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WO2025106913A1 - Peptides destinés à favoriser la néovascularisation et la cicatrisation de plaies, et méthodes les utilisant - Google Patents

Peptides destinés à favoriser la néovascularisation et la cicatrisation de plaies, et méthodes les utilisant Download PDF

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WO2025106913A1
WO2025106913A1 PCT/US2024/056265 US2024056265W WO2025106913A1 WO 2025106913 A1 WO2025106913 A1 WO 2025106913A1 US 2024056265 W US2024056265 W US 2024056265W WO 2025106913 A1 WO2025106913 A1 WO 2025106913A1
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residues
peptide
wound
compound
vegfr2
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Mehran Sadeghi
Jae-Joon Jung
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Yale University
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Yale University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
    • 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
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/25—Peptides having up to 20 amino acids in a defined sequence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors

Definitions

  • This invention was made with government support under HL114703, HL112992. and HL162792 awarded by National Institutes of Health. This invention was made with government support under I0-BX001750 awarded by Department of Veterans Affairs. The government has certain rights in the invention.
  • Neovascularization is the process of forming new blood vessels.
  • Neovascularization includes: vasculogenesis, the de novo formation of blood vessels; angiogenesis, the formation of new vessels from pre-existing vessels; and arteriogenesis, the remodeling of existing vasculature to create collateral arteries.
  • vasculogenesis the de novo formation of blood vessels
  • angiogenesis the formation of new vessels from pre-existing vessels
  • arteriogenesis the remodeling of existing vasculature to create collateral arteries.
  • the present invention is directed to the following non-limiting embodiments:
  • the present invention is directed to a method of promoting neovascularization and/or hair growth in a subject in need thereof.
  • the method comprises: administering to the subject a compound comprising a polypeptide that promotes neovascularization and/or hair growth.
  • the polypeptide comprises a first peptide comprising from about 6 amino acid residues to about 14 amino acid residues, 80% or more of the amino residues of the first peptide are leucine residues, and the compound does not comprise a peptide sequence that has a length longer than about 14 amino acids and that has more than about 80% of the amino acid residues as leucine residues.
  • the polypeptide comprises the amino acid set forth in SEQ ID NO:5. 6, 8, or 9.
  • the compound is soluble in water or insoluble in water.
  • the compound is soluble in water, and the compound further comprises a hydrophilic motif.
  • the hydrophilic motif is at least one selected from the group consisting of: a second peptide which is hydrophilic; and one or more polyethylene glycol (PEG) groups.
  • the peptide comprises residues 1-66, residues 39-66, residues 51-66, or residues 51-61 of the sequence set forth in SEQ ID NO:15.
  • the method is a method of promoting neovascularization, and wherein the method promotes in the subject at least one selected from the group consisting of angiogenesis and arteriogenesis.
  • the subject suffers from a chronic wound healing disorder, and wherein the method promotes neovascularization in or near at least one chronic wound of the subject.
  • the subject suffers from an acute wound, and wherein the method promotes neovascularization which speeds up the healing of the acute wound.
  • the chronic wound comprises at least one of the following:
  • an ulcer optionally a diabetic ulcer such as a diabetic foot ulcer, an arterial ulcer, a venous ulcer, a pressure ulcer, or an ischemic ulcer; or
  • the compound reduces phosphatase activity exerted by protein-tyrosine phosphatase IB (PTP1B) and/or T-cell protein tyrosine phosphatase (TC- PTP) on vascular endothelial grow th factor receptor 2 (VEGFR2).
  • PTP1B protein-tyrosine phosphatase IB
  • TC- PTP T-cell protein tyrosine phosphatase
  • VAGFR2 vascular endothelial grow th factor receptor 2
  • the method further comprises administering to the subject an effective amount of vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • the present invention is directed to a method of promoting wound healing in a subject in need thereof.
  • the method comprises administering to the subject an effective amount of a compound comprises a polypeptide.
  • the polypeptide comprises a first peptide comprising from about 6 amino acid residues to about 14 amino acid residues, 80% or more of the amino residues of the first peptide are leucine residues, and the compound does not comprise a peptide sequence that has a length longer than about 14 amino acids and that has more than about 80% of the amino acid residues as leucine residues.
  • the polypeptide comprises an amino acid set forth in SEQ ID NO: 5, 6, 8 or 9.
  • the compound is soluble in w ater or insoluble in water.
  • the compound is soluble in water, and the compound further comprises a hydrophilic motif.
  • the hydrophilic motif is at least one selected from the group consisting of: (a) a second peptide which is hydrophilic; and (b) one or more polyethylene glycol (PEG) groups.
  • the peptide comprises residues 1-66, residues 39-66, residues 51-66, or residues 51-61 of the sequence set forth in SEQ ID NO: 15.
  • the method promotes wound healing by promoting in the subject at least one selected from the group consisting of angiogenesis and arteriogenesis.
  • the wound is a chronic wound or an acute wound.
  • the chronic wound comprises at least one of the following:
  • an ulcer optionally a diabetic ulcer such as a diabetic foot ulcer, an arterial ulcer, a venous ulcer, a pressure ulcer, or an ischemic ulcer; or
  • the compound reduces phosphatase activity exerted by protein-tyrosine phosphatase IB (PTP1B) and/or T-cell protein ty rosine phosphatase (TC- PTP) on vascular endothelial growth factor receptor 2 (VEGFR2).
  • PTP1B protein-tyrosine phosphatase IB
  • TC- PTP T-cell protein ty rosine phosphatase
  • VAGFR2 vascular endothelial growth factor receptor 2
  • the method further comprises administering to the subject an effective amount of vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • the present invention is directed to a compound comprising a polypeptide and a hydrophilic motif, or a salt or solvate thereof.
  • the polypeptide comprises a first peptide comprising from about 6 amino acid residues to about 14 amino acid residues, 80% or more of the amino residues of the first peptide are leucine residues, and the compound does not comprise a peptide sequence that has a length longer than about 14 amino acids and that has more than about 80% of the amino acid residues as leucine residues.
  • the polypeptide comprises an amino acid set forth in SEQ ID NO: 5, 6, 8 or 9.
  • the hydrophilic motif is at least one selected from the group consisting of:
  • the present invention is directed to a wound dressing.
  • the wound dressing comprises a base dressing material, and a compound comprising a polypeptide.
  • the polypeptide comprises an amino acid set forth in SEQ ID NO: 5, 6, 8 or 9.
  • the compound is soluble in water or insoluble in water.
  • the compound is soluble in water, and wherein the compound further comprises a hydrophilic motif.
  • the hydrophilic motif is at least one selected from the group consisting of:
  • the peptide comprises residues 1-66, residues 39-66, residues 51-66, or residues 51-61 of the sequence set forth in SEQ ID NO:15.
  • the base dressing material comprises at least one selected from the group consisting of an alginate dressing material, a biosynthetic dressing material, a collagen dressing material, a composite dressing material, a contact layer dressing material, a gauze, a hydrocolloid dressing material, a hydrofiber dressing material, a hydrogel, a hydropolymer dressing material, a polyurethane foam dressing material, a skin substitute dressing material, a superabsorbent dressing material, a transparent film dressing material, a wound filler, a wound pouch.
  • the compound comprising the poly peptide is on a surface of or impregnated into the base dressing material.
  • the wound dressing further comprises an adhesive substrate.
  • Figs. 1A-1E demonstrates that the endothelial and smooth muscle cell-derived neuropilin-like molecule (ESDN, also called DCBLD2) signal sequence is the key part for its interaction with vascular endothelial growth factor receptor 2 (VEGFR2), in accordance with some embodiments.
  • Fig. 1A Schematic representation of the constructs named with Roman numerals I to VIII. Construct I represents the full-length ESDN fused with an heme agglutinin (HA) tag. Construct II represents the extracellular part of ESDN fused with an HA tag.
  • HA heme agglutinin
  • Construct III, IV, and V represent different ESDN key domains (CUB, LCCL, Discoidin) connected to ESDN signal sequence and fused with an HA tag, respectively.
  • Construct VI and VII represent the signal sequence of ESDN (VI)/ Shrew 1 (VII) fused with green fluorescent protein (GFP) and HA tag, and construct VIII represents GFP-HA alone as a negative control.
  • Fig. IB Interaction of key ESDN domains with VEGFR2.
  • Construct I- V co-transfected with VEGFR2 to HEK 293T cells, respectively. Western blot analysis of VEGFR2 confirmed their successful expression (upper panel).
  • FIG. 1C Effect of key ESDN domains on VEGF- mediated ERK phosphorylation.
  • MLECs_ transfected with constructs I-V upper panel.
  • Western blot analysis of HA tag to confirm protein expression of each construct (lower panel).
  • Fig. ID Interaction of ESDN signal sequence with VEGFR2.
  • Fig. IE Effect of ESDN signal sequence on VEGF-mediated ERK phosphorylation.
  • HUVEs human umbilical vein endothelial cells
  • Figs. 2A-2E demonstrate that the traC subdomain is the key part of ESDN SS that interacts with VEGFR2, in accordance with some embodiments.
  • Fig. 2A The predicted ’NtraC’ subdomain combination of ESDN signal sequence (residues 1-66 of SEQ ID NO: 1).
  • Figs. 2B-2C Significant differences between ESDN signal sequence subdomains. Hydrophobicity analysis performed by ProtScale (Fig. 2B) and transmembrane helices prediction performed by DeepTMHMM (Fig. 2C).
  • Fig. 2D Schematic diagram of constructs named with roman numerals I to IV.
  • Construct I- III represent full length ESDN signal sequence / the N subdomain / the traC subdomain fused with GFP and HA tags, respectively.
  • Construct IV represents GFP-HA alone as a negative control.
  • Fig. 2E Interaction of key ESDN signal sequence subdomains with VEGFR2. Construct I to IV products coimmunoprecipitation with VEGFR2 in HEK 293T cells.
  • Figs. 3A-3F demonstrate that traC increases VEGF/VEGFR2 signaling in endothelial cells, in accordance with some embodiments.
  • Figs. 3A-3B Immunofluorescence staining of traC treated HUVECs. FITC-traC is in green. Nuclei was stained with DAPI in blue. VEGFR2 was stained in red. Arrowheads indicate traC co-localized with VEGFR2 on cell membrane. Scale bar: 20pm.
  • Fig. 3C Western blot example and quantification of the effect of traC on VEGF/VEGFR2 signaling.
  • Serum starved HUVECs were treated with 1 pM traC / 1% DMSO for 15min, followed by lOng/ml VEGF treatment for 5min or not.
  • the levels ofVEGFR2 Tyri l 75 phosphorylation, ERK Thr202/Tyr204 phosphorylation, and Akt Ser473 phosphorylation, and their corresponding total proteins were detected. **p ⁇ 0.01, ***p ⁇ 0.001.
  • Fig. 3D Western blot example and quantification of the effect of different concentrations of traC on VEGF/VEGFR2 signaling.
  • Serum starved HUVECs were treated with 0/0.1/lpM traC for 15min, followed by lOng/ml VEGF stimulation for 5min or not. *p ⁇ 0.1, **p ⁇ 0.01.
  • Fig. 3E Western blot example and quantification of VEGF/VEGFR2 signaling in WT / ESDN deficient HUVECs following treatment with / without traC / VEGF. *p ⁇ 0.1.
  • Fig. 3F traC's effect on the interaction between VEGFR2 and phosphatases in HUVECs. VEGFR2 immunoprecipitation was followed by Western blot detection of PTP1B and TCPTP.
  • Fig. 4 Specificity of traC-VEGFR2 interaction, in accordance with some embodiments. Following overexpression of different tyrosine kinase receptors in HEK 293 cells, their expression was confirmed by Western blotting (Fig. 4, right panel). After adding FITC-traC for 15 minutes to the cultures, VEGFR2. but not VEGFR1 or PDGFRp. could be co- immunoprecipitated with FITC-traC (Fig. 4, left panel), [0055] Figs. 5A-5B demonstrate that traC promotes in vivo matrigel angiogenesis, in accordance with some embodiments. Fig.
  • FIG. 5A Examples of matrigel plugs harvested 5 days after 1 pM traC / 1% DMSO in 300pL growth factor reduced matrigel with or without 2 ug/ml VEGF-A were subcutaneously injected into the opposite iliac regions of C57BL/6J mice.
  • Fig. 5B Examples and quantification of CD31 immunofluorescence staining (in red) of plug sections. Scale bar: 200pm. **p ⁇ 0.01.
  • Figs. 6A-6B demonstrate that traC promotes in vivo cornea angiogenesis, in accordance with some embodiments.
  • Fig. 6A Examples of cornea pictures taken before tissue collection on the 13th day. Small pellets containing traC / DMSO with or without VEGF were implanted into a 1 mm pocket incision in the cornea about 1-1.3 mm from the limbus.
  • Fig. 6B Examples and quantification of CD31 whole mount immunostaining (in red) of cornea tissue. The white dashed circles represent the location where the pellets were implanted. Scale bar: 500 pm. ****p ⁇ 0.0001.
  • FIGs. 7A-7D demonstrate that traC facilitates ear wound healing, in accordance with some embodiments.
  • Fig. 7A Schematic diagram of the ear wound healing model. A 2mm diameter circular wound was punched in the center of both ears. FITC-traC 10 pg / ear or 1% DMSO were injected around the ear wounds of each mouse every other day for 20 days.
  • Fig. 7B Typical pictures of ear wounds of albino mice on the 1 st and 20 th days.
  • Fig. 7D Examples and quantification of CD31 immunostaining (in red) of ear tissues collected on day 8 and day 20. FITC-traC is seen in green. Nuclei were stained with DAPI in blue. Scale bar: 300 pm. *p ⁇ 0. 1.
  • Figs. 8A-8G Poly-leucine interact with VEGFR2, in accordance with some embodiments.
  • Fig. 8A Co-Immunoprecipitation of traC / traC part 1(CSNSSSFSMPLF, residues 39-50 of the sequence set forth in SEQ ID NO: 1) / traC part 2 (LLLLLVLLLLLEDAGA, residues 51-66 of the sequence set forth in SEQ ID NO: 1) with VEGFR2.
  • Fig. 8A Co-Immunoprecipitation of traC / traC part 1(CSNSSSFSMPLF, residues 39-50 of the sequence set forth in SEQ ID NO: 1) / traC part 2 (LLLLLVLLLLLEDAGA, residues 51-66 of the sequence set forth in SEQ ID NO: 1) with VEGFR2.
  • Fig. 8A Co-Immunoprecipitation of traC / traC part 1(CSNSSSFSMPLF, residues 39-50 of the sequence set forth
  • Fig. 8B Co-Immunoprecipitation of traC part 2 (LLLLLVLLLLLEDAGA, residues 51-66 of the sequence set forth in SEQ ID NO:1) / LLLLLVLLLLL (residues 51-61 of the sequence set forth in SEQ ID NO: 1) / ED AGA (residues 62-66 of the sequence set forth in SEQ ID NO: 1) / GFP (negative control) with VEGFR2.
  • Fig. 8C Summary of the interaction between traC segments and VEGFR2.
  • Fig. 8D Co-Immunoprecipitation of LLLLLVLLLLL (residues 51-61 of the sequence set forth in SEQ ID NO: 1) /LLLLLLLLL (9L) I LLLLLLLLLLLLLLLL (16L) with VEGFR2.
  • Fig. 8E Summary' of the interaction between traC part 2 segments and VEGFR2.
  • Fig. 8F Co-Immunoprecipitation of 7L / 8L / 9L / 10L / 11 L / 12L peptides with VEGFR2.
  • Fig. 8G Western blot of the effect of a 10L synthetic peptide (FITC-L10 connected to EDAGA to increase water solubility ) at 1 pM and 10 pM on VEGF signaling in endothelial cells.
  • 10L synthetic peptide FITC-L10 connected to EDAGA to increase water solubility
  • Fig. 9 recites of the sequences of the TracC, scrambled (Scr, SEQ ID NO:7), and hydrophobic control (SEQ ID NO: 6) peptides, in accordance with some embodiments.
  • the sequences shown in Fig. 9 is also listed below:
  • Figs. 10A-10C illustrate certain aspects of the effect of Scrambled (Scr) peptide and traC on VEGF induced VEGFR2 signaling in HUVEC, in accordance with some embodiments.
  • HUVECs were treated with DMSO, Scr peptide, or traC peptide for 15 min followed by 5 mins of VEGF(10 ng/ml) treatment.
  • traC peptide enhances VEGF- induced VEGFR2 and ERK phosphorylation compared to DMSO.
  • Scr peptide increases VEGF-induced VEGFR2 phosphory lation (albeit less than traC) compared to DMSO.
  • Fig. 10A western blot panels.
  • Fig. 10B quantification of p-VEGFR2/Total VEGFR2.
  • Figs. 11 A-l IB illustrate certain aspects of the effect of traC in comparison with DMSO on VEGF induced VEGFR2 signaling in HUVEC, in accordance with some embodiments.
  • HUVECs were treated with DMSO, or traC peptide for 15 min followed by VEGF(10 ng/ml).
  • traC peptide enhances VEGF-induced VEGFR2, AKT and ERK phosphorylation compared to DMSO.
  • N 3.
  • Fig. 11 A Western blot panels.
  • Fig. 11B quantification bar charts of pVEGFR2, pERK and pAKT.
  • Fig. 12 demonstrates the different kinetics of VEGF-induced VEGFR2 signaling in HUVEC in the presence of scrambled (Scr) peptide or traC. in accordance with some embodiments.
  • HUVECs were treated with scr peptide or traC peptide for 15 min followed by 0-30mins of VEGF(10ng/ml).
  • traC and Scr peptides increase VEGFR2 phosphorylation with different time courses.
  • Figs. 13A-13E demonstrate the different kinetics of VEGF-induced VEGFR2 signaling in HUVEC in the presence of control peptide (CP) or traC, in accordance with some embodiments.
  • HUVECs were treated with CP or traC for 1 min followed by 0- 30mins of VEGF(10ng/ml) treatment.
  • traC and CP increase VEGFR2 phosphorylation with different time courses.
  • Figs. 14A-14B demonstrate that FITC-traC, but not FITC-CP or FITC-Scr peptides, are internalized in HUVEC, in accordance with some embodiments.
  • HUVECs were treated with Scr, CP or traC for 15 min followed by 0-30mins of VEGF(10ng/ml) treatment. After washing the cells, the FITC signal was only detectable in FITC-traC treated cells.
  • a separate gel with the three peptides showed similar protein and fluorescence intensities on the gel and after transfer to a membrane.
  • Figs. 15A-15G demonstrate that the DCBLD2 SS traC subdomain is involved in DCBLD2 interaction with VEGFR2, in accordance with some embodiments.
  • Fig. 15 A Schematic representation of constructs I-VIIE each with various combinations of DCBLD2 domains tagged with HA (I-V), DCBLD2, or Shrew- 1 SS fused with GFP and HA tags (VI, VII) or GFP-HA alone (VIII).
  • Fig. 15B Co-immunoprecipitation of the products of constructs I-V with VEGFR2 following transfection of each construct with VEGFR2 (or transfection of construct I alone without VEGFR2) in HEK 293T cells.
  • VEGFR2 immunoprecipitation is followed by Western blotting for HA and VEGFR2.
  • Fig. 15C Western blot analysis of the effects of constructs I-V on VEGF-induced ERK phosphorylation following their transfection in Dcbld2 ⁇ ⁇ murine lung endothelial cells transfected.
  • Fig. 15D Co-immunoprecipitation of the products of constructs VI-VIII with VEGFR2 following their transfection in HEK 293T cells.
  • HA immunoprecipitation is followed by Western blotting for HA and VEGFR2.
  • Fig. 15E NtraC model of DCBLD2 SS. Fig.
  • FIG. 15F Schematic representation of constructs IX-XI encompassing different DCBLD2 SS subdomains fused with GFP and HA tags (IX-XI) or GFP-HA alone (VIII).
  • Fig. 15G Coimmunoprecipitation of the products of constructs IX-XI with VEGFR2 following their transfection in HEK 293T cells. HA immunoprecipitation is followed by Western blotting for HA and VEGFR2.
  • SS signal sequence; TMD: transmembrane domain.
  • HA Hemagglutinin
  • the sequence shown in Fig. 15E is:
  • Figs. 16A-16F demonstrate that FITC-traC enhances VEGF/VEGFR2 signaling in endothelial cells, in accordance with some embodiments.
  • Fig. 16E Co-immunoprecipitation of FITC-traC with VEGFR2 but not PDGFRp following the addition of FITC-traC to endothelial cell cultures. FITC immunoprecipitation is followed by Western blotting for VEGFR2. PDGFRP, or FITC.
  • Fig. 16F Effect of FITC-traC on protein tyrosine phosphatases PTP1B and TCPTP interaction with VEGFR2 in endothelial cells. VEGFR2 immunoprecipitation is followed by Western blotting for VEGFR2, PTP1B or TCPTP.
  • PTP1B Protein tyrosine phosphatase IB
  • TCPTP T cell protein tyrosine phosphatase, ns: not significant
  • Statistical significance was determined by two-way ANOVA and Tukey’s multiple comparison post hoc test (Fig. 16C) or by repeated measures one-way ANOVA and Holm-Sidak’s multiple comparisons post hoc test (Fig. 16D).
  • Fig. 17A-17G demonstrate that the DCBLD2 SS FITC-traC peptide promotes VEGF and ischemia-induced angiogenesis in vivo, in accordance with some embodiments.
  • FIG. 17D Illustrative laser Doppler images and quantification of hindlimb blood flow recovery after femoral artery ligation in mice treated with FITC-traC compared to control animals.
  • L ligated
  • NL nonligated.
  • Figs. 18 A-l 8E demonstrate that a non-limiting poly leucine decapeptide mimics the effect of DCBLD2 SS traC on VEGF signaling, in accordance with some embodiments.
  • Fig. 18 A Co-immunoprecipitation of the products of HA-tagged traC Part 1 or Part 2 constructs with VEGFR2 following their co-transfection in HEK 293T cells. HA immunoprecipitation is followed by western blotting for HA and VEGFR2.
  • Fig. 18 A Co-immunoprecipitation of the products of HA-tagged traC Part 1 or Part 2 constructs with VEGFR2 following their co-transfection in HEK 293T cells. HA immunoprecipitation is followed by western blotting for HA and VEGFR2.
  • FIG. 18B Co-immunoprecipitation of the products of HA-tagged traC Part 2 or its derivative constructs or a control HA-tagged GFP with VEGFR2 following their co-transfection in HEK 293T cells. HA immunoprecipitation is followed by western blotting for HA and VEGFR2.
  • FIG. 18C Co-immunoprecipitation of the products of HA- tagged traC Part 2 L5VL5 sequence, or 9 or 16 poly-L-leucine constructs with VEGFR2 following their co-transfection in HEK 293T cells. HA immunoprecipitation is followed by western blotting for HA and VEGFR2. Fig.
  • FIG. 18D Co-immunoprecipitation of the products of HA- tagged polyleucine constructs with VEGFR2 following their co-transfection in HEK 293T cells. HA immunoprecipitation is followed by western blotting for HA and VEGFR2.
  • Fig.19 illustrates certain aspects of the expression of DCBLD2 constructs in HEK 293T cells, in accordance with some embodiments.
  • FIGs. 20A-20B illustrate certain aspects of the signal peptide cleavage prediction, in accordance with some embodiments.
  • SignalP 6.0 prediction of signal peptide cleavage sites of DCBLD2 (Fig. 20A) and SHREW1 (Fig. 20B). showing the low probability of DCBLD2
  • Fig. 21 illustrates certain aspects of the DCBLD2 SS cleavage determination, in accordance with some embodiments.
  • Fig. 22 demonstrates that VEGF-induced ERK phosphorylation in Dcbld2-/- MLEC transfected with DCBLD2-SS-GFP-HA or GFP-HA constructs, in accordance with some embodiments.
  • Fig. 23 illustrates certain aspects of the hydrophobicity analysis of DCBLD2 SS subdomains by ProtScale, in accordance with some embodiments.
  • the sequence shown in Fig. 23 is also produced below-:
  • Fig. 24 illustrates certain aspects of the DeepTMHMM prediction of transmembrane regions within DCBLD2 SS subdomains, in accordance with some embodiments.
  • FIG. 26 illustrates certain aspects of the FITC-traC dose-response on VEGF signaling in endothelial cells, in accordance with some embodiments.
  • Illustrative Western blot and quantitative analysis of VEGF-induced VEGFR2, AKT, and ERK phosphorylation (n 4). *: P ⁇ 0.05. **: P ⁇ 0.01. Statistical significance was determined by two-way ANOVA and Tukey’s multiple comparison post hoc test.
  • Fig. 27 provides the Western blots showing the effect of pre-heating (120 °C, 50 minutes) on FITC-traC potentiation of VEGF signaling in endothelial cells, in accordance with some embodiments.
  • Fig. 28 provides the Western blots showing the effect of FITC-antennapedia and FITC-traC on VEGF signaling in endothelial cells, in accordance with some embodiments.
  • Fig. 29 illustrates certain aspects of the specificity of FITC-traC interaction with VEGFR2, in accordance with some embodiments. Co-immunoprecipitation of FITC-traC with VEGFR2 but not VEGFR1 or PDGFRJ3 following the addition of FITC-traC to HEK293T cells cotransfected with VEGFR1, VEGFR2, or PDGFR . FITC immunoprecipitation is follow ed by Western blotting for VEGFR2, VEGFR1, PDGFRty or FITC.
  • Figs. 31 A-3 IB show an alignment of rat, mouse and human DCBLD2 protein sequences, in accordance with some embodiments.
  • the mouse and rat sequences contain a L9 sequence instead of the L5VL5 sequence in human DCBLD2.
  • the sequences shown in Figs. 31A-31B are also listed below:
  • first and second features are formed in direct contact
  • additional features may be formed between the first and second features, such that the first and second features may not be in direct contact
  • present disclosure may repeat reference numerals and/or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and/or configurations discussed.
  • a "disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
  • a disease or disorder is "alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.
  • co-administered and “co-administration” as relating to a subject refer to administering to the subject a compound and/or composition of the disclosure along with a compound and/or composition that may also treat or prevent a disease or disorder contemplated herein.
  • the co-administered compounds and/or compositions are administered separately, or in any kind of combination as part of a single therapeutic approach.
  • the co-administered compound and/or composition may be formulated in any kind of combinations as mixtures of solids and liquids under a variety of solid, gel, and liquid formulations, and as a solution.
  • composition refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition facilitates administration of the compound to a patient.
  • Multiple techniques of administering a compound exist in the art including, but not limited to, subcutaneous, intravenous, oral, aerosol, inhalational, rectal, vaginal, transdermal. intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical. ophthalmic, pulmonary, and topical administration.
  • the term "pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non- toxic, z.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • the term "pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the patient such that it may perform its intended function.
  • a pharmaceutically acceptable material, composition or carrier such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the patient such that it may perform its intended function.
  • Each carrier must be “acceptable” in the sense of being compatible w ith the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the patient.
  • compositions that may sen e as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives.
  • pharmaceutically acceptable carrier also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the patient.
  • pharmaceutically acceptable carrier may further include a pharmaceutically acceptable salt of the compound useful within the disclosure.
  • the language “pharmaceutically acceptable salt” refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof.
  • a “pharmaceutically effective amount,” “therapeutically effective amount,” or “effective amount” of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.
  • prevent means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.
  • the terms “subject” and “individual” and “patient” can be used interchangeably and may refer to a human or non-human mammal or a bird.
  • Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals.
  • the subject is human.
  • treatment is defined as the application or administration of a therapeutic agent, i. e. , a compound useful within the disclosure (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a disease or disorder and/or a symptom of a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder and/or the symptoms of the disease or disorder.
  • a therapeutic agent i. e. , a compound useful within the disclosure (alone or in combination with another pharmaceutical agent
  • a therapeutic agent i. e. , a compound useful within the disclosure (alone or in combination with another pharmaceutical agent
  • an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications)
  • Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • ESDN endothelial and smooth muscle cell- derived neuropilin-like molecule
  • VEGFR2 vascular endothelial growth factor receptor 2
  • Mapping studies of the signal sequence led to the discovery that the minimum essential sequence in the signal sequence is an eleven-residue long section including ten leucine residues and one valine residues (see e.g., Figs. 8D-8E).
  • any peptides that include a section of certain length range, and has a sufficiently high leucine content are able to interact with VEGFR2 and activate VEGFR2 signaling (see e.g., Figs. 8D-8G).
  • mice that were administered some of the peptides herein topically grew hair faster . It has been reported that VEGF pathway and VEGF-mediated angiogenesis promote hair growth, as well. (Yano et al., J Clin Invest. 2001 Feb;107(4):409-17).
  • the present invention is directed to a method of promoting neovascularization in a subject in need thereof. In some aspects, the present invention is directed to a method of promoting hair grow th in a subject in need thereof. [00104] In some embodiments, the method includes: administering to the subject a compound including a polypeptide. In some embodiments, the compound and/or the polypeptide is/are capable of activating VEGFR2 signaling. In some embodiments, the polypeptide includes a first peptide capable for activating VEGFR2 signaling. In some embodiments, the first peptide contain a poly -leucine. It is worth noting that nucleic acids encoding the polypeptide herein are considered within the scope of the present invention. In some embodiments, the compound is formulated as a composition, such as a pharmaceutical composition with one or more pharmaceutically acceptable carriers.
  • the first peptide ranges in length from about 6 amino acid residues to about 14 amino acid residues, such as about 7 amino acid residues to about 12 amino acid residues, or about 8 amino acid residues to about 11 amino acid residues.
  • the peptide has a length of about 6 amino acid residues, about 7 amino acid residues, about 8 amino acid residues, about 9 amino acid residues, about 10 amino acid residues, about 1 1 amino acid residues, about 12 amino acid residues, about 13 amino acid residues, about 14 amino acid residues, or any ranges therebetween.
  • the percentage of the amino acid residues in the first peptide that are leucine residues is 80% or higher, such as 85% or higher. 90% or higher. 95% or higher, or 100%.
  • the first peptide comprises no non-leucine amino acid residue (i.e., all residues of the first peptide are leucine.
  • the first peptide comprises one non-leucine amino acid residue.
  • the first peptide comprises two independently selected non-leucine amino acid residue.
  • the first peptide comprises three independently selected non-leucine amino acid residue.
  • the first peptide comprises four independently selected non-leucine amino acid residue.
  • the first peptide comprises five independently selected non-leucine amino acid residue.
  • the compound does not comprise a peptide sequence that has a length longer than about 14 amino acids and that has more than 80% or higher (such as 85% or higher, 90% or higher, 95% or higher, or 100%) of the amino acid residues as leucine residues.
  • the compound does not comprise a peptide sequence that includes more than 14 continuous leucine residues.
  • a polypeptide can include one, two, or more of the polyleucine sections, as long as the poly-leucine sections are not close enough to each other to create an uninterrupted poly -leucine section of 14 amino acids or more. Accordingly, in some embodiments, the polypeptide herein comprises a plurality of the first peptides as contemplated herein.
  • the compound is soluble in water.
  • soluble in water means having a solubility of 1 mg/ml or larger, such as 2 mg/ml or larger, 5 mg/ml or larger, 10 mg/ml or larger, 20 mg/ml or larger, 50 mg/ml or larger, 100 mg/ml or larger, 200 mg/ml or larger, or 500 mg/ml or larger.
  • the compound further includes a hydrophilic motif.
  • the hydrophilic motif increases solubility of the compound in water.
  • the hydrophilic motif is a second peptide which is hydrophilic.
  • the hydrophilic motif includes one or more peptides, for example, attached to either or both of the N-terminal and the C-terminal of the first peptide.
  • the hydrophilic motif and the first peptide are of the same peptide chain in which all of the amino acid residues are linked via peptide bonds.
  • the hydrophilic motif and the first peptide are linked via chemical bonds other than peptide bonds.
  • the present study has discovered that the "traC" peptide, which includes only 17 residues apart from the poly-leucine section is able to interact and activate VEGFR2 (see e.g., Fig. 3C-E).
  • the present study has further discovered that even a protein as large as the green fluorescence protein (GFP), which composed of 238 amino acids and has a molecular weight of 27 kDa (i. e. , significantly larger than the poly-leucine section), can be attached to the polyleucine section without affecting the interaction between the poly-leucine section and VEGFR2 (see e.g., Figs. 2D-2E).
  • GFP green fluorescence protein
  • the hydrophilic motif is not particularly limited.
  • hydrophilic motif brings a percentage of hydrophilic residues in the polypeptide of the compound to about 20% or higher, such as about 30% or higher, about 40% or higher, about 50% or higher, or about 60% or higher.
  • the hydrophilic motif has a tertiary structure such that some of the hydrophobic amino acid residues are buried inside the tertiary' structure.
  • the percentage of hydrophilic residues in the polypeptide of the compound may be lower than 20% as the percentage of the residues exposed on the surface of the tertiary' structure, and therefore the overall hydrophilicity, are higher than the raw number indicates.
  • the hydrophilic motif includes one or more poly ethylene glycol (PEG) groups, one or more saccharide groups such as chitosan groups, one of more bis-ethylene glycol groups, tn-ethylene glycol group, sulfonation (such as on the tyrosine residues).
  • PEG poly ethylene glycol
  • saccharide groups such as chitosan groups
  • tn-ethylene glycol group tn-ethylene glycol group
  • sulfonation such as on the tyrosine residues.
  • Other non-limiting hydrophilic motifs that can be attached to increase the hydrophilicity' include those described in US 2004/0147728 Al, the entirety' of the reference is hereby incorporated herein by reference.
  • the hydrophilic motifs are attached to the peptide via a covalent bond.
  • the compound is not soluble or has low solubility in water. Since transdermal or transcutaneous applications would benefit from hydrophobicity' which is an inherent characteristic of peptides rich in leucine residues, in some embodiments, the peptide is not attached to a hydrophilic motif, or is attached to a hydrophobic motif.
  • the compound can be formulated with a delivery vehicle.
  • the delivery vehicle is a particle having a hydrophilic outer surface, and a hydrophobic inner side for containing hydrophobic compounds.
  • vehicles for delivering compounds such as hydrophobic compounds with low solubility in water includes pluronic gel. methylcellulose, hydroxyethylcellulose, various scaffolds, Matrigel, or nanoparticles such as liposomes and micelles.
  • the polypeptide and/or first peptide of the compound includes the complete or part of the amino acid sequence of endothelial and smooth muscle cell-derived neuropilin-like molecule (ESDN) protein.
  • ESDN is the protein having the sequence set forth in SEQ ID NO: 15, isoforms thereof or orthologs in nonhuman animals.
  • the polypeptide and/or first peptide includes residues 1-66 of the sequence set forth in SEQ ID NO: 15. In some embodiments, the polypeptide and/or first peptide includes residues 39-66 of the sequence set forth in SEQ ID NO: 15. In some embodiments, the polypeptide and/or first peptide includes residues 51-66 of the sequence set forth in SEQ ID NO: 15. In some embodiments, the polypeptide and/or first peptide includes residues 51-61 of the sequence set forth in SEQ ID NO: 15.
  • the method promotes at least one selected from the group consisting of angiogenesis and arteriogenesis in the subject.
  • Impaired wound revascularization is a hallmark of chronic wounds. Accordingly, in some embodiments, the subject suffers from a chronic wound. In some embodiments, the method promotes neovascularization in or near the chronic wound.
  • the term "chronic wound” refers to a wound that has shown no significant progress toward healing (such as failed to achieve sufficient healing) in a prolonged time period, such as about 7 days or longer, about 10 days or longer, about 2 weeks or longer, about 15 days or longer, about 20 days or longer, about 3 weeks or longer, about 4 weeks or longer, or about 30 days or longer.
  • Non-limiting examples of chronic wounds includes nonhealing wounds; infected wounds, such as infected surgical wounds or infected traumatic wounds; or ulcers, such as diabetic ulcer (e.g., diabetic foot ulcer), arterial ulcers, venous ulcers, pressure ulcers, ischemic ulcers, wounds associated with immunosuppression or radiation, and the like.
  • infected wounds such as infected surgical wounds or infected traumatic wounds
  • ulcers such as diabetic ulcer (e.g., diabetic foot ulcer), arterial ulcers, venous ulcers, pressure ulcers, ischemic ulcers, wounds associated with immunosuppression or radiation, and the like.
  • the subject is suffering from an acute wound, which would benefit from faster healings, as well.
  • peptides including poly-leucine are able to compete with protein-tyrosine phosphatase IB (PTP1B) or T-cell protein ty rosine phosphatase (TCPTP) for interaction with vascular endothelial growth factor receptor 2 (VEGFR2), thereby reducing the phosphate activity exert on VEGFR2 by PTP1B or TCPTP. Since PTP1B and TCPTP counter the tyrosine phosphorylation of VEGFR2 required for kinase activation, peptides including poly-leucine are able to activate VEGFR2. Accordingly, in some embodiments, compound reduces phosphatase activity exerted on VEGFR2 by PTP1B or TCPTP.
  • the method further includes administering to the subject an effective amount of vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • the subject is a mammal, such as a human.
  • ESDN endothelial and smooth muscle cell- derived neuropilin-like molecule
  • VEGFR2 vascular endothelial growth factor receptor 2
  • Figs. 7A-7D vascular endothelial growth factor receptor 2
  • Mapping studies of the signal sequence led to the discovery' that the minimum essential sequence in the signal sequence is an eleven-residue long section including ten leucine residues and one valine residues (see e.g., Figs. 8C-8E).
  • the present specification is directed to a method of promoting wound healing in a subject in need thereof.
  • the method includes administering to the subject an effective amount of a compound and/or composition that promotes wound healing.
  • the compound, the composition, as well as the formulations thereof is the same as or similar to those detailed elsewhere herein, such as in the "Method of Promoting Neovascularization" section.
  • the type of the wound and/or the nature of the subject are the same as or similar to those detailed elsewhere herein, such as in the "Method of Promoting Neovascularization and/or Hair Growth” section.
  • the method further includes administering to the subject an effective amount of vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • compounds such as polypeptides
  • leucine rich peptides of certain length ranges herein are able to promote neovascularization and wound healing.
  • the compounds herein can be administered topically, and are therefore suitable for being included in wound dressings that are placed directly against the wound to speed up wound healing.
  • the present invention is directed to a wound dressing.
  • the wound dressing includes a base dressing material and a compound for promoting neovascularization and/or wound healing.
  • the compound for promoting neovascularization and/or wound healing is the same as or similar to those described in the "‘Method of Promoting Neovascularization and/or Hair Growth’’ section.
  • the base dressing material suitable for the wound dressing is not limited, as long as the base dressing material allows the compound attached thereto to be in contact with the wound when the wound dressing is applied.
  • None limiting examples of base dressing material includes alginate dressing materials, biosynthetic dressing materials, collagen dressing materials, composite dressing materials, contact layer dressing materials, gauzes, hydrocolloid dressing materials, hydrofiber dressing materials, hydrogels, hydropolymer dressing materials, polyurethane foam dressing materials, skin substitute dressing materials, superabsorbent dressing materials, transparent film dressing materials, wound filles, wound pouches, or the like.
  • the compound including the polypeptide herein is on a surface of or impregnated into the base dressing material.
  • the wound dressing further includes an adhesive substrate, such that the wound dressing can be used as adhesive bandage, such as a Band-Aid.
  • the method of treating, ameliorating, and/or preventing the disease/disorder includes administering to the subject the effective amount of at least one compound and/or composition contemplated within the disclosure.
  • the composition for treating the disease/disorder herein includes at least one compound and/or composition contemplated within the disclosure.
  • the subject is further administered at least one additional agent that treats, ameliorates, and/or prevents a disease and/or disorder contemplated herein.
  • the compound and the at least one additional agent are coadministered to the subject.
  • the compound and the at least one additional agent are co-formulated.
  • the compounds contemplated within the disclosure are intended to be useful in combination with one or more additional compounds.
  • additional compounds may comprise compounds of the present disclosure and/or at least one additional agent for treating the disease/disorder, and/or at least one additional agent that treats one or more diseases or disorders contemplated herein.
  • a synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 1 14:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.
  • suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 1 14:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.
  • the regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations contemplated within the disclosure may be administered to the subject either prior to or after the onset of a disease and/or disorder contemplated herein. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations contemplated within the disclosure may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • compositions contemplated within the disclosure may be carried out using known procedures, at dosages and for periods of time effective to treat a disease and/or disorder contemplated herein in the patient.
  • An effective amount of the therapeutic compound necessary' to achieve a therapeutic effect may vary' according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound contemplated within the disclosure to treat a disease and/or disorder contemplated herein in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • an effective dose range for a therapeutic compound contemplated within the disclosure is from about 0.001 and 5,000 mg/kg of body weight/per day.
  • One of ordinary' skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions contemplated within the disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.
  • a medical doctor e.g, physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • physician or veterinarian could start doses of the compounds contemplated within the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the patients to be treated: each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • the dosage unit forms contemplated within the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease and/or disorder contemplated herein.
  • the compounds of the disclosure are formulated as a composition with one or more pharmaceutically acceptable excipients or carriers.
  • the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of a compound of the disclosure and a pharmaceutically acceptable carrier.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more.
  • the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.
  • Compounds of the disclosure for administration may be in the range of from about 1 pg to about 10.000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9.000 mg, about 75 pg to about 8.500 mg, about 150 pg to about 7.500 mg, about 200 pg to about 7,000 mg, about 3050 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg. about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.
  • the dose of a compound of the disclosure is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 10.000 mg, or less than about 8.000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.
  • a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg. or less than about 25 mg, or less than about 20 mg, or less than about 15 mg. or less than about 10 mg, or less than about 5 mg. or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
  • the present disclosure is directed to a packaged pharmaceutical composition
  • a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of the disease/disorder herein in a patient.
  • Formulations may be employed in admixtures with conventional excipients, z.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for intracranially, oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
  • routes of administration of any of the compositions of the disclosure include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical.
  • the compounds for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans )urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary. intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.
  • compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets.
  • excipients include, for example an inert diluent such as lactose: granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
  • the tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
  • the compounds of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g, cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g, magnesium stearate, talc, or silica); disintegrates (e.g, sodium starch gly collate); or wetting agents (e.g., sodium lauryl sulphate).
  • the tablets may be coated using suitable methods and coating materials such as OPADRYTM film coating systems available from Colorcon, West Point. Pa.
  • Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions.
  • the liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g, sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g, lecithin or acacia); non-aqueous vehicles (e.g, almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).
  • suspending agents e.g, sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agent e.g, lecithin or acacia
  • non-aqueous vehicles e.g, almond oil, oily esters or ethyl alcohol
  • preservatives e.g., methyl or propyl p-hydroxy benzoates or sorbic acid
  • the present disclosure also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the disclosure, and a further layer providing for the immediate release of another medication.
  • a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.
  • the compounds of the disclosure may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion.
  • Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used.
  • Additional dosage forms of this disclosure include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos.
  • the formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may. although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
  • the period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds.
  • the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the compounds of the disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
  • pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profdes of the drug after drug administration.
  • immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
  • short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
  • rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
  • the therapeutically effective amount or dose of a compound of the present disclosure depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of disease/disorder in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.
  • a suitable dose of a compound of the present disclosure may be in the range of from about 0.01 mg to about 5,000 mg per day. such as from about 0. 1 mg to about 1,000 mg. for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.
  • the dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.
  • the amount of compound dosed per day may be administered, in non-limiting examples, every day, every' other day, every 2 days, every' 3 days, every' 4 days, or every 5 days.
  • a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the administration of the modulator of the disclosure is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (z.e., a "drug holiday").
  • the length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days. 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days.
  • the dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the patient's condition, to a level at which the improved disease is retained.
  • patients require intermittent treatment on a longterm basis upon any recurrence of symptoms and/or infection.
  • the compounds for use in the method of the disclosure may be formulated in unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population).
  • the dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LDso and EDso.
  • Capsid assembly modulators exhibiting high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human.
  • the dosage of such capsid assembly modulators lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity.
  • the dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
  • Example 1-1 Post-targeting functions of a long signal sequence in angiogenesis
  • Angiogenesis the process of new blood vessel formation from existing vessels, plays a key role in a number of physiological processes, including development and growth, adult organ regeneration, and wound healing .
  • Angiogenesis is tightly controlled through numerous stimulators and inhibitors and its dysregulation, whether insufficient or excessive, contributes to a wide variety of diseases, ranging from malignancy, ischemic heart and peripheral artery disease, and neurodegeneration to proliferative retinopathy. Accordingly, modulation of this process provides several therapeutic opportunities.
  • DCBLD2 Discoidin, CUB and LCCL domain containing 2
  • ESDN endothelial and smooth muscle cell-derived neuropilin-like protein
  • CLCP1 LCCL domain containing 2
  • DCBLD2 is highly conserved among vertebrates and closely resembles the structure of neuropilms, major co-receptors for vascular endothelial cell growth factor (VEGF) receptors (VEGFRs), which play a critical role in angiogenesis.
  • VEGF vascular endothelial cell growth factor
  • VAGFRs vascular endothelial cell growth factor
  • DCBLD2 is upregulated in human, rat, and mouse remodeling arteries, and regulates VEGF-induced endothelial cell (EC) proliferation, migration, and signal transduction, as well as developmental and adult angiogenesis.
  • SS signal sequences
  • SP signal peptide
  • a typical SP has a tripartite structure with a hydrophobic, a-helical core (h region) surrounded by an N-terminal polar n region, and a C-terminal c region that contains the signal peptidase cleavage site.
  • exceptionally long SS may have a bipartite 'NtraC domain structure with potentially functional N-terminal (N-domain, ‘N’) and C-terminal (C-domain, ‘C’) domains connected by a turn-rich linker area (transition area, ‘tra’).
  • the 66 amino acid SS of DCBLD2 is predicted to fit the ’NtraC' model.
  • the present study identified the DCBLD2 SS, and more specifically its 'traC segment, as a key domain of DCBLD2 that interacts with VEGFR2 and promotes VEGF/VEGFR2 signaling in EC. Furthermore, an exogenous synthetic traC peptide promoted angiogenesis in vivo. The smallest unit in DCBLD2 SS that interacted with VEGFR2 was the 5LV5L sequence. As such, in addition to establishing a novel posttargeting function of signal sequences in regulating growth factor signaling, these findings introduce the DCBLD2 traC and its derivatives as promising candidates for therapeutic angiogenesis.
  • Example 1-2 The ESDN (DCBLD2) signal sequence is involved in interaction with VEGFR2
  • Distinct functional and structural unites of DCBLD2 include an unusually long SS, which in tandem with CUB, LCCL, and Discoidin domains form the extracellular segment.
  • the transmembrane and intracellular domains comprise the rest of the molecule.
  • the present study generated multiple plasmids containing various combinations of DCBLD2 domains linked to a C-terminal hemagglutinin (HA) tag (Fig. 1 A I-V).
  • Example 1-3 The traC subdomain of ESDN SS interacts with VEGFR2
  • the DCBLD2 SS 'N' subdomain contains 38 amino acid residues, and the 24 amino acid C-terminal residues constitute the ‘C’ subdomain, with those two separated by a short ‘tra’ subdomain (Fig. 2A).
  • Example 1-4 FITC-TraC enhances VEGF/VEGFR2 signaling in endothelial cells
  • the present study sought to investigate whether a synthetic traC peptide, labeled with FITC for tracking purposes, can modulate VEGF signaling in EC.
  • the present study followed by fluorescence imaging the fate of the labeled peptide added to EC culture media.
  • FITC-traC was rapidly taken up by HUVEC with the peptide detected in the cytoplasm as early as 1 minute after its addition to the medium.
  • cytoplasmic accumulation of the peptide increased over time and by 15 minutes it was also detectable in the cell membrane (Fig. 3A), where it co-localized with VEFR2 (Fig. 3B). Accordingly, the 15-minute time point was selected for the next set of studies.
  • the present study evaluated the effect of synthetic DCBLD2 traC on VEGF signaling. Pretreatment of HUVEC with FITC-traC peptide (IpM in 1% DMSO, 15 minutes) significantly increased VEGF-induced VEGFR2, ERK and AKT phosphorylation compared to HUVEC pre-treated with vehicle control (Fig. 3C).
  • FITC-traC peptide A dose-response study showed that the effect of FITC-traC peptide on VEGF signaling is concentrati on-dependent (Fig.3D). Of note, higher concentrations of FITC-traC induced AKT and ERK phosphorylation independent of VEGF. Pre-heating of FITC-traC peptide abrogated its effect on VEGF signaling. To explore the molecular mechanisms of this observation, the present study first determined whether the effect of FITC-traC is simply related to its cell penetrating property 7 . A similar set of experiments showed that unlike FITC-traC, pre-treatment of HUVEC with a classical cell penetrating peptide, antennapedia. does not affect downstream VEGF signaling.
  • DCBLD2 siRNA significantly reduced VEGF-induced VEGFR2, ERK and AKT phosphorylation in HUVEC (Fig. 3E). This impaired VEGF/VEGFR2 signaling was fully restored upon pre-treatment of cells with FITC-traC peptide (Fig. 3E).
  • VEGFR2 interaction with FITC-traC w confirmed following overexpression of different tyrosine kinase receptors in HEK 293 cells.
  • VEGFR2 but not PDGFRP, could be immunoprecipitated from FITC-traC, when the cells were pretreated with the peptide for 15 minutes (Fig. 4), indicating that like endogenous DCBLD2, the exogenous peptide interacts with VEGFR2.
  • Example 1-5 DCBLD2 SS traC peptide promotes VEGF-induced angiogenesis in vivo
  • the effect of DCBLD2 SS traC peptide on in vivo response to exogenous VEGF was evaluated in matrigel plug and cornea micropocket angiogenesis assays. Visual inspection of matrigel plugs showed to contain more neovascularization in the presence of FITC-traC and VEGF, compared to VEGF alone (Fig. 5A).
  • FITC-traC enhanced angiogenesis in the presence of VEGF. This difference was readily detectable on visual inspection of corneas and was confirmed by CD31 immunostaining (Fig. 6A-B).
  • Example 1-6 A poly leucine decapeptide mimics the effect of DCBLD2 SS traC on VEGF signaling
  • DCBLD2 SS traC peptide in therapeutic applications, the present study sought to determine its minimal sequence that interacts with VEGFR2.
  • the DCBLD2 SS traC was split into two parts with the C-terminal part 2 (LLLLLVLLLLLEDAGA) encompassing the poly-leucine sequence.
  • LLLLLVLLLLLEDAGA C-terminal part 2
  • CSNSSSFSMPLF GFP-HA-tagged Part 1
  • Part 2 but not Part 1 could be co-immunoprecipitated with VEGFR2 (Fig. 8A).
  • Part 2 was then split into two sub-parts: LLLLLVLLLLL (5LV5L) and ED AGA, each connected to a C-terminal GFP-HA tag.
  • LLLLLVLLLLL 5LV5L
  • ED AGA ED AGA
  • the exceptionally long signal sequence of DCBLD2/ESDN contains a hydrophobic and transmembrane subdomain, ‘traC’, which specifically interacts with VEGFR2.
  • ‘traC’ promotes VEGF/VEGFR2 signaling in EC and enhances in vivo angiogenesis and promotes ear wound healing.
  • the minimum essential sequence of traC for interacting with VEGFR2 is the polyleucine segment.
  • Example 1-7 traC peptide effect on VEGFR2 signaling in comparison with control peptide and scrambled peptide in endothelial cells
  • FIG. 10A-13E treating human umbilical vein endothelial cells (HUVEC) with traC peptide resulted in the activation of VEGFR2 signaling (upregulation of p-VEGFR2, p-ERK and p-AKT).
  • the scrambled peptide and control peptide also increased VEGF signaling in endothelial cells, albeit to a smaller intensity and different kinetics of activation, than the traC peptide.
  • Example 1-8 traC but not control peptide and scrambled peptide, is internalized by endothelial cells
  • traC peptide upon contact with HUVEC, traC peptide is internalized.
  • a control peptide having similar sequence to traC (CSNSSSFSMPIFIIIIIVIIIIIEDAGA, SEQ ID NOV, leucine residues of traC are replaced with isoleucine residues) and a scramble w ere not.
  • Reagents [00207] Recombinant Human VEGF 165 was obtained from R&D System (Catalog No. 293-VE). The antibodies for phospho-VEGFR2 (Tyrl 175, 19A10, Catalog No. 2478S), total VEGFR2 (55B11, Catalog No. 2479S), phospho-ERK 1/2 (Thr202/Tyr204, Catalog No.
  • the anti-PTPlB antibody (EP1837Y, Catalog No. ab52650) was obtained from Abeam.
  • HEK 293T cells were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Catalog No. 11965-092) with 10% heat-inactivated fetal Bovine serum (FBS. Sigma- Aldrich, Catalog No. 12306C) and 1% penicillin-streptomycin (Gibco, Catalog No. 15140- 122).
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS heat-inactivated fetal Bovine serum
  • penicillin-streptomycin Gibco, Catalog No. 15140- 122).
  • Endothelial cells were cultured on 0.5% gelatin (Alfa Aesar, Catalog No. J62699) coated plates in Medium 199 (Gibco, Catalog No. 11150-059) supplemented with 20% FBS.
  • HUVECs w ere provided by the tissue culture core laboratory of Yale Vascular Biology and Therapeutics Program.
  • RNAiMAX Reagent Thermo Fisher Scientific, Catalog No. 13778-150
  • ESDN siRNA Horizon, Catalog No. L-016714-00-0005
  • non-targeting control siRNA Horizon, Catalog No. D-001810-01-05
  • the cells were then starv ed in Medium 199 with 1% FBS for 2 hours and treated with / without traC (luM for 15min, unless stated otherwise) or VEGF (10 ng/ml for lOmin, unless stated otherwise) as indicated.
  • the traC peptide was synthesized by Thermo Fisher Scientific following a sequence of FITC- Acp-CSNSSSFSMPLFLLLLLVLLLEDAG (SEQ ID NO: 19), with a purity of over 90%.
  • the formula of this peptide is C141H231N29O39S2, with a molecular w eight of 3410.09.
  • the traC peptide is very hydrophobic, so the peptide was initially dissolved in 100% DMSO.
  • Equal amounts of protein were resolved by SDS-PAGE and transferred onto an Immun-Blot PVDF membrane (Bio-Rad, Catalog No. 1620174).
  • the membranes were probed with primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies.
  • the membranes probed with phosphorylated protein antibodies were stripped using restore plus Western blot stripping buffer (Thermo Fisher Scientific, Catalog No. 46430) and re-probed with total protein antibodies.
  • the blots were imaged using Bio-Rad ChemiDoc MP Imaging System and quantified using Image J software.
  • Cell lysates (containing 500pg protein) were incubated with 50ul (1.5mg) Dynabeads protein A (Invitrogen, Catalog No. 10006D) for one hour at 4°C, after which the beads were removed by magnet to clear the lysate. The precleared lysates were then incubated with respective Dynabeads-antibody complex at 4°C overnight and washed gently with washing buffer three times. After eluting the target antigen from the immunocomplexes, the beads were removed with a magnet, and the supernatant was stored for further analysis.
  • Dynabeads protein A Invitrogen, Catalog No. 10006D
  • Cornea pocket assay was performed as described in Tang et al. (J Vis Exp. 2011(54).) with minor modifications. Using a micro-knife (Ambler Surgical, Catalog No. 3401E) a 1 mm incision was made in the cornea about 1-1.3 mm from the limbus. The small pellets freshly prepared by mixing poly-HEMA (Sigma, Catalog No. P3932). sucralfate (Cayman Chemical, Catalog No. 27882), with combinations of recombinant human VEGF 165 and traC /DMSO (1%) were applied to the pockets, which were allowed to be closed over the pellets.
  • corneas were collected and cut into four equal sectors, followed by whole mount immunostaining using antibodies against CD31 (R&D Systems, Catalog No. AF3628). Corneas were then flat mounted using ProLong Glass Antifade Mountant (Thermo Fisher Scientific, Catalog No. P36980) on a glass slide (epithelium up) and sealed under a #1.5 thick coverslip for imaging. Quantification of angiogenesis was performed by defining the fraction of CD31 positive area in each sector.
  • a finger loop ear punch was used to perforate 2mm diameter circular wounds in the center of both ears of albino mice.
  • FITC- traC (10 pg) dissolved in lOpl PBS was injected into the left ear wound edge every other day, and 1% DMSO in lOpl PBS was injected into the right ear as control.
  • the two ears of each mouse were photographed daily to track their healing. After 20 days, the animals were euthanized, and ear tissues were harvested. For each ear, the wound sections with the largest diameters were imaged and used for measuring the size of the remaining hole.
  • DCBLD2 The neuropilin-like, Discoidin. CUB and LCCL domain containing 2
  • SS unusually long signal sequence
  • C C-terminal subdomains
  • tra transition subdomain
  • DCBLD2 SS interacted with VEGFR2 and promoted VEGF signaling.
  • SS was not cleaved in the mature DCBLD2 and its hydrophobic transmembrane ’traC' segment, but not the ‘N’ subdomain, was involved in DCBLD2-VEGFR2 interaction.
  • the smallest unit in DCBLD2 SS that interacts with VEGFR2 was the L5VL5 sequence. Even after the central valine was removed, the LIO sequence mimicked the DCBLD2 SS traC’s effect on VEGF- signaling, while shorter or longer poly-leucine sequences were less effective.
  • a synthetic traC peptide enhanced VEGF signaling in vitro, promoted VEGF-induced angiogenesis in vivo, and improved blood flow recovery following hindlimb ischemia.
  • DCBLD2 SS along with its derivative peptides can promote VEGFR2 signaling and angiogenesis.
  • Synthetic peptides based on DCBLD2 SS hold promise as therapeutic agents for regulating angiogenesis.
  • these findings refine the traditional view of signal sequences as mere targeting elements, revealing a role in cellular signaling. This opens new avenues for research and therapeutic strategies.
  • SS signal sequences
  • SP signal peptide
  • a typical SP has a tripartite structure with a hydrophobic, a-helical core (/? region) surrounded by an N-terminal polar n region, and a C-terminal c region that contains the signal peptidase cleavage site.
  • exceptionally long SS may have a bipartite ‘NtraC’ domain structure with potentially functional N-terminal (N-domain, ‘N’) and C-terminal (C-domain, ‘C’) domains connected by a turn-rich linker area (transition area. ‘ tra’).
  • DCBLD2 CUB and LCCL domain containing 2
  • ESDN endothelial and smooth muscle cell- derived neuropilin-like protein
  • CLCP1 vascular endothelial cell growth factor receptors
  • DCBLD2 is upregulated in human, rat, and mouse remodeling arteries, and regulates VEGF- induced endothelial cell (EC) proliferation, migration, and signal transduction, as well as developmental and adult angiogenesis.
  • EC VEGF- induced endothelial cell
  • Angiogenesis the process of new blood vessel formation from existing vessels, plays a key role in a number of physiological processes, including development and growth, adult organ regeneration, and wound healing. Angiogenesis is tightly controlled through numerous stimulators and inhibitors and its dysregulation, whether insufficient or excessive, contributes to a wide variety of diseases, including malignancy, ischemic heart and peripheral artery disease, neurodegeneration, and proliferative retinopathy. Accordingly, modulation of this process provides several therapeutic opportunities.
  • the present study identified the DCBLD2 SS, and more specifically its ‘traC’ segment, as a key domain of DCBLD2 that interacts with VEGFR2 and promotes VEGF/VEGFR2 signaling in EC. Furthermore, an exogenous synthetic FITC-traC peptide promoted angiogenesis in vivo.
  • the smallest unit in DCBLD2 SS that interacted with VEGFR2 was the L5VL5 sequence. Even after the middle valine was removed, the LIO sequence could still mimic the effect of DCBLD2 SS traC on VEGF signaling.
  • these findings introduce the DCBLD2 traC and its derivatives as promising candidates for therapeutic angiogenesis.
  • HEK 293T cells were maintained in Dulbecco's Modified Eagle Medium (DMEM. Gibco) supplemented with 10% heat inactivated fetal bovine serum (FBS, Sigma Aldrich) and 1% penicillin/ streptomycin (Gibco).
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS heat inactivated fetal bovine serum
  • Gibco penicillin/ streptomycin
  • endothelial cell culture primary' human umbilical vein endothelial cells (HUVECs) were obtained from Yale Vascular Biology & Therapeutics Program Tissue Culture program and cultured on gelatin-coated cultureware with Medium 199 (Gibco) supplemented with 20% FBS, 1% endothelial cell growth supplement (ECGS), 2 mM L- glutamine, and 1% penicillin/streptomycin.
  • MLEC were isolated from 4-week-old wild-type and Dcbld2 ' "mice. Mice were anesthetized and lungs harvested, rinsed in Hank’s Balanced Salt Solution (HBSS, Sigma Aldrich), cut into small pieces, and subjected to enzy matic digestion with a Img/ml collagenase solution for 45 minutes. The tissue solution was then mechanically disrupted, passed through a 70 pm strainer, and cells were collected by centrifugation at 350g for 5 minutes and resuspended in complete Medium 199. CD31 -positive cells were separated using immunomagnetic separation with CD31 -conjugated beads, followed by detachment of the cells from the beads and cultured until 70-80% confluent.
  • HBSS Hank’s Balanced Salt Solution
  • Fig. 15A and 15F The constructs used in this study' are schematically shown in Fig. 15A and 15F.
  • the genes encoding different protein fragments were cloned into a pCDNA3.0 plasmid between Hindlll and Kpnl restriction sites. All constructs were confirmed by PCR or DNA sequencing. Transfection of cells with plasmid DNA was carried out using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer’s instructions. 24 hours after transfection, cells w ere washed and cultured in DMEM supplemented with 10% FBS for another 24 hours before use.
  • the blots were developed using an ECL substrate (Thermo Fisher).
  • ECL substrate Thermo Fisher.
  • DynabeadsTM Protein A Immunoprecipitation Kit Invitrogen was used. All steps were carried out according to the manufacturer’s protocol. After completing the protocol, beads were removed with a magnet and the resultant supernatant was collected for analysis.
  • FITC-L10 and FITC-scramble peptide with a sequence of [FITC][Ahx]-LLLLLLLLLLEDAGA(SEQ ID NO: 16)-[COOH] and [FITC][Ahx]-LSLESALMLFLCLDLSGVFLSLPLSNL(SEQ ID NO: 10)-[COOH] respectively, were obtained from Thermo Fisher Scientific. Mass spectrum and HPLC data reported a molecular mass of 2094.58 Da and 3412.10 Da for FITC-L10 and FITC-scramble respectively and a purity of >98% for both. Antennapedia peptide was obtained from GeneScript, with mass spectrometry' and HPLC analysis reporting a molecular mass of 2749.28 Da and a purity of 96.6%.
  • Matrigel plug angiogenesis assay [00236] One pmol of traC, with or without 2 pg/ml VEGF, was mixed into 300 gil of growth factor-reduced Matrigel (Coming) at 4°C. The mixture was subcutaneously injected into the opposite iliac regions of 8-10-w ? eek-old C57BL/6J WT mice. Five days post-injection, mice were euthanized and Matrigel plugs were collected and embedded in OCT.
  • Coming growth factor-reduced Matrigel
  • the plugs were frozen, sectioned, and stained with goat polyclonal anti-CD31 (R&D Systems) overnight at 4°C, followed by Alexa Fluor 594-labeled chicken anti-goat IgG (Thermo Fisher) for 1 hour at room temperature. Sections were imaged using a widefield microscope (Teica DMi8). When imaging, areas containing the highest CD31 staining at the edge of the sections were imaged and the percentage of the CD31 -positive area relative to the total imaged area was quantified blindly and reported.
  • Pellets were prepared 24 hours before implantation. Initially , 5 pl of a 12% (w/v) solution of poly(2-hydroxy ethyl methacrylate) (poly-HEMA; Sigma Aldrich) dissolved in ethanol and 1 pl of a 10% (w/v) sucralfate solution (Cayman Chemical) dissolved in PBS were mixed.
  • poly-HEMA poly(2-hydroxy ethyl methacrylate)
  • the resulting pellets contained either 60 ng of FITC-traC, 80 ng of VEGF, a combination of both, or the vehicle controls.
  • Implantation of the pellets was performed on 12-week-old C57BL/6J mice (Jackson Laboratory). Briefly, using a micro-knife (Ambler Surgical), a 1 mm incision was made on the cornea, approximately 1 mm from the limbus, into w hich a pellet was inserted. Each mouse was implanted with two pellets, one under each cornea. 13 days post implantation, corneas were harvested and fixed in 4% paraformaldehyde on ice for 1 hour, followed by permeabilization and blocking with a 5% BSA solution containing 0.
  • Osmotic mini pumps (Alzet) w ere primed and prepared to deliver 1.74mg/day of traC for 7 days to the ligated limb based on a pumping rate of 1.05ml/hr, according to the manufacturer’s instructions.
  • the osmotic mini pump was subcutaneously inserted into the back of the mouse, and a catheter (Alzet) connected to the pump was guided and secured adjacent to the ligated thigh.
  • the animals were randomly assigned to treatment or control groups.
  • Laser Doppler flow images of the foot w ere acquired before, immediately after surgery, and on days 3, 5, and 7 post-surgery.
  • mice After 7 days of treatment, the mice were euthanized and the thigh and the leg were collected in OCT, frozen, and sectioned. Four sections, approximately 2 mm spaced apart, from each thigh were stained for CD31. The percentage of the CD31 -positive area w as calculated for each of the four sections relative to the total area of each thigh and the results were averaged to obtain a representative percentage of the CD31 -positive area within each thigh. Quantification of cdhS gene expression was performed using RT-PCR with Gapdh used as the housekeeping gene. Results were reported as 2" act . Fifty sections, each 5 pm thick, were collected at 8 locations along the thigh or the leg at 1 mm intervals and pooled for each animal.
  • Total mRNA was isolated using the RNeasy kit (Qiagen), and reverse transcription (RT) was carried out with the QuantiTect RT kit (Qiagen). Real-time PCR was conducted in triplicates using cDNA with TaqMan gene expression assays, following the manufacturer's instructions. Tissue analysis was performed blinded to treatment status.
  • Results are expressed as the mean ⁇ standard deviation. All statistical analyses were performed using GraphPad Prism 10. Data were checked for normality with the Shapiro-Wilk test. For data that did not pass the Shapiro-Wilk test, non-parametric tests were used. Statistical significance was determined by a two-way analysis of variance (ANOVA, followed by Tukey’s post hoc test, for multiple groups, and a student t-test or a Mann- Whitney U test for comparison between two groups. A p-value ⁇ 0.05 was considered significant.
  • Example 2-3 The DCBLD2 SS traC subdomain is involved in DCBLD2 interaction with VEGFR2
  • Distinct functional and structural units of DCBLD2 include an unusually long SS, which in tandem with CUB, LCCL, and Discoidin domains form the extracellular segment. A transmembrane domain and an intracellular segment comprise the rest of the molecule.
  • the present study generated several plasmids expressing various combinations of different DCBLD2 domains linked to a C-terminal hemagglutinin (HA) tag (Fig. 15A I-V. Upon co-transfection with VEGFR2 in HEK 293T cells each of these constructs expressed the appropriate-size protein (Fig. 19).
  • the present study transfected HEK 293T cells with a dually tagged DCBLD2 plasmid with N-terminal FLAG and C-terminal HA tags. Immunoprecipitation of the product with an anti-HA antibody followed by FLAG and DCBLD2 immunoblotting showed a single (non-cleaved) product (Figs. 18A-18E).
  • the DCBLD2 SS ’N’ subdomain contains 38 amino acid residues, and the 24 amino acid C-terminal residues constitute the ‘C’ subdomain, with those two separated by a short ‘tra’ subdomain (Fig. 15E).
  • Example 2-4 FITC-TraC enhances VEGF/VEGFR2 signaling in EC
  • the present study sought to investigate whether a synthetic traC peptide, labeled with FITC for tracking purposes, can modulate VEGF signaling in EC.
  • the present study followed by fluorescence imaging the fate of the labeled peptide added to EC culture media.
  • FITC-traC was rapidly taken up by HUVEC with the peptide detected in the cytoplasm as early as 1 minute after its addition to the medium.
  • the cytoplasmic accumulation of the peptide increased over time and by 15 minutes it was also detectable in the cell membrane (Fig. 16A), where VEGFR2 is also located (Fig. 16B). Accordingly, the 15-minute time point was selected for the next set of studies.
  • VEGFR2 but not PDGFR0, could be coimmunoprecipitated with FITC-traC, when HUVEC w ere pretreated with the peptide for 15 minutes (Fig. 16E), indicating that like endogenous DCBLD2, the exogenous peptide interacts with VEGFR2.
  • the specificity of VEGFR2 interaction with FITC-traC was confirmed following co-transfection of different tyrosine kinase receptors in HEK 293 cells (Fig. 29).
  • Example 2-5 DCBLD2 SS FITC-traC peptide promotes VEGF and ischemia-induced angiogenesis in vivo
  • Example 2-6 A poly leucine decapeptide mimics the effect of DCBLD2 SS traC on VEGF signaling
  • the DCBLD2 SS traC was split into two parts with an N-terminal part 1 (CSNSSSFSMPLF, SEQ ID NO: 17), and a C-terminal part 2 (LLLLLVLLLLLEDAGA, SEQ ID NO: 1) encompassing the poly-leucine sequence.
  • CSNSSSFSMPLF N-terminal part 1
  • LLLLLVLLLLLEDAGA C-terminal part 2
  • Part 2 was then split into two sub-parts: LLLLLVLLLLL (L5VL5) (SEQ ID NO:5) and ED AGA (SEQ ID NO:3), each connected to a C-terminal GFP-HA tag.
  • L5VL5 SEQ ID NO:5
  • ED AGA SEQ ID NO:3
  • DCBLD2 regulates developmental and adult angiogenesis by engaging in a macromolecular complex with VEGFR2, which promotes VEGF signaling by dissociating VEGFR2 away from its negative regulators of downstream signaling, VE- cadherin and protein tyrosine phosphatases. PTP1 and TC-PTP.
  • the present study identified the hydrophobic 'traC’ subdomain of the DCBLD2 SS, and more specifically, its L5VL5 segment, as a key component of the DCBLD2 interaction with VEGFR2.
  • DCBLD2 SS is not cleaved and therefore, may function as a signal anchor sequence. This would indicate that DCBLD2 is a double-pass transmembrane protein with cytosolic N and C terminals. Transmembrane domains are often engaged in protein-protein interactions and regulate the activity of the parent molecule. Similarly, the DCBLD2 SS and more specifically, its traC subdomain is involved in DCBLD2-VEGFR2 interaction and modulates VEGF signaling.
  • the present study further determined that the smallest unit in the DCBLD2 SS that can interact with VEGFR2 is the L5VL5 sequence.
  • Most short ploy-leucine transmembrane domains containing single amino acid substitutions can bind to and activate PDGFR(3. Minor changes in the composition of these sequences (e.g., changing the placement of a single side chain methyl group) can alter their partner selectivity.
  • the LIO sequence could also interact with VEGFR2.
  • shorter or longer poly-leucines were less effective in this regard, raising the possibility that the size of the poly -leucine sequence is one of the fundamental principles that determine such interactions.
  • the exogenous FITC-traC and its derivative polyleucine peptide mimicked the effect of endogenous DCBLD2 in interacting with VEGFR2 and regulating VEGF signaling.
  • VEGFR2 is a main receptor for VEGF, the protot pic pro-angiogenesis grow th factor, in endothelial cells.
  • Angiogenesis is a key process during development, growth, and adulthood. Inadequate neovessel formation is a major pathologic feature in ischemia-related cardiovascular diseases and contributes to poor wound healing. In other pathological settings, e.g., diabetic retinopathy and malignancy, angiogenesis is improperly exaggerated. As such, there is considerable interest in regulators of VEGF signal transduction. FITC-traC promoted angiogenesis, not only when induced by exogenous VEGF, but also in the presence of ischemia, suggesting that it may be of value in treating ischemic diseases.
  • FITC- traC also improved hindlimb blood flow recovery within a few days after femoral artery ligation in mice. This is in line with the finding that deletion of DCBLD2 impairs blood flow recovery in the same animal model, and may indicate that in addition to regulating angiogenesis, DCBLD2 and its SS derivatives may regulate collateral recruitment or arteriogenesis.
  • the role of DCBLD2 SS in related signaling pathways and cell types will be the subject of future studies, which could also identify the specific VEGFR2 sequences/domains that interact with traC.
  • the DCBLD2 SS has a novel post-targeting function in interacting with and regulating VEGFR2 signaling and promoting angiogenesis through its transmembrane traC subdomain. This effect is narrowed down to the L5VL5 and LIO sequences.
  • the present invention is directed to the following non-limiting embodiments:
  • Embodiment 1 A method of promoting neovascularization and/or hair growth in a subject in need thereof, the method comprising: administering to the subject a compound comprising a polypeptide, wherein at least one of the following applies:
  • the polypeptide comprises a first peptide comprising from about 6 amino acid residues to about 14 amino acid residues, 80% or more of the amino residues of the first peptide are leucine residues, optionally wherein the polypeptide comprise a plurality of independently selected first peptides, and the compound does not comprise a peptide sequence that has a length longer than about 14 amino acids and that has more than about 80% of the amino acid residues as leucine residues, or
  • polypeptide comprises the amino acid set forth in SEQ ID NO:5, 6, 8. or 9.
  • Embodiment 2 The method of Embodiment 1 , wherein the compound is soluble in water or insoluble in water.
  • Embodiment 3 The method of any one of Embodiments 1-2, wherein the compound is soluble in water, and wherein the compound further comprises a hydrophilic motif.
  • Embodiment 4 The method of Embodiment 3, wherein the hydrophilic motif is at least one selected from the group consisting of:
  • Embodiment 5 The method of any one of Embodiments 1-4, wherein the peptide comprises residues 1-66, residues 39-66, residues 51-66, or residues 51-61 of the sequence set forth in SEQ ID NO: 15.
  • Embodiment 6 The method of any one of Embodiments 1-5, which is a method of promoting neovascularization, and wherein the method promotes in the subject at least one selected from the group consisting of angiogenesis and arteriogenesis.
  • Embodiment 7 The method of any one of Embodiments 1-6, which is a method of promoting neovascularization, wherein at least one of the following applies:
  • the subject suffers from a chronic wound healing disorder, and wherein the method promotes neovascularization in or near at least one chronic wound of the subject;
  • Embodiment 8 The method of Embodiment 7, wherein the chronic wound comprises at least one of the following:
  • an ulcer optionally a diabetic ulcer such as a diabetic foot ulcer, an arterial ulcer, a venous ulcer, a pressure ulcer, or an ischemic ulcer; or
  • Embodiment 9 The method of any one of Embodiments 1-8, wherein the compound reduces phosphatase activity exerted by protein-tyrosine phosphatase IB (PTP1B) and/or T- cell protein tyrosine phosphatase (TC-PTP) on vascular endothelial growth factor receptor 2 (VEGFR2).
  • PTP1B protein-tyrosine phosphatase IB
  • TC-PTP T- cell protein tyrosine phosphatase
  • VAGFR2 vascular endothelial growth factor receptor 2
  • Embodiment 10 The method of any one of Embodiments 1-9, wherein the method further comprises administering to the subject an effective amount of vascular endothelial growth factor (VEGR).
  • VEGR vascular endothelial growth factor
  • Embodiment 11 A method of promoting wound healing in a subject in need thereof, the method comprising: administering to the subject a compound comprising a polypeptide, wherein at least one of the following applies:
  • the polypeptide comprises a first peptide comprising from about 6 amino acid residues to about 14 amino acid residues, 80% or more of the amino residues of the first peptide are leucine residues, optionally wherein the polypeptide comprise a plurality of independently selected first peptides, and the compound does not comprise a peptide sequence that has a length longer than about 14 amino acids and that has more than about 80% of the amino acid residues as leucine residues, or
  • the polypeptide comprises an amino acid set forth in SEQ ID NO:5. 6, 8 or 9.
  • Embodiment 12 The method of Embodiment 11, wherein the compound is soluble in water or insoluble in water.
  • Embodiment 13 The method of any one of Embodiments 11-12, wherein the compound is soluble in water, and wherein the compound further comprises a hydrophilic motif.
  • Embodiment 14 The method of Embodiment 13, wherein the hydrophilic motif is at least one selected from the group consisting of:
  • Embodiment 15 The method of any one of Embodiments 11-14, wherein the peptide comprises residues 1-66, residues 39-66, residues 51-66, or residues 51-61 of the sequence set forth in SEQ ID NO: 15.
  • Embodiment 16 The method of any one of Embodiments 11-15, wherein the method promotes wound healing by promoting in the subject at least one selected from the group consisting of angiogenesis and arteriogenesis.
  • Embodiment 17 The method of any one of Embodiments 11-16, wherein the wound is a chronic wound or an acute wound.
  • Embodiment 18 The method of Embodiment 17, wherein the chronic wound comprises at least one of the following:
  • an ulcer optionally a diabetic ulcer such as a diabetic foot ulcer, an arterial ulcer, a venous ulcer, a pressure ulcer, or an ischemic ulcer; or
  • Embodiment 19 The method of any one of Embodiments 11-18, wherein the compound reduces phosphatase activity exerted by protein-tyrosine phosphatase IB (PTP1B) and/or T-cell protein tyrosine phosphatase (TC-PTP) on vascular endothelial grow th factor receptor 2 (VEGFR2).
  • PTP1B protein-tyrosine phosphatase IB
  • TC-PTP T-cell protein tyrosine phosphatase
  • Embodiment 20 The method of any one of Embodiments 11-19, wherein the method further comprises administering to the subject an effective amount of vascular endothelial grow th factor (VEGR).
  • VEGR vascular endothelial grow th factor
  • Embodiment 21 A compound comprising a polypeptide and a hydrophilic motif, wherein at least one of the following applies:
  • the polypeptide comprises a first peptide comprising from about 6 amino acid residues to about 14 amino acid residues, 80% or more of the amino residues of the first peptide are leucine residues, and the compound does not comprise a peptide sequence that has a length longer than about 14 amino acids and that has more than about 80% of the amino acid residues as leucine residues,
  • the polypeptide comprises an amino acid set forth in SEQ ID NO:5, 6, 8 or 9. or a salt or solvate thereof.
  • Embodiment 22 The compound of Embodiment 21, wherein the hydrophilic motif is at least one selected from the group consisting of:
  • Embodiment 23 The compound of Embodiment 21 or 22, wherein the polypeptide comprise a plurality of independently selected first peptides.
  • Embodiment 24 A wound dressing, comprising a base dressing material, and a compound comprising a polypeptide, wherein at least one of the following applies:
  • the polypeptide comprises a first peptide comprising from about 6 amino acid residues to about 14 amino acid residues. 80% or more of the amino residues of the first peptide are leucine residues, and the compound does not comprise a peptide sequence that has a length longer than about 14 amino acids and that has more than about 80% of the amino acid residues as leucine residues,
  • the polypeptide comprises an amino acid set forth in SEQ ID NO:5. 6, 8 or 9.
  • Embodiment 25 The wound dressing of Embodiment 24, wherein the compound is soluble in water or insoluble in water.
  • Embodiment 26 The wound dressing of any one of Embodiments 24-25, wherein the compound is soluble in water, and wherein the compound further comprises a hydrophilic motif.
  • Embodiment 27 The wound dressing of any one of Embodiments 24-26, wherein the hydrophilic motif is at least one selected from the group consisting of:
  • Embodiment 28 The wound dressing of any one of Embodiments 24-27, wherein the peptide comprises residues 1-66, residues 39-66, residues 51-66, or residues 51-61 of the sequence set forth in SEQ ID NO: 15.
  • Embodiment 29 The wound dressing of any one of Embodiments 24-28, wherein the base dressing material comprises at least one selected from the group consisting of an alginate dressing material, a biosynthetic dressing material, a collagen dressing material, a composite dressing material, a contact layer dressing material, a gauze, a hydrocolloid dressing material, a hydrofiber dressing material, a hydrogel, a hydropolymer dressing material, a polyurethane foam dressing material, a skin substitute dressing material, a superabsorbent dressing material, a transparent film dressing material, a wound filler, a wound pouch.
  • the base dressing material comprises at least one selected from the group consisting of an alginate dressing material, a biosynthetic dressing material, a collagen dressing material, a composite dressing material, a contact layer dressing material, a gauze, a hydrocolloid dressing material, a hydrofiber dressing material, a hydrogel, a hydropolymer dressing material, a polyurethane foam dressing material, a skin substitute dressing material
  • Embodiment 30 The wound dressing of any one of Embodiments 24-29. wherein the compound comprising the polypeptide is on a surface of or impregnated into the base dressing material.
  • Embodiment 31 The wound dressing of any one of Embodiments 24-30, further comprises an adhesive substrate.

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Abstract

L'invention concerne une méthode destinée à la promotion de la néovascularisation chez un sujet et/ou une méthode destinée à la cicatrisation des plaies chez un sujet. Les deux méthodes consistent à administrer au sujet un composé contenant un peptide, qui comprend une section poly-leucine ayant une longueur allant d'environ 6 résidus d'acides aminés à environ 14 résidus d'acides aminés, et une teneur en leucine supérieure ou égale à 80 %.
PCT/US2024/056265 2023-11-15 2024-11-15 Peptides destinés à favoriser la néovascularisation et la cicatrisation de plaies, et méthodes les utilisant Pending WO2025106913A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002022815A1 (fr) * 2000-09-11 2002-03-21 Hyseq, Inc. Procedes et substances relatifs aux polypeptides et polynucleotides de type neuropiline
US20050202481A1 (en) * 2001-12-27 2005-09-15 Ono Pharmaceutical Co., Ltd. Novel polypeptide ESDN, polynuleotides encoding the polypeptide, and utility of the polypeptide

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002022815A1 (fr) * 2000-09-11 2002-03-21 Hyseq, Inc. Procedes et substances relatifs aux polypeptides et polynucleotides de type neuropiline
US20050202481A1 (en) * 2001-12-27 2005-09-15 Ono Pharmaceutical Co., Ltd. Novel polypeptide ESDN, polynuleotides encoding the polypeptide, and utility of the polypeptide

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