[go: up one dir, main page]

WO2019010533A1 - Traitement de troubles oculaires - Google Patents

Traitement de troubles oculaires Download PDF

Info

Publication number
WO2019010533A1
WO2019010533A1 PCT/AU2018/050717 AU2018050717W WO2019010533A1 WO 2019010533 A1 WO2019010533 A1 WO 2019010533A1 AU 2018050717 W AU2018050717 W AU 2018050717W WO 2019010533 A1 WO2019010533 A1 WO 2019010533A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
chimeric
fusion protein
amino acid
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/AU2018/050717
Other languages
English (en)
Inventor
Garry SHOOTER
Derek Van Lonkhuyzen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Invert Graphite Ltd
Original Assignee
Factor Therapeutics Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2017902737A external-priority patent/AU2017902737A0/en
Application filed by Factor Therapeutics Ltd filed Critical Factor Therapeutics Ltd
Publication of WO2019010533A1 publication Critical patent/WO2019010533A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present invention relates to methods, compositions and kits for the treatment of ocular conditions.
  • the methods, compositions and kits are particularly useful for, but not limited to, the treatment of ocular conditions by promoting proliferation and/or migration of corneal epithelial cells.
  • the cornea serves a dual function: first, as a key tissue through which light is refracted and transmitted for vision, and second, as a protective barrier to the external environment.
  • the very superficial layer of the cornea consists of a multilayered squamous epithelium and its integrity and transparency is critical for sight.
  • a compromised ocular surface renders the cornea liable to vision-threatening infection, neovascularization, scarring, and melting, with the risk of perforation and loss of the eye.
  • Resurfacing of the corneal epithelium after a corneal wound forms occurs in three phases: migration of adjacent intact epithelial cells to cover the injured zone, proliferation of the migrated monolayer to reestablish epithelial thickness, and differentiation of the reconstruct epithelium to restore structure and function.
  • the initial stage is characterized by cells attaching and spreading over the denuded substratum which typically occurs by epithelial "sliding."
  • An important aspect of this process is cell attachment to the extracellular matrix (ECM).
  • ECM extracellular matrix
  • Most corneal epithelial wounds are promptly repaired via the above-mentioned endogenous processes. However, in some individuals healing is delayed, wounds persevere, and a condition collectively known as persistent epithelial defects (PEDs) can develop.
  • PEDs persistent epithelial defects
  • PEDs are characterized by ulcers with delayed (>2 weeks) healing and can recur once healed, particularly when the basement membrane (BM) is lost. They arise from a variety of insults, including trauma, tear-film disorders, neoplasia and its treatment, corneal graft, surgery, chemical and thermal burns, or in neurotrophic, diabetic, and herpetic keratopathies, and can also accompany immunological disorders.
  • the present invention provides a method for treating or preventing a corneal wound in an individual in need thereof, the method comprising administering:
  • the first peptide comprises, consists essentially of or consists of an amino acid sequence of a growth factor, or at least a domain of a growth factor, that is capable of binding to and activating its cognate growth factor receptor.
  • the growth factor receptor is the insulin like growth factor I receptor (IGF-IR) or epidermal growth factor receptor (EGFR).
  • the first peptide comprises, consists essentially of or consists of an amino acid sequence of a full length growth factor.
  • the first peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 22 or 24.
  • the second peptide comprises an integrin binding sequence such as RGD.
  • the second peptide comprises, consists essentially of or consists of an amino acid sequence of an integrin-binding domain of vitronectin (VN) or fibronectin (FN).
  • the integrin-binding domain of VN may have an amino acid sequence of SEQ ID NO: 23.
  • the peptide does not comprise an amino acid sequence of the heparin binding domain of VN.
  • the second peptide comprises, consists essentially of or consists of an amino acid sequence of a full length VN or FN.
  • the present invention provides a method for increasing the rate of corneal wound healing in an individual, the method comprising administering: (iii) a first peptide capable of binding to and activating a growth factor receptor; and
  • the present invention provides a method for treating or preventing a corneal wound in an individual in need thereof, the method comprising administering:
  • a growth factor or at least a domain of a growth factor which is capable of binding to and activating a cognate growth factor receptor
  • vitronectin VN
  • fibronectin FN
  • at least an integrin-binding domain of VN or FN thereby treating a corneal wound in the individual.
  • the corneal wound is a corneal epithelial wound.
  • the growth factor is selected from insulin like growth factor (IGF)-I or epidermal growth factor (EGF).
  • IGF insulin like growth factor
  • EGF epidermal growth factor
  • the method further comprises administering a further peptide capable of binding to and activating a growth factor receptor that is different from the growth factor receptor that the first peptide binds to and activates.
  • the first peptide and further peptide do not activate the same growth factor receptor.
  • the first peptide may be capable of binding to and activating an IGF-I receptor, and the further peptide may be capable of binding to and activating an EGF receptor.
  • the method comprises administering two or more growth factors, or domains of two or more growth factors which are capable of binding to their respective cognate growth factor receptors.
  • the growth factors are IGF-I and EGF.
  • the method comprises administering IGF-I and EGF.
  • a second peptide capable of binding to an integrin are administered simultaneously or sequentially.
  • a second peptide capable of binding to an integrin are covalently linked.
  • the covalent linker comprises, consists essentially of or consists of amino acids. More preferably, the amino acids are selected from glycine and serine. In any embodiment of the invention, the covalent linker may be chosen from SEQ ID NO: 25, 26, 27, 28, 29, 30, 31 or 32.
  • the first peptide and second peptide are covalently linked and comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 18, 19 or 20.
  • any method of the present invention when two or more peptides or growth factors, or domains of two or more growth factors which are capable of binding and activating their respective cognate growth factor receptors, are present they may be covalently linked.
  • only one peptide, growth factor, or at least a domain of a growth factor which is capable of binding to and activating a cognate growth factor receptor may be covalently linked to a peptide capable of binding to an integrin or at least an integrin-binding domain of VN or FN, and any further growth factor is administered simultaneously or sequentially.
  • the corneal wound may result from surgical and non-surgical trauma or abrasion, tear-film disorders, severe dry eye, neoplasia and its treatment, corneal graft, diabetic keratopathy, neuropathic keratopathy, thermal or chemical burns, herpetic epithelial keratitis, injury caused by ocular anti-viral or -microbial agents, or immunological disorders.
  • the corneal wound may be a persistent epithelial defect (PED).
  • the present invention provides use of: (i) a first peptide capable of binding to and activating a growth factor receptor; and
  • a second peptide capable of binding to an integrin in the manufacture of a medicament for the treatment or prevention of a corneal wound in an individual in need thereof.
  • the present invention also provides a pharmaceutical composition comprising:
  • composition does not contain IGF binding protein 3
  • the composition is IGFBP-3 free.
  • the pharmaceutical composition does not contain any IGF binding proteins (IGFBPs). In other words, the composition is IGFBP free.
  • an IGFBP-3 is not administered to the individual.
  • no IGF binding proteins are administered to the individual.
  • the present invention provides a chimeric or fusion protein comprising a first peptide joined directly or through a linker to a second peptide, wherein the first peptide is capable of binding to and activating an EGF receptor, and the second peptide is capable of binding to an integrin.
  • the present invention provides a chimeric or fusion protein comprising a first peptide joined directly or through a linker to a second peptide, wherein the first peptide comprises part of, or all of an amino acid sequence that is the same as, or homologous to an epidermal growth factor (EGF), and the second peptide comprises part of, or all of an amino acid sequence that is the same as, or homologous to the sequence of an vitronectin (VN) or fibronectin (FN).
  • EGF epidermal growth factor
  • VN vitronectin
  • FN fibronectin
  • the first peptide may comprise, consist essentially of or consist of an amino acid sequence of a receptor binding domain of EGF.
  • EGF is human EGF.
  • the second peptide may comprise, consist essentially of or consist of an amino acid sequence of an integrin-binding domain of VN or FN.
  • VN or FN is human VN or FN.
  • the chimeric or fusion protein may comprise a secretion signal peptide of VN and amino acids 1 to 64 of VN. More preferably, the chimeric or fusion protein comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 19.
  • the present invention provides a chimeric or fusion protein comprising a first peptide, second peptide and third peptide joined directly or through a linker, wherein the first peptide comprises part of, or all of an amino acid sequence that is the same as, or homologous to an epidermal growth factor (EGF), the second peptide comprises part of, or all of an amino acid sequence that is the same as, or homologous to the sequence of an vitronectin (VN) or fibronectin (FN), and the third peptide comprises part of, or all of an amino acid sequence that is the same as, or homologous to an IGF.
  • EGF epidermal growth factor
  • VN vitronectin
  • FN fibronectin
  • the IGF may be either IGF-I or IGF-I I.
  • the third peptide comprises, consists essentially of or consists of an amino acid sequence of an IGF-I receptor binding domain of either IGF-I or IGF-I I.
  • the IGF is human IGF.
  • the chimeric or fusion protein comprises the secretion signal peptide of VN and amino acids 1 to 64 of VN.
  • the chimeric or fusion protein comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 20.
  • the first peptide and second peptide may be arranged such that the first peptide is N-terminal to the second peptide. In one embodiment, the peptides are arranged from N to C-terminal: first peptide and second peptide. In any aspect, within the chimeric or fusion protein the first, second and third peptides may be arranged such that the first peptide is N-terminal to the second peptide, and the second peptide is N-terminal to the third peptide. In one embodiment, the peptides are arranged from N to C-terminal: first peptide, second peptide and third peptide. In yet another aspect, the invention provides a composition comprising a chimeric or fusion protein as broadly described above, optionally a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention also provides a method of treating or preventing a corneal wound in an individual in need thereof, the method comprising administering to the individual a chimeric or fusion protein as described above, or a composition as described above, thereby treating or preventing a corneal wound in the individual.
  • the invention further provides the use of a chimeric or fusion protein as described above, or a composition as described above, in the manufacture of a medicament for treating or preventing a corneal wound in an individual in need thereof.
  • the invention also provides a nucleic acid molecule comprising, consisting essentially of or consisting of a nucleotide sequence encoding a chimeric or fusion protein as described above, optionally operatively linked to at least one regulatory element.
  • the nucleic acid molecule comprises, consists essentially of or consists of a nucleotide sequence encoding a chimeric or fusion protein comprising, consisting essentially of or consisting of amino acid sequences shown in SEQ ID NO: 22 and 23, optionally further comprising or consisting of amino acid sequences shown in SEQ ID NO: 21 and any one or more of SEQ ID NOs: 25 to 33.
  • the nucleic acid molecule comprises, consists essentially of or consists of a nucleotide sequence encoding a chimeric or fusion protein comprising, consisting essentially of or consisting of amino acid sequences shown in SEQ ID NO: 22, 23 and 24, optionally further comprising or consisting of amino acid sequences shown in SEQ ID NO: 21 and any one or more of SEQ ID NOs: 25 to 33.
  • the nucleic acid molecule comprises, consists essentially of or consists of nucleotide sequences shown in SEQ ID NO: 5 and 6, optionally further comprising, consisting essentially of or consisting of nucleotide sequences shown in SEQ ID NO: 4 and any one or more of SEQ ID NOs: 8 to 16.
  • the nucleic acid molecule comprises or consists essentially of nucleotide sequences shown in SEQ ID NO: 5, 6 and 7, optionally further comprising, consisting essentially of or consisting of nucleotide sequences shown in SEQ ID NO: 4 and any one or more of SEQ ID NOs: 8 to 16.
  • the nucleotide sequence may comprise SEQ ID NO: 1 , 2 or 3, or any other nucleotide sequence as shown in Table 3A, or encode for VF003 or VF004 as described herein.
  • the invention further provides a vector including such a nucleic acid molecule, as well as a prokaryotic or eukaryotic cell including such a nucleic acid molecule.
  • the invention also provides a method of treating a corneal wound in an individual in need thereof, the method comprising administering to the individual a nucleic acid molecule as described above, a vector as described above, or a prokaryotic or eukaryotic cell as described above.
  • the invention further provides the use of a nucleic acid molecule as described above, a vector as described above, or a prokaryotic or eukaryotic cell as described above, in the manufacture of a medicament for treating a corneal wound in an individual in need thereof.
  • the administration of the first and second peptide, or chimeric or fusion protein does not result in significant ocular phimosis or corneal neovascularisation. In one embodiment, the administration of the first and second peptide, or chimeric or fusion protein, does not result in any clinically observable ocular phimosis or corneal neovascularisation.
  • Figure 1 VF001 (VN:IGF-I) purification from Sf9 conditioned media.
  • Conditioned media from expression cultures was passed onto Q-Sepharose IEX and eluted therefrom using high salt. The subsequent sample was then applied to Ni2+-NTA IMAC resin and eluted with 250 mM Imidazole. Purification samples were interrogated by Silver Stain SDS-PAGE (A) and Western blot (B) using a- VN (red) and a-IGF-l (green) Ab's.
  • VF003 VN:EGF purification from Sf9 conditioned media.
  • Conditioned media from expression cultures was passed onto Q-Sepharose IEX and eluted therefrom using high salt. The subsequent sample was then applied to Ni2+-NTA IMAC resin and eluted with 250 mM Imidazole. Purification samples were interrogated by Silver Stain SDS-PAGE (A) and Western blot (B) using a- VN (red) and a-EGF (green) Ab's.
  • VF004 (EGF:VN:IGF-I) purification from Sf9 conditioned media
  • Conditioned media from expression cultures was passed onto Q-Sepharose IEX and eluted therefrom using high salt. The subsequent sample was then applied to Ni2+-NTA IMAC resin and eluted with 250 mM Imidazole. Purification samples were interrogated by Silver Stain SDS-PAGE (A) and Western blot (B) using a- VN (red) and a-IGF-l (green) Ab's and (C) with a-EGF only (green).
  • BSA Bovine Serum Albumin
  • HCE-T cell proliferation in response to chimeric and control treatments.
  • Cell proliferation in HCE-T cells was measured after 48 hours in response to chimeric proteins and control treatments by the MTS method.
  • VN: IGF-I VF001
  • V+E VN + EGF
  • VN:EGF VF003
  • V+E+l VN + EGF + IGF-I
  • EGF:VN: IGF-I VF004.
  • VF4-P03 was included as a good manufacturing practice (GMP)-grade VF001 reference material that is manufactured using a Pichia pastoris expression system.
  • HCE-T cell migration in response to chimeric and control treatments.
  • Cell migration in HCE-T cells was measured after 48 hours in response to chimeric proteins and control treatments by the fence method.
  • VN: IGF-I VF001
  • V+E VN + EGF
  • VN:EGF VF003
  • V+E+l VN + EGF + IGF-I
  • EGF:VN: IGF-I VF004.
  • FIG. 7 Stained HCE-T cells. Example of image analysis after fixing and staining of migrated HCE-T cells.
  • FIG. 9 HCEC migration (Fence) in response to chimeric and control treatments.
  • Cell migration in HCECs was measured after 48 hours in response to chimeric proteins and control treatments by the fence method.
  • Data were analysed using a One-Way ANOVA with Tukey's post-hoc test and each treatment compared to each other. All treatments were found to induce HCEC migration significantly above Basal medium (not noted on plot), * indicates a significant decrease in migration compared to equimolar VF001 (p ⁇ 0.05) and # indicates significant increase in migration above GM (p ⁇ 0.05).
  • Figure 10 Efficacy of best performing test articles as measured by fluorescein staining. Fastest wound healing was noticed with VF003, then VF004, followed by VF001 . Least resolved area was observed with Placebo treatment group. (Images for additional test groups are given in Figure 4). Figure 11. Diffuse areas of fluorescein staining were observed with VF004
  • FIG. 12 Corneal transparency and clarity as imaged at Day 5: The rate of resolution of corneal transparency and clarity seems almost the same in all groups but is best with VF003 (high concentration).
  • Figure 13 Representative images of fluorescein staining across treatment groups. The most significant change in the epithelial defects were observed at Day 4 and Day 5 with fastest healing observed in the VF003 high and low concentration groups. VF004 and VF001 treatments also showed better re-epithelialization than the placebo (vehicle alone) group.
  • Figure 14 Representative images of corneal transparency and clarity across treatment groups.
  • Figure 15 Change in epithelial wound area between Day 0 and Day 5. A predictable dose-response benefit was observed with the high dose treatments performing better than the low doses in all 3 treatment groups.
  • Figure 16 Corneal epithelial wound healing expressed as a percentage of the initial wound area with time.
  • FIG. 17 Relative changes in wound area across time - day 3.5. Corneal epithelial wound healing commenced from day 2 in the placebo group. However, wound healing rates increased on Day 3 for the VF treatment groups.
  • FIG. 18 Relative changes in wound area across time - day 4. Corneal epithelial wound healing commenced from day 2 in the placebo group. However, wound healing rates increased on Day 3 for the VF treatment groups. On Day 4 reduction in wound area was significantly superior for VF003, VF004 (high dose) and VF001 (high dose) treatments as compared to the placebo group.
  • FIG. 19 Relative changes in wound area across time - day 5. Corneal epithelial wound healing commenced from day 2 in the placebo group. However, wound healing rates increased on Day 3 for the VF treatment groups. On Day 4 and Day 5, reduction in wound area was significantly superior for VF003, VF004 (high dose) and VF001 (high dose) treatments as compared to the placebo group.
  • Figure 20 Relative healing rates by linear regression. Linear regression of wound areas from Day 0 to Day 5 was used as a measure of healing rates with higher values (steeper slope) indicating faster healing rates. This analysis indicates superior healing rates in VF003 (high and low dose) and VF004 (high) treatment groups. Detailed description of the embodiments
  • peptides capable of binding to and activating a growth factor receptor and peptides capable of binding to an integrin can stimulate proliferation and/or migration of corneal epithelial cells. This clearly identifies an application in the treatment of corneal wounds.
  • the peptides can exert their effects when covalently linked or when separate, distinct molecules (i.e. not covalently linked).
  • peptides, fusion and chimeric proteins of the invention have been shown to stimulate primary human corneal epithelial cell migration, increase corneal wound healing and increase the rate of corneal wound healing in vivo.
  • An aspect of the present invention includes the presence of or use of peptides capable of binding to and activating a growth factor receptor.
  • a growth factor receptor mediates cellular growth, proliferation, and/or cellular differentiation upon activation.
  • growth factor receptors are the insulin like growth factor I receptor (IGF-IR) or epidermal growth factor receptor (EGFR). Typically, these receptors are activated on the binding growth factors such as IGF-I, IGF-II or EGF.
  • the IGF-family of proteins is comprised of two growth factor receptor ligands (IGF-I and IGF-II) and six 'classic' IGF binding proteins (IGFBPs). Together these proteins regulate a range of cellular responses throughout the body especially those associated with tissue development and regeneration.
  • the primary action of IGF is mediated by binding to its specific receptor, the insulin-like growth factor I receptor (IGF1 R), which is present on many cell types in many tissues. Binding to the IGF-IR, a receptor tyrosine kinase, initiates intracellular signalling.
  • IGF-I and IGF-II bind to at least two cell surface receptors: the IGF-I receptor (IGF-IR), and the insulin receptor.
  • IGF-I is one of the most potent activators of the AKT signalling pathway, a stimulator of cell growth and proliferation, and a potent inhibitor of programmed cell death.
  • the IGF-IR signals through multiple pathways including phosphatidylinositol-3 kinase (PI3K) and its downstream partner, the mammalian target of rapamycin (mTOR).
  • PI3K phosphatidylinositol-3 kinase
  • mTOR mammalian target of rapamycin
  • Epidermal growth factor EGF is a growth factor that stimulates cell growth, proliferation, and differentiation by binding to its cognate receptor EGFR. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface. This stimulates ligand-induced dimerization, activating the intrinsic protein-tyrosine kinase activity of the receptor. The tyrosine kinase activity, in turn, initiates a signal transduction cascade that results in
  • the binding of a peptide, domain or growth factor to a cognate receptor may be measured by any routine means in the art including BIAcore analysis (Forbes et al., 2002, Eur J Biochem. 269: 961 -968), ligand radiobinding assays, liquid phase ligand binding assays or solid phase ligand binding assays.
  • Activity of the cognate receptor may be determined by routine means in the art including assessment of downstream signalling pathway activation (ie phosphorylation of Akt) or assessment of PI3K activity.
  • Activation of the IGF-IR or insulin receptor may be measured as described in Denley, et al. 2004 Mol. Endocrinol. 18(10): 2502-2512.
  • a second peptide which comprises, consists essentially of or consists of an amino acid sequence of an integrin- binding domain of vitronectin (VN) or fibronectin (FN).
  • VN is a multi-functional 75 kDa glycoprotein that is found in the circulation and numerous tissues and forms a major component of the ECM. VN binds to a number of ligands including integrin receptors, heparin, plasminogen, plasminogen activator inhibitor-1 (PAI-1 ), thrombin-anti-thrombin (TAT) complexes, glycosaminoglycans, collagen, complement, and the urokinase plasminogen activator receptor (uPAR) Cellular responses to VN are mediated via a v integrins ( ⁇ ⁇ ⁇ 3 and a v Ps) which recognise an Arg-Gly-Asp (RGD) sequence adjacent to the protein's N-terminus.
  • integrin receptors heparin
  • plasminogen plasminogen activator inhibitor-1
  • TAT thrombin-anti-thrombin
  • uPAR urokinase plasminogen activator receptor
  • Fibronectin is a high-molecular weight ( ⁇ 440kDa) glycoprotein of the extracellular matrix that binds to membrane-spanning receptor proteins called integrins. Fibronectin exists as a protein dimer, consisting of two nearly identical monomers linked by a pair of disulfide bonds.
  • a "peptide capable of binding to an integrin" refers to a peptide that includes, but not limited to, an amino acid sequence of VN and FN that mediates binding to an integrin such as ⁇ ⁇ ⁇ 3 and ⁇ ⁇ ⁇ 5.
  • the binding of FN or VN to integrins can be determined by any number of known methods including solid-phase binding assays, FN or VN binding assays, or surface plasmon resonance (SPR) on Biacore.
  • the peptide comprises an integrin binding sequence of RGD.
  • the integrin-binding domain of VN may have an amino acid sequence of SEQ ID NO: 23.
  • the peptide does not comprise an amino acid sequence of the heparin binding domain of VN.
  • the first peptide comprises, consists essentially of or consists of an amino acid sequence of a full length VN or FN.
  • the C-terminal residue of the first peptide may be covalently linked to the N-terminal residue of the second peptide, or the N-terminal residue of the first peptide may be covalently linked to the C- terminal residue of the second peptide.
  • the first peptide and the second peptide are said to be "directly linked” or "adjacent”.
  • a suitable covalent linker is preferably selected from glycine and serine residues.
  • the chimeric or fusion protein includes a linker for linking first, second and/or third peptides.
  • the linker may be any linker able to join two or more peptides, including both amino acid and non-amino acid linkers.
  • the linker is non-immunogenic. Suitable linkers may be at least, or equal to, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 and 25 amino acids in length, although any linker that allows each peptide to bind to its cognate growth factor receptor or integrin is contemplated.
  • the linker sequence comprises one or more glycine residues and one or more serine residues.
  • Particular examples of linker sequences may be selected from; (Gly 4 Ser) 4 (SEQ ID NO:25) ; Gly 4 Ser Gly 4 (SEQ ID NO:26); Gly 4 Ser Gly 4 Ser Gly 4 Ser Gly 4 (SEQ ID NO:27); Gly 4 Ser (SEQ ID NO:28); Gly 4 Ser 3 (SEQ ID NO:29) and (Gly 4 Ser) 3 (SEQ ID NO:30), although without limitation thereto.
  • the linker sequence includes a Plasmin Cleavage Recognition Site, such as Leu lie Lys Met Lys Pro (SEQ ID NO:31 ). In yet another embodiment, the linker sequence includes a Collagenase-3 Cleavage Recognition Site, such as Gin Pro Gin Gly Leu Ala Lys (SEQ ID NO:32).
  • nucleic acid molecule including a nucleotide sequence encoding a chimeric or fusion protein as broadly described above, optionally operatively linked to at least one regulatory element.
  • the nucleic acid is isolated, purified, recombinant or synthetic.
  • operably linked or “operably connected” is meant that said regulatory nucleotide sequence(s) is/are positioned relative to the nucleic acid of the invention to initiate, regulate or otherwise control transcription of the nucleic acid, or translation of a protein encoded by the nucleic acid.
  • Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.
  • said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, splice donor/acceptor sequences and enhancer or activator sequences.
  • Constitutive promoters such as CMV, RSV, adenovirus, SV40 and human elongation factor promoters
  • inducible/repressible promoters such as tet- repressible promoters and IPTG-, metallothionine- or ecdysone-inducible promoters
  • promoters may be hybrid promoters that combine elements of more than one promoter.
  • the recombinant nucleic acid may be operably linked to one or more regulatory sequences in an expression vector.
  • An "expression vector" may be either a self-replicating extrachromosomal vector such as a plasm id, or a vector that integrates into a host genome.
  • viral vectors such as vaccinia
  • viral vectors useful in gene therapy include adenovirus and adenovirus-associated viruses (AAV) such as described in Braun-Falco et al., 1999, Gene Ther. 6: 432, retroviral and lentiviral vectors such as described in Buchshacher et al., 2000, Blood, 2499 and vectors derived from herpes simplex virus and cytomegalovirus.
  • AAV adenovirus and adenovirus-associated viruses
  • the expression construct may also include a fusion partner (typically provided by the expression vector) so that the recombinant protein of the invention is expressed as a fusion polypeptide with said fusion partner.
  • a fusion partner typically provided by the expression vector
  • the main advantage of fusion partners is that they assist identification and/or purification of said fusion protein.
  • fusion partners include, but are not limited to, glutathione-S-transferase (GST), Fe portion of human IgG, maltose binding protein (MBP) and hexahistidine (His 6 ), which are particularly useful for isolation of the fusion protein by affinity chromatography.
  • GST glutathione-S-transferase
  • MBP maltose binding protein
  • His 6 hexahistidine
  • relevant matrices for affinity chromatography are glutathione-, amylose-, and nickel- or cobalt-conjugated resins respectively.
  • Many such matrices are available in "kit” form, such as the QIAexpress system (Qiagen) useful with (His 6 ) fusion partners and the Pharmacia GST purification system.
  • a reference to a "homologue" of a peptide or polypeptide is a reference to a peptide or polypeptide having an amino acid sequence that shares homology or that is homologous to, or that has identity with the amino acid sequence of the first-mentioned peptide or polypeptide, preferably at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. Sequence identity refers to exact matches between the amino acids of two sequences which are being compared.
  • Such a homologue may derive from a naturally occurring variant or isolate of, for example, an IGF, EGF, VN or FN.
  • it may be a "conservative-substitution" variant of a peptide or polypeptide in which one or more amino acid residues have been changed without altering the overall conformation and function of the peptide or polypeptide; including, but by no means limited to, replacement of an amino acid with one having similar properties.
  • polar/hydrophilic amino acids which may be interchangeable include asparagine, glutamine, serine, cysteine, threonine, lysine, arginine, histidine, aspartic acid and glutamic acid; nonpolar/hydrophobic amino acids which may be interchangeable include glycine, alanine, valine, leucine, isoleucine, proline, tyrosine, phenylalanine, tryptophan and methionine; acidic amino acids which may be interchangeable include aspartic acid and glutamic acid and basic amino acids which may be interchangeable include histidine, lysine and arginine.
  • conservative-substitution variants have less than 20, more preferably less than 15, more preferably less than 10, and most preferably less than 5 amino acid changes.
  • the peptides described herein may have conservative substitutions of at least one amino acid residue. Preferably, this conservative substitution does not alter the overall conformation or function of the peptide.
  • the conservative substitution comprises a replacement of an amino acid with another having one or more similar properties. Table 1 outlines the properties of each of the amino acids.
  • the peptides described herein may have non-, or unnatural amino acids incorporated. Unless otherwise specified, any amino acid may be natural or non-natural / unconventional. Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t- butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6- methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • Table 2 A list of unnatural / non-conventional amino acids contemplated herein is shown in Table 2.
  • Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyric acid Abu L-N-methylalanine Nmala ⁇ -amino-a-methylbutyrate Mgabu L-N-methylarginine Nmarg am inocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile
  • D-a-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg D-a-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
  • heterologous protein or “chimeric or fusion protein” are used to refer to a protein that is composed of functional units, domains, sequences or regions of amino acids derived from different sources or that are derived from the same source and that have been assembled so as to have an organisation that is distinguished from that observed in a molecule from which the unit, domain, sequence or region is derived or related to.
  • a method of treating a corneal wound in an individual in need thereof comprising administering to the individual a chimeric or fusion protein as described herein.
  • wound refers to an injury, such as an ulcer or lesion, as a result of a disease or disorder, or as a result of an accident, incident or surgical procedure.
  • a 'corneal wound' includes all ocular surface conditions with impairment of the corneal epithelia that would benefit from a topical treatment that stimulates corneal epithelial cell proliferation and migration.
  • Such conditions may result from surgical and non-surgical trauma or abrasion, tear-film disorders, severe dry eye, neoplasia and its treatment, corneal graft, diabetic keratopathy, neuropathic keratopathy, thermal or chemical burns, herpetic epithelial keratitis, injury caused by ocular anti-viral or -microbial agents, or immunological disorders.
  • the wound may be an abrasion, which is caused by contact of the cornea with foreign bodies (e.g. sand) or contact lenses.
  • the wound may be a corneal wound (including specifically a corneal epithelial wound, together with or without other wound or injury) that is a result of an alkali injury i.e. an alkali-induced wound.
  • the condition to be treated that results in a corneal wound may therefore result from surgical and non-surgical trauma or abrasion, severe dry eye, diabetic keratopathy, neuropathic keratopathy, thermal or chemical burns, herpetic epithelial keratitis, extensive contact lens wear, injury caused by ocular anti-viral or -microbial agents.
  • the treatment stimulates corneal epithelial cell proliferation and/or migration to promote repair and/or healing of the cornea.
  • a corneal wound may also include a persistent epithelial defect (PED). PEDs are characterized by ulcers with delayed (>2 weeks) healing and can recur once healed, particularly when the basement membrane (BM) is lost.
  • PED persistent epithelial defect
  • Corneal epithelial cells aid in maintaining a stable tear film, and secrete the epithelial basement membrane which is critical in corneal healing. Corneal epithelial cells are constantly turned over as the outermost cells are shed into the tear film. The entire epithelium is turned over in approximately seven to 10 days. This process is accelerated during wound healing and generally leads to rapid healing for corneal injuries that only involve the epithelial cells.
  • the process of corneal epithelial wound healing can be divided into phases that occur in sequence, but may overlap in time.
  • the three phases are: migration of adjacent intact epithelial cells to cover the injured zone, proliferation of the migrated monolayer to reestablish epithelial thickness, and differentiation of the reconstruct epithelium to restore structure and function.
  • the initial stage is characterized by cells attaching and spreading over the denuded substratum which typically occurs by epithelial "sliding."
  • An important aspect of this process is cell attachment to the extracellular matrix (ECM), which is mediated by integrins. Integrins and their ligands are components of structures known as focal contacts which facilitate cell-to-ECM interactions during wound healing.
  • Suitable assay systems used to determine corneal wound healing include mechanically wounding confluent epithelial cells by passing a pipette tip through the monolayer. The rate of repair (% wound recovery) is monitored using an inverted phase-contrast microscope and wound recovery compared with cells grown on uncoated dishes over a given period of time. Alternatively, a fenestrated/barrier assay may be employed. In these experiments, epithelial cells are resuspended in media and dispensed into adjacent chambers within a l-Dish (ibidi, Kunststoff, Germany).
  • Epithelial cell proliferation may be determined by any number of routine methods in the art including a colorimetric 5-bromo-2-deoxyuridine (BrdU) ELISA which detects BrdU incorporation into newly synthesized DNA, the MTS method as shown in Example 3, or the MTT assay.
  • the MTS method of assessing proliferation is based on the reduction of MTS tetrazolium compound by viable cells to generate a coloured formazan product that is soluble in cell culture media.
  • the MTT assay measures conversion of methyl-thiazolyldiphenyl-tetrazolium bromide (MTT) by mitochondrial enzymes.
  • MTT methyl-thiazolyldiphenyl-tetrazolium bromide
  • Symptoms of a corneal wound may include blurred vision, eye pain or stinging and burning in the eye, feeling like something is in your eye (may be caused by a scratch or something in your eye), light sensitivity, redness of the eye, swollen eyelids, watery eyes or increased tearing.
  • An individual in need of treatment by a method, use, chimeric or fusion protein, or composition of the invention may display one or more of these symptoms.
  • corneal wounds may be diagnosed by physical eye examination. Obvious signs include evidence of penetrating trauma, infection, and significant vision loss. In corneal abrasion, the pupil is typically round and central, and conjunctival injection may be present. Ciliary spasm causing miosis, pain, and ciliary flush (injection of ciliary vessels surrounding the cornea) may indicate traumatic ulceris.
  • a corneal opacity or infiltrate may occur with corneal ulcers or infection.
  • Fluorescein staining can help to identify a corneal epithelial wound.
  • a drop of topical anesthetic (proparacaine 0.5%) is applied directly into the eye or on a fluorescein strip.
  • the patient's lower lid is pulled down, and the fluorescein strip is lightly touched to the bulbar conjunctiva.
  • the dye spreads over the cornea as the patient blinks, and stains any exposed basement membrane of the epithelium.
  • an abrasion may stain yellow. Illumination with cobalt blue light shows the defect as green.
  • Traumatic corneal wounds typically have linear or geographic shapes. If contact lenses are involved, the abrasion may have several punctate lesions that coalesce into a round, central defect.
  • a branching (dendritic) appearance suggests herpetic keratitis.
  • Multiple vertical lines on the superior cornea suggest a foreign body under the upper eyelid.
  • the existence of, or improvement in, treatment of or prevention of a corneal wound may be by any clinically or biochemically relevant method of the individual or a biopsy therefrom.
  • a parameter measured may be epithelial cell migration or proliferation, or lack of yellow light staining in the presence of Fluorescein.
  • existence of, or improvement in, treatment may also result in a reduction or lessening of one or more symptoms as described herein.
  • preventing or “prevention” is intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).
  • Biological and physiological parameters for identifying such patients are provided herein and are also well known by physicians.
  • the methods or uses of the invention may prevent or delay a corneal wound from developing into a persistent epithelial defect.
  • treatment includes the application or administration of peptides, chimeric or fusion protein, or composition of the invention to a subject with the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition.
  • treating refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being.
  • the phrase 'therapeutically effective amount' generally refers to an amount of one or more peptides, chimeric or fusion proteins of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.
  • the disease or disorder is a corneal wound, preferably a corneal epithelial wound.
  • a peptide, chimeric or fusions protein, or composition of the present invention may be present in an ophthalmic composition, which is a composition suitable for administration to the eye.
  • ophthalmic compositions according to the invention are suspensions, ointments, sustained release formulations, gels or solutions suitable for application as an eye drop.
  • the composition may be loaded onto, or impregnated into, a contact lens or other suitable biomaterial.
  • the pharmaceutical compositions according to the present invention will be formulated for topical administration or for sustained release delivery.
  • the composition of the present invention is in a form suitable for administration to the eye.
  • compositions are generally preferred, based on ease of formulation, as well as a subject's ability to easily administer such compositions by means of instilling one to two drops of the solutions in the affected eyes.
  • the compositions may also be suspensions, viscous or semi-viscous gels, or other types of solid or semi-solid compositions, or those appropriate for sustained release.
  • any of a variety of carriers may be used in the compositions of the present invention including water, mixtures of water and water-miscible solvents, such as Ci to C 7 alkanols, vegetable oils or mineral oils comprising from 0.5 to 5% non-toxic water- soluble polymers, gelling products, such as gelatin, alginates, pectins, tragacanth, karaya gum, xanthan gum, carrageenin, agar and acacia, and their derivatives, starch derivatives, such as starch acetate and hydroxypropyl starch, cellulose and its derivatives and also other synthetic products, such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, preferably cross-linked polyacrylic acid, such as neutral Carbopol, or mixtures of those polymers, naturally- occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, ka
  • the composition according to the present invention may comprise at least one gelling agent.
  • Gelling agents suitable for use in pharmaceutical compositions are well known to those of ordinary skill in the art and include, for example, xanthan gum and its derivatives, carbomer and its derivatives, acrylate based copolymers and cross polymers, sodium polyacrylate and its derivatives, cellulose and its derivatives, and starch and agar and their derivatives.
  • the selection of the gelling agent according to the present invention is important in providing a clear gel.
  • the amount of gelling agent added to the composition may be readily determined by one of ordinary skill in the art with a minimum of experimentation, and will depend upon factors known to those skilled in the art, such as the properties of the gelling agent and the desired properties of the pharmaceutical composition.
  • Additional ingredients that may be included in the pharmaceutical composition of the invention include tonicity enhancers, preservatives, solubilizers, stabilizers, nontoxic excipients, demulcents, sequestering agents, pH adjusting agents, co-solvents and viscosity building agents.
  • tonicity enhancers preferably to a physiological pH
  • buffers may especially be useful.
  • the pH of the present solutions should be maintained within the range of between 4 to 8, preferably 6 to 7.5. It will be understood by a person of ordinary skill in the art that any pH that is compatible with the ocular surface is suitable.
  • Suitable buffers may be added, such as boric acid, sodium borate, potassium citrate, citric acid, sodium bicarbonate, TRIS, disodium edetate (EDTA) and various mixed phosphate buffers (including combinations of Na 2 HP0 4 , NaH 2 P0 4 and KH 2 P0 4 ) and mixtures thereof.
  • buffers will be used in concentrations ranging from about 0.05 to 0.5 M.
  • Tonicity is adjusted if needed typically by tonicity enhancing agents.
  • Such agents may, for example, be of ionic and/or non-ionic type.
  • ionic tonicity enhancers are alkali metal or earth metal halides, such as, for example, CaCI 2 , KBr, KCI, LiCI, Nal, NaBr or NaCI, Na 2 S0 4 or boric acid.
  • Non-ionic tonicity enhancing agents are, for example, urea, glycerol, sorbitol, mannitol, propylene glycol, or dextrose.
  • the aqueous solutions of the present invention are typically adjusted with tonicity agents to approximate the osmotic pressure of normal lachrymal fluids.
  • compositions of the invention additionally comprise a preservative.
  • a preservative may typically be selected from a quaternary ammonium compound such as benzalkonium chloride (N-benzyl-N-(C 8 -Ci 8 alkyl)-N. N- dimethylammonium chloride), benzoxonium chloride or the like.
  • preservatives different from quaternary ammonium salts are alkyl-mercury salts of thiosalicylic acid, such as, for example, thiomersal, phenylmercuric nitrate, phenylmercuric acetate or phenylmercuric borate, sodium perborate, sodium chlorite, parabens, such as, for example, methylparaben or propylparaben, sodium benzoate, salicylic acid, alcohols, such as, for example, chlorobutanol, benzyl alcohol or phenyl ethanol, guanidine derivatives, such as, for example, chlorohexidine or polyhexamethylene biguanide, sodium perborate, GermalTM or sorbic acid.
  • thiosalicylic acid such as, for example, thiomersal, phenylmercuric nitrate, phenylmercuric acetate or phenylmercuric borate
  • Preferred preservatives are quaternary ammonium compounds, in particular benzalkonium chloride or its derivative such as Polyquad (see US patent number 4,407,791 ), alkyl- mercury salts and parabens. Where appropriate, a sufficient amount of preservative is added to the ophthalmic composition to ensure protection against secondary contaminations during use caused by bacteria and fungi.
  • compositions of this invention do not include a preservative. Such formulations would be particularly useful for subjects who wear contact lenses.
  • composition of the invention may additionally require the presence of a solubilizer, in particular if the active or the inactive ingredients tends to form a suspension or an emulsion.
  • a solubilizer suitable for an above concerned composition is for example selected from the group consisting of tyloxapol, fatty acid glycerol polyethylene glycol esters, fatty acid polyethylene glycol esters, polyethylene glycols, glycerol ethers, a cyclodextrin (for example alpha-, beta- or gamma-cyclodextrin, e.g.
  • a specific example of an especially preferred solubilizer is a reaction product of castor oil and ethylene oxide, for example the commercial products Cremophor EL ® or Cremophor RH40 ® .
  • solubilizers that are tolerated extremely well by the eye.
  • Another preferred solubilizer is selected from tyloxapol and from a cyclodextrin.
  • concentration used depends especially on the concentration of the active ingredient.
  • the amount added is typically sufficient to solubilize the active ingredient.
  • the compositions may comprise further non-toxic excipients, such as, for example, emulsifiers, wetting agents or fillers, such as, for example, the polyethylene glycols designated 200, 300, 400 and 600, or Carbowax designated 1000, 1500, 4000, 6000 and 10000.
  • excipient added is in accordance with the particular requirements and it will be understood by a person of ordinary skill in the art what types and amounts of excipients and other additives may be present in a composition such that the composition is compatible with the eye.
  • Other compounds may also be added to the compositions of the present invention to increase the viscosity of the carrier.
  • viscosity enhancing agents include, but are not limited to: polysaccharides, such as hyaluronic acid and its salts, chondroitin sulfate and its salts, dextrans, various polymers of the cellulose family; vinyl polymers; and acrylic acid polymers.
  • compositions of the present invention may contain other active ingredients that are effective in the treatment of wounds e.g. growth factors, cleansers and antibiotics. Generally, these active ingredients and treatments are provided in a combined amount effective to promote the healing of a wound. This may involve administering the composition of the present invention and the active ingredient/treatment at the same time or at times close enough such that the administration results in an overlap of the desired effect. Alternatively, the composition of the present invention may precede or follow other treatments. The composition may be administered in any way that is deemed suitable by a person of ordinary skill in the art. The pharmaceutical composition may be administered topically.
  • composition of the invention may be administered in single or multiple doses and for any length of time until the wound is either completely healed or until the desired level of wound healing has been achieved.
  • dosage amount, dosage regime and length of treatment will depend on factors such as, for example, the wound type, the location of the wound and the health of the subject.
  • the treatment required will depend on factors such as the extent of the ocular surface damaged, the degree of intraocular penetration by the chemical agent, and the concentration and nature of the agent involved.
  • the composition is administered every half hour or hourly, up to, for example, eight times a day.
  • the kit or "article of manufacture” may comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, blister pack, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a peptide, chimeric or fusion protein, or pharmaceutical composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the label or package insert indicates that the peptide, chimeric or fusion protein, or pharmaceutical composition is used for treating the condition of choice.
  • the label or package insert includes instructions for use and indicates that the therapeutic composition can be used to treat a corneal wound.
  • the kit may comprise (a) a peptide, chimeric or fusion protein, or pharmaceutical composition; and (b) a second container with a second active principle or ingredient contained therein.
  • the kit in this embodiment of the invention may further comprise a package insert indicating that a peptide, chimeric or fusion protein, or pharmaceutical composition and other active principle can be used to treat a corneal wound.
  • the kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer
  • VF001 VN: IGF-I
  • VF003 VN:EGF
  • VF004 EGF:VN: IGF-I
  • VN, IGF-I and EGF gene DNA sequences (NCBI accession # AF382388, # X03563 and # AY548762 respectively) were codon-optimised for expression in S. frugiperda and synthesised by GeneArt (Regensburg, Germany). The coding sequences were then cloned into the ⁇ /5-His expression vector (Invitrogen) incorporating a poly-histidine affinity tag to aid in purification. A truncated section of VN comprising the first 249 nucleotides (coding for the secretion signal peptide and amino acids 1 -64 of the mature protein sequence) was amplified by polymerase chain reaction (PCR) simultaneously incorporating restriction enzyme sites for insertion into the expression vector.
  • PCR polymerase chain reaction
  • a nucleotide sequence encoding a (Gly 4 Ser) 4 amino acid linker was inserted via site-directed mutagenesis PCR between protein domain coding sequences and a smaller (Gly 4 SerGly 4 ) linker inserted 5' of the poly-histidine tag was also inserted by the same methods. Further rounds of site-directed mutagenesis PCR were used to delete superfluous restriction enzymes sites in the final coding sequence.
  • DNA and protein sequences according to VF001 are as follows: DNA: atggctcctctgcgtcctctgctgatcctggctctgctggcttgggtggccctggctgaccaggag tcttgcaagggacgttgcaccgagggtttcaacgtggacaagaagtgccagtgcgacgagctgtgctc ctactaccagtcaccgcaccgctgagtgcaagcctcaggtgacccgtggtgacgtgtt caccatgcccgaggacgagtacactgtgtacgacgacggcgaggaggcgaggaggagagaggcgaggagagaggcgaggagagaataataattccaataat ggt
  • VN Signal VN; IGF-I; Linker, 6xHis tag
  • VF003 The DNA and protein sequences according to VF003 are as follows:
  • VF004 The DNA and protein sequences according to VF004 are as follows:
  • VN Signal
  • a sequence of 3 amino acids may be included between the VN signal and EGF to ensure that no EGF sequence is cleaved.
  • An example of a 3 amino acid sequence is GTS (e.g. nucleotide sequence is gggacgtct).
  • VN signal atggctcctctgcgtcctctgctgatcctggctctgctggcttgggtg SEQ ID NO:4 gccctggct
  • EGF aactccgactccgaatgccccctgtcccacgacggttactgcctg SEQ ID NO:5 cacgacggtgtctgcatgtacatcgaggctctggacaagtacgct tgcaactgcgtggtgggctacatcggcgagcgttgccagtaccgt gacctgaagtggtgggagctgcgtttgtttgtgt
  • IGF-II SEQ ID NO:17 gcttaccgccccagtgagaccctgtgcggcggggagctggtgga caccctccagttcgtctgtggggaccgcggcttctacttcagcagg cccgcaagccgtgtgagccgtcgcagccctggcatcgttgagga gtgctgtttccgcagctgtgacctggccctctggagacgtactgtg ctacccccgccaagtccgagtaaa
  • Linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO:25 Linker GGGGS GGGG SEQ ID NO:26
  • Clones in the ⁇ /5-His vector were used to transfect Sf9 insect cells and transiently- expressed secreted protein was detected in the conditioned media, as assessed by immunoblotting. Briefly, the samples were resolved on SDS-PAGE under reducing conditions and the proteins were transferred onto a nitrocellulose membrane using a semi-dry transfer method. The membrane was interrogated with poly-clonal anti-VN, anti-IGF-l and anti-EGF antibodies, and the target protein species were then visualized using enhanced chemiluminescence following the manufacturer's instructions (GE Healthcare, Buckingham- shire, UK).
  • chimeric proteins Purification of the chimeric proteins was based on Ni-NTA Superflow Agarose (QIAGEN, Australia) affinity chromatography performed according to the manufacturer's instructions. The chimeric proteins were monitored throughout the purification process by SDS-PAGE and Western blot as above. Clarified culture media was loaded onto a 20 mL Q-Sepharose Superflow (GE Healthcare, Buckinghamshire, UK) column equilibrated with dd H 2 0 and bound material was eluted with 100 mL of 50 mM NaH 2 P0 4 , 600 mM NaCI, 10 mM Imidazole, pH 8.5 in order to concentrate the sample.
  • Q-Sepharose Superflow GE Healthcare, Buckinghamshire, UK
  • the complete Q- Sepharose eluate was diluted 1 :1 with 50 mM NaH 2 P0 4 , 10 mM Imidazole pH 8.5 to reduce the NaCI concentration to 300 mM.
  • the diluted sample was then loaded onto a 2 ml_ Ni-NTA Superflow Agarose column equilibrated with 50 mM NaH 2 P0 4 , 10 mM Imidazole, 300 mM pH 8.5. After loading, the column was washed with 30 ml_ of equilibration buffer containing 20 mM Imidazole. Bound proteins were eluted with 50 mM NaH 2 P0 4 , 250 mM Imidazole, 300 mM pH 8.5.
  • Example 2 This study was conducted to generate sufficient quantities of VF001 (VN: IGF-I),
  • VF003 VN:EGF
  • VF004 EGF-I chimeric proteins in Sf9 (insect) cells.
  • Sf9 cells (Invitrogen) were transfected with expression vectors encoding VF001 , VF003 and VF004 constructs. These cells were cultured in SF900-II SFM (Gibco - Thermo Fisher Scientific). Suspension cultures were undertaken using flat bottom vent cap Erlenmeyer flasks of varying volumes sourced from Corning. For purification of the expressed proteins, Q-Sepharose resin was sourced from GE Healthcare while Ni2+- NTA resin was purchased from QIAGEN. Protein quantitation was performed using a BCA kit from Pierce (Thermo Fisher Scientific).
  • VF003 and VF004 are currently only available in the Sf9 insect expression system.
  • Transfected cells for each construct were revived and cultured as per standard procedures (Van Lonkhuyzen et al., 2007, Growth Factors, 25(5): 295-308). Briefly, Sf9 cell stocks of VF001 , VF003 and VF004 were thawed, added to T25 flasks containing 5 mL of SF-900II SFM (Gibco - Thermo Fisher Scientific) and incubated at 28°C until confluent. Cells were then dislodged by cell scraper and sloughing before transfer to T75 flasks in 12 mL SF-900II SFM.
  • SF-900II SFM Gibco - Thermo Fisher Scientific
  • VF001 , VF003 and VF004 Purification of VF001 , VF003 and VF004 from expression culture conditioned media
  • FIG. 1 shows the results of the VF001 (VN: IGF-I) purification procedure.
  • the samples demonstrate a prominent band of the expected molecular weight (MW - 27 kDa) in the first Q-Sepharose elution sample (Q-E1 ) which is seen to be depleted in the Ni2+-NTA flow-through (F/T). While a very small amount of this band can be seen in the Ni2+-NTA wash (Ni-W), there is an obvious high density band in Ni2+-NTA elution (Ni- E * ) samples 1 -6 ( Figure 1A).
  • VF001 , VF003 and VF004 gave final concentrations of 14.39 pg/pL, 1 1 .99 g/ L and 9.58 g/ L respectively. As each sample is in a volume of 1 mL this gave a total yield of 14.39 mg of VF001 , 1 1 .99 mg VF003 and 9.58 mg VF004, in each case from 3 L of Sf9 conditioned media.
  • VF001 , VF003 and VF004 recombinant proteins were successfully expressed and purified using IEX and IMAC methods.
  • VF001 VN: IGF-I
  • VF003 VN:EGF
  • VF004 EGF:VN: IGF-I
  • VF001 , VF003 and VF004 were generated previously as detailed in Examples 1 and 2.
  • Native human vitronectin (VN) was sourced from Promega, while IGF-I was purchased from Gro-Pep and EGF from Thermo-Fisher Scientific (Invitrogen).
  • the Human Corneal Epithelial cell line HCE-T was obtained from the Queensland Eye Institute, Brisbane under the permission of Dr. Damien Harkin.
  • HCE-T cells were cultured in DMEM (high-glucose, + L-glutamine, + pyruvate) with the addition of 10% FCS (Thermo-Fisher Scientific - Gibco) and 1 % Penicillin/Streptomycin (Thermo- Fisher Scientific - Invitrogen). In developing proliferation assays, MTS reagent was used (Promega).
  • proteins were functionally assessed in cell proliferation assays based on the MTS method. Briefly, proteins (VF001 , VF003 and VF004, along with controls including VN alone, IGF-I alone, EGF alone, VN+IGF-I, VN+EGF, VN+IGF-I+EGF and M-VF4-P03 (GMP-grade VF001 ) were added to the wells of a 96 well plate at various doses (15 nM, 50 nM and 150 nM for controls and 50 nM, 150 nM and 450 nM for VF001 , VF003, VF004 and M-VF4-P03 - GMP-grade VF001 ) and incubated for a period of 3 hours (See Table 6 for sample concentration summary).
  • Serum starved (4 hours) and harvested (TrypLE) HCE-T cells were added to pre- incubated proteins/protein mixtures in plate wells and incubated at 37°C for 48 hours, after which MTS reagent was added and cells incubated for a further 2 hours to allow for product development.
  • HCE-T cells were treated with various concentrations of test articles including chimeric proteins and controls. Following 48 hours of treatment in SFM, MTS reagent was added to the well to assess cell proliferation. The results of this assay are depicted in Figure 5 and are expressed as corrected (SFM control subtracted) absorbance readings. The assays were performed in a 'solution phase' where treatments were added to wells in a 2x concentration and cells added on top in an equal volume in SFM, the wells were not washed between treatment addition and cell seeding.
  • VF4-P03 was included as a good manufacturing practice (GMP)-grade VF001 reference material that is manufactured using a Pichia pastoris expression system.
  • IGF-I As expected IGF-I promoted HCE-T proliferation to a significant degree over the negative control (SFM) at all concentrations tested (15 nM, 50 nM and 150 nM). Interestingly, VN alone was inhibitory in function.
  • the combination of VN + IGF-I (V+l in figure) produced similar results to IGF-I alone (not significantly different at any concentration) while VF001 as VF4-P03 (GMP-grade VF001 ) (VF4 in figure) and insect expressed VN: IGF-I also produced cell proliferation to a similar extent when compared to IGF-I alone, indicating no effect on function of the IGF-I portion by the attached VN domain.
  • VF001 , VF003 and VF004 were assessed for their ability to induce cell proliferation in HCE-T cells by the MTS method. The results showed that VF001 and VF004 were equivalent to each other and the IGF-I alone control. Thus, it is concluded that the chimeric proteins containing IGF-I retain the full function of the IGF-I domain. VF003 (VN:EGF) was unable to stimulate cell proliferation to the same degree however, this was not unexpected and the migration data outlined in Example 3 may provide a beneficial aspect to the inclusion of EGF in the chimeric constructs.
  • VF001 VN: IGF- I
  • VF003 VN:EGF
  • VF004 EGF:VN: IGF-I
  • VF001 , VF003 and VF004 were generated previously as detailed in Examples 1 and 2.
  • Native human vitronectin (VN) was sourced from Promega, while IGF-I was purchased from Gro-Pep and EGF from Thermo-Fisher Scientific (Invitrogen).
  • the Human Corneal Epithelial cell line HCE-T was obtained from the Queensland Eye Institute, Brisbane under the permission of Dr. Damien Harkin. HCE-T cells were cultured in DMEM (high-glucose, + L-glutamine, + pyruvate) with the addition of 10% FCS (Thermo Fisher Scientific - Gibco) and 1 % Penicillin/Streptomycin (Thermo Fisher Scientific - Invitrogen).
  • Migration fence rings were sourced from Aix Scientific (Germany). In developing migration assays, PFA and Crystal Violet (Sigma Aldrich) were used.
  • HCE-T cell migration was performed using the Fence method to assess the ability of treatments to stimulate outward migration of cells over a surface. After the migration period cells were fixed, stained and imaged. The image was analysed for the area taken up by the cells as a complete colony with the boundary tracing the outer reaches of cells (see Figure 7 for example of analysis image). Area data were collected and the average SFM result was subtracted from each other treatment within each individual experiment and these corrected data pooled together from each experiment for the final result as shown in Figure 6.
  • VN and IGF-I were found to be potent stimulators of HCE-T migration at all concentrations and such responses were replicated with all treatments containing IGF-I - VF4-P03, VN: IGF- I/VF001 , VN + EGF + IGF-I and EGF:VN: IGFI/ VF004 with each concentration of these treatments resulting in migration significantly (p ⁇ 0.05) above SFM.
  • the lowest concentrations of VN + EGF and VN:EGF/VF003 produced responses significantly (p ⁇ 0.05) above SFM, however, higher doses did not maintain this level of stimulation.
  • Comparison of the VF proteins showed that VF4-P03, VF001 and VF004 were similar to each other at respective concentrations. Furthermore, although VF003 results were lower than those observed with VF001 , only at 450 nM was a significant (p ⁇ 0.05) reduction in response detected.
  • the Fence assay was chosen for migration as this better represented the desired action of cells regenerating in vivo, that is across a surface rather than through a membrane/tissue as would have been assessed using a Transwell® system for example.
  • the data from the assay demonstrate that VN alone is unable to stimulate HCE-T migration, however, the addition of IGF-I as a combination treatment or in the form of a chimeric protein such as VF001 or VF004 resulted in significant increases in migratory responses.
  • Example 5 This study was conducted to assess the motogenic potential of VF001 (VN: IGF-
  • VF003 VN:EGF
  • VF004 EGF:IGF-I
  • Examples 1 through 2 GMP-grade VF001 Drug Substance, M-VF4-P03, was provided by Eurogentec. Native human vitronectin (VN) was sourced from Promega, while IGF-I was purchased from Gro-Pep and EGF from Thermo-Fisher Scientific (Invitrogen). Primary Human Corneal Epithelial Cells (HCEC) were obtained from the American Type Culture Collection (ATCC, #PCS-700-010).
  • HCECs were cultured in Corneal Epithelial Basal Medium (ATCC, #PCS-700-030) supplemented with Corneal Epithelial Growth Kit (ATCC, #PCS-700-040) and 1 % Penicillin/Streptomycin (Thermo-Fisher Scientific - Invitrogen).
  • MTS reagent was used (Promega).
  • Migration fence rings were sourced from Aix Scientific (Germany).
  • PFA and Crystal Violet (Sigma Aldrich) were used.
  • Accutase Cell Dissociation Solution was purchased from Thermo Fisher Scientific.
  • VF001 , VF003 and VF004 proteins were functionally assessed in primary HCEC proliferation assays based on the MTS method.
  • a basal medium formulation of Corneal Epithelial Basal Medium supplemented with Apo-transferrin, Epinephrine, Extract-P, hydrocortisone and L-glutamine (lacking rh Insulin and CE growth factors) was employed as the 'serum-free' negative control and all treatments were prepared in this formulation (termed; Basal medium).
  • proteins VF001 , VF003 and VF004, along with controls including VN alone, IGF-I alone, EGF alone, VN+IGF-I, VN+EGF, VN+IGF-I+EGF and M-VF4-P03 (Internal VF001 Drug Substance Reference Batch) were added to the wells of a 96 well plate at various doses (15 nM for controls and 15 nM, 50 nM, 150 nM and 450 nM for VF001 , VF003, VF004 and M-VF4-P03) and incubated for a period of 3 hours (See Table 8 for sample concentration summary).
  • Serum starved (3 hours) and harvested (TrypLE) HCECs were added to pre- incubated proteins/protein mixtures in plate wells and incubated at 37°C for 72 hours, after which MTS reagent was added and cells incubated for a further 2 hours to allow for product development.
  • VF001 , VF003 and VF004 proteins were further assessed in HCEC migration assays based on the Fence method. Briefly, fence seeding rings (Aix Scientific) were inserted into each well and allowed to seal over 1 hr at 37°C. Serum starved (3 hours) HCECs were harvested using TryPLE (Thermo Fisher Scientific), seeded into the inner chamber at 4 x 10 4 cells/well in basal medium and allowed to attach over 2 hours at 37°C.
  • Monolayers of HCECs were cultured in growth media (Corneal Epithelial Basal Medium + Corneal Epithelial Growth Kit + 1 % Pen/Strep) within T75 flasks (x6, one for each treatment; Basal medium, Growth medium, VF001 , VF4-P03, VF003 and VF004 each at 50 nM) until cultures were ⁇ 80% confluent. Cells were then serum-starved for 3 hours in Basal medium after which treatments were added. Each treatment was applied to an individual T75 flask of HCECs and the cells incubated with treatments for 72 hours. At the completion of the treatments period, cells were harvested using Accutase Cell Dissociation Solution at RT and held separately per treatment.
  • the cells were fixed in ice-cold Fix buffer on ice for 1 hr. Once fixed, cells were pelleted, washed 2x with DPBS, split into seven (7) equal aliquots per treatment and stained with primary Ab's; a-Pax-6 (1 :50), oc- CK3 (1 : 100), OC-CK12 (1 : 100) and a-CK13 (1 :200) in ice-cold Stain Buffer for 1 hr on ice. Samples of 'unstained' control cell preparations were included for each treatment, these underwent all incubation, wash and centrifugation steps but were incubated in the absence of primary Ab.
  • the FACS Celesta (BD Biosciences) was initiated and prepared as per the manufacturer and facilitator instructions. Cell preparations were run on the machine with detection for side and forward scatter (SSC and FSC) along with appropriate lasers (AF488 and BV421 compatible lasers). Laser intensities were optimised to contain the whole cell fraction within detection parameters and for unstained cells to display fluorescence intensities of ⁇ 10 2 units.
  • HCECs were treated with various concentrations of test articles including chimeric proteins and controls according to the above defined methods. Following 72 hours of treatment with test articles and controls in Basal medium, MTS reagent was added to the wells to measure cell proliferation. The results of this assay are depicted in Figure 8 and are expressed as corrected (Basal medium control subtracted) absorbance readings. The assays were performed in a 'solution phase' where treatments were added to wells in a 2x concentration and cells added on top in an equal volume in Basal medium, the wells were not washed between treatment addition and cell seeding. Dissimilar to observations with HCE-T cells, the vitronectin (VN) control stimulated limited proliferation in these primary cells, however, it was the least potent treatments outside of Basal medium alone.
  • VN vitronectin
  • IGF-I promoted HCEC proliferation while the combination of VN + IGF-I (V+l in figure) produced similar results to a slightly lesser extent.
  • VF4-P03 batch control and VF001 produced cell proliferation equivalent to IGF-I alone, indicating no clear synergistic effects for the IGF-1 and Vitronectin portions of VF001 in this analysis. It was not expected that EGF would stimulate HCEC cell proliferation and the data supported this as EGF alone and VN + EGF stimulated responses similar to VN alone.
  • VF003 responses were improved over EGF alone and VN+EGF, however were still trailing those of IGF-I, VN+IGF-I, VF4-P03 and VF001 .
  • Motogenic potential of VF001 , VF003 and VF004 in primary corneal epithelial cells is a registered trademark of VF001 , VF003 and VF004 in primary corneal epithelial cells.
  • HCEC migration was performed using the fence method to assess the ability of treatments (as per Table 9) to stimulate outward migration of cells over a surface. After the migration period, cells were fixed, stained and imaged. The image was analysed for the area taken up by the cells as a complete colony with the boundary tracing the outer reaches of cells. Area data were collected and the average Basal medium result was subtracted from each other treatment within each individual experiment and these corrected data pooled together from each experiment for the final result as shown in Figure 9. All treatments were found to be significantly above the Basal medium control. Interestingly, the VN alone treatment did not stimulate high levels of migration and was, as per the proliferation assay, the least effective treatment within the experiments.
  • VN and IGF-I were found to be a potent stimulator of HCEC migration to the extent of being significantly above GM (#). Even more potent than the VN+IGF-I combination was the VF001 treatments, providing the greatest migration stimulus of the experiments.
  • VF001 afforded the greatest proliferative response in the experiments above analysis here was made on the basis of comparison with molar equivalent VF001 treatments. All treatments except VF004 50 nM were found to be significantly below the VF001 levels ( * ), indicating once more that VF001 offers the greatest potential in stimulating HCEC cell motility.
  • VF001 VN: IGF-I
  • VF003 VN:EGF
  • VF004 EGF:VN:IGF-I
  • mice were performed in accordance with the statement for the use of animals in ophthalmic and vision research approved by the Association for Research in Vision and Ophthalmology. The guidelines of the Animal Ethics Committee of the Singhealth Singapore Association for Assessment and Accreditation of Laboratory Animal Care were also satisfied. Mice were sedated by intraperitoneal injection of ketamine (80 mg /kg body weight) and Xylazine (10 mg/kg) combination. 1 -2 drops of 1 % xylocaine were applied for topical anesthesia to reduce the discomfort.
  • Test articles 1 , 2 and 3 were supplied and tested at low concentration (28 ⁇ g/mL) and high concentration (280 ⁇ g /ml_) where they remained frozen ( ⁇ -20°C) and thawed only immediately prior to use.
  • Slit-lamp imaging will be performed 2x daily from Day 1 -3 (to capture any fast healing reaction from the tested solutions), and once daily on Days 0, 4 and 5.
  • the key variable to be evaluated was the corneal wound area. After central cornea wounding, cornea was photographed by fluorescein staining under cobalt blue light at different time points. The wound area was expressed as a percentage of total wounded corneal area. On Day 6, animals were sacrificed and ocular tissues were collected and stored for H&E staining.
  • Formulations comprise 3 distinct recombinant fusion proteins generated using yeast (Pichia pastoris) expression system. Each contain a 64 amino acid domain derived from vitronectin which includes a collagen-binding domain and a cell attachment site (RGD integrin binding motif). These test articles differ in the covalently linked growth factor (either IGF-1 or EGF or both IGF-1 and EGF). All protein-based test articles were formulated at a low concentration (28 ⁇ g/mL) and high concentration (280 ⁇ g/mL) in Dulbecco's PBS pH 7.2. Consequently, Dulbecco's PBS represents the vehicle or negative control for this study.
  • VF001 Vitronectin (1 -64) - (Gly 4 Ser) 4 - IGF-1
  • VF003 Vitronectin (1 -64) - (Gly 4 Ser) 4 - EGF
  • VF004 EGF - (Gly 4 Ser) 4 - Vitronectin (1 -64) - (Gly 4 Ser) 4 - IGF-1
  • DPBS 2.67 mM Potassium Chloride (KCI), 1 .47 mM Potassium Phosphate monobasic (KH 2 PO 4 ), 138 mM Sodium Chloride (NaCI), 8.06 mM Sodium Phosphate dibasic (Na 2 HPO 4 -7H 2 O).
  • Wound healing area was quantified by ImageJ software. Comparisons at each time point between control and test groups were conducted using Student t-test. Significance was set at 0.05 level.
  • Epithelial Defect as per Fluorescein Staining 1 .
  • the area of epithelial defect after 5 days post-induction of corneal wounds seems smallest with VF003 treatments, then VF004, followed by VF001 .
  • the group treated with Dulbecco's PBS shows the least effect in resolving the epithelial defect as reflected in the area stained with fluorescein at days 4 and 5. ( Figures 10 & 13).
  • Clinical examination 1 No ocular phimosis detected in all groups.
  • the study results show that corneal epithelial wound healing (as measured by area reduction) started from day 2 for the placebo group, however the rate of change for the placebo group decreased following Day 2.
  • the corneal wound healing was slow between Day 0 and Day 2.
  • the VF001 , VF003 and VF004 treatments showed more rapid reductions in wound areas.
  • statistically significant differences in mean wound area was observed between VF001 , VF003 and VF004 treatments and the placebo group.
  • VF003 (high and low dose) VF004 (high dose) and VF001 (high dose) performed better than the placebo.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Ophthalmology & Optometry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Diabetes (AREA)
  • Endocrinology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des procédés, des compositions et des kits destinés au traitement de troubles oculaires. Les procédés, les compositions et les kits sont particulièrement utiles, mais sans y être limités, dans le traitement d'états pathologiques oculaires en favorisant la prolifération et/ou la migration de cellules épithéliales cornéennes. L'invention concerne également des chimères d'un premier peptide qui se lie et active un récepteur EGF, un second peptide choisi parmi la vitronectine, la fibronectine ou des parties de celui-ci qui se lient à une intégrine et un troisième peptide qui est identique ou homologue à IGF. L'invention concerne en outre des acides nucléiques codant pour les chimères et des méthodes de traitement utilisant les chimères.
PCT/AU2018/050717 2017-07-12 2018-07-12 Traitement de troubles oculaires Ceased WO2019010533A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2017902737 2017-07-12
AU2017902737A AU2017902737A0 (en) 2017-07-12 Treatment of ocular conditions

Publications (1)

Publication Number Publication Date
WO2019010533A1 true WO2019010533A1 (fr) 2019-01-17

Family

ID=65000941

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2018/050717 Ceased WO2019010533A1 (fr) 2017-07-12 2018-07-12 Traitement de troubles oculaires

Country Status (1)

Country Link
WO (1) WO2019010533A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0240031B1 (fr) * 1986-04-04 1993-09-01 Allergan, Inc Préparation visqueuse à utiliser pour des plaies de la cornée
WO2004069871A1 (fr) * 2003-02-05 2004-08-19 Queensland University Of Technology Complexes a facteurs de croissance et modulation de la migration et de la croissance cellulaires
WO2011063477A1 (fr) * 2009-11-30 2011-06-03 Queensland University Of Technology Chimères de fibronectine/facteur de croissance
US8871709B2 (en) * 2003-02-05 2014-10-28 Queensland University of Technolgy Synthetic chimeric proteins comprising epidermal growth factor and vitronectin

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0240031B1 (fr) * 1986-04-04 1993-09-01 Allergan, Inc Préparation visqueuse à utiliser pour des plaies de la cornée
WO2004069871A1 (fr) * 2003-02-05 2004-08-19 Queensland University Of Technology Complexes a facteurs de croissance et modulation de la migration et de la croissance cellulaires
US8871709B2 (en) * 2003-02-05 2014-10-28 Queensland University of Technolgy Synthetic chimeric proteins comprising epidermal growth factor and vitronectin
WO2011063477A1 (fr) * 2009-11-30 2011-06-03 Queensland University Of Technology Chimères de fibronectine/facteur de croissance

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PARAPURAM, S.K. ET AL.: "The integrin needle in the stromal haystack: emerging role in corneal physiology and pathology", J. CELL . COMMUN. SIGNAL., vol. 8, no. 2, June 2014 (2014-06-01), pages 113 - 124, XP035307538, Retrieved from the Internet <URL:doi:10.1007/s12079-014-0230-1> *
SCHULTZ, G. ET AL.: "Effects of growth factors on corneal wound healing", ACTA OPHTHALMOL SUPPL., vol. 70, no. S202, March 1992 (1992-03-01), pages 60 - 66, XP055564929, Retrieved from the Internet <URL:https://doi.org/10.1111/j.1755-3768.1992.tb02170.x> *
VAN LONKHUYZEN, D.R. ET AL.: "Chimeric vitronectin:insulin-like growth factor proteins enhance cell growth and migration through co-activation of receptors", GROWTH FACTORS, vol. 25, no. 5, October 2007 (2007-10-01), pages 295 - 308, XP008105538, Retrieved from the Internet <URL:doi:10.1080/08977190701803752> *

Similar Documents

Publication Publication Date Title
JP7097930B2 (ja) エンドグリンポリペプチドおよびその使用
US10596223B2 (en) JNK inhibitor molecules for treatment of various diseases
JP6585720B2 (ja) 眼疾患治療のペプチド及びこれを含む眼疾患治療用組成物
US20120315256A1 (en) Use of transforming growth factor - beta 1 (tgf-b1) inhibitor peptides for the treatment of corneal fibrosis and/or haze
CN102827253B (zh) 一种抑制炎症反应的小分子多肽及其应用
KR20010015868A (ko) 피치아 효모 발현계를 사용하여 항혈관형성 단백질인엔도스타틴, 안지오스타틴 또는 레스틴을 생성시키는 방법
WO2013079213A1 (fr) Utilisation d&#39;inhibiteurs peptidiques pouvant pénétrer dans des cellules de la voie de transduction de signal jnk pour le traitement du syndrome de sécheresse oculaire
US20210386837A1 (en) Active low molecular weight variants of angiotensin converting enzyme 2 (ace2) for the treatment of diseases and conditions of the eye
US11370816B2 (en) Histatins and method of use thereof
JP2024009210A (ja) Ｃｘｃｒ３の切断可能な活性化因子および使用方法
EP1357930A2 (fr) Glycoproteine riche en histidine
AU2018372396A1 (en) Composition for promoting goblet cell proliferation or mucin secretion comprising thymosin beta 4 or derivative thereof as active ingredient
WO2019010533A1 (fr) Traitement de troubles oculaires
EP3618851A2 (fr) Expression de g nes induite par la norrin et son utilisation pour traiter une maladie
IL302804A (en) Treatment of ocular diseases
CN104861058A (zh) 一类新的具有神经保护功能的多肽
WO2017221011A1 (fr) Traitement de maladie oculaire
AU2022403614A1 (en) Il-38 variants
Qi et al. Tissue Inhibitor of Metalloproteinases-3 Peptides Inhibit Angiogenesis and Choroidal
WO2019168697A1 (fr) Surexpression de foxc1 pour traiter la vascularisation cornéenne
AU2002304352A1 (en) Histidine-rich glycoprotein
HK1223556A1 (zh) Il-22二聚体在制备用於治疗胰腺炎的药物中的用途
WO2014090948A1 (fr) Serpin spn4a et ses dérivés biologiquement actifs destinés à être utilisés dans le traitement du cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18832024

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18832024

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 18832024

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 26/08/2020)