be achieved by any suitable method of synthesis. Thus, heterodetic linkages may include, but are not limited to formation via disulphide, cysteine, alkylene or sulphide bridges. In one embodiment, the peptide is capable of forming at least one intramolecular cysteine bridge.
In one embodiment, the agent comprises or consists of a peptide comprising or consisting of an amino acid sequence of the general formula:
XsXeSXyXsYGLR (SEQ ID NO: 1) wherein:
Xs is D or G;
Xe is I or G;
X7 is V or L; and Xa is V or A.
In one embodiment, the peptide comprises or consists of an amino acid sequence of the general formula:
VDX2X3X4GX5X6SX7X8YGLR (SEQ ID NO: 2) wherein:
X2 is T or V;
X3 is Y or P;
X4 is D or N;
Xs is D or G;
Xe is I or G;
X7 is V or L; and
X8 is V or A.
In one embodiment, the peptide comprises or consists of an amino acid sequence of the general formula:
VDTYX4GX5X6SX7X8YGLR (SEQ ID NO: 3) wherein:
X4 is D or N;
X5 is D or G;
X6 is I or G;
X7 is V or L; and
X8 is V or A.
In one embodiment, the peptide comprises or consists of an amino acid sequence of the general formula:
VDTYDGZ7Z8SZIOZIIYGLR (SEQ ID NO: 4) wherein:
X5 is D or G;
X8 is I or G;
X7 is V or L; and
X8 is V or A.
In one embodiment, the peptide comprises or consists of an amino acid sequence of the general formula:
VDTYDGZ7Z8SVVYGLR (SEQ ID NO: 5) wherein:
Xs is D or G; and
X8 is I or G; In one embodiment, the agent comprises or consists of a peptide comprising or consisting of an amino acid sequence of the general formula:
X14LX15YGIK (SEQ ID NO: 105) wherein:
X14 is E or G; and
X15 is S or T.
In one embodiment, the peptide comprises or consists of an amino acid sequence of the general formula:
KX9LAX10X11X12X13IX14LX15YGIK (SEQ ID NO: 106) wherein:
X9 is C, P or G;
X is E or G;
Xu is C, D or I;
X12 is D, I, S or G;
X13 is S, D or G;
X14 is E or G; and
X15 is S or T.
In one embodiment, the peptide comprises or consists of an amino acid sequence of the general formula:
KX9LAX10X11X12X13IX14LSYGIK (SEQ ID NO: 107) wherein:
X9 is C, P or G;
X10 is E or G;
Xu is C, I or absent;
X12 is D, G or absent;
X13 is S, G or absent; and
X14 is E or G.
In one embodiment, the peptide comprises or consists of an amino acid sequence of the general formula:
KX9LAX10IX14LSYGIK (SEQ ID NO: 108) wherein:
X9 is C, P or G; Xio is E or G; and
X14 is E or G.
In one embodiment, the peptide comprises or consisted of the amino acid sequence IELSYGIK (SEQ ID NO: 109).
In one embodiment, the peptide comprises or consists of VDTYDGGISVVYGLR (SEQ ID NO: 6). In one embodiment, the peptide comprises or consists of AEIDSIELSYGIK (SEQ ID NO: 110). In one embodiment, the peptide comprises or consists of VDTYDGDISVVYGLR (SEQ ID NO: 7). In one embodiment, the peptide comprises or consists of DTYDGDISVVYGLR (SEQ ID NO: 8). In one embodiment, the peptide comprises or consists of TYDGDISVVYGLRS (SEQ ID NO: 9). In one embodiment, the peptide comprises or consists of TYDGDISVVYGLR (SEQ ID NO: 10). In one embodiment, the peptide comprises or consists of YDGDISWYGLRS (SEQ ID NO: 11). In one embodiment, the peptide comprises or consists of YDGDISVVYGLR (SEQ ID NO: 12). In one embodiment, the peptide comprises or consists of DGDISVVYGLRS (SEQ ID NO: 13). In one embodiment, the peptide comprises or consists of DGDISVVYGLR (SEQ ID NO: 14). In one embodiment, the peptide comprises or consists of GDISVVYGLRS (SEQ ID NO: 15). In one embodiment, the peptide comprises or consists of GDISVVYGLR (SEQ ID NO: 16). In one embodiment, the peptide comprises or consists of DISVVYGLRS (SEQ ID NO: 17). In one embodiment, the peptide comprises or consists of DISVVYGLR (SEQ ID NO: 18). In one embodiment, the peptide comprises or consists of VDVPNGDISLAYGLR (SEQ ID NO: 19). In one embodiment, the peptide comprises or consists of DVPNGDISLAYGLRS (SEQ ID NO: 20). In one embodiment, the peptide comprises or consists of DVPNGDISLAYGLR (SEQ ID NO: 21). In one embodiment, the peptide comprises or consists of VPNGDISLAYGLRS (SEQ ID NO: 22). In one embodiment, the peptide comprises or consists of VPNGDISLAYGLR (SEQ ID NO: 23). In one embodiment, the peptide comprises or consists of PNGDISLAYGLRS (SEQ ID NO: 24). In one embodiment, the peptide comprises or consists of PNGDISLAYGLR (SEQ ID NO: 25). In one embodiment, the peptide comprises or consists of NGDISLAYGLRS (SEQ ID NO: 26). In one embodiment, the peptide comprises or consists of NGDISLAYGLR (SEQ ID NO: 27). In one embodiment, the peptide comprises or consists of GDISLAYGLRS (SEQ ID NO: 28). In one embodiment, the peptide comprises or consists of GDISLAYGLR (SEQ ID NO: 29). In one embodiment, the peptide comprises or consists of DISLAYGLRS (SEQ ID NO: 30). In one embodiment, the peptide comprises or consists of DISLAYGLR (SEQ ID NO: 31). In one embodiment, the peptide comprises or consists of VDTYDGDGSVVYGLR (SEQ ID NO: 32). In one embodiment, the peptide comprises or consists of VDVPEGDISLAYGLR (SEQ ID NO: 33). In one embodiment, the peptide comprises or consists of KPLAEIDSIELSYGIK (SEQ ID NO: 111). In one embodiment, the peptide comprises or consists of KCLAECDSIELSYGIK (Cyclic) (SEQ ID NO: 112). In one embodiment, the peptide comprises or consists of KPLAEDISIELSYGIK (SEQ ID NO: 113). In one embodiment, the peptide comprises or consists of KPLAEISDIELSYGIK (SEQ ID NO: 114). In one embodiment, the peptide comprises or consists of KPLAEIGDIELSYGIK (SEQ ID NO: 115). In one embodiment, the peptide comprises or consists of KPLAEGDIELSYGIK (SEQ ID NO: 116). In one embodiment, the peptide comprises or consists of KPLAEIELSYGIK (SEQ ID NO: 117). In one embodiment, the peptide comprises or consists of KPLAEIDSIELTYGIK (SEQ ID NO: 118). In one embodiment, the peptide comprises or consists of KPLAEIDGIELSYGIK (SEQ ID NO: 119). In one embodiment, the peptide comprises or consists of KPLAEIDGIELTYGIK (SEQ ID NO: 120). In one embodiment, the peptide comprises or consists of KPLAEIGSIELSYGIK (SEQ ID NO: 121). In one embodiment, the peptide comprises or consists of KGLAEIDSIELSYGIK (SEQ ID NO: 122). In one embodiment, the peptide comprises or consists of KPLAGIDSIGLSYGIK (SEQ ID NO: 123). In one embodiment, the peptide comprises or consists of Cyclic KCLAEIDSCELSYGIK (SEQ ID NO: 124). In one embodiment, the peptide comprises or consists of LAEIDSIELSYGIK (SEQ ID NO: 125). In one embodiment, the peptide comprises or consists of EIDSIELSYGIK (SEQ ID NO: 126). In one embodiment, the peptide comprises or consists of IDSIELSYGIK (SEQ ID NO: 127). In one embodiment, the peptide comprises or consists of DSIELSYGIK (SEQ ID NO: 128). In one embodiment, the peptide comprises or consists of SIELSYGIK (SEQ ID NO: 129). In one embodiment, the peptide comprises or consists of IELSYGIK (SEQ ID NO: 109). In one embodiment, the peptide comprises or consists of VDTYDGDISVVYGL (SEQ ID NO: 34). In one embodiment, the peptide comprises or consists of VDTYDGDISVVYG (SEQ ID NO: 35). In one embodiment, the peptide comprises or consists of VDTYDGDISVVY (SEQ ID NO: 36). In one embodiment, the peptide comprises or consists of VDTYDGDISVV (SEQ ID NO: 37). In one embodiment, the peptide comprises or consists of VDTYDGDISV (SEQ ID NO: 38). In one embodiment, the peptide comprises or consists of VDTYDGDIS (SEQ ID NO: 39). In one embodiment, the peptide comprises or consists of VDTYDGRGDSVVYGLR (SEQ ID NO: 40). In one embodiment, the peptide comprises or consists of VDVPNGDISLAYGL (SEQ ID NO: 41). In one embodiment, the peptide comprises or consists of VDVPNGDISLAYG (SEQ ID NO: 42). In one embodiment, the peptide comprises or consists of VDVPNGDISLA (SEQ ID NO: 43). In one embodiment, the peptide comprises or consists of VDVPNGDIS (SEQ ID NO: 44). In one embodiment, the peptide comprises or consists of GDPNDGRGDSVVYGLR (SEQ ID NO: 45 In one embodiment, the peptide comprises or consists of LDGLVRAYDNISPVG (SEQ ID NO: 46). In one embodiment, the peptide comprises or consists of GDPNGDISVVYGLR (SEQ ID NO: 47). In one embodiment, the peptide comprises or consists of VDVPNGDISLAYRLR (SEQ ID NO: 48). In one embodiment, the peptide comprises or consists of VDVPEGDISLAYRLR (SEQ ID NO: 49). In one embodiment, the peptide comprises or consists of V(beta-D)TYDGDISVVYGLR (SEQ ID NO: 50). In one embodiment, the peptide comprises or consists of VDTY(beta-D)GDISVVYGLR (SEQ ID NO: 51 In one embodiment, the peptide comprises or consists of VDTYDG(beta- D)ISVVYGLR (SEQ ID NO: 52). In one embodiment, the peptide comprises or consists of CLAEIDSC (Cyclic) (SEQ ID NO: 130). In one embodiment, the peptide comprises or consists of CFKPLAEIDSIECSYGIK (Cyclic) (SEQ ID NO: 131). In one embodiment, the peptide comprises or consists of Cyclic CFKPL EIDSIEC (SEQ ID NO: 132). In one embodiment, the peptide comprises or consists of KPLAEIDSIELSYGI (SEQ ID NO: 133). In one embodiment, the peptide comprises or consists of KPLAEIDSIELSYG (SEQ ID NO: 134). In one embodiment, the peptide comprises or consists of KPLAEIDSIELSY (SEQ ID NO: 135), KPLAEIDSIELS (SEQ ID NO: 136). In one embodiment, the peptide comprises or consists of KPLAEIDSIEL (SEQ ID NO: 137). In one embodiment, the peptide comprises or consists of KPLAEIDSIE (SEQ ID NO: 138).
Salts and prodrugs
The agent as defined herein can be in the form of a pharmaceutically acceptable salt or prodrug of said agent. In one embodiment, the agent as defined herein can be formulated as a pharmaceutically acceptable addition salt or hydrate of said agent, such as but not limited to K+, Na+, as well as non-salt e.g. H+.
Modifications
In one embodiment, the agent is a chemical derivative of a peptide. Chemical derivatives of one or more amino acids may be achieved by reaction with a functional side group. Such derivatives include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulphonyl groups, carboxybenzoxy groups, f-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are those peptides which contain naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5- hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine and ornithine for lysine. Derivatives also include peptides containing one or more additions or deletions as long as the requisite activity is maintained. Other included modifications are amidation, amino terminal acylation (e.g. acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g. with ammonia or methylamine), and the like terminal modifications.
In one embodiment, the agent is further modified such by glycosylation, PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.
In some embodiments, the agent is further conjugated to a moiety, which may be selected from the group consisting of polyethylene glycol (PEG), monosaccharides, fluorophores, chromophores, radioactive compounds, and cell-penetrating peptides. In one embodiment, the fluorophore is selected from the group consisting of Lucifer yellow, biotin, 5,6-carboxyltetramethylrhodamine (TAMRA), indodicarbocyanine (C5) Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 647, ATTO 488, ATTO 532, 6- carboxyfluorescein (6-FAM), Alexa Fluor® 350, DY-415, ATTO 425, ATTO 465, Bodipy® FL, fluorescein isothiocyanate, Oregon Green® 488, Oregon Green® 514, Rhodamine Green™, 5’-Tetrachloro-Fluorescein, ATTO 520, 6-carboxy-4',5'-dichloro- 2',7'-dimethoxyfluoresceine, Yakima Yellow™ dyes, Bodipy® 530/550, hexachlorofluorescein, Alexa Fluor® 555, DY-549, Bodipy® TMR-X, cyanine phosphoramidites (cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7.5), ATTO 550, Rhodamine Red™, ATTO 565, Carboxy-X-Rhodamine, Texas Red (Sulforhodamine 101 acid chloride), LightCycler® Red 610, ATTO 594, DY-480-XL, DY-610, ATTO 610, LightCycler® Red 640, Bodipy 630/650, ATTO 633, Bodipy 650/665, ATTO 647N, DY- 649, LightCycler® Red 670, ATTO 680, LightCycler® Red 705, DY-682, ATTO 700, ATTO 740, DY-782, IRD 700, IRD 800, CAL Fluor® Gold 540 nm, CAL Fluor® Gold 522 nm, CAL Fluor® Gold 544 nm , CAL Fluor® Orange 560 nm, CAL Fluor® Orange 538 nm, CAL Fluor® Orange 559 nm, CAL Fluor® Red 590 nm, CAL Fluor® Red 569 nm, CAL Fluor® Red 591 nm, CAL Fluor® Red 610 nm, CAL Fluor® Red 590 nm, CAL Fluor® Red 610 nm, CAL Fluor® Red 635 nm, Quasar® 570 nm, Quasar® 548 nm, Quasar® 566 nm (Cy 3), Quasar® 670 nm, Quasar® 647 nm, Quasar® 670 nm, Quasar® 705 nm, Quasar® 690 nm, Quasar® 705 nm (Cy 5.5), Pulsar® 650 Dyes, SuperRox® Dyes.). In one embodiment, the agent further comprises a detectable moiety, such as a moiety that is detectable by an imaging technique such as SPECT, PET, MRI, optical or ultrasound imaging. In one embodiment, the detectable moiety comprises or consists of a radioisotope, such as selected from the group consisting of 99mTc, 111 In, 67Ga, 58Ga, 72As, 89Zr, 123l and 201TI.
In one embodiment, the peptide comprises or consists of a fusion. For example, the peptide may comprise a fusion of two amino acid sequences as disclosed herein.
The term ‘fusion’ of a peptide relates to an amino acid sequence fused to another peptide. For example, the said peptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said peptide. Examples of such fusions are well known to those skilled in the art. Similarly, the said peptide may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well-known Myc tag epitope. Fusions to any variant or derivative of said peptide are also included in the scope of the disclosure. Alternatively, the fused portion may be a lipophilic molecule or peptide domain that is capable of promoting cellular uptake of the polypeptide, as known to those skilled in the art.
Peptide length
In one embodiment, said peptide is of no more than no more than 85, such as no more than 80, such as no more than 75, such as no more than 70, such as no more than 65, such as no more than 60, such as nor more than 55, such as no more than 50, such as no more than 55, such as no more than 40 amino acids, such as no more than 35, such as no more than 30, such as no more than 28, such as no more than 26, such as no more than 24, such as no more than 22, such as no more than 20, such as no more than 19, such as no more than 18, such as no more than 17, such as no more than 16, such as no more than 15, such as no more than 14, such as no more than 13, such as no more than 12, such as no more than 11, such as no more than 10 amino acids in length.
In one embodiment, the peptide comprises at least 2 additional amino acids, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 15 or such as at least 20 amino acids conjugated to the N- or C-terminus of the peptide.
In one embodiment, said peptide is between 5 and 30 amino acids in length, such as between 5 and 20, such as between 8 and 20, such as between 8 and 18, such as between 10 and 16 amino acids in length.
In one embodiment, when
Figure imgf000026_0001
is T, then the peptide comprises no more than 25 amino acid residues.
In yet another embodiment, the agent is a fragment of a peptide described herein, and the fragment comprises 15 or fewer amino acids in length, such as fewer than 14 amino acids, such as fewer than 13 amino acids, such as fewer than 12 amino acids, such as fewer than 11 amino acids, such as fewer than 10 amino acids, such as fewer than 9 amino acids, such as fewer than 8 amino acids, such as fewer than 7 amino acids, such as fewer than 6 amino acids, such as fewer than 5 amino acids in length.
By “fragment”, at least 5 contiguous amino acids of the amino acid sequence are included, for example at least 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 contiguous amino acids of the amino acid sequence. The fragment may be 15 or fewer amino acids in length, for example 14, 13, 12, 11 , 10, 9, 8, 7, 6 or 5 amino acids in length.
Variants
In one embodiment, the agent is a variant of a peptide as described herein, wherein the variant comprises or consists of a sequence wherein any one amino acid has been altered for another proteinogenic or non-proteinogenic amino acid, with the proviso that no more than five amino acids are so altered. The term “variant” refers to a peptide that does not share 100% amino acid sequence identity with the parent peptide, i.e. one or more amino acids must be mutated. “Mutated” refers to altering an amino acid at a specified position in the parent peptide. For example, an amino acid at a specified position may be deleted, altered, substituted or may be the site of an insertion/addition of one or more amino acids. It will be appreciated by persons skilled in the art that the substitutions may be conservative or non-conservative.
In one embodiment, said peptide variant comprises or consists of a sequence wherein no more than five amino acids are altered for another proteinogenic or non- proteinogenic amino acid, such as no more than 4 amino acids, such as no more than 3 amino acids, such as no more than 2 amino acids, such as no more than 1 amino acid is altered. In one embodiment, one or more amino acids are conservatively substituted. “Conservatively substituted” refers to a substitution of one amino acid with another with similar properties (size, hydrophobicity, etc.), such that the function of the peptide is not significantly altered. Thus, by “conservative substitutions” is intended combinations such as Gly, Ala; Vai, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
In one embodiment, the peptide has one additional amino acid. In one embodiment, said peptide comprises or consists of one or more additional amino acids, inserted at the N- and/or C-terminus and/or internally within the sequence. In one embodiment, at least 2 additional amino acids, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 15 or such as at least 20 additional amino acids are inserted.
Polynucleotides, vectors and cells
In embodiment, the agent is a polynucleotide encoding upon expression a peptide as described herein. In embodiment, the agent is a vector comprising a polynucleotide as described herein. In embodiment, the agent is a cell comprising a polynucleotide or a vector as described herein. Compositions and coatings
In one embodiment, the agent is in a composition. In one embodiment, the composition is a pharmaceutical composition. In one embodiment, the composition is a cosmetic composition. In one embodiment, the composition is a coating.
Coatings on various implants are known in the art. Applications in humans include central venous catheters, coronary stents, ventricular assist devices, extracorporeal blood circuits, blood sampling devices, and vascular grafts. Such coatings can be in a gel or non-gel form. As used herein, a "coating comprising the agent" includes that the agent adsorbed to the surface, bonded to the surface, and imbedded in the polymer surface.
Implants
In one aspect, the present invention relates to an implant comprising the agent described herein. In one embodiment, the implant is coated with a composition comprising the agent. Thus, the agent may for example be adsorbed to the surface, bonded to the surface, and imbedded in the polymer surface of the implant.
In one embodiment, the implant is of a biomaterial, such as bone.
In one embodiment, the implant is a medical device, such as a stent.
Medical devices
In one aspect, the present invention relates to a medical device comprising the agent as defined herein. In one embodiment, the medical device is a stent. In one embodiment, the medical device is a wound dressing. In one embodiment, the medical device is a mesh.
Medical use
A normal wound healing process typically includes the stages of 1) haemostasis via platelets, fibrin, and proteoglycans; 2) inflammation comprising recruitment of neutrophils, macrophages, and lymphocytes; 3) proliferation comprising formation of fibroblasts, epithelial cells, collagen, and endothelial cells; and 4) remodelling including collagen fibril cross-linking and scar maturation. The mode of action of the disclosed polypeptides as outlined herein is likely to play a favourable role in the inflammation, proliferation, and remodelling stages of wound healing through the interaction with the NRP-1 co-receptors such as VEGFR and several others. An interplay of the agents of the disclosure with several important growth factors can therefore improve the wound healing process.
In one embodiment, the composition of the disclosure is a pharmaceutical composition.
In one embodiment, the composition of the disclosure is a cosmetic composition.
In one embodiment, the composition is a coating composition.
Tissue meshes have found use in facilitating wound healing. One embodiment of the present disclosure provides for a mesh comprising an agent of the disclosure. One embodiment of the disclosure provides for a stent comprising an agent of the disclosure. One embodiment of the present disclosure provides for a wound dressing comprising the agent of the disclosure.
The agents of the present disclosure were found to increase vascularisation/induce angiogenesis, and to induce healing of wounds. One embodiment of the disclosure provides for the agent of the disclosure for use in the treatment and/or regeneration of a tissue defect in a subject. One embodiment of the disclosure provides for an agent of the disclosure for use in the treatment and/or promoting healing of a tissue defect.
As shown in the examples herein, the agents of the disclosure induce healing of epithelial tissue. In one embodiment of the disclosure, the tissue defect is an epithelial tissue defect. In one embodiment of the disclosure, the tissue defect is a skin defect. In one embodiment of the disclosure, the tissue is epithelium. In one embodiment of the disclosure, the tissue is epithelial cells. In one embodiment of the disclosure, the tissue is skin.
One embodiment of the disclosure provides for the agent of the disclosure for use in the prevention of fibrosis of the skin in a subject. In one embodiment, the skin defect is a wound, such as an open wound. In one embodiment, the wound is a hard-to-heal wound, such as a chronic wound. In one embodiment, the wound is a surgical wound.
In one embodiment, the wound is associated with venous insufficiency, arterial insufficiency, diabetic complications, sickle cell anaemia, or is a pressure-related wound.
In one embodiment of the disclosure, the diabetic complication is due to diabetes mellitus, such as Type 1 diabetes mellitus, such as Type 1 diabetes mellitus with foot ulcer and/or Type 1 diabetes mellitus with other skin ulcer, or Type 2 diabetes mellitus, such as Type 2 diabetes mellitus with foot ulcer and/or Type 2 diabetes mellitus with other skin ulcer. In one embodiment of the disclosure, the diabetes mellitus presents with gangrene.
In one embodiment of the disclosure, the diabetes mellitus is associated with a disease of the connective tissue, such as fibrosis. In one embodiment, the diabetes mellitus presents with fibrosis. In one embodiment, the diabetes mellitus is associated with fibrosis. On embodiment of the disclosure provides for an agent of the disclosure for prevention or treatment of fibrosis. In one embodiment of the disclosure, the fibrosis is associated with diabetes mellitus.
In one embodiment of the disclosure, the wound is a dermal ulcer, or a skin defect due to vascular insufficiency or diabetic affection of the blood vessels.
In one embodiment, the tissue defect is a diabetic complication. In one embodiment, the diabetic complication is a diabetes mellitus related eye condition. In one embodiment, the diabetic complication is retinopathy.
In one embodiment of the disclosure, the tissue defect is damage to the eye and/or orbit, such as injury (trauma) of the eye and/or orbit.
In one embodiment, the skin defect is a skin disorder provoked by external factors. In one embodiment, the skin disorder provoked by external factors is pressure ulceration, dermatoses provoked by friction or mechanical stress, dermatoses due to foreign bodies, dermatoses provoked or exacerbated by exposure to cold, dermatoses provoked by heat or electricity, dermatoses provoked by light or UV radiation, dermatoses due to ionizing radiation, allergic contact dermatitis, photo-allergic contact dermatitis, irritant contact dermatitis, allergic contact urticarial, protein contact dermatitis, allergic contact sensitisation, phototoxic reactions to skin contact with photoactive agents, cutaneous reactions to venomous or noxious animals.
In one embodiment, the skin defect is an inflammatory dermatosis. In one embodiment, the inflammatory dermatosis is dermatitis, eczema, atopic dermatitis, papulosquamous dermatoses, urticaria, angioedema or other urticarial disorders, inflammatory erythemas and other reactive inflammatory dermatoses, immunobullous diseases of the skin, cutaneous lupus erythematosus, scarring or sclerosing inflammatory dermatoses.
In one embodiment, the skin defect is sclerosis. In one embodiment, the sclerosis is systemic sclerosis (scleroderma), such as circumscribed scleroderma.
In one embodiment, the skin defect is a postprocedural disorder of the skin. In one embodiment of the disclosure, the postprocedural disorder of the skin is an unsatisfactory surgical scar of skin, a cutaneous flap necrosis, a myocutaneous flap necrosis, a skin graft failure, a composite graft failure. In one embodiment, the postprocedural disorder of the skin is a surgical incision.
In one embodiment, the skin defect is a genetic and/or developmental disorder affecting the skin. In one embodiment, the genetic and/or developmental disorder affecting the skin is epidermolysis bullosa, or genodermatosis, such as pemphigus genodermatosis.
In one embodiment, the epidermolysis bullosa is selected from the group consisting of epidermolysis bullosa simplex, junctional epidermolysis bullosa, dystrophic epidermolysis bullosa, recessive dystrophic epidermolysis bullosa, syndromic epidermolysis bullosa and epidermolysis bullosa.
In one embodiment, the skin defect is an external defect. In one embodiment, the skin defect is an open defect. In one embodiment, the skin defect is an inflamed wound. In one embodiment, the skin defect is infected. Infected wounds can be difficult to heal due to formation of biofilm, e.g. from bacteria.
In one embodiment, the skin defect is an injury or trauma to internal or external tissue, such as injury or trauma to the epidermis and/or dermis of the skin. In one embodiment, the skin defect is an acute defect or a chronic defect. In one embodiment, the skin defect is full thickness skin defect.
In one embodiment, the wound is an acute wound or a chronic wound. In one embodiment, the acute wound is an incision, laceration, abrasion graze or burn, a puncture wound, a penetration wound or a wound due to dermatologic diseases such as psoriasis, acne and eczema. In one embodiment, the chronic wound is a venous ulcer, a diabetic ulcer, a pressure ulcer, corneal ulcer, digestive ulcer or wounds as a result of ischemia and radiation poisoning.
In one embodiment, the wound is a non-pressure chronic ulcer, such as a nonpressure chronic ulcer of lower limb.
In one embodiment, the agent is administered topically, orally, subcutaneously, or systemically. In one embodiment, the administration is by instillation.
One embodiment of the disclosure provides for an agent of the disclosure for use in the treatment of a disease of the skin, such as injury of external cause.
In one embodiment, the skin defect is a disease of the skin, such as injury of external cause.
In one embodiment, the disease of the skin is selected from the group consisting of: a. a skin disorder involving specific cutaneous structures, such as disorders of cutaneous blood and lymphatic vessels, such as dermatoses resulting from vascular insufficiency, such as ischaemic ulceration of the skin, and b. a skin disorder associated with pregnancy, the neonatal period, and infancy, such as dermatoses of infancy, such as erythrodermas of infancy (Leiner’s disease). In one embodiment, the injury of external cause is selected from the group consisting of: a. injury to the head, such as intracranial injury, such as diffuse brain injury, such as cerebral contusion, and b. injury to unspecified part of trunk, limb, or body region, such as other injuries of spine or trunk, such as injury of the spinal cord.
In one embodiment, the subject is suffering from diabetes mellitus.
In one embodiment, the subject is suffering from stoma. In one embodiment, the wound is generated as part of internal acidic fluids.
In one embodiment, the subject is a mammal. In one embodiment, the mammal is a human.
One embodiment of the disclosure provides for a method for treatment and/or regeneration of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent of the disclosure to an subject in need thereof.
One embodiment of the disclosure provides for a method for treating and/or promoting healing of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent of the disclosure to an subject in need thereof.
One embodiment of the disclosure provides for a method for regeneration of skin at the site of a skin defect and/or for reducing scar formation resulting from healing of a skin defect in a subject, said method comprising administering an agent of the disclosure.
One embodiment of the disclosure provides for a use of an agent of the disclosure for the manufacture of a medicament for the treatment and/or regeneration of a skin defect in a subject. One embodiment of the disclosure provides for a use of an agent of the disclosure for the manufacture of a medicament for the treating and/or promoting healing of a tissue defect in a subject.
One embodiment of the disclosure provides for a use of an agent of the disclosure for the manufacture of a medicament for the treatment of a skin defect in a subject.
One embodiment provides for a use of an agent of the disclosure for reducing scar formation resulting from healing of a skin defect in a subject.
One embodiment provides for a use of an agent of the disclosure for promoting the healing of a skin defect in a subject.
One embodiment of the disclosure provides a use of an agent of the disclosure for promoting vascularisation in a healing tissue, such as skin healing from a skin defect, such as a wound.
One embodiment provides for a use of an agent of the disclosure in combination with a tissue meshing technique, for promoting healing of a skin defect, such as a wound.
One embodiment provides for a use of an agent of the disclosure for promoting grafting of skin.
One embodiment of the disclosure provides for a method for inserting a stent in a subject, said method comprising administering an agent of the disclosure. In one embodiment, the stent is coated with the agent of the disclosure.
In one embodiment, the agent of the disclosure is part of a cosmetic composition.
In one embodiment, the peptide comprises or consists of the amino acid sequence VDTYDGDISVVYGLR (SEQ ID NO: 7), and the tissue defect is a wound.
In one embodiment, the peptide comprises or consists of the amino acid sequence VDTYDGGISVVYGLR (SEQ ID NO: 6), and the tissue defect is a wound. In one embodiment, the peptide comprises or consists of the amino acid sequence AEIDSIELSYGIK (SEQ ID NO: 110), and the tissue defect is a wound.
In one embodiment, the peptide comprises or consists of the amino acid sequence KPLAEIDSIELSYGIK (SEQ ID NO: 111), and the tissue defect is a wound.
One aspect of the disclosure provides for the agent of the disclosure for use in the prevention of fibrosis of the skin in a subject.
One aspect of the disclosure provides for an agent of the disclosure for use in the treatment of a disease of the skin, such as injury of external cause.
One aspect of the disclosure provides for a method for treatment and/or regeneration of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent of the disclosure to an subject in need thereof.
One aspect of the disclosure provides for a method for treating and/or promoting healing of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent of the disclosure to an subject in need thereof.
One aspect provides for a use of an agent of the disclosure for reducing scar formation resulting from healing of a skin defect in a subject.
One aspect of the disclosure provides a use of an agent of the disclosure for promoting vascularisation in a healing tissue, such as skin healing from a skin defect, such as a wound.
Items
1. An agent comprising: a) a peptide selected from the group consisting of:
(i) a peptide comprising or consisting of an amino acid sequence of the general formula:
XsXeSXyXsYGLR (SEQ ID NO: 1) wherein:
X5 is D or G; Xe is I or G;
X? is V or L;
Xs is V or A;
(ii) a peptide comprising or consisting of an amino acid sequence of the general formula:
X14LX15YGIK (SEQ ID NO: 105) wherein:
X14 is E or G;
X15 is S or T;
(iii) a peptide comprising or consisting of an amino acid sequence selected from the group consisting of:
VDTYDGDISVVYGL (SEQ ID NO: 34),
VDTYDGDISVVYG (SEQ ID NO: 35),
VDTYDGDISVVY (SEQ ID NO: 36),
VDTYDGDISVV (SEQ ID NO: 37),
VDTYDGDISV (SEQ ID NO: 38),
VDTYDGDIS (SEQ ID NO: 39),
VDTYDGRGDSVVYGLR (SEQ ID NO: 40),
VDVPNGDISLAYGL (SEQ ID NO: 41),
VDVPNGDISLAYG (SEQ ID NO: 42),
VDVPNGDISLA (SEQ ID NO: 43),
VDVPNGDIS (SEQ ID NO: 44),
GDPNDGRGDSVVYGLR (SEQ ID NO: 45),
LDGLVRAYDNISPVG (SEQ ID NO: 46),
GDPNGDISVVYGLR (SEQ ID NO: 47)
VDVPNGDISLAYRLR (SEQ ID NO: 48),
VDVPEGDISLAYRLR (SEQ ID NO: 49),
V(beta-D)TYDGDISVVYGLR (SEQ ID NO: 50),
VDTY(beta-D)GDISVVYGLR (SEQ ID NO: 51),
VDTYDG(beta-D)ISVVYGLR (SEQ ID NO: 52);
CLAEIDSC (Cyclic) (SEQ ID NO: 130),
CFKPLAEIDSIECSYGIK (Cyclic) (SEQ ID NO: 131),
Cyclic CFKPLAEIDSIEC (SEQ ID NO: 132),
KPLAEIDSIELSYGI (SEQ ID NO: 133),
KPLAEIDSIELSYG (SEQ ID NO: 134), KPLAEIDSIELSY (SEQ ID NO: 135), KPLAEIDSIELS (SEQ ID NO: 136), KPLAEIDSIEL (SEQ ID NO: 137), and KPLAEIDSIE (SEQ ID NO: 138); b) a polynucleotide encoding upon expression, the peptide of a); c) a vector comprising the polynucleotide of b); or d) a cell comprising the polynucleotide of b), or the vector of c). The agent according to item 1 , wherein the peptide comprises an amino acid sequence of the general formula:
VDX2X3X4GX5X6SX7X8YGLR (SEQ ID NO: 2) wherein: X2 is T or V;
X3 is Y or P;
X4 is D or N;
X5 is D or G;
Xs is I or G;
X7 is V or L; and X8 is V or A. The agent according to any one of the preceding items, wherein the peptide comprises an amino acid sequence of the general formula: VDTYX4GX5X6SX7X8YGLR (SEQ ID NO: 3) wherein:
X4 is D or N;
Xs is D or G;
X8 is I or G;
X7 is V or L; and Xs is V or A. The agent according to any one of the preceding items, wherein the peptide comprises an amino acid sequence of the general formula: VDTYDGZ7Z8SZioZnYGLR (SEQ ID NO: 4) wherein:
Xs is D or G;
Xs is I or G;
X7 is V or L; and
Xs is V or A. The agent according to any one of the preceding items, wherein the peptide comprises an amino acid sequence of the general formula: VDTYDGZ7Z8SVVYGLR (SEQ ID NO: 5) wherein:
X5 is D or G; and X5 is I or G; The agent according to item 1 , wherein the peptide comprises an amino acid sequence of the general formula:
KX9LAX10X11X12X13IX14LX15YGIK (SEQ ID NO: 106) wherein:
X9 is C, P or G;
X is E or G;
Xn is C, D or I;
X12 is D, I, S or G;
X13 is S, D or G;
X14 is E or G; and X15 is S or T. The agent according to item 1 , wherein the peptide comprises an amino acid sequence of the general formula:
KX9LAX10X11X12X13IX14LSYGIK (SEQ ID NO: 107) wherein:
Xg is C, P or G;
X is E or G;
X11 is C, I or absent;
X12 is D, G or absent;
X13 is S, G or absent; and
X14 is E or G. 8. The agent according to item 1 , wherein the peptide comprises an amino acid sequence of the general formula:
KX9LAX10IX14LSYGIK (SEQ ID NO: 108) wherein:
X9 is C, P or G;
X is E or G; and X14 is E or G.
9. The agent according to item 1 , wherein the peptide comprises the amino acid sequence IELSYGIK (SEQ ID NO: 109).
10. The agent according to item 1 , with the proviso that if X14 is T, the peptide comprises no more than 25 amino acid residues.
11. The agent according to item 1 , wherein the peptide comprises or consists of an amino acid sequence selected from the group consisting of: VDTYDGGISVVYGLR (SEQ ID NO: 6),
VDTYDGDISVVYGLR (SEQ ID NO: 7), DTYDGDISVVYGLR (SEQ ID NO: 8), TYDGDISVVYGLRS (SEQ ID NO: 9), TYDGDISVVYGLR (SEQ ID NO: 10), YDGDISVVYGLRS (SEQ ID NO: 11), YDGDISVVYGLR (SEQ ID NO: 12), DGDISVVYGLRS (SEQ ID NO: 13), DGDISVVYGLR (SEQ ID NO: 14), GDISVVYGLRS (SEQ ID NO: 15), GDISVVYGLR (SEQ ID NO: 16), DISVVYGLRS (SEQ ID NO: 17), DISVVYGLR (SEQ ID NO: 18), VDVPNGDISLAYGLR (SEQ ID NO: 19), DVPNGDISLAYGLRS (SEQ ID NO: 20), DVPNGDISLAYGLR (SEQ ID NO: 21), VPNGDISLAYGLRS (SEQ ID NO: 22), VPNGDISLAYGLR (SEQ ID NO: 23), PNGDISLAYGLRS (SEQ ID NO: 24),
PNGDISLAYGLR (SEQ ID NO: 25),
NGDISLAYGLRS (SEQ ID NO: 26),
NGDISLAYGLR (SEQ ID NO: 27),
GDISLAYGLRS (SEQ ID NO: 28),
GDISLAYGLR (SEQ ID NO: 29),
DISLAYGLRS (SEQ ID NO: 30), DISLAYGLR (SEQ ID NO: 31), VDTYDGDGSVVYGLR (SEQ ID NO: 32), VDVPEGDISLAYGLR (SEQ ID NO: 33).
AEIDSIELSYGIK (SEQ ID NO: 110),
KPLAEIDSIELSYGIK (SEQ ID NO: 111),
KCLAECDSIELSYGIK (Cyclic) (SEQ ID NO: 112),
KPLAEDISIELSYGIK (SEQ ID NO: 113),
KPLAEISDIELSYGIK (SEQ ID NO: 114),
KPLAEIGDIELSYGIK (SEQ ID NO: 115),
KPLAEGDIELSYGIK (SEQ ID NO: 116), KPLAEIELSYGIK (SEQ ID NO: 117), KPLAEIDSIELTYGIK (SEQ ID NO: 118), KPLAEIDGIELSYGIK (SEQ ID NO: 119), KPLAEIDGIELTYGIK (SEQ ID NO: 120), KPLAEIGSIELSYGIK (SEQ ID NO: 121), KGLAEIDSIELSYGIK (SEQ ID NO: 122), KPLAGIDSIGLSYGIK (SEQ ID NO: 123), Cyclic KCLAEIDSCELSYGIK (SEQ ID NO: 124), LAEIDSIELSYGIK (SEQ ID NO: 125), EIDSIELSYGIK (SEQ ID NO: 126), IDSIELSYGIK (SEQ ID NO: 127), DSIELSYGIK (SEQ ID NO: 128), SIELSYGIK (SEQ ID NO: 129), and IELSYGIK (SEQ ID NO: 109). The agent according to item 1 , wherein the peptide comprises or consists of an amino acid sequence VDTYDGGISVVYGLR (SEQ ID NO: 6). 13. The agent according to item 1 , wherein the peptide comprises or consists of an amino acid sequence VDTYDGDISVVYGLR (SEQ ID NO: 7).
14. The agent according to item 1, wherein the peptide comprises or consists of an amino acid sequence AEIDSIELSYGIK (SEQ ID NO: 110).
15. The agent according to any one of the preceding items, wherein the peptide comprises no more than 85, such as no more than 80, such as no more than 75, such as no more than 70, such as no more than 65, such as no more than 60, such as nor more than 55, such as no more than 50, such as no more than 55, such as no more than 40 amino acids, such as no more than 35, such as no more than 30, such as no more than 28, such as no more than 26, such as no more than 24, such as no more than 22, such as no more than 20, such as no more than 19, such as no more than 18, such as no more than 17, such as no more than 16, such as no more than 15, such as no more than 14, such as no more than 13, such as no more than 12, such as no more than 11 , such as no more than 10 amino acids.
16. The agent according to any one of the preceding items, wherein the peptide comprises at least 2 additional amino acids, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 15 or such as at least 20 amino acids conjugated to the N- or C-terminus of the peptide.
17. The agent according to any one of the preceding items, wherein the agent is non- naturally occurring.
18. The agent according to any one of the preceding items, wherein the agent is conjugated to a moiety.
19. The agent according to item 18, wherein the moiety is selected from the group consisting of polyethylene glycol (PEG), monosaccharides, fluorophores, chromophores, radioactive compounds, and cell-penetrating peptides. 20. The agent according to any one of the preceding items, wherein the agent is further modified such as by glycosylation, PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.
21. The agent according to any one of the preceding items, wherein the agent comprises or consists of a tandem repeat comprising two or more repeat units.
22. The agent according to item 21, wherein the repeat unit comprises or consists of the amino acid sequence of any one or more of the sequences as described in the preceding items.
23. The agent according to any of the preceding items, wherein the agent is fused to another polypeptide.
24. The agent according to item 23, wherein the said polypeptide is selected from the group consisting of glutathione-S-transferase (GST) and protein A.
25. The agent according to any of the preceding items, wherein the agent is fused to a tag.
26. The agent according to item 25, wherein the tag is an oligo-histidine tag.
27. The agent according to any of the preceding items, wherein the agent is cyclic.
28. The agent according to any of the preceding items, wherein the agent is capable of forming at least one intramolecular cysteine bridge.
29. The agent according to any one of the preceding items, wherein the agent is a variant of the peptide, wherein the variant comprises or consists of a sequence wherein any one amino acid has been altered for another proteinogenic or non- proteinogenic amino acid, with the proviso that no more than five amino acids are so altered.
30. The agent according to item 29, wherein the variant comprises or consists of a sequence wherein no more than five amino acids are altered for another proteinogenic or non-proteinogenic amino acid, such as no more than 4 amino acids, such as no more than 3 amino acids, such as no more than 2 amino acids, such as no more than 1 amino acid is altered.
31. The agent according to any one of the preceding items, wherein one or more amino acids are conservatively substituted.
32. The agent according to any one of the preceding items, wherein the peptide comprises or consists of one or more additional amino acids, inserted at the N- and/or C-terminus and/or internally within the sequence.
33. The agent according to any one of the preceding items, wherein the peptide has one additional amino acid.
34. The agent according to any of the preceding items, wherein the agent further comprises a detectable moiety.
35. The agent according to item 34, wherein the detectable moiety comprises or consists of a radioisotope.
36. The agent according to item 35, wherein the radioisotope is selected from the group consisting of 99mTc, 111ln, 67Ga, 68Ga, 72As,89Zr, 123l and 201TI.
37. The agent according to item 34, wherein the detectable moiety is detectable by an imaging technique such as SPECT, PET, MRI, optical or ultrasound imaging.
38. A composition comprising the agent according to any one of the preceding items.
39. The composition according to item 38, wherein the composition is a pharmaceutical composition.
40. The composition according to item 38, wherein the composition is a cosmetic composition. 41. The composition according to any one of the preceding items, wherein the composition is a coating composition.
42. A medical device comprising an agent according to any one of the preceding items.
43. The medical device according to item 42, wherein the medical device is a wound dressing.
44. The medical device according to item 42, wherein the medical device is a mesh.
45. The medical device according to item 42, wherein the medical device is a stent.
46. A mesh comprising an agent according to any one of the preceding items.
47. A stent comprising an agent according to any one of the preceding items.
48. A wound dressing comprising the agent according to any one of the preceding items.
49. The agent according to any one of the preceding items for use in the treatment and/or regeneration of a tissue defect in a subject.
50. The agent according to any one of the preceding items for use in the treatment and/or promoting healing of a tissue defect.
51. The agent for use according to any one of the preceding items, wherein the tissue defect is a skin defect.
52. The agent according to any one of the preceding items for use in the prevention of fibrosis of the skin in a subject.
53. The agent for use according to any one of the preceding items, wherein the skin defect is a wound, such as an open wound. 54. The agent for use according to any one of the preceding items, wherein the wound is a hard-to-heal wound, such as a chronic wound.
55. The agent for use according to any one of the preceding items, wherein the wound is a surgical wound.
56. The agent for use according to any one of the preceding items, wherein the wound is associated with venous insufficiency, arterial insufficiency, diabetic complications, sickle cell anaemia, or is a pressure-related wound.
57. The agent for use according to any one of the preceding items, wherein the diabetic complication is due to diabetes mellitus, such as Type 1 diabetes mellitus, such as Type 1 diabetes mellitus with foot ulcer and/or Type 1 diabetes mellitus with other skin ulcer, or Type 2 diabetes mellitus, such as Type 2 diabetes mellitus with foot ulcer and/or Type 2 diabetes mellitus with other skin ulcer.
58. The agent for use according to any one of the preceding items, wherein the diabetes mellitus presents with gangrene.
59. The agent for use according to any one of the preceding items, wherein the diabetes mellitus is associated with a disease of the connective tissue, such as fibrosis.
60. The agent for use according to any one of the preceding items, wherein the wound is a dermal ulcer, or a skin defect due to vascular insufficiency or diabetic affection of the blood vessels.
61. The agent for use according to any one of the preceding items, wherein the tissue defect is a diabetic complication.
62. The agent for use according to any one of the preceding items, wherein the diabetic complication is a diabetes mellitus related eye condition.
63. The agent for use according to any one of the preceding items, wherein the diabetic complication is retinopathy. The agent for use according to any one of the preceding items, wherein the tissue defect is damage to the eye and/or orbit, such as injury (trauma) of the eye and/or orbit. The agent for use according to any one of the preceding items, wherein the skin defect is a skin disorder provoked by external factors. The agent for use according to any one of the preceding items, wherein the skin disorder provoked by external factors is pressure ulceration, dermatoses provoked by friction or mechanical stress, dermatoses due to foreign bodies, dermatoses provoked or exacerbated by exposure to cold, dermatoses provoked by heat or electricity, dermatoses provoked by light or UV radiation, dermatoses due to ionizing radiation, allergic contact dermatitis, photo-allergic contact dermatitis, irritant contact dermatitis, allergic contact urticarial, protein contact dermatitis, allergic contact sensitisation, phototoxic reactions to skin contact with photoactive agents, cutaneous reactions to venomous or noxious animals. The agent for use according to any one of the preceding items, wherein the skin defect is an inflammatory dermatosis. The agent for use according to any one of the preceding items, wherein the inflammatory dermatosis is dermatitis, eczema, atopic dermatitis, papulosquamous dermatoses, urticaria, angioedema or other urticarial disorders, inflammatory erythemas and other reactive inflammatory dermatoses, immunobullous diseases of the skin, cutaneous lupus erythematosus, scarring or sclerosing inflammatory dermatoses. The agent for use according to any one of the preceding items, wherein the skin defect is sclerosis. The agent for use according to any one of the preceding items, wherein the sclerosis is systemic sclerosis (scleroderma), such as circumscribed scleroderma. 71. The agent for use according to any one of the preceding items, wherein the skin defect is a postprocedural disorder of the skin.
72. The agent for use according to any one of the preceding items, wherein the postprocedural disorder of the skin is an unsatisfactory surgical scar of skin, a cutaneous flap necrosis, a myocutaneous flap necrosis, a skin graft failure, a composite graft failure.
73. The agent for use according to any one of the preceding items, wherein the postprocedural disorder of the skin is a surgical incision.
74. The agent for use according to any one of the preceding items, wherein the skin defect is a genetic and/or developmental disorder affecting the skin.
75. The agent for use according to any one of the preceding items, wherein the genetic and/or developmental disorder affecting the skin is epidermolysis bullosa, or genodermatosis, such as pemphigus genodermatosis.
76. The agent for use according to any one of the preceding items, wherein the epidermolysis bullosa is selected from the group consisting of epidermolysis bullosa simplex, junctional epidermolysis bullosa, dystrophic epidermolysis bullosa, recessive dystrophic epidermolysis bullosa, syndromic epidermolysis bullosa and epidermolysis bullosa.
77. The agent for use according to any one of the preceding items, wherein the skin defect is an external defect.
78. The agent for use according to any one of the preceding items, wherein the skin defect is an open defect.
79. The agent for use according to any one of the preceding items, wherein the skin defect is an inflamed wound.
80. The agent for use according to any one of the preceding items, wherein the skin defect is infected. 81. The agent for use according to any one of the preceding items, wherein the skin defect is an injury or trauma to internal or external tissue, such as injury or trauma to the epidermis and/or dermis of the skin.
82. The agent for use according to any one of the preceding items, wherein the skin defect is an acute defect or a chronic defect.
83. The agent for use according to any one of the preceding items, wherein the skin defect is full thickness skin defect.
84. The agent for use according to any one of the preceding items, wherein the wound is an acute wound or a chronic wound.
85. The agent for use according to any one of the preceding items, wherein the acute wound is an incision, laceration, abrasion graze or burn, a puncture wound, a penetration wound or a wound due to dermatologic diseases such as psoriasis, acne and eczema.
86. The agent for use according to any one of the preceding items, wherein the chronic wound is a venous ulcer, a diabetic ulcer, a pressure ulcer, corneal ulcer, digestive ulcer or wounds as a result of ischemia and radiation poisoning.
87. The agent for use according to any one of the preceding items, wherein the wound is a non-pressure chronic ulcer, such as a non-pressure chronic ulcer of lower limb.
88. The agent for use according to any one of the preceding items, wherein the agent is administered topically, orally, subcutaneously, or systemically.
89. The agent according to any one of the preceding items for use in the treatment of a disease of the skin, such as injury of external cause.
90. The agent for use according to any one of the preceding items, wherein the disease of the skin is selected from the group consisting of: a. a skin disorder involving specific cutaneous structures, such as disorders of cutaneous blood and lymphatic vessels, such as dermatoses resulting from vascular insufficiency, such as ischaemic ulceration of the skin, and b. a skin disorder associated with pregnancy, the neonatal period, and infancy, such as dermatoses of infancy, such as erythrodermas of infancy (Leiner’s disease).
91. The agent for use according to any one of the preceding items, wherein the injury of external cause is selected from the group consisting of: a. injury to the head, such as intracranial injury, such as diffuse brain injury, such as cerebral contusion, and b. injury to unspecified part of trunk, limb, or body region, such as other injuries of spine or trunk, such as injury of the spinal cord.
92. The agent for use according to any one of the preceding items, wherein the subject is suffering from diabetes mellitus.
93. The agent for use according to any one of the preceding items, wherein the subject is suffering from stoma.
94. The agent for use according to any one of the preceding items, wherein the subject is a mammal.
95. The agent for use according to item 94, wherein the mammal is a human.
96. A method for treatment and/or regeneration of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent according to any one of the preceding items to an subject in need thereof.
97. A method for treating and/or promoting healing of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent according to any one of the preceding items to an subject in need thereof. 98. A method for regeneration of skin at the site of a skin defect and/or for reducing scar formation resulting from healing of a skin defect in a subject, said method comprising administering an agent according to any one of the preceding items.
99. Use of an agent according to any one of the preceding items for the manufacture of a medicament for the treatment and/or regeneration of a skin defect in a subject.
100. Use of an agent according to any one of the preceding items for the manufacture of a medicament for the treating and/or promoting healing of a tissue defect in a subject.
101. Use of an agent according to any one of the preceding items for the manufacture of a medicament for the treatment of a skin defect in a subject.
102. Use of an agent according to any one of the preceding items for reducing scar formation resulting from healing of a skin defect in a subject.
103. Use of an agent according to any one of the preceding items for promoting the healing of a skin defect in a subject.
104. Use of an agent according to any one of the preceding items for promoting vascularisation in a healing tissue, such as skin healing from a skin defect, such as a wound.
105. Use of an agent according to any one of the preceding items in combination with a tissue meshing technique, for promoting healing of a skin defect, such as a wound.
106. Use of an agent according to any one of the preceding items for promoting grafting of skin.
107. A method for inserting a stent in a subject, said method comprising administering an agent according to any one of the preceding items. 108. The method according to item 107, wherein the stent is coated with the agent according to any one of the preceding items.
109. The use according to any one of the preceding items, wherein the agent is part of a cosmetic composition.
Examples
Example 1: Polypeptides of the disclosure stimulates endothelial and vascular smooth muscle cell proliferation Methods
Cell proliferation was evaluated with BrdU Cell Proliferation Elisa Kit (Abeam, UK). After incubation at 37 °C overnight with respective complete medium, for NRP-1 knock down-relative experiments, HUVECs and HCASMCs on 96-well plates were transfected with siRNA/ siRNA-NC directly; for test polypeptide stimulations, HUVECs and HCASMCs were starved with low-serum condition for additional 24 hours and 48 hours before treatments. At 32 hours after transfection or polypeptide stimulations, cells were cultured with BrdU reagent for another 16 hours to determine cell proliferation. Subsequent steps were performed as manufacturer’s instructions and data were read by Wallac 1420 Victor 2 (Perkin Elmer, USA).
For stimulation of endothelial cell proliferation, human umbilical cord endothelial cells were first treated with a scramble non-coding siRNA (NC) or siRNA for NRP-1 , subsequently they were treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and DNA synthesis analyzed by measuring the uptake of BrdU.
Results
The results are shown in Figure 1 for FOL26 and Figure 18 for FOL56. Both test polypeptides induced a significant increase in proliferation of the HUVECs and HCASMCs.
FOL26 stimulation of endothelial cell proliferation is NRP-1 dependent (Figure 10). Conclusion
The polypeptides of the disclosure stimulates endothelial and vascular smooth muscle cell proliferation.
Example 2 Polypeptides of the disclosure induces tube formation in HUVECs Methods
Cells were transfected for 48 hours or performed with 0.5% supplement starvation for 24 hours on 6-well plates as described above and dissociated by Accutase (Gibco, USA) to obtain cell suspension. Geltrex reduced growth factor basement membrane matrix ((Invitrogen, CA, USA) was thawed at 4°C overnight and added into a 96-well plate (50 pl/well) next. The coated 96-well plate was placed in the incubator more than 30 mins for further use and the cell suspension was seeded into it at a density of 1.5 x 104/well. For NRP-1 silencing experiments, cells were cultured with complete medium. For polypeptide treatment experiments, cells were stimulated with complete medium plus the different concentration of polypeptide. For NRP-1 rescued experiments, cells transfected with siRNA were cultured with complete medium and NRP-1 stimulations. After a 24-hour incubation, images were recorded by an inverted microscope (Nikon, Eclipse TE2000-U, Japan) and the sum length of master segments of vessels in each image were quantified using Angiogenesis Analyzer plug-in ImageJ software program (pixel/unit). Data were analyzed with photographs from 8-14 wells per condition.
Human umbilical cord endothelial cells were first treated with a scramble non-coding siRNA (NC) or siRNA for NRP-1 , subsequently treated with FOL26 in concentrations of 10 nM, 100 nM, and 1000 nM, and endothelial tube formation and the total master segemnts were measured using ImageJ. gene expression of PECAM-1 (CD31) analyzed.
Results
The results are shown in Figure 2 for FOL26 and in Figure 19 for FOL56. The test polypeptides significantly increased tube formation in the HUVECs. The polypeptides also increased expression of PECAM-1. Figure 12 shows that FOL26 increased tube formation compared to control in a NRP-1 dependent way.
Conclusion
Polypeptides of the disclosure induces tube formation in HUVECs. Example 3: Polypeptides of the disclosure induces wound healing in a scratch wound assay on HUVECs and HCASMCs
Methods
Ibidi Culture-Insert 2 well (Ibidi, Nanterre, France) was used to assess the ability of wound healing in vitro. Briefly, one silicone culture-insert (cut 2 well inlet) was placed in the one well of 24-well plate and 3.5x104 cells were seeded into each inlet with 70 pl complete medium. For NRP-1 silencing and NRP-1 rescued experiments, cells were transfected with siRNA/siRNA-NC for 48 hours before seeding. For test polypeptide treatment experiments, cells were starved with respective low-serum medium for 24 hours and 48 hours after adhering in the inlet. The cell-free gap between two inlets was created by removing the culture-insert, followed by washing with DPBS gently to remove suspended cells. Subsequently, cells in respective medium containing 2% supplements with/without test polypeptide were recorded at appropriate time-points. Images were taken using an inverted microscope (Nikon, Eclipse TE2000-U, Japan) and the percentage of wound closure was calculated with MRI-Wound-Healing plug-in ImageJ software program: wound closure % =1 - (wound area at Tx I wound area at To)] * 100 (To is the wound area at 0 h, and Tx is the wound area at corresponding hours after creating the gap (pixel/unit). Data were quantified with pictures from 8-20 wells per group.
Results
The results are shown in Figures 3 and 4 for FOL26 and Figure 20 and 21 for FOL56. Both polypeptides stimulated wound closure in both HUVECs and HCASMCs.
Conclusion
Polypeptides of the disclosure induces wound healing in a scratch wound assay on HUVECs and HCASMCs.
Example 4: Polypeptides of the disclosure prevents apoptosis in HUVECs and HCASMCs
Methods
Human Umbilical Vein Endothelial Cells (HUVECs) and Human Coronary Artery Smooth Muscle Cells (HCASMCs) were purchased from Thermo Fisher. Cells were grown in Medium 200 containing 2% Low Serum Growth Supplement and Medium 231 with 5% Smooth Muscle Growth Supplement (Invitrogen, CA, USA) respectively with addition of Antibiotic-Antimycotic (1%, Invitrogen, CA, USA). Test polypeptide and sFasL (PeproTech, NJ, USA)) were dissolved in the respective complete medium.
Caspase-Gio 3/7 Assay (Promega, USA) was performed to evaluate cell apoptosis. HUVECs and HCASMCs were seeded into 96-well plates at a density of 3-5 x 103 per well. For additional NRP-1 on cells transfected siRNA, NRP-1 was added 24 hours after transfection and the mixture of Caspase-Gio 3/7 was added 48 hours after polypeptide stimulations. Subsequent steps were performed as manufacturer’s instructions using the FOL26 and FOL56 as test polypeptides. Data were read by Wallac 1420 Victor 2 (Perkin Elmer, USA).
Results
The results are shown in Figure 5 for FOL26 and Figure 22 for FOL56 on both HUVECs and HCASMCs. Both polypeptides reduced sFasL-induced apoptosis in the cell lines.
Conclusion
Polypeptides of the disclosure prevents apoptosis in HUVECs and HCASMCs.
Example 5: Effect on gene expression of polypeptides of the disclosure in HUVECs and HCASMCs
Methods
Cells on 6-well plates were washed with cold DPBS (Gibco, USA) and homogenized in Trizol reagent (Invitrogen, CA, USA). Total RNA was extracted using PureLink RNA Mini Kit (Invitrogen, CA, USA), followed by nucleic acid quantification with NanoDrop 2000c (Thermo Fisher Scientific, USA). To determine mRNA expression of target genes, real-time qPCR was performed using by KAPA SYBR FAST One-Step qRT- PCR Master Mix Kit (KAPA Biosystems, USA) on LightCycler 480 system (Roche, Mannheim, Germany). All primers were purchased from miScript Primer Assays (Qiagen, Valencia, CA) and GAPDH mRNA expression of each sample was used for normalization. Cells on 6-well plates were washed with cold DPBS and lysed with the mixture of lysis buffer and protease inhibitors on ice, then centrifuged at 15,000g for 20 min at 4 °C. Supernatant was performed to measure the protein concentration using BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Protein sample was loaded onto SDS-PAGE gels and separated with electrophoresis, followed by transferred to PVDF membranes (Millipore, USA). Membranes were blocked with 5% nonfat milk. Primary antibodies (Abeam, UK) and secondary antibodies were performed to incubate with membranes at 4 °C overnight and at room temperature for 1 hour respectively.
Results
The results are shown in Figures 6-9 for FOL26 and Figures 23-26 for FOL56. Table 1 shows various important biological mediators in a wound healing process.
Table 1 : Growth Factors and Cytokines Affecting Various Steps in Wound Healing.
Figure imgf000055_0001
Conclusion
Polypeptides of the disclosure induces a milieu inducing wound healing by TNF and IL- 6 expression likely to promote fibroblast migration and proliferation, collagen secretion, angiogenesis, and induce correct wound healing structure by inducing cell remodelling through MMP expression.
Example 6: Healing of wounds in combination with tissue meshing.
Methods
It is contemplated that a study of the effect of the polypeptide the disclosure on wound healing in combination with tissue meshing could be carried out as follows: A PVDF mesh material surface modified by FOL-peptides +/- NRP siRNA and vehicle will be developed. Three different FOL-peptide concentrations to be bound to the provided active sites of the mesh surfaces. Mice are randomized to five groups. Bilateral of the abdominal midline one of the five different meshes is to be implanted subcutaneously after a wound is induced in each animal (FOL-peptides in three concentrations, FOL- peptide + siNRP-1 , and vehicle). Immunohistochemistry of VEGF, c-MET, NRP-1 , TNF, IL-6, and MMP 7, 21 and 90 days after mesh implantation will be analyzed. The collagen type l/lll ratio will be analyzed by cross polarization microscopy to determine the quality of mesh integration.
Results
It is contemplated that tissue meshes having a coating of a polypeptide of the disclosure, and/or having a polypeptide dispersed within it will effect faster and/or more complete healing of a wound, compared to tissue meshes without the polypeptide of the disclosure.
Example 7: Effect of test polypeptides in acute and pathological wound healing Methods
The pharmacological profile of the test polypeptides has been tested using two models, with full-thickness human abdominal skin cultured ex-vivo under normal conditions (WCM medium only, 37 °C, 5% CO2,) representing “acute wound healing” and under extreme culture conditions (VCM, high glucose, H2O2, 37 °C, 5% CO2, 5% O2) representing “pathological wound healing” (Figure 13) (Monasterium Labs, Prof. Ralf Paus, Munster, Germany).
Results
The results are shown in Figure 14. Promotion of skin re-epithelization in the acute wound healing model was observed after once topical application of FOL-005, 100nM; FOL-056, 100nM and 300nM (Figures 14 and 15), but not with FGL-005, 300nM and FOL-026 100nM and 300nM. The full wound closure was also confirmed with FOL-005, 100nM as outlined in Table 2.
Furthermore, promotion of skin re-epithelization in the pathological wound healing model was observed after topical application of FOL-005, 100nM; and FOL-026 100nM and 300nM, FOL-056, 100nM and 300nM (Figure 14 and 16), but not with FOL-005, 300nM.
Table 2: Number of wound closure observed in acute wound healing-pooled data at 72 hours timer point*. *No full wound closure was observed in the pathological punches. The wound closure in day 6 time point from the first experiment were excluded from this table.
Figure imgf000057_0001
Additionally, increased proliferation of epidermal keratinocytes (Ki67+ cells) and decreased apoptosis of keratinocytes (TUNEL+ cells) was observed with FOL-005 300nM (Figure 17). There are strong indications that the OPN-derived FOL peptides have wound healing promoting properties under acute and pathological conditions. Both the single and the repeated topical applications of FOL peptide on human skin grafts with partial thickness wound under pathological conditions increase the epithelial wound area. Additionally, the repeated doses of FOL peptide seem to “protect” the cellular integrity of the tissue due to the increased proliferation and decreased apoptosis of keratinocytes adjacent to the wound bed under pathological conditions.
Conclusion
Polypeptides of the disclosure induce full wound closure.
Example 8: Effect of FOL-026 peptide on endothelial cell function and gene expression Method
The impact of FOL-026 peptide on human umbilical vascular endothelial cells (HUVECs) proliferation (figure 27a) and apoptosis (figure 27c) induced with low-oxygen condition was determined by BrdU uptake (n=8-9 per group) and active caspase-3/- 7 (n=7-10 per group) separately. The ROS level (figure27b) activated by 50ug/ml oxidized Low-density Lipoprotein (oxLDL) for 2 hours in cells pre-incubated with FOL- 026 peptides was assessed using H2O2 measurement (n=4-8 per group). Representative pictures of endothelial migration into an in vitro scratch injury were shown (figure 28a) and the wound closure rate (figure 28b) in HUVECs stimulated with increasing concentration of FOL-026 peptide was quantified by image J software (n=8- 9 per group). Scale bar =150um. Data are presented as means ± SEM and acquired from 3 to 4 independent replicate tests. *P<0.05, **P<0.01 , ****P<0.0001 by one-way ANOVA and Dunnett’s post-hoc test.
Results The results shown in figure 27a indicates that the proliferation of on human umbilical vascular endothelial cells increases with the addition of the FOL-026 peptide. FOL-026 also alleviates the effect of low oxygen induced apoptosis in HUVECs and can reduce the amount of ROS levels induced by oxLDL (figure 27c and 27b).
Figure 2a and 2b shows that addition of FOL-026 accelerates HUVEC wound closure. Conclusions
The FOL-026 peptide has a stimulatory effect on the proliferation of endothelial cells.
Example 9: Effect of FOL-026 peptide on angiogenesis in vitro and vivo Method
Representative images of tube formation in HUVECs treated with FOL-026 peptides are shown in figure 29a. Quantified measurement of total length (figure 29b), total master segments length (figure 29c), total branching length (figure 29d) and total segments length (figure 29e) were performed to evaluate angiogenesis in vitro (n=7 per group for figure 29 b-e).
The quantification of vessel density was assessed with CD31+ positive area (figure 30a, n=13 per group).
Western blot analysis of phosphorylated AKT (pAKT-T308), AKT, phosphorylated ERK1/2 (p-ERK1/2) and ERK1/2 in endothelial cells stimulated with FOL-26 peptides for 48 hours (figure 30b). Quantification of phosphorylation intensities normalized to respective total expressions (figure 30c-d, n=3 per group).
Data are presented as means ± SEM and acquired from 3 to 4 independent replicate tests. *P<0.05, **P<0.01, ***P<0.001 , ****P<0.0001 by one-way ANOVA and Dunnett’s post-hoc test.
Results
Figures 30 a-d shows the positive effect FOL-026 has on tube formation. It demonstrates the increased vessel density effect of FOL-026 and how FOL-026 induces upregulation of angiogenesis related genes.
Conclusion
The FOL-026 peptide affects angiogenesis both in vitro and in vivo.
Example 10: Effect of NR P-1 knock-down on endothelial cell function induced with
FOL-026 peptide
Method The efficiency of small interfering RNA transfection for 48 hours in HUVECs was confirmed by qRT-PCR (n=4) (figure 31a). Effect of FOL-026 peptide stimulation on cell proliferation (figure 31b) and cell apoptosis at 0.1% oxygen (figure 31c) in HUVECs transfected with siRNA-NC or siNRP-1 was evaluated by BrdU incorporation (n=4-7 per group) and active caspase-3/-7 ((n=4-7 per group). Representative pictures of endothelial cells at 0 and 5 hours after scratch wounding are shown in 32a. The wound closure rate (figure 32b) of siRNA-NC/ siNRP-1 -transfected HUVECs stimulated with increasing concentration of FOL-026 peptide was quantified by image J software (n=9- 12 per group). Scale bar =150um. Data are presented as means ± SEM and acquired from 3 to 4 independent replicate tests. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by Student’s t test for A, two-way ANOVA followed with Sidak’s and Dunnett's multiple comparisons test for B, C and E.
Results
Figures 31a-c shows that reducing the NRP-1 expression blocks the effect of FOL-026 Figures 32a-b show that FOL-026 has a positive effect on wound closure in siRNA-NC/ siNRP-1-transfected HUVECs.
Conclusion
FOL-026 stimulates wound closure in HUVECs by binding to NRP-1
Example 11: Effect of NRP-1 knock-down on tube formation induced with FOL-026 peptide Method
Tube formation with rising dose of FOL-026 peptide treatment in siRNA-NC or siNRP-1 group (figure 33a) and total length (figure 33b), total master segments length (figure 33c), total branching length (figure 33d) and total segments length (figure 33e) was calculated using Angiogenesis Analyzer plug-in Imaged (n=7-12 per group). Scale bar =150um. Data are presented as means ± SEM and acquired from 3 to 4 independent replicate tests. *P<0.05, **P<0.01 , ***P<0.001, ****P<0.0001 by two-way ANOVA followed with Sidak’s and Dunnett's multiple comparisons test.
Results and conclusion
The FOL-026 peptide induce tube formation in siRNA-NC and siNRP-1. Example 12: Effect of FOL-026 peptide on smooth muscle cell function
Method
The effect of FOL-026 peptide treatment on smooth muscle cell proliferation (figure 34a) and apoptosis (figure 34c) induced with low-oxygen condition was evaluated with the uptake of BrdU (n=10 per group) and active caspase-3/-7 (n=6-8 per group) separately. H2O2 measurement (figure 34b) of smooth muscle cells pre-incubated with FOL-26 peptides after 50ug/ml oxidized Low-density Lipoprotein (oxLDL) stimulation for 2 hours (n=6-7 per group). Representative images of scratch wound assay in vitro were shown (figure 35a) and the ability of wound healing (figure 35b) in human coronary smooth muscle cells (HCASMCs) treated with FOL-026 peptides was measured by image J software (n=10 per group). Scale bar =150um. Data are presented as means ± SEM and acquired from 3 to 4 independent replicate tests. *P<0.05, **P<0.01, ***P<0.001, ****P< 0.0001 by one-way ANOVA and Dunnett’s post- hoc test.
Results and conclusion
FOL-026 induces smooth muscle cell proliferation, inhibits apoptosis, reduces ROS formation and accelerates wound closure
Example 13: Effect of NRP-1 knock-down on smooth muscle cell function induced with FOL-026 peptide Method qRT-PCR analysis of NRP-1 mRNA expression in HCASMCs with small interfering RNA transfection for 48 hours (n=4) (figure 36a). The assessment of BrdU incorporation (figure 36b, n=10-11) and caspase-3/-7 activation (figure 36c, n=3-10) in siRNA-NC/ siNRP-1-transfected HUVECs treated with indicated concentration of FOL- 026 peptide. Representative images of smooth muscle cells at 0 and 5 hours after scratch wounding were shown (figure 37a). The wound closure rate (figure 37b) of HCASMCs in siRNA-NC and siNRP-1 group stimulated with increasing concentration of FOL-026 peptide was quantified by image J software (n=9-12 per group). Scale bar =150um. Data are presented as means ± SEM and acquired from 3 to 4 independent replicate tests. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by Student’s t test for A, two-way ANOVA followed with Sidak’s and Dunnett's multiple comparisons test. Results and conclusion
FOL-026 has a positive effect on wound closure in siRNA-NC/ si NRP-1 -transfected HUVECs. Example 14: The FOL-005 peptide stimulates endothelial and smooth muscle cell proliferation and wound healing ability
Methods
The endothelial cell proliferation (figure 38a) was assessed with BrdU uptake in human umbilical vascular endothelial cells (HUVECs) stimulated with increasing concentration of FOL-005 peptide for 48 hours (n=7-10 per group).
The wound closure rate (figure 38c) in HUVECs with indicated dose of FOL-005 treatment was quantified by image J software (n=12-14 per group) and representative images of at 0 and 6 hours after scratch wounding were displayed (figure 38b).
The effect of FOL-005 peptide on smooth muscle cell proliferation (figure 39a) was determined with BrdU incorporation in peptide-treated human coronary smooth muscle cells (HCASMCs). Scratch wound assay (figure 39b) of HCASMCs incubated with FOL-005 peptides for 5 hours was measured by image J software (n=10-12 per group) and representative pictures of smooth muscle cell migration into an in vitro scratch injury were shown (figure 39c). Scale bar =150um. Data are presented as means ± SEM and acquired from 3 to 4 independent replicate tests, p values were displayed as figure and calculated with One-way ANOVA and Dunnett’s post-hoc test.
Results
The results shown in figure 38a indicates that the proliferation of human umbilical vascular endothelial cells (HUVECs) increases with the addition of the FOL-005 peptide. The scratch wound assay in figure 38b and 38c indicates that the FOL-005 peptide accelerate HUVEC wound closure.
The results shown in figure 39a indicates that the proliferation of human coronary smooth muscle cells (HCASMCs) increases with the addition of the FOL-005 peptide. The scratch wound assay in figure 39b and 39c indicates that the FOL-005 peptide accelerate HCASMC wound closure.
Conclusion
The FOL-005 peptide has a positive effect on endothelial and smooth muscle cell proliferation and wound healing ability.
Example 15: The effect of FOL-005 peptide on tube formation Methods
Tube formation in human umbilical vascular endothelial cells (HUVECs) treated with FOL-005 peptides are shown in figure 40a. Quantified measurement of total master segments length figure 40b was used to evaluate angiogenesis in vitro (n=7 per group). Scale bar =150um. Data are presented as means ± SEM and acquired from 3 to 4 independent replicate tests, p values were displayed as figure and calculated with Oneway ANOVA and Dunnett’s post-hoc test in B, two-way ANOVA followed with Sidak’s and Dunnett's multiple comparisons test for D.
Results
The results shown in figure 40a and 40b indicates that addition of the FOL-005 peptide increases the tube formation in human umbilical vascular endothelial cells (HUVECs) Conclusion FOL-005 peptide addition has a stimulatory effect on endothelial tube formation.
Sequence overview
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
References
Gottrup F., A specialized wound-healing center concept: importance of a multidisciplinary department structure and surgical treatment facilities in the treatment of chronic wounds. The American Journal of Surgery. 2004;187(5):S38-S43.
Kirker K. R., James G. A., In vitro studies evaluating the effects of biofilms on woundhealing cells: a review. APMIS. 2017; 125(4): 344-52.
Frykberg R. G., Banks J., Challenges in the Treatment of Chronic Wounds. Adv Wound Care (New Rochelle). 2015;4(9):560-82.

Claims

Claims
1. An agent comprising: a) a peptide selected from the group consisting of:
(i) a peptide comprising or consisting of an amino acid sequence of the general formula:
X5X6SX7X8YGLR (SEQ ID NO: 1) wherein: X5 is D or G; X6 is I or G; X7 is V or L; X8 is V or A;
(ii) a peptide comprising or consisting of an amino acid sequence of the general formula:
X14LX15YGIK (SEQ ID NO: 105) wherein: X14 is E or G; X15 is S or T;
(iii) a peptide comprising or consisting of an amino acid sequence selected from the group consisting of: VDTYDGDISVVYGL (SEQ ID NO: 34), VDTYDGDISVVYG (SEQ ID NO: 35),
VDTYDGDISVVY (SEQ ID NO: 36), VDTYDGDISVV (SEQ ID NO: 37), VDTYDGDISV (SEQ ID NO: 38), VDTYDGDIS (SEQ ID NO: 39), VDTYDGRGDSWYGLR (SEQ ID NO: 40), VDVPNGDISLAYGL (SEQ ID NO: 41), VDVPNGDISLAYG (SEQ ID NO: 42), VDVPNGDISLA (SEQ ID NO: 43), VDVPNGDIS (SEQ ID NO: 44), GDPNDGRGDSVVYGLR (SEQ ID NO: 45), LDGLVRAYDNISPVG (SEQ ID NO: 46), GDPNGDISVVYGLR (SEQ ID NO: 47) VDVPNGDISLAYRLR (SEQ ID NO: 48), VDVPEGDISLAYRLR (SEQ ID NO: 49), V(beta-D)TYDGDISVVYGLR (SEQ ID NO: 50), VDTY(beta-D)GDISVVYGLR (SEQ ID NO: 51), VDTYDG(beta-D)ISVVYGLR (SEQ ID NO: 52); CLAEIDSC (Cyclic) (SEQ ID NO: 130), CFKPLAEIDSIECSYGIK (Cyclic) (SEQ ID NO: 131), Cyclic CFKPLAEIDSIEC (SEQ ID NO: 132), KPLAEIDSIELSYGI (SEQ ID NO: 133), KPLAEIDSIELSYG (SEQ ID NO: 134), KPLAEIDSIELSY (SEQ ID NO: 135), KPLAEIDSIELS (SEQ ID NO: 136), KPLAEIDSIEL (SEQ ID NO: 137), and KPLAEIDSIE (SEQ ID NO: 138); b) a polynucleotide encoding upon expression, the peptide of a); c) a vector comprising the polynucleotide of b); or d) a cell comprising the polynucleotide of b), or the vector of c) for use in treating and/or promoting healing of a tissue defect. The agent for use according to claim 1 , wherein the peptide comprises an amino acid sequence of the general formula:
VDX2X3X4GX5X6SX7X8YGLR (SEQ ID NO: 2) wherein: X2 is T or V; X3 is Y or P;
X4 is D or N; Xs is D or G; Xs is I or G;
X7 is V or L; and Xs is V or A. The agent for use according to any one of the preceding claims, wherein the peptide comprises an amino acid sequence of the general formula: VDTYX4GX5X6SX7X8YGLR (SEQ ID NO: 3) wherein:
X4 is D or N; Xs is D or G;
Xs is I or G;
X7 is V or L; and Xs is V or A. The agent for use according to any one of the preceding claims, wherein the peptide comprises an amino acid sequence of the general formula: VDTYDGZ7Z8SZioZnYGLR (SEQ ID NO: 4) wherein:
X5 is D or G;
X5 is I or G;
X7 is V or L; and Xs is V or A. The agent for use according to any one of the preceding claims, wherein the peptide comprises an amino acid sequence of the general formula: VDTYDGZ7Z8SVVYGLR (SEQ ID NO: 5) wherein:
X5 is D or G; and Xs is I or G; The agent for use according to claim 1 , wherein the peptide comprises an amino acid sequence of the general formula: KX9LAX10X11X12X13IX14LX15YGIK (SEQ ID NO: 106) wherein:
X9 is C, P or G;
X10 is E or G;
Xn is C, D or I;
X12 is D, I, S or G;
X13 is S, D or G;
X14 is E or G; and X15 is S or T. The agent for use according to claim 1 , wherein the peptide comprises an amino acid sequence of the general formula: KX9LAX10X11X12X13IX14LSYGIK (SEQ ID NO: 107) wherein: Xg is C, P or G; X10 is E or G;
X11 is C, I or absent;
X12 is D, G or absent;
X13 is S, G or absent; and X14 is E or G.
8. The agent for use according to claim 1 , wherein the peptide comprises an amino acid sequence of the general formula:
KX9LAX10IX14LSYGI K (SEQ ID NO: 108) wherein: X9 is C, P or G; X is E or G; and X14 is E or G.
9. The agent for use according to claim 1 , wherein the peptide comprises the amino acid sequence IELSYGIK (SEQ ID NO: 109).
10. The agent according to claim 1 , with the proviso that if X14 is T, the peptide comprises no more than 25 amino acid residues.
11. The agent for use according to claim 1 , wherein the peptide comprises or consists of an amino acid sequence selected from the group consisting of: VDTYDGDISVVYGLR (SEQ ID NO: 7)
VDTYDGGISVVYGLR (SEQ ID NO: 6),
DTYDGDISVVYGLR (SEQ ID NO: 8), TYDGDISVVYGLRS (SEQ ID NO: 9), TYDGDISVVYGLR (SEQ ID NO: 10), YDGDISVVYGLRS (SEQ ID NO: 11), YDGDISVVYGLR (SEQ ID NO: 12), DGDISVVYGLRS (SEQ ID NO: 13), DGDISVVYGLR (SEQ ID NO: 14), GDISVVYGLRS (SEQ ID NO: 15), GDISVVYGLR (SEQ ID NO: 16), DISVVYGLRS (SEQ ID NO: 17), DISVVYGLR (SEQ ID NO: 18), VDVPNGDISLAYGLR (SEQ ID NO: 19), DVPNGDISLAYGLRS (SEQ ID NO: 20), DVPNGDISLAYGLR (SEQ ID NO: 21), VPNGDISLAYGLRS (SEQ ID NO: 22), VPNGDISLAYGLR (SEQ ID NO: 23), PNGDISLAYGLRS (SEQ ID NO: 24), PNGDISLAYGLR (SEQ ID NO: 25), NGDISLAYGLRS (SEQ ID NO: 26), NGDISLAYGLR (SEQ ID NO: 27), GDISLAYGLRS (SEQ ID NO: 28), GDISLAYGLR (SEQ ID NO: 29), DISLAYGLRS (SEQ ID NO: 30), DISLAYGLR (SEQ ID NO: 31), VDTYDGDGSVVYGLR (SEQ ID NO: 32), VDVPEGDISLAYGLR (SEQ ID NO: 33). AEIDSIELSYGIK (SEQ ID NO: 110), KPLAEIDSIELSYGIK (SEQ ID NO: 111), KCLAECDSIELSYGIK (Cyclic) (SEQ ID NO: 112), KPLAEDISIELSYGIK (SEQ ID NO: 113), KPLAEISDIELSYGIK (SEQ ID NO: 114), KPLAEIGDIELSYGIK (SEQ ID NO: 115), KPLAEGDIELSYGIK (SEQ ID NO: 116), KPLAEIELSYGIK (SEQ ID NO: 117), KPLAEIDSIELTYGIK (SEQ ID NO: 118), KPLAEIDGIELSYGIK (SEQ ID NO: 119), KPLAEIDGIELTYGIK (SEQ ID NO: 120), KPLAEIGSIELSYGIK (SEQ ID NO: 121), KGLAEIDSIELSYGIK (SEQ ID NO: 122), KPLAGIDSIGLSYGIK (SEQ ID NO: 123), Cyclic KCLAEIDSCELSYGIK (SEQ ID NO: 124), LAEIDSIELSYGIK (SEQ ID NO: 125), EIDSIELSYGIK (SEQ ID NO: 126), IDSIELSYGIK (SEQ ID NO: 127), DSIELSYGIK (SEQ ID NO: 128), SIELSYGIK (SEQ ID NO: 129), and IELSYGIK (SEQ ID NO: 109). The agent for us eaccording to claim 1 , wherein the peptide comprises or consists of the amino acid sequence VDTYDGDISWYGLR (SEQ ID NO: 7). The agent for use according to claim 1 , wherein the peptide comprises or consists of the amino acid sequence VDTYDGGISVVYGLR (SEQ ID NO: 6). The agent for use according to claim 1 , wherein the peptide comprises or consists of the amino acid sequence AEIDSIELSYGIK (SEQ ID NO: 110). The agent for use according to claim 1 , wherein the peptide comprises or consists of the amino acid sequence KPLAEIDSIELSYGIK (SEQ ID NO: 111). The agent for use according to any one of the preceding claims, wherein the peptide comprises no more than 85, such as no more than 80, such as no more than 75, such as no more than 70, such as no more than 65, such as no more than 60, such as nor more than 55, such as no more than 50, such as no more than 55, such as no more than 40 amino acids, such as no more than 35, such as no more than 30, such as no more than 28, such as no more than 26, such as no more than 24, such as no more than 22, such as no more than 20, such as no more than 19, such as no more than 18, such as no more than 17, such as no more than 16, such as no more than 15, such as no more than 14, such as no more than 13, such as no more than 12, such as no more than 11 , such as no more than 10 amino acids. The agent for use according to any one of the preceding claims, wherein the peptide comprises at least 2 additional amino acids, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 15 or such as at least 20 amino acids conjugated to the N- or C-terminus of the peptide. 18. The agent for use according to any one of the preceding claims, wherein the agent is non-naturally occurring.
19. The agent for use according to any one of the preceding claims, wherein the agent is conjugated to a moiety.
20. The agent for use according to claim 19, wherein the moiety is selected from the group consisting of polyethylene glycol (PEG), monosaccharides, fluorophores, chromophores, radioactive compounds, and cell-penetrating peptides.
21. The agent for use according to any one of the preceding claims, wherein the agent is further modified such as by glycosylation, PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.
22. The agent for use according to any one of the preceding claims, wherein the agent comprises or consists of a tandem repeat comprising two or more repeat units.
23. The agent for use according to claim 22, wherein the repeat unit comprises or consists of the amino acid sequence of any one or more of the sequences as described in the preceding claims.
24. The agent for use according to any of the preceding claims, wherein the agent is fused to another polypeptide.
25. The agent for use according to claim 24, wherein the said polypeptide is selected from the group consisting of glutathione-S-transferase (GST) and protein A.
26. The agent for use according to any of the preceding claims, wherein the agent is fused to a tag.
27. The agent for use according to claim 26, wherein the tag is an oligo-histidine tag.
28. The agent for use according to any of the preceding claims, wherein the agent is cyclic. The agent for use according to any of the preceding claims, wherein the agent is capable of forming at least one intramolecular cysteine bridge. The agent for use according to any one of the preceding claims, wherein the agent is a variant of the peptide, wherein the variant comprises or consists of a sequence wherein any one amino acid has been altered for another proteinogenic or non- proteinogenic amino acid, with the proviso that no more than five amino acids are so altered. The agent for use according to claim 30, wherein the variant comprises or consists of a sequence wherein no more than five amino acids are altered for another proteinogenic or non-proteinogenic amino acid, such as no more than 4 amino acids, such as no more than 3 amino acids, such as no more than 2 amino acids, such as no more than 1 amino acid is altered. The agent for use according to any one of the preceding claims, wherein one or more amino acids are conservatively substituted. The agent for use according to any one of the preceding claims, wherein the peptide comprises or consists of one or more additional amino acids, inserted at the N- and/or C-terminus and/or internally within the sequence. The agent for use according to any one of the preceding claims, wherein the peptide has one additional amino acid. The agent for use according to any of the preceding claims, wherein the agent further comprises a detectable moiety. The agent for use according to any one of the preceding claims, wherein the agent is in a composition. The agent for use according to claim 36, wherein the composition is a pharmaceutical composition. The agent for use according to claim 36, wherein the composition is a cosmetic composition. The agent for use according to any one of claims 36 to 38, wherein the composition is a coating composition. The agent for use according to any one of the preceding claims, wherein the tissue defect is a skin defect. The agent for use according to claim 40, wherein the skin defect is a wound, such as an open wound. The agent for use according to claim 41 , wherein the wound is a hard-to-heal wound, such as a chronic wound. The agent for use according to claim 41 , wherein the wound is a surgical wound. The agent for use according to claim 41 , wherein the wound is associated with venous insufficiency, arterial insufficiency, diabetic complications, sickle cell anaemia, or is a pressure-related wound. The agent for use according to claim 44, wherein the diabetic complication is due to diabetes mellitus, such as Type 1 diabetes mellitus, such as Type 1 diabetes mellitus with foot ulcer and/or Type 1 diabetes mellitus with other skin ulcer, or Type 2 diabetes mellitus, such as Type 2 diabetes mellitus with foot ulcer and/or Type 2 diabetes mellitus with other skin ulcer. The agent for use according to claim 45, wherein the diabetes mellitus presents with gangrene. The agent for use according to claim 45, wherein the diabetes mellitus is associated with a disease of the connective tissue, such as fibrosis. The agent for use according to claim 41 , wherein the wound is a dermal ulcer, or a skin defect due to vascular insufficiency or diabetic affection of the blood vessels. 49. The agent for use according to any one of claims 1 to 38, wherein the tissue defect is a diabetic complication.
50. The agent for use according to claim 49, wherein the diabetic complication is a diabetes mellitus related eye condition.
51. The agent for use according to claim 49, wherein the diabetic complication is retinopathy.
52. The agent for use according to any one of claims 1 to 38, wherein the tissue defect is damage to the eye and/or orbit, such as injury (trauma) of the eye and/or orbit.
53. The agent for use according to claim 40, wherein the skin defect is a skin disorder provoked by external factors.
54. The agent for use according to claim 53, wherein the skin disorder provoked by external factors is pressure ulceration, dermatoses provoked by friction or mechanical stress, dermatoses due to foreign bodies, dermatoses provoked or exacerbated by exposure to cold, dermatoses provoked by heat or electricity, dermatoses provoked by light or UV radiation, dermatoses due to ionizing radiation, allergic contact dermatitis, photo-allergic contact dermatitis, irritant contact dermatitis, allergic contact urticarial, protein contact dermatitis, allergic contact sensitisation, phototoxic reactions to skin contact with photoactive agents, cutaneous reactions to venomous or noxious animals.
55. The agent for use according to claim 40, wherein the skin defect is a disease of the skin, such as injury of external cause.
56. The agent for use according to claim 40, wherein the skin defect is a disease of the skin is selected from the group consisting of: a. a skin disorder involving specific cutaneous structures, such as disorders of cutaneous blood and lymphatic vessels, such as dermatoses resulting from vascular insufficiency, such as ischaemic ulceration of the skin, and b. a skin disorder associated with pregnancy, the neonatal period, and infancy, such as dermatoses of infancy, such as erythrodermas of infancy (Leiner’s disease).
57. The agent for use according to claim 55, wherein the injury of external cause is selected from the group consisting of: a. injury to the head, such as intracranial injury, such as diffuse brain injury, such as cerebral contusion, and b. injury to unspecified part of trunk, limb, or body region, such as other injuries of spine or trunk, such as injury of the spinal cord.
58. The agent for use according to claim 40, wherein the skin defect is an inflammatory dermatosis.
59. The agent for use according to claim 58, wherein the inflammatory dermatosis is dermatitis, eczema, atopic dermatitis, papulosquamous dermatoses, urticaria, angioedema or other urticarial disorders, inflammatory erythemas and other reactive inflammatory dermatoses, immunobullous diseases of the skin, cutaneous lupus erythematosus, scarring or sclerosing inflammatory dermatoses.
60. The agent for use according to claim 40, wherein the skin defect is sclerosis.
61. The agent for use according to claim 60, wherein the sclerosis is systemic sclerosis (scleroderma), such as circumscribed scleroderma.
62. The agent for use according to claim 40, wherein the skin defect is a postprocedural disorder of the skin.
63. The agent for use according to claim 62, wherein the postprocedural disorder of the skin is an unsatisfactory surgical scar of skin, a cutaneous flap necrosis, a myocutaneous flap necrosis, a skin graft failure, a composite graft failure.
64. The agent for use according to claim 62, wherein the postprocedural disorder of the skin is a surgical incision. The agent for use according to claim 40, wherein the skin defect is a genetic and/or developmental disorder affecting the skin. The agent for use according to claim 65, wherein the genetic and/or developmental disorder affecting the skin is epidermolysis bullosa, or genodermatosis, such as pemphigus genodermatosis. The agent for use according to claim 66, wherein the epidermolysis bullosa is selected from the group consisting of epidermolysis bullosa simplex, junctional epidermolysis bullosa, dystrophic epidermolysis bullosa, recessive dystrophic epidermolysis bullosa, syndromic epidermolysis bullosa and epidermolysis bullosa. The agent for use according to claim 40, wherein the skin defect is an external defect. The agent for use according to claim 40, wherein the skin defect is an open defect. The agent for use according to claim 40, wherein the skin defect is an inflamed wound. The agent for use according to any one of claims 40 to 70, wherein the skin defect is infected. The agent for use according to claim 40, wherein the skin defect is an injury or trauma to internal or external tissue, such as injury or trauma to the epidermis and/or dermis of the skin. The agent for use according to any one of claims 40 to 72, wherein the skin defect is an acute defect or a chronic defect. The agent for use according to any one of claims 40 to 72, wherein the skin defect is full thickness skin defect. The agent for use according to any one of claims 41 to 74, wherein the wound is an acute wound or a chronic wound. 76. The agent for use according to claim 75, wherein the acute wound is an incision, laceration, abrasion graze or burn, a puncture wound, a penetration wound or a wound due to dermatologic diseases such as psoriasis, acne and eczema.
77. The agent for use according to claim 75, wherein the chronic wound is a venous ulcer, a diabetic ulcer, a pressure ulcer, corneal ulcer, digestive ulcer or wounds as a result of ischemia and radiation poisoning.
78. The agent for use according to claim 41 , wherein the wound is a non-pressure chronic ulcer, such as a non-pressure chronic ulcer of lower limb.
79. The agent for use according to claim 1 , wherein the peptide comprises or consists of the amino acid sequence VDTYDGDISVVYGLR (SEQ ID NO: 7), and the tissue defect is a wound.
80. The agent for use according to claim 1, wherein the peptide comprises or consists of the amino acid sequence VDTYDGGISVVYGLR (SEQ ID NO: 6), and the tissue defect is a wound.
81. The agent for use according to claim 1 , wherein the peptide comprises or consists of the amino acid sequence AEIDSIELSYGIK (SEQ ID NO: 110), and the tissue defect is a wound.
82. The agent for use according to claim 1 , wherein the peptide comprises or consists of the amino acid sequence KPLAEIDSIELSYGIK (SEQ ID NO: 111), and the tissue defect is a wound.
83. The agent for use according to any one of the preceding claims, wherein the agent is administered topically, orally, subcutaneously, or systemically.
84. The agent for use according to any one of the preceding claims, wherein the subject is suffering from diabetes mellitus. The agent for use according to any one of the preceding claims, wherein the subject is suffering from stoma. The agent for use according to any one of the preceding claims, wherein the subject is a mammal. The agent for use according to claim 86, wherein the mammal is a human. A method for treatment and/or regeneration of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent as defined in any one of claims 1 to 39 to an subject in need thereof. A method for treating and/or promoting healing of a tissue defect in a subject, the method comprising administering a therapeutically effective amount of an agent as defined in any one of claims 1 to 39 to an subject in need thereof. A method for regeneration of skin at the site of a skin defect and/or for reducing scar formation resulting from healing of a skin defect in a subject, said method comprising administering an agent as defined in any one of claims 1 to 39. Use of an agent as defined in any one of claims 1 to 39 for the manufacture of a medicament for the treatment and/or regeneration of a skin defect in a subject. Use of an agent defined in any one of claims 1 to 39 for the manufacture of a medicament for the treating and/or promoting healing of a tissue defect in a subject. Use of an agent defined in any one of claims 1 to 39 for the manufacture of a medicament for the treatment of a skin defect in a subject. Use of an agent defined in any one of claims 1 to 39 for reducing scar formation resulting from healing of a skin defect in a subject. Use of an agent defined in any one of claims 1 to 39 for promoting the healing of a skin defect in a subject. Use of an agent defined in any one of claims 1 to 39 for promoting vascularisation in a healing tissue, such as skin healing from a skin defect, such as a wound. Use of an agent defined in any one of claims 1 to 39 in combination with a tissue meshing technique, for promoting healing of a skin defect, such as a wound. Use of an agent as defined in any one of claims 1 to 39 for promoting grafting of skin. A method for inserting a stent in a subject, said method comprising administering an agent as defined in any one of claims 1 to 39. . The method according to claim 99, wherein the stent is coated with the agent according to any one of the preceding claims. . The use according to any one of the preceding claims, wherein the agent is part of a cosmetic composition. . A medical device comprising a peptide selected from the group consisting of:
(i) a peptide comprising or consisting of an amino acid sequence of the general formula:
XsXeSXyXgYGLR (SEQ ID NO: 1) wherein:
X5 is D or G;
Xs is I or G;
X? is V or L;
Xs is V or A;
(ii) a peptide comprising or consisting of an amino acid sequence of the general formula:
X14LX15YGIK (SEQ ID NO: 105) wherein:
X14 is E or G;
X15 is S or T; (iii) a peptide comprising or consisting of an amino acid sequence selected from the group consisting of:
VDTYDGDISVVYGL (SEQ ID NO: 34),
VDTYDGDISVVYG (SEQ ID NO: 35),
VDTYDGDISVVY (SEQ ID NO: 36),
VDTYDGDISVV (SEQ ID NO: 37),
VDTYDGDISV (SEQ ID NO: 38),
VDTYDGDIS (SEQ ID NO: 39),
VDTYDGRGDSVVYGLR (SEQ ID NO: 40),
VDVPNGDISLAYGL (SEQ ID NO: 41),
VDVPNGDISLAYG (SEQ ID NO: 42),
VDVPNGDISLA (SEQ ID NO: 43),
VDVPNGDIS (SEQ ID NO: 44),
GDPNDGRGDSVVYGLR (SEQ ID NO: 45),
LDGLVRAYDNISPVG (SEQ ID NO: 46),
GDPNGDISVVYGLR (SEQ ID NO: 47)
VDVPNGDISLAYRLR (SEQ ID NO: 48),
VDVPEGDISLAYRLR (SEQ ID NO: 49),
V(beta-D)TYDGDISVVYGLR (SEQ ID NO: 50),
VDTY(beta-D)GDISVVYGLR (SEQ ID NO: 51),
VDTYDG(beta-D)ISVVYGLR (SEQ ID NO: 52);
CLAEIDSC (Cyclic) (SEQ ID NO: 130),
CFKPLAEIDSIECSYGIK (Cyclic) (SEQ ID NO: 131),
Cyclic CFKPLAEIDSIEC (SEQ ID NO: 132),
KPLAEIDSIELSYGI (SEQ ID NO: 133),
KPLAEIDSIELSYG (SEQ ID NO: 134),
KPLAEIDSIELSY (SEQ ID NO: 135),
KPLAEIDSIELS (SEQ ID NO: 136),
KPLAEIDSIEL (SEQ ID NO: 137), and
KPLAEIDSIE (SEQ ID NO: 138).
103. The medical device according to claim 102, wherein the medical device is a mesh.
104. The medical device according to claim 102, wherein the medical device is a stent. . The medical device according to claim 102, wherein the medical device is a wound dressing.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6482410B1 (en) * 1994-09-16 2002-11-19 The Scripps Research Institute Cytotactin derivatives that stimulate attachment and neurite outgrowth, and methods of making same
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WO2018202870A1 (en) * 2017-05-04 2018-11-08 Follicum Ab Peptides for treatment of diabetes
WO2020094797A1 (en) * 2018-11-07 2020-05-14 Follicum Ab Peptide fragments for treatment of diabetes
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WO2013021212A2 (en) * 2011-08-10 2013-02-14 Follicum Ab Novel compositions and uses thereof
WO2018202870A1 (en) * 2017-05-04 2018-11-08 Follicum Ab Peptides for treatment of diabetes
WO2020094797A1 (en) * 2018-11-07 2020-05-14 Follicum Ab Peptide fragments for treatment of diabetes
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GOTTRUP F.: "A specialized wound-healing center concept: importance of a multidisciplinary department structure and surgical treatment facilities in the treatment of chronic wounds", THE AMERICAN JOURNAL OF SURGERY, vol. 187, no. 5, 2004, pages S38 - S43
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