WO2013073968A2 - Agents de modulation de la signalisation cellulaire - Google Patents

Agents de modulation de la signalisation cellulaire Download PDF

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Publication number
WO2013073968A2
WO2013073968A2 PCT/NZ2012/000164 NZ2012000164W WO2013073968A2 WO 2013073968 A2 WO2013073968 A2 WO 2013073968A2 NZ 2012000164 W NZ2012000164 W NZ 2012000164W WO 2013073968 A2 WO2013073968 A2 WO 2013073968A2
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seq
peptide
amino acid
serine
acid sequence
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PCT/NZ2012/000164
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WO2013073968A3 (fr
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Keryn Johnson
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Industrial Research Limited
Meat & Livestock Australia Limited
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Priority claimed from AU2011903730A external-priority patent/AU2011903730A0/en
Application filed by Industrial Research Limited, Meat & Livestock Australia Limited filed Critical Industrial Research Limited
Publication of WO2013073968A2 publication Critical patent/WO2013073968A2/fr
Publication of WO2013073968A3 publication Critical patent/WO2013073968A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to methods and peptides for the modulation of gap junction communication between cells.
  • Connexins are cell membrane proteins that oligomerize to form a channel known as a gap junction which directly connects the cytoplasm of two neighboring cells. Each gap junction is formed by two connexons that dock with one another across an extracellular space, one connexon being provided by each cell. Each connexon is formed by 6 connexin subunits. In vertebrates, gap junctions are formed by proteins from the connexin family, which is composed of 21 members in humans (Goodenough and Paul 2003; Saez et al, 2003; Sohl and Willecke. 2004). Connexins are differentially expressed in tissues with some being significantly expressed in only a few tissues and some, like Cx43, being more widespread. Gap junctions play significant regulatory roles in embryonic development, electrical coupling, apoptosis, differentiation, tissue homeostasis and metabolic transport (Goodenough and Paul 2003 ;
  • Connexin43 (Cx43).
  • Gap junctions are dynamic channels that are regulated in response to changes in the cellular environment, and by protein interactions and phosphorylation. Gap junctions coordinate cell-to-cell communication within tissues by allowing for the transfer of molecules less than 1000 Daltons between cells, including ions, amino acids, nucleotides, second messengers (e.g., Ca 2+ , cAMP, cGMP, IP 3 ) and other metabolites (Loewenstein and Azarnia, 1988; Saez et al, 2003; Simon et al, 1998; Willecke et al, 2002), and so can modulate function and activity of cells.
  • second messengers e.g., Ca 2+ , cAMP, cGMP, IP 3
  • Gap junction communication has a central role in many physiological functions and has, for example, been reported to be important in the passage of electrical impulses such as in the heart, and for wound healing, bone growth and deposition, and in physiological disease and conditions such as pain (both acute and chronic pain, and nerve pain), heart disease,
  • Modulation of gap junction communication also has application to suppression of abnormal cardiac arrhythmias.
  • Connexin43 (Cx43) is phosphorylated at multiple different serine residues during its life cycle.
  • Cx43 is phosphorylated soon after synthesis and its phosphorylation changes as it traffics through the endoplasmic reticulum and Golgi apparatus to the plasma membrane ultimately forming into a gap junction structure.
  • Many reports have indicated changes in "phosphorylation” based on these mobility shifts and others that occur in response to growth factors or other biological effectors.
  • Phospho specific and epitope specific antibodies have been utilized to show when and where phosphorylation events occur during the Cx43 life cycle.
  • phosphorylation at S364 and or S365 is involved in forming the CxP43 PI isoform which is believed to regulate trafficking to or within the plasma membrane.
  • the CxP43 isoform is phosphorylated at S325, S328, and/or S330, this phosphorylation pattern only being present in gap junctions.
  • Treatment with protein kinase C activators leads to phosphorylation at S368, S279/S282 and S262 with a shift in mobility, the shift being dependent on MAPK activity but not phosphorylation at S279/S282.
  • the modulation of gap junction communication may be obtained by uncoupling connexons such as by increasing cell membrane fluidity with the use of long chain alcohols, or by phosphorylation of gap junctions, or blocking of connexon docking.
  • connexons such as by increasing cell membrane fluidity with the use of long chain alcohols, or by phosphorylation of gap junctions, or blocking of connexon docking.
  • indirect or direct inhibitors of gap junctions have also been reported (e.g., flufenamic acid and
  • a method for modulating gap junction communication in mammalian cells comprising treating the cells with an effective amount of a peptide having the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having at least 60% sequence identity with the amino acid sequence and being at least 4 amino acids in length.
  • the invention in another aspect relates to a method for modulating gap junction communication in mammalian cells, comprising treating the cells with an effective amount of a peptide having the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having at least 60% sequence identity with the amino acid sequence, wherein the peptide is 4 to 12 amino acids in length.
  • the invention in another aspect relates to a method for modulating gap junction communication in mammalian cells, comprising treating the cells with an effective amount of a peptide consisting of the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having at least 60% sequence identity with the amino acid sequence.
  • the variant is 5 amino acids in length.
  • variant forms of the peptide in accordance with the invention may either inhibit or up-regulate gap junction communication.
  • modulating gap junction communication in mammalian cells is inhibiting or up regulating gap junction communication between mammalian cells or in a mammal. In a preferred embodiment, modulating gap junction communication in mammalian cells is inhibiting gap junction communication between mammalian cells or in the mammal.
  • a variant form of the FHPSS (SEQ ID NO: 3) peptide comprises the amino acid sequence Z-X-P/L-p/n-X', wherein:
  • Z is an amino acid selected from phenylalanine (F), tyrosine (Y), serine (S), threonine (T), asparagine (N), glutamine (Q), tryptophan (W) and alanine (A);
  • X is an amino acid selected from histidine (H), lysine ( ), arginine (R) and alanine (A), valine (V), isoleucine (I), serine (S), threonine (T), or is not present;
  • P/L is proline (P), alanine (A), valine (V), or leucine (L);
  • p/n is a polar amino acid (p) or a non-polar amino acid (n);
  • X' is an amino acid selected from serine (S), threonine (T), asparagine (N) and alanine (A), or is not present.
  • a variant form of the FHPSS (SEQ ID NO: 3) peptide that inhibits gap junction communication comprises the amino acid sequence Z-X-P/L-p/n-X', wherein:
  • Z is an amino acid selected from phenylalanine (F), serine (S), asparagine (N), tryptophan (W), alanine (A), lysine (K), glutamic acid (E), arginine (R), and isoleucine (I);
  • X is an amino acid selected from histidine (H), lysine (K), cysteine (C), leucine (L), serine (S), threonine (T), proline (P), glutamine (Q), glycine (G), asparagine (N) or is not present;
  • P/L is selected from methionine (M), proline (P), glutamic acid (E), glycine (G), phenylalanine (F), histidine (H), serine (S), aspartic acid (D), tyrosine (Y), threonine (T) alanine (A), valine (V), and lysine (K);
  • p/n is a polar amino acid (p) or a non-polar amino acid (n);
  • X' is an amino acid selected from serine (S), threonine (T), asparagine (N), lysine (K) and alanine (A), or is not present.
  • modulation of cellular gap junction communication in accordance with the invention has direct application to the prophylaxis or treatment of a range of diseases or conditions and all such uses are expressly encompassed.
  • a method for promoting angiogenesis and/or wound healing in a mammal comprising administering to the mammal an effective amount of a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater and being at least 4 amino acids in length.
  • the invention in another aspect relates to a method for promoting angiogenesis and/or wound healing in a mammal, comprising administering to the mammal an effective amount of a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater, wherein the peptide is 4 to 12 amino acids in length.
  • a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater, wherein the peptide is 4 to 12 amino acids in length.
  • the invention in another aspect relates to a method for promoting angiogenesis and/or wound healing in a mammal, comprising administering to the mammal an effective amount of a peptide consisting of the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater.
  • a method for reducing scarring from a wound in a mammal comprising treating the wound with an effective amount of a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater and being at least 4 amino acids in length.
  • the invention in another aspect relates to a method for reducing scarring from a wound in a mammal, comprising treating the wound with an effective amount of a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater, wherein the peptide is 4 to 12 amino acids in length.
  • a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater, wherein the peptide is 4 to 12 amino acids in length.
  • the invention in another aspect relates to a method for reducing scarring from a wound in a mammal, comprising treating the wound with an effective amount of a peptide consisting of the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater.
  • a method for prophylaxis or treatment of pain in a mammal comprising administering to the mammal an effective amount of a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater and being at least 4 amino acids in length.
  • the invention in another aspect relates to a method for prophylaxis or treatment of pain in a mammal comprising administering to the mammal an effective amount of a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater, wherein the peptide is 4 to 12 amino acids in length.
  • a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater, wherein the peptide is 4 to 12 amino acids in length.
  • the invention in another aspect relates to a method for prophylaxis or treatment of pain in a mammal comprising administering to the mammal an effective amount of a peptide consisting of the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater.
  • a method for prophylaxis or treatment of inflammation in a mammal comprising treating the mammal with an effective amount of a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof haying 60% sequence identity with the amino acid sequence or greater and being at least 4 amino acids in length.
  • the invention in another aspect relates to a method for prophylaxis or treatment of inflammation in a mammal, comprising treating the mammal with an effective amount of a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater, wherein the peptide is 4 to 12 amino acids in length.
  • a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater, wherein the peptide is 4 to 12 amino acids in length.
  • a method for prophylaxis or treatment of inflammation in a mammal comprising treating the mammal with an effective amount of a peptide consisting of the amino acid sequence FHPSS (SEQ ID NO: 3), or a variant form thereof having 60% sequence identity with the amino acid sequence or greater.
  • the peptide or variant thereof is selected from the peptides shown in any of Tables 1 to 4.
  • the invention in another aspect relates to a method for inhibiting gap junction
  • FHPIN SEQ ID NO: 2
  • FHPSS SEQ ID NO: 3
  • Ac- FHPSS-NH 2 SEQ ID NO: 4
  • AHPSS SEQ ID NO: 5
  • FKPSS SEQ ID NO: 6
  • FH(P1030)S SEQ ID NO: 7
  • KNMSS SEQ ID NO: 8
  • FH(P1030)SN SEQ ID NO: 9
  • ECEGSS SEQ ID NO: 10
  • SLFSS SEQ ID NO: 1 1)
  • FH(P1030)AS SEQ ID NO: 12
  • FHPS SEQ ID NO: 13
  • NLVSS SEQ ID NO: 14
  • VSVSS SEQ ID NO: 15
  • FH(P1016)SA SEQ ID NO: 16
  • APPSS SEQ ID NO: 17
  • AHHS SEQ ID NO: 18
  • FQESS SEQ ID NO: 19
  • FK(P1016)SS (SEQ ID NO: 27), Ac-WHPSS-NH 2 (SEQ ID NO: 29), VEGSS (SEQ ID NO: 30), WH(P1030)SS (SEQ ID NO: 31), IT ASS (SEQ ID NO: 32), VHHSS (SEQ ID NO: 33), FHPIS (SEQ ID NO: 34), RGVSS (SEQ ID NO: 35), and VTTSS (SEQ ID NO: 36).
  • the peptide or variant thereof consists of the amino acid sequence FHPIN (SEQ ID NO: 2) or FHPSS (SEQ ID NO: 3).
  • the peptide or variant thereof is an isolated peptide. In another embodiment the peptide is substantially purified.
  • isolated means that the peptide or variant thereof is separated from its natural cellular environment, if any.
  • the peptide or variant thereof is separated from amino acid sequences normally associated with the peptide in nature, if any. More preferably the peptide or variant thereof is separated from flanking amino acid sequences normally associated with the peptide in nature, if any.
  • a "substantially purified" peptide or variant thereof comprises at least 50% of the total peptides in a preparation.
  • the peptide or variant thereof comprises at least 60% of the total peptides, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, most preferably at least 99% of the total peptides.
  • the variant form has at least 80% sequence identity with the specified amino acid sequence.
  • a method for modulating gap junction communication in mammalian cells comprising treating the cells with a peptide comprising the amino acid sequence FLSS (SEQ ID NO: 26), or a variant form thereof having at least 75% sequence identity with the amino acid sequence and being at least 4 amino acids in length.
  • the invention in another aspect relates to a method for modulating gap junction communication in mammalian cells, comprising treating the cells with a peptide comprising the amino acid sequence FLSS (SEQ ID NO: 26), or a variant form thereof having at least 75% sequence identity with the amino acid sequence, wherein the peptide is 4 to 12 amino acids in length.
  • a peptide comprising the amino acid sequence FLSS (SEQ ID NO: 26), or a variant form thereof having at least 75% sequence identity with the amino acid sequence, wherein the peptide is 4 to 12 amino acids in length.
  • the invention in another aspect relates to a method for modulating gap junction communication in mammalian cells, comprising treating the cells with a peptide consisting of the amino acid sequence FLSS (SEQ ID NO: 26), or a variant form thereof having at least 75% sequence identity with the amino acid sequence.
  • a method for modulating gap junction communication in mammalian cells comprising treating the cells with a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN- NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence and being at least 4 amino acids in length.
  • the invention in another aspect relates to a method for modulating gap junction communication in mammalian cells, comprising treating the cells with a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN- NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence, wherein the peptide is 4 to 12 amino acids in length.
  • a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN- NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence, wherein the peptide is 4 to 12 amino acids in length.
  • the invention in another aspect relates to a method for modulating gap junction communication in mammalian cells, comprising treating the cells with a peptide consisting of the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN- NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence.
  • a peptide consisting of the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN- NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence.
  • the invention in another aspect relates to a method for reducing mammalian cell migration and/or proliferation comprising contacting mammalian cells or a mammal with a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO:108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence and being at least 4 amino acids in length.
  • a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO:108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence and being at least 4 amino acids in length.
  • the invention in another aspect relates to a method for reducing mammalian cell migration and/or proliferation comprising contacting mammalian cells or a mammal with a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence wherein the peptide is 4 to 12 amino acids in length.
  • a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence wherein the peptide is 4 to 12 amino acids in length.
  • the invention in another aspect relates to a method for reducing mammalian cell migration and/or proliferation comprising contacting mammalian cells or a mammal with a peptide consisting of the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO:108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence.
  • a peptide consisting of the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO:108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence.
  • the invention in another aspect relates to a method for treating cancer in a mammalian cell or in a mammal comprising contacting the mammalian cells or mammal with a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence and being at least 4 amino acids in length.
  • a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence and being at least 4 amino acids in length.
  • the invention in another aspect relates to a method for treating cancer in a mammalian cell or in a mammal comprising contacting the mammalian cells or mammal with a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence, wherein the peptide is 4 to 12 amino acids in length.
  • a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence, wherein the peptide is 4 to 12 amino acids in length.
  • the invention in another aspect relates to a method for treating cancer in a mammalian cell or in a mammal comprising contacting the mammalian cells or mammal with a peptide consisting of the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO.108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence.
  • a peptide consisting of the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO.108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) or a variant form thereof having at least 60% sequence identity with the amino acid sequence.
  • a variant form thereof has at least 80% sequence identity with the specified amino acid sequence. In one embodiment of the above aspects the variant is 4 or 5 amino acids in length.
  • the peptide consists of the amino acid sequence FHPSA (SEQ ID NO: 108) or YPSS (SEQ ID NO: 105) or Ac-YHPSN-NH 2 (SEQ ID NO: 109).
  • the peptide is an isolated peptide. In another embodiment the peptide is substantially purified.
  • a variant form of the FHPSS (SEQ ID NO: 3) peptide that up- regulates gap junction communication comprises the amino acid sequence Z-X-P/L-p/n-X', wherein:
  • Z is an amino acid selected from phenylalanine (F), glycine (G), tyrosine (Y), serine (S), asparagine (N), tryptophan (W), alanine (A), lysine (K), glutamic acid (E), arginine (R), valine (V), aspartic acid (D), glutamine (Q), threonine (T), and isoleucine (I);
  • X is an amino acid selected from valine (V), histidine (H), proline (P), cysteine (C), threonine (T), serine (S), lysine (K), aspartic acid (D), arginine (R), asparagine (N), glutamic acid (E), leucine (L), glycine (G), isoleucine (I) and alanine (A), or is not present;
  • P/L is selected from proline (P), glutamic acid (E), serine (S), aspartic acid (D), tryptophan (W), glycine (G), isoleucine (I), methionine (M), leucine (L), histidine (H), alanine (A), asparagine (N), lysine (K), glutamine (Q), valine (V), and alanine (A);
  • p/n is a polar amino acid (p) or a non-polar amino acid (n);
  • X' is an amino acid selected from serine (S), asparagine (N), isoleucine (I), lysine (K), glutamic acid (E) and alanine (A), or is not present.
  • the variant form is a peptide consisting of the amino acid sequence FHPSA (SEQ ID NO: 108), YPSS (SEQ ID NO: 105) or Ac-YHPSN-N2 (SEQ ID NO: 109).
  • the invention relates to a method for up-regulating gap junction communication in mammalian cells, comprising treating the cells with a peptide selected from the group of peptides consisting of DRMSS (SEQ ID NO: 54), GPPSS (SEQ ID NO: 55), APESS (SEQ ID NO: 56), YH(P1016)SN (SEQ ID NO: 57), Ac-FRPSS-NH 2 (SEQ ID NO: 58), KCDWSS (SEQ ID NO: 59), VTGSS (SEQ ID NO: 60), NSPSS (SEQ ID NO: 61), FKISI (SEQ ID NO: 62), DKMSS (SEQ ID NO: 63), GDMSS (SEQ ID NO: 64), GRLSS (SEQ ID NO: 65), FKPVN (SEQ ID NO: 66), YH(P1030)SN (SEQ ID NO: 67), FHASS (SEQ ID NO: 68), FRPSS (SEQ ID NO: 69), Ac
  • AH(P1016)SS (SEQ ID NO: 80), ADQSS (SEQ ID NO: 81), TPLNSS (SEQ ID NO: 82), GVPSS (SEQ ID NO: 83), VTVSS (SEQ ID NO: 84), FH(P1016)AS (SEQ ID NO: 86), GGGPSS (SEQ ID NO: 87), AHHSS (SEQ ID NO: 88), Ac-YHPSS-NH 2 (SEQ ID NO: 89), SPASS (SEQ ID NO: 90), APPSSS (SEQ ID NO: 91), IKISS (SEQ ID NO: 92), Ac-FHPTN- NH 2 (SEQ ID NO: 93), Ac-FHPSN-NH 2 (SEQ ID NO: 94), FAPSS (SEQ ID NO: 95), NTGSS (SEQ ID NO: 96), WPSS (SEQ ID NO: 97), DTQSS (SEQ ID NO: 98), FH(P1030)IN (SEQ ID NO:
  • a peptide including the amino acid sequence FHPSS (SEQ ID NO: 3) or a variant form thereof comprising the amino acid sequence Z-X-P/L-p/n-X', wherein:
  • Z is an amino acid selected from phenylalanine (F), tyrosine (Y), serine (S), threonine (T), asparagine (N), glutamine (Q), tryptophan (W) and alanine (A);
  • X is an amino acid selected from histidine (H), lysine (K), arginine (R), alanine (A), valine (V), isoleucine (I), serine (S), threonine (T), or is not present;
  • P/L is proline (P), alanine (A), valine (V), or leucine (L);
  • p/n is a polar amino acid (p) or a non-polar amino acid (n);
  • X' is an amino acid selected from serine (S), threonine (T), asparagine (N) and alanine (A), or is not present; and
  • peptide or the variant form thereof is at least 4 amino acids in length.
  • a variant form of the FHPSS (SEQ ID NO: 3) peptide comprises the amino acid sequence Z-X-P/L-p/n-X', wherein:
  • Z is an amino acid selected from phenylalanine (F), serine (S), asparagine (N), tryptophan (W), alanine (A), lysine (K), glutamic acid (E), arginine (R), isoleucine (I) and proline (P).
  • X is an amino acid selected from histidine (H), lysine (K), cysteine (C), leucine (L), serine (S), threonine (T), proline (P), glutamine (Q), glycine (G), asparagine (N) or is not present;
  • P/L is selected from methionine (M), proline (P), glutamic acid (E), glycine (G), phenylalanine (F), histidine (H), serine (S), aspartic acid (D), tyrosine (Y), threonine (T) alanine (A), valine (V), and lysine (K);
  • p/n is a polar amino acid (p) or a non-polar amino acid (n);
  • X' is an amino acid selected from serine (S), threonine (T), asparagine (N), lysine ( ) and alanine (A), or is not present.
  • S serine
  • T threonine
  • N asparagine
  • N lysine
  • A alanine
  • a peptide comprising the amino acid sequence FLSS (SEQ ID NO: 26) or YPSS (SEQ ID NO: 105), or a variant form thereof having at least 75% sequence identity with the amino acid sequence and being at least 4 amino acids in length.
  • the variant form of the peptide comprises the amino acid sequence Z-P/L-p/n-X', and:
  • Z is phenylalanine (F), tyrosine (Y), serine (S), threonine (T), asparagine (N), glutamine (Q), tryptophan (W) or alanine (A);
  • P/L is proline (P), alanine (A), valine (V), or leucine (L);
  • p/n is a polar amino acid or a non-polar amino acid
  • X' is an amino acid selected from serine (S), threonine (T) and asparagine (N) and alanine (A).
  • the variant form of the FHPSS (SEQ ID NO: 3) peptide that up regulates gap junction communication may comprise the amino acid sequence Z-X-P/L-p/n-X', wherein:
  • Z is an amino acid selected from phenylalanine (F), glycine (G), tyrosine (Y), serine (S), asparagine (N), tryptophan (W), alanine (A), lysine (K), glutamic acid (E), arginine (R), valine (V), aspartic acid (D), glutamine (Q), threonine (T), and isoleucine (I);
  • X is an amino acid selected from valine (V), histidine (H), proline (P), cysteine (C), threonine (T), serine (S), lysine ( ), aspartic acid (D), arginine (R), asparagine (N), glutamic acid (E), leucine (L), glycine (G), isoleucine (I) and alanine (A), or is not present;
  • P/L is selected from proline (P), glutamic acid (E), serine (S), aspartic acid (D), tryptophan (W), glycine (G), isoleucine (I), methionine (M), leucine (L), histidine (H), alanine (A), asparagine (N), lysine (K), glutamine (Q), valine (V), and alanine (A);
  • p/n is a polar amino acid (p) or a non-polar amino acid (n);
  • X' is an amino acid selected from serine (S), asparagine (N), isoleucine (I), lysine (K), glutamic acid (E) and alanine (A), or is not present.
  • the peptide FHPSS (SEQ ID NO: 3) or a variant form thereof in accordance with the invention may be coupled to a facilitator moiety for facilitating passage of the peptide or the variant form thereof into cells as described herein.
  • a peptide agent comprising a peptide or variant form thereof embodied by the invention for modulating cellular gap junction communication, the peptide or variant form being coupled to a facilitator moiety for facilitating passage of the peptide or the variant form thereof into mammalian cells.
  • composition comprising a peptide, variant form thereof, or a peptide agent embodied by the invention together with a
  • a peptide, variant form thereof or peptide agent embodied by the invention or utilised in a method of the invention may be in the form or a pharmaceutically acceptable salt.
  • Divalent metal cation salts are particularly preferred (e.g., calcium or magnesium salts).
  • Such salts can be provided in formulations comprising an absorbent.
  • Such formulations can be provided on, or impregnated in, dressings.
  • dressings comprising or coated with such formulations may facilitate slow release of the peptide to the wound and/or reduce the frequency with which the dressing needs to be changed or replaced.
  • a dressing for topical application to the skin of a mammal comprising a peptide or variant form thereof embodied by the invention, or a peptide agent embodied by the invention, together with at least one absorbent preparation.
  • the absorbent is a polysaccharide preparation and preferably, an alginate preparation.
  • a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3) for use in modulating gap junction communication in mammalian cells, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater and being at least 4 amino acids in length.
  • a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3) for use in modulating gap junction communication in mammalian cells, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater, wherein the peptide is 4 to 12 amino acids in length.
  • a peptide consisting of the amino acid sequence FHPSS (SEQ ID NO: 3) for use in modulating gap junction communication in mammalian cells, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater.
  • a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3) for use in the prophylaxis or treatment of a disease or condition in a mammal, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater and being at least 4 amino acids in length.
  • a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3) for use in the prophylaxis or treatment of a disease or condition in a mammal, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater, wherein the peptide is 4 to 12 amino acids in length.
  • a peptide consisting of the amino acid sequence FHPSS (SEQ ID NO: 3) for use in the prophylaxis or treatment of a disease or condition in a mammal, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater.
  • a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3) for use in promoting angiogenesis, wound healing, or bone density and/or bone strength in a mammal, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater and being at least 4 amino acids in length.
  • a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3) for use in promoting angiogenesis, wound healing, or bone density and/or bone strength in a mammal, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater, wherein the peptide is 4 to 12 amino acids in length.
  • a peptide consisting of the amino acid sequence FHPSS (SEQ ID NO: 3) for use in promoting angiogenesis, wound healing, or bone density and/or bone strength in a mammal, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater.
  • a peptide comprising the amino acid sequence FLSS (SEQ ID NO: 26) for use in modulating gap junction communication in mammalian cells, or a variant form of the peptide having 75% sequence identity with the amino acid sequence or greater and being at least 4 amino acids in length.
  • a peptide comprising the amino acid sequence FLSS (SEQ ID NO: 26) for use in modulating gap junction communication in mammalian cells, or a variant form of the peptide having 75% sequence identity with the amino acid sequence or greater wherein the peptide is 4 to 12 amino acids in length.
  • a peptide consisting of the amino acid sequence FLSS (SEQ ID NO: 26) for use in modulating gap junction communication in mammalian cells, or a variant form of the peptide having 75% sequence identity with the amino acid sequence or greater.
  • a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) for use in treating cancer in a mammal, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater and being at least 4 amino acids in length.
  • a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) for use in treating cancer in a mammal, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater, and wherein the peptide is 4 to 12 amino acids in length.
  • a peptide consisting of the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) for use in treating cancer in a mammal, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater.
  • a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) for use in treating cancer in a mammal, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater and being at least 4 amino acids in length.
  • a peptide comprising the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) for use in treating cancer in a mammal, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater, wherein the peptide is 4 to 12 amino acids in length.
  • a peptide consisting of the amino acid sequence YPSS (SEQ ID NO: 105) or FHPSA (SEQ ID NO: 108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) for use in treating cancer in a mammal, or a variant form of the peptide having 60% sequence identity with the amino acid sequence or greater.
  • the variant form has 80% sequence identity with the specified amino acid sequence or greater.
  • a peptide or variant form thereof embodied by the invention or utilised in a method of the invention is from 4 to 12 amino acids in length although longer peptides may also be utilised. In one embodiment of the above aspects the variant is 4 or 5 amino acids in length.
  • longer peptides e.g., generally longer than 12 amino acids in length
  • peptides having a length greater than 4 or 5 amino acids that are embodied by the invention or may be utilised in a method of the invention include peptides from the C-terminal end of oA crystallin proteins such as SREEKPSSAPSS (SEQ ID NO: 1) (e.g., from ovine or bovine source) which may be cleaved to release the peptide SAPSS (SEQ ID NO: 101), SREEKPTSAPSS (SEQ ID NO: 1) (e.g., sourced from chicken) which may cleaved to release the peptide SAPSS (SEQ ID NO: 101), and the peptide NSAVSS (SEQ ID NO: 110) which may be cleaved to release the peptide SAVSS (SEQ ID NO: 111).
  • SREEKPSSAPSS SEQ ID NO: 1
  • SREEKPTSAPSS SEQ ID NO: 1
  • SEQ ID NO: 1 e.g., sourced from chicken
  • a peptide embodied by the invention or utilised in a method of the invention may also have one or more post translational or post synthesis modifications.
  • modification(s) include modification of the N- and/or C-terminal end of the peptide (e.g., by replacing, converting or substituting terminal carboxyl and/or amine groups to form or provide other substituent groups), and phosphorylation of one or more amino acids.
  • the phosphorylation of one or more of serine (S), threonine (T), and/or tyrosine (Y) amino acid(s) of a peptide embodied by the invention or useful in a method of the invention may affect the modulation of gap junction communication in cells and is expressly encompassed.
  • S serine
  • T threonine
  • phosphorylated peptides may interact with kinase and/or phosphatase enzymes to modulate the phosphorylation events that control the function of gap junctions.
  • an amino acid providing a phosphorylation site of a peptide such as a serine residue as described herein may be substituted with another amino acid that can be phosphorylated such as threonine, and all such substitutions are also expressly encompassed.
  • the FHPSS (SEQ ID NO: 3) peptide or variant form thereof may have particular application to modulation of gap junctions formed entirely or partially by one or more connexin43 (Cx43) sub-units.
  • the peptide or variant form thereof is an N- acetyl peptide. In one embodiment of the above aspects the peptide or variant form thereof comprises an aminated C-terminal amino acid residue. In one embodiment of the above aspects the peptide or variant form thereof is an N-acetyl peptide comprising an aminated C-terminal amino acid residue.
  • the peptide or variant thereof has one of the amino acids in each of positions 1 to 5, and activity, as set out in Table 2.
  • peptide is used interchangeably herein with “polypeptide” unless it is otherwise clear from the context in which it is used that “peptide” is intended.
  • Figure 1 shows amino acid sequence alignment of crystallin peptides
  • Figure 2 is a graph showing inhibition of gap junction communication by the peptide FHPSS (SEQ ID NO: 3);
  • Figure 3 is a graph showing dose response inhibition of gap junction communication by the peptide FHPSS (SEQ ID NO: 3);
  • Figure 4 is a graph showing a peptide FHPSS (SEQ ID NO: 3) and alginate dressing promotes wound closure;
  • Figure 5 is a graph showing enhanced collagen formation in a wound treated with the peptide FHPSS (SEQ ID NO: 3) and alginate dressing;
  • Figure 6 is a graph showing reduced scarring from wounds treated with the peptide FHPSS (SEQ ID NO: 3) and alginate dressing compared to other wound treatments
  • Figure 7 is a graph showing reduced TNFa in wounds treated with the peptide FHPSS (SEQ ID NO: 3) and alginate dressing as a measure of the anti-inflammatory activity of the peptide;
  • Figure 8 is a graph showing a reduction in CD68 staining in wounds treated with the j peptide FHPSS (SEQ ID NO: 3) and alginate dressing;
  • Figure 9 shows immuno-stained smooth muscle cell actin (SMA) in a wound treated with the peptide FHPSS (SEQ ID NO: 3) and alginate dressing compared to other treatments;
  • Figure 10 is graph showing SMA area of wounds treated with the peptide FHPSS (SEQ ID NO: 3) and alginate dressing compared to the other treatments;
  • Figure 11 is a graph showing the level of CD34 staining of blood vessels in wounds treated with the peptide FHPSS (SEQ ID NO: 3) and alginate dressing compared to the other treatments;
  • Figure 12 shows E-cadherin immuno-stained wound tissue at Day 14 and
  • Figure 14 is a graph showing modulation of gap junction communication by a range of peptides in accordance with embodiments of the invention.
  • the peptide utilised in a method embodied by the invention can be a fragment of a native polypeptide, or be a recombinant or synthetic peptide.
  • a recombinant or synthetic peptide can have an identical amino acid sequence to that of the native fragment or one or more amino acid differences compared to the native fragment.
  • the amino acid changes can comprise the i addition, deletion and/or substitution of one or more amino acids. Inversion of amino acids and other mutational changes that result in modification of the native peptide sequence are also encompassed.
  • a recombinant or synthetic peptide can comprise an amino acid or amino acids not encoded by the genetic code. For example, D-amino acids rather than L-amino acids can be utilized to inhibit endopeptidase degradation of the peptide in vivo.
  • substitution of an amino acid can be a conservative or non-conservative substitution.
  • any amino acid substitution will be a conservative amino acid substitution.
  • conservative amino acid substitution is to be taken in the normally accepted sense of replacing an amino acid residue with another amino acid having similar properties which substantially does not adversely affect the cell gap junction communication modulation activity of the peptide.
  • a conservative amino acid substitution can involve substitution of a basic amino acid such as arginine with another basic amino acid such as lysine.
  • a cysteine residue can be replaced with serine, or a non-polar amino acid may be substituted with another non-polar amino acid such as alanine.
  • the identification of amino acids amenable to substitution or deletion may be determined on the basis of the teaching provided herein and by routine trial and experimentation well within the skill of the addressee.
  • a variant form of a peptide in accordance with one or more embodiments of the invention can be provided by introducing nucleotide change(s) in a nucleic acid sequence such that the desired amino acid changes are achieved upon expression of the nucleic acid in a host cell.
  • a variant form of the FHPSS (SEQ ID NO: 3) peptide embodied by the invention and/or useful in a method embodied by the invention may have amino acid sequence identity with the amino acid sequence FHPSS (SEQ ID NO: 3) of at least 60% or greater, more usually at least about 75% or greater and more usually, at least 80%.
  • a variant form the FHPSS (SEQ ID NO: 3) peptide that is longer than 4 amino acids in length may have an overall sequence identity with a peptide including the FHPSS (SEQ ID NO: 3) sequence (e.g., a native or parent peptide) of at least 60% or greater, such as at least about 65%, 70%, 75%, 80%, 85%, 90% or 95% or greater, and all sequence identities and ranges thereof within those enumerated above are herein expressly encompassed.
  • a peptide embodied by the invention and/or useful in a method embodied by the invention may have amino acid sequence identity with the amino acid sequence FLSS (SEQ ID NO: 26) or YPSS (SEQ ID NO: 105) of at least 75%.
  • a peptide longer than 4 amino acids in length may have an overall sequence identity compared to a peptide including the FLSS (SEQ ID NO: 26) or YPSS (SEQ ID NO: 105) sequence (e.g., a native or parent peptide) of at least 60%, and more usually at least about 65%, 70%, 75%, 80%, 85%, 90% or 95% or greater, and all sequence identities and ranges thereof within those enumerated above are herein expressly encompassed.
  • sequence identity between amino acid sequences is determined by comparing amino acids at each position in the sequences when optimally aligned for the purpose of comparison.
  • the sequences are considered identical at a position if the amino acids at that position are the same.
  • a gap that is a position in an alignment where an amino acid residue is present in one sequence but not the other, is regarded as a position with non-identical residues. Alignment of sequences may be performed using any suitable program or algorithm such as for instance, by the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970).
  • Computer assisted sequence alignment can be conveniently performed using standard software programs such as GAP which is part of the Wisconsin Package Version 10.1 (Genetics Computer Group, Madison, Wisconsin, United States) using the default scoring matrix with a gap creation penalty of 50 and a gap extension penalty of 3, although sequences can be aligned for comparison manually or by any suitable algorithm and/or computer assisted method.
  • Computer assisted alignment tools are also available to evaluate functional amino acid identity between sequences where one or more amino acid changes between the sequences occur (e.g., conservative amino acid changes) but wherein the amino acid substitution(s) are functionally equivalent such that the peptide retains functional activity of the parent or native peptide.
  • a peptide that differs from the peptide FHPSS (SEQ ID NO: 3) in one or more amino acids will nevertheless have at least 60% functional amino acid identity when optimally aligned for sequence comparison with the FHPSS (SEQ ID NO: 3) sequence and desirably, at least 75%, 80% or 100% functional amino acid sequence identity with the FHPSS (SEQ ID NO: 3) sequence as applicable.
  • the peptide will be a C-terminal fragment of a crystallin polypeptide, and more preferably a fragment of the C-terminal end of a crystallin polypeptide selected from the group consisting of 3B2 crystallin, ⁇ 3 crystallin and oA crystallin
  • Bovine and ovine eye lens is a useful source of oA crystallin and ⁇ 2 crystallin proteins.
  • the FHPSS (SEQ ID NO: 3) peptide may comprise the 5 amino acid C- terminal fragment of bovine and ovine ⁇ 2 crystallin (e.g., see GenBank accession No.
  • FHPIN (8B2 crystallin (Tetraodon nigroviridis)
  • bovine aA crystallin The amino acid sequence for bovine aA crystallin is for instance provided by GenBank Accession No. NP 776714 crystallin, alpha A [Bos taurus], and is also highly conserved.
  • bovine aA crystallin has 98% amino acid sequence identity with mouse, hamster and rat aA crystallin (Hay and Petrash., 1987).
  • variant forms of the peptide in accordance with the invention may either inhibit or up-regulate gap junction communication.
  • the FHPSS (SEQ ID NO: 3) peptide can the inhibit gap junction communication in cells.
  • the YPSS (SEQ ID NO: 105), FHPSA (SEQ ID NO: 108) or Ac-YHPSN-NH 2 (SEQ ID NO: 109) variant form of the peptide can up-regulate gap junction communication between cells.
  • the finding that differential modulation of gap junction communication in cells can be obtained by variant forms of the FHPSS (SEQ ID NO: 3) amino acid lends itself to the prophylaxis or treatment of various diseases and conditions as described further below.
  • modulating gap junction communication in a mammal is inhibiting or up regulating gap junction communication in the mammal.
  • modulating gap junction communication in the mammal is inhibiting gap junction communication in the mammal.
  • the inventor believes that the activity observed relates to three convergent pathways which are involved in regulating gap junction formation and stability which involves the binding of PDZ2 domain of ZO with the C-terminal tail of Cx43 and phosphorylation of Cx43 at Ser368 by PRKCG (SEQ ID NO: 1 12) which induces disassembly of gap junction plaques and inhibition of gap junction communication.
  • Inhibition of PRKCG (SEQ ID NO: 112) and peptide binding to Cx43 C-terminal tail around the motifs QRPSS (SEQ ID NO: 1 13) and SRASS (SEQ ID NO: 104) and inhibition of phosphorylation of Ser368 and promotion of phosphorylation of Ser372 can also modulate the formation of gap junction communication which stimulates gap junction communication.
  • the modulation of gap junction communication through regulation of phosphorylation and stabilisation of connexin proteins in the plasma membrane through the interaction with ZO PDZ2 domain and displacement of the Cx43 C-terminal tail peptide results in the inhibition of gap junction communication.
  • the FLSS (SEQ ID NO: 26) peptide constitutes the terminal 4 amino acids of bovine and ovine B/33 crystallin monomer polypeptide chains and has high sequence identity to the corresponding sequence FPSS (SEQ ID NO: 78) of human B03 crystallin monomer polypeptide, the use of both of which is expressly encompassed herein.
  • the QA crystallin C-terminal peptide sequence SAPSS (SEQ ID NO: 101) can inhibit gap junction communication in accordance with an embodiment of the invention as shown by stimulation of angiogenesis in an aortic ring model whereas its phosphorylation product
  • S*APS*S inhibits angiogenesis in the same model and promotes gap junction communication, wherein * is a phosphate moiety and one or both of the serine residues indicated may be phosphorylated.
  • the stimulation of gap junction communication may, for example, prevent or inhibit cancer metastasis, and reduce cell migration and proliferation and so is suitable for use in applications where these physiological cellular functions are stimulated e.g., as in cancer.
  • gap junction communication is important in the maintenance of a normal cardiac rhythm promotion or stabilisation of gap junctions is useful for the treatment of arrhythmias. Maturation of the skin keratinocytes requires gap junction communication and it is also expected that acceleration of skin maturation may be enhanced through promoting gap junction
  • gap junction communication whereas the initial inhibition of gap junction communication accelerates the earlier stages of wound healing but may delay the later stages of healing especially around skin maturation.
  • amino acid sequence Z-X-P/L-p/n-X' encompasses the peptides listed in
  • the group of polar amino acids typically comprises serine (S), threonine (T), tyrosine (Y), asparagine (N) and glutamine (Q).
  • the group of non-polar amino acids typically comprises glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), cysteine (C), methionine (M), phenylalanine (F), and tryptophan (W).
  • p/n is an amino acid other than cysteine (C) thereby avoiding the potential for di-sulfide linkages forming between peptides.
  • C cysteine
  • p/n is selected from the group consisting of serine (S), valine (V), isoleucine (I) and threonine (T).
  • a variant form of the peptide FHPSS comprises, or consists of, the amino acid sequence Z-X-P/L-p/n-X', and is at least 4 amino acids in length, wherein:
  • Z is phenylalanine (F), tyrosine (Y), tryptophan (W), alanine (A), or serine (S);
  • X is histidine (H), lysine (K), or alanine (A), or is not present;
  • P/L is proline (P), leucine (L), or valine (V);
  • p/n is serine (S), isoleucine (I), alanine (A), valine (V) or threonine (T); and
  • X' is serine (S), asparagine (N), alanine (A), or is not present.
  • Z is phenylalanine (F), tyrosine (Y), tryptophan (W), or alanine (A);
  • X is histidine (H) or lysine (K), or is not present;
  • P/L is proline (P) or leucine (L); p/n is serine (S), isoleucine (I), or alanine (A); and
  • X' is serine (S), asparagine (N) or alanine (A).
  • the variant form of the FHPSS (SEQ ID NO: 3) peptide may, for example, comprise, or consist of, the amino acid sequence Z-X-P/L-p/n-X', and is at least 4 amino acids in length, wherein:
  • Z is phenylalanine (F) or alanine (A);
  • X is histidine (H), lysine (K), or is not present;
  • P/L is proline (P) or leucine (L);
  • p/n is selected from the group of amino acids consisting of serine (S), isoleucine (I), or alanine (A); and
  • X' is serine (S), asparagine (N), or is not present.
  • Z is phenylalanine (F).
  • X is histidine (H).
  • a variant form of the FHPSS (SEQ ID NO: 3) peptide that inhibits gap junction communication may comprise the amino acid sequence Z-X-P/L-p/n-X', wherein:
  • Z is an amino acid selected from phenylalanine (F), serine (S), asparagine (N), tryptophan (W), alanine (A), lysine (K), glutamic acid (E), arginine (R), and isoleucine (I);
  • X is an amino acid selected from histidine (H), lysine (K), cysteine (C), leucine (L), serine (S), threonine (T), proline (P), glutamine (Q), glycine (G), asparagine (N) or is not present;
  • P/L is selected from methionine (M), proline (P), glutamic acid (E), glycine (G), phenylalanine (F), histidine (H), serine (S), aspartic acid (D), tyrosine (Y), threonine (T) alanine (A), valine (V), and lysine (K);
  • p/n is a polar amino acid (p) or a non-polar amino acid (n);
  • X' is an amino acid selected from serine (S), threonine (T), asparagine (N), lysine ( ) and alanine (A), or is not present.
  • the peptide FLSS (SEQ ID NO: 26) is an example of this embodiment in which amino acid X is not present.
  • the variant form of the FHPSS (SEQ ID NO: 3) peptide may, for example, comprise, or consist of, the amino acid consensus sequence Z-X-P/L-p/n-X', and is at least 4 amino acids in length, wherein:
  • Z is phenylalanine (F), tryptophan (W) or tyrosine (Y); X is histidine (H) or is not present;
  • P/L is proline (P);
  • X' is serine (S), asparagine (N) or alanine (A).
  • a variant form of the FHPSS (SEQ ID NO: 3) peptide that up- regulates gap junction communication comprises the amino acid sequence Z-X-P/L-p/n-X', wherein:
  • Z is an amino acid selected from phenylalanine (F), glycine (G), tyrosine (Y), serine (S), asparagine (N), tryptophan (W), alanine (A), lysine (K), glutamic acid (E), arginine (R), valine (V), aspartic acid (D), glutamine (Q), threonine (T), and isoleucine (I);
  • X is an amino acid selected from valine (V), histidine (H), proline (P), cysteine (C), threonine (T), serine (S), lysine (K), aspartic acid (D), arginine (R), asparagine (N), glutamic acid (E), leucine (L), glycine (G), isoleucine (I) and alanine (A), or is not present;
  • P/L is selected from proline (P), glutamic acid (E), serine (S), aspartic acid (D), tryptophan (W), glycine (G), isoleucine (I), methionine (M), leucine (L), histidine (H), alanine (A), asparagine (N), lysine (K), glutamine (Q), valine (V), and alanine (A);
  • p/n is a polar amino acid (p) or a non-polar amino acid (n);
  • X' is an amino acid selected from serine (S), asparagine (N), isoleucine (I), lysine (K), glutamic acid (E) and alanine (A), or is not present.
  • the variant form is a peptide consisting of the amino acid sequence YPSS (SEQ ID NO: 105), FHPSA (SEQ ID NO: 108) or Ac-YHPSN-NH 2 (SEQ ID NO: 109).
  • Peptide fragments useful in embodiments of the invention may be prepared by partial hydrolysis of the parent polypeptide/protein (e.g., by partial protease digestion such as by elastase or chymotrypsin) and purified by any suitable purification technique including, but not limited to filtration and chromatography (e.g., RP-HPLC) protocols.
  • suitable purification techniques including, but not limited to filtration and chromatography (e.g., RP-HPLC) protocols.
  • Alternative purification methods that may be used include divalent cation (e.g., calcium) selective precipitation or IMAC chromatography.
  • a peptide according to the invention is prepared in an isolated or substantially purified form for use in a method of the invention.
  • the FHPSS (SEQ ID NO: 3) C-terminal end of bovine ⁇ 2 crystallin can be cleaved free by elastase I as follows RDMQWHQRGA (SEQ ID NO: 28) / FHPSS (SEQ ID NO: 3) wherein "/" indicates the cleavage point.
  • the cleavage point of human B 32 crystallin for release of the C-terminal peptide FHPSN (SEQ ID NO: 47) is as follows: WHQRGA (SEQ ID NO: 85)/FHPSN (SEQ ID NO: 47).
  • WHQRGA SEQ ID NO: 85
  • FHPSN SEQ ID NO: 47
  • Nucleic acid encoding a fusion protein can be provided by joining separate DNA fragments encoding the peptide and, for example, a lipophilic amino acid sequence for enhancing the lipophilic characteristics of the protein by employing blunt-ended termini and oligonucleotide linkers, digestion to provide staggered termini and ligation of cohesive ends as required.
  • Host cells that can be transfected for expression of recombinant peptides and fusion proteins as described herein include bacteria such as E. coli, Bacillus strains (e.g., B. subtilis), Streptomyces and Pseudomonas bacterial strains, yeast such as Saccharomyces and Pichia, insect cells, avian cells and mammalian cells such as Chinese Hamster Ovary cells (CHO), COS, HeLa, HaRas, WI38, SW480, and NIH3T3 cells.
  • bacteria such as E. coli, Bacillus strains (e.g., B. subtilis), Streptomyces and Pseudomonas bacterial strains, yeast such as Saccharomyces and Pichia, insect cells, avian cells and mammalian cells such as Chinese Hamster Ovary cells (CHO), COS, HeLa, HaRas, WI38, SW480, and NIH3T3 cells.
  • bacteria such as E.
  • the host cells are cultured in a suitable culture medium under conditions for expression of the introduced nucleic acid (typically in an appropriate expression vector) prior to purification of the expressed product from the host cells, and/or supernatants as required using standard purification techniques known in the art or as described herein.
  • a suitable culture medium under conditions for expression of the introduced nucleic acid (typically in an appropriate expression vector) prior to purification of the expressed product from the host cells, and/or supernatants as required using standard purification techniques known in the art or as described herein.
  • a peptide in accordance with the invention or utilised in a method of the invention can also be modified by coupling one or more proteinaceous or non-proteinaceous moieties to the protein to improve solubility, lipophilic characteristics, stability, biological half-life, or for instance to act as a label for subsequent detection or the like. Modifications can also result from post-translational or post-synthesis modification such as by the attachment of carbohydrate moieties, or chemical reaction(s) resulting in structural modification(s) (e.g., the alkylation or acetylation of one or more amino acid residues or other changes involving the formation of chemical bonds).
  • the peptide can have one or more modifications selected from the group consisting of methylation, phosphorylation, oxidation of tyrosine and/or tryptophan residues, glycosylation, and S- methylcysteine covalent attachment.
  • a peptide may be PEGylated, succinylated, acetylated (Ac) or otherwise modified at its N- and/or C-terminal ends to render it less resistant to degradation by proteases in vivo or to inhibit their clearance from the circulation via the kidneys.
  • Particularly desirable such modifications include acetylation of the N- terminal end of the peptide and/or coupling of an amine (-NH 2 ) group to the C-terminal end of the peptide.
  • the peptide or variant form thereof according to the invention is an N-acetyl peptide.
  • the peptide or variant form thereof according to the invention comprises an aminated C-terminal amino acid residue.
  • the peptide or variant form thereof according to the invention is an N-acetyl peptide comprising an aminated C-terminal amino acid residue.
  • a peptide embodied by the invention or utilised in a method of the invention can be a fragment of naturally occurring protein (e.g., a crystallin protein), or a recombinant or synthetic peptide, and will generally have a length of from 4 amino acids (e.g., FLSS (SEQ ID NO: 26) or YPSS (SEQ ID NO: 105)) or 5 amino acids (e.g., FHPSS (SEQ ID NO: 3), FHPSA (SEQ ID NO: 108) or YHPSN (SEQ ID NO: 103)) up to 12 amino acids or more.
  • the peptide will have a length up to 12 amino acids or less and more generally, a length of 11, 10, 9, 8, 7, 6, 5 or 4 amino acids.
  • a peptide may for example, be any length in the range of from 4 or 5 amino acids up to 25 amino acids (e.g., 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, or 13 amino acids), or up to 30, 35, or 40, 41, 42, 43, 44 or 45 amino acids or more, and all peptides within the ranges specifically recited above are expressly encompassed.
  • the peptide will be 40 amino acids or less in length.
  • longer peptides embodied by the invention or utilised in a method of the invention will include at least one cleave site for being hydrolysed or enzymatically cleaved (e.g., a protease cleavage site) in vivo to release one or more shorter peptides for modulating gap junction communication.
  • the shorter "active peptides" will generally be 4 to 8 amino acids in length (i.e., 4, 5, 6, 7 or 8 amino acids in length).
  • a peptide utilised in a method embodied by the invention that is 25, 30, 35 or 40 amino acids in length or more, will generally be other than a fragment of a native crystallin protein.
  • the active peptide will have a molecular weight of up to about 1000 Da, e.g., a molecular weight of about 950Da or less, 900 Da or less, 850 Da or less, 800 Da or less, 700 Da or less, or 600 Da or less.
  • peptides comprising one or more repeats of the FHPSS (SEQ ID NO: 3), YPSS (SEQ ID NO: 105), FLSS (SEQ ID NO: 26) or Ac-YHPSN-NH 2 (SEQ ID NO: 109) amino acid sequence, or variant form thereof, can be utilized in a method of the invention (i.e., a multimer form of the peptide such as (FHPSS)n (SEQ ID NO: 3), wherein n is the number of repeats of the peptide (typically 1 to 3).
  • the peptide repeats may be linked together by respective intervening (e.g., amino acid or other) linker
  • a peptide utilized in a method embodied by the invention may be inherently permeable to the outer plasma membrane of cells.
  • the peptide may be coupled to a "facilitator moiety" for facilitating passage or translocation of the active peptide across the outer plasma membrane into the cytoplasm of cells.
  • facilitator moieties include carrier peptides which have the capacity to deliver cargo molecules across cell membranes in an energy-independent manner.
  • Carrier peptides that are known in the art include penetratin and variants or fragments thereof, human immunodeficiency virus Tat derived peptide, transportan derived peptide, and signal peptides.
  • a signal peptide used in a method of the invention be a complete signal peptide, and fragments thereof which retain the ability to pass across the outer cellular membrane to effect delivery of the cargo peptide to which it is coupled may be utilized.
  • Cationic peptides can also be used successfully to transfer macromolecules into living cells (e.g., a 15 mer arginine peptide), and the invention extends to the use of such cationic peptides in embodiments of the invention.
  • the facilitator moiety can be a lipid moiety or other non- peptide moiety which enhances cell membrane solubility of the peptide active to which it is coupled for passage of that peptide into cells.
  • the lipid moiety can for instance be selected from triglycerides, including mixed triglycerides. Fatty acids and particularly, C 16 - C 20 fatty acids can also be used. Preferably the fatty acid will be a saturated fatty acid and most usually, stearic acid.
  • the invention is not limited to the use of any particular facilitator moiety, and any such moiety that provides the desired cell membrane solubility and which is physiologically acceptable can be used.
  • peptide dendrimer framework can be utilized as a facilitator moiety in accordance with the invention.
  • Peptide dendrimers in at least some embodiments may present multiple units of the peptide coupled to a branched framework of polyamino acids (typically lysine branching units).
  • the dendrimer will typically have at least 2 or 3 layers/generations of amino acid branching units, the units of the peptide being coupled to the outermost
  • the dendrimer framework may, for example, include enzyme cleave sites for release of the peptide or variant form thereof within the cytoplasm of target cells.
  • Suitable peptide dendrimer framework to which a peptide as described herein can be coupled, and methods for the provision of peptide dendrimers, are for instance described in Lee et al, 2005; Sadler and Tarn, 2002; and Cloninger, 2002, the entire contents of which are incorporated herein in their entirety by reference.
  • the communication in accordance with the invention can be linked to a selected facilitator moiety in any conventionally known manner.
  • the peptide can be linked directly to a carrier peptide through an amino acid linker sequence by a peptide bond or by a non-peptide covalent bond using a cross-linking reagent.
  • chemical ligation methods may be used to create a covalent bond between the carboxy terminal amino acid of the carrier peptide or a linker amino acid sequence, and the peptide active (e.g., FHPSS) (SEQ ID NO: 3).
  • FHPSS peptide active
  • Evaluation of the log P ratio of a peptide is useful in assessing whether cell entry of the peptide would be enhanced by coupling the peptide to a facilitator moiety as described herein.
  • the determination of the log P ratio of a peptide is well within the scope of the skilled addressee, e.g., see Mo and Colby, 2010, the contents of which is also incorporated herein in its entirety by reference.
  • the peptide may be released from a larger peptide (or e.g., from a peptide agent, facilitator moiety, dendrimer or the like) for modulation of gap junction communication between cells in accordance with
  • the release or the peptide can for instance occur at the site of topical administration (e.g., by enzymatic cleavage such as by elastase I present at a wound site) either within the cytoplasm of cells or at the plasma membrane for passage or translocation of the active peptide into the cytoplasm of cells.
  • a peptide, dendrimer or the like as described herein can be adapted or designed to include one or more enzyme cleavage sites for release of the active peptide and all such embodiments are within the scope of the invention.
  • human leukocytes are recruited to wound sites and a long polypeptide incorporating the peptide active can be administered to the wound wherein the polypeptide is cleaved by leukocyte elastase hydrolysis to release a peptide comprising the amino acid sequence FHPSS (SEQ ID NO: 3) or a variant form thereof for passage across the outer cell membrane into epithelial cells.
  • FHPSS amino acid sequence FHPSS
  • Peptides released at the surface of the target cell that include a facilitator moiety for facilitating passage of the released peptide across the outer cell membrane of the cell can also be adapted (e.g., by including one or more protease cleave sites) for being cleaved or hydrolysed in the cytoplasm of the cell to release the active peptide(s) from the facilitator moiety to permit the active peptides to effect modulation of gap junction communication.
  • the invention extends to the administration of full length native polypeptides and proteins such as full length or truncated forms of crystallin polypeptides and proteins (e.g., aA, B/32, and B/33 crystallin polypeptides) and variant forms of the foregoing for subsequent cleavage and release or the active peptide(s) to effect modulation of gap junction communication as described herein.
  • full length native polypeptides and proteins such as full length or truncated forms of crystallin polypeptides and proteins (e.g., aA, B/32, and B/33 crystallin polypeptides) and variant forms of the foregoing for subsequent cleavage and release or the active peptide(s) to effect modulation of gap junction communication as described herein.
  • liposomes ghost bacterial cells, mini-cells, caveospheres, synthetic polymer agents, ultra- centrifuged nanoparticles and other anucleate nanoparticles may be loaded with peptides or peptide agents for delivery of the cargo to target tissue/cells (e.g., via labelling of the minicells, caveospheres or nanoparticles with bi-specific antibodies, targeting peptides or the like).
  • cell to cell communication is vital.
  • gap junctions modulating the activity of neighbouring cells.
  • Protein expression and phosphorylation are two main control points of gap junction function. Turnover of gap junctions is quite rapid within 4-6 hours highlighting the importance of these proteins in cellular function.
  • Propagation of cellular damage to neighbouring cells can occur through gap junction communication leading to death of neighbouring cells and thereby generating a larger wound than the initial injury.
  • the ability to down regulate cellular gap junction can occur through gap junction communication leading to death of neighbouring cells and thereby generating a larger wound than the initial injury.
  • inflammatory cell numbers in the wound bed are low due to their inability to exit out of blood vessels to the wound as this involves gap junction communication.
  • decreased gap junction communication leads to higher rates of cellular migration and proliferation because cellular contact inhibition is down regulated due to the failure of cells to communicate with each other.
  • Stimulation of angiogenesis is another attribute of down regulation of gap junction communication in wound healing as endothelial cells are connected to each other via gap junctions. Indeed, cellular independence is obtained through down regulation of gap junction communication which leads to accelerated tissue regeneration and regenerative healing.
  • gap junction regulation is important in maintaining a normal heart rhythm as gap junctions in heart cells allow the contract signal to be sufficiently transmitted, allowing the heart muscle cells to contract in tandem.
  • Gap junction communication is also vital in retina and neurons. Experimental data, for instance, show strong gap junction expression in astrocytes. Moreover, mutations in the gap junction genes Cx43 and Cx56.6 cause white matter
  • the skin is another vital organ that relies heavily on gap communication between cells to control cellular differentiation and proliferation, and loss of gap junction communication can lead to disease. Further, continued inhibition of gap junction communication can lead to a delay in wound closure as keratinocyte populations remain immature until they have fully formed gap junctions (e.g., which may reduce scar tissue formation). In contrast, stimulation of gap junction communication during the later stages of epithelialisation is believed to accelerate the formation of the barrier function of the skin.
  • Cxs connexins
  • Tissue sites and wounds that may be treated include acute and chronic wounds, burns including burns arising from exposure to ionizing radiation, chemical wounds, surgical wounds, oral wounds, skin and muscle trauma, open skin wounds, diabetic skin sores including diabetic foot ulcers, diabetic naturopathic foot ulcers, ischemic tissue including ischemic naturopathic foot ulcers, venous stasis ulcers, pressure sores, and hypoxic tissue.
  • Examples of ischemic and hypoxic tissues include ischemic heart tissue and hypoxic tissues associated with stroke.
  • Conditions in which the wound healing process may be promoted by treatment in accordance with the invention include in circumstances of delayed wound healing in which healing is impaired or prevented by for example due to tissue hypoxia or repeated trauma, or systemic causes such as diabetes and vascular disease.
  • a peptide comprising the FHPSS (SEQ ID NO: 3) amino acid sequence or a variant form thereof in accordance with the invention may be administered to a mammal for any further disease or condition for which modulation of cellular gap junction communication is desirable, such as (in this case involving inhibition of gap junction communication) pain, cell proliferative disorders such as cancer, benign growths, and hyper-proliferative skin conditions such as scleroderma and psoriasis, for diseases and conditions involving inflammation, cataract, glaucoma, and age-related macular degeneration, for scar reduction, spinal cord injury, cardiac injury, or corneal injury.
  • any further disease or condition for which modulation of cellular gap junction communication is desirable such as (in this case involving inhibition of gap junction communication) pain, cell proliferative disorders such as cancer, benign growths, and hyper-proliferative skin conditions such as scleroderma and psoriasis, for diseases and conditions involving inflammation, cataract, glaucoma, and age-related
  • Inhibition of cell migration has particular application to the inhibition of cancer metastases.
  • the prophylaxis or treatment of pain in accordance with methods embodied by the invention encompasses both acute and chronic pain as may result from trauma, injury or surgery, including but not limited to bone joint pain and neuropathic pain.
  • Neuropathic pain typically develops when peripheral nerves are damaged such as during surgical procedures, diabetes and diabetic related conditions such as diabetic retinopathy, chemotherapy or, for example, viral infection (e.g., shingles), and is a major factor causing impaired quality of life for millions of people worldwide.
  • Inflammation is involved in a wide range of physiological responses and disorders, examples of which include ulcerative inflammation, arthritic conditions, bone joint related inflammation, atherosclerosis, and inflammation of muscle, tendon and soft tissues.
  • arthritic conditions include inflammatory arthritis, lupus, osteoarthritis and
  • gap junction communication has also been reported to increase bone density and bone strength (Jorgensen et al, 2004), and the modulation of gap junction communication may have application to the prophylaxis or treatment of osteoporosis.
  • gap junction communication such as by using a peptide as described herein (e.g., phosphorylated SAPSS (SEQ ID NO: 101) peptide, YPSS (SEQ ID NO: 105), FHPSA (SEQ ID NO: 108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) but not limited thereto)) in accordance with embodiments of the invention
  • a peptide as described herein e.g., phosphorylated SAPSS (SEQ ID NO: 101) peptide, YPSS (SEQ ID NO: 105), FHPSA (SEQ ID NO: 108), or Ac-YHPSN-NH 2 (SEQ ID NO: 109) but not limited thereto
  • several disease states may be moderated that are associated with loss of cell function due to loss of cellular gap junction communication.
  • Conditions where stimulation of gap junction communication may provide therapeutic benefit include cardiovascular related conditions such arrhythmia, bradyarrhythmia, atherosclerosis, thrombosis and thrombotic conditions, angina pectoris, acute myocardial infarction, congestive heart failure, hypertensive heart disease, and cardiomyopathies, stroke, nerve injury and trauma, brain ischemia, and chronic neurodegenerative disorders. Gap junctions also appear to control renal circulation and vascular responses to control blood pressure.
  • Co-ordinated electrical activation of the heart is maintained by intercellular coupling of cardiomyocytes via gap junctional channels located in the intercalated disks. These channels consist of two hexameric hemichannels, docked to each other, provided by either of the adjacent cells. Thus, a complete gap junction channel is made from 12 protein subunits, the connexins. While 21 isoforms of connexins are presently known, cardiomyocytes typically are coupled by Cx43 (most abundant), Cx40 or Cx45. Some years ago, antiarrhythmic peptides were discovered and synthesised, which were shown to increase macroscopic gap junction conductance (electrical coupling) and enhance dye transfer (metabolic coupling).
  • the peptide AAP10 (H-Gly-Ala-Gly- Hyp-Pro-Tyr-CONH 2 ; wherein Hyp is the amino acid hydroxyproline) is one such example and has a horseshoe-like spatial structure.
  • Antiarrhythmic peptides act on Cx43 and Cx45 gap junctions but not on Cx40 channels, and AAP10 peptide has been shown to enhance intercellular communication in rat, rabbit and human cardiomyocytes.
  • Antiarrhythmic peptides like AAP 10 are effective against ventricular tachyarrhythmias, such as late ischaemic (type IB) ventricular fibrillation, and CaCl 2 or aconitine-induced arrhythmia.
  • the effect of antiarrhythmic peptides is higher in partially uncoupled cells and is related to maintained Cx43 phosphorylation, while arrhythmogenic conditions like ischemia result in Cx43 dephosphorylation and intercellular decoupling.
  • the tyrosine amino acid of the AAP10 peptide H-Gly-Ala-Gly-Hyp-Pro-Tyr-CONH 2 is a potential phosphorylation site and phosphorylation of this site may result in modulation of gap junction function as shown by the SAPSS (SEQ ID NO: 101) peptide.
  • gap junction communication is promoted when the SAPSS (SEQ ID NO: 101) peptide is phosphorylated.
  • the modulation of cell communication in the prophylaxis or treatment of diseases or conditions is, for example, described in International Patent Applications WO 2009/010733, WO 2009/012224 and WO 2002/254033, the contents of which are incorporated in their entirety by reference.
  • endothelial cell types that may be induced to proliferate and/or migrate by peptides in accordance with the invention include human umbilical vein endothelial cells, human microvascular endothelial cells, and bovine aorta endothelial cells.
  • a peptide e.g., the peptide FHPSS (SEQ ID NO: 3), FLSS (SEQ ID NO: 26), YPSS (SEQ ID NO: 105), FHPSA (SEQ ID NO: 108) or Ac-YHPSN-NH 2 (SEQ ID NO: 109)
  • a peptide agent as described herein can be administered in accordance with embodiments of the invention to a subject in need of such treatment alone or be coadministered with one or more other therapeutic agents.
  • the peptide can be co administered in combination with one or more other therapeutic agents conventionally used for modulating gap junction communication, inhibiting inflammation, promoting angiogenesis, wound healing, and/or inhibiting wound scarring or the like.
  • co administered is meant simultaneous administration in the same formulation or in two different formulations by the same or different routes, or sequential administration by the same or different routes, whereby the peptide and other therapeutic agent(s) exhibit overlapping therapeutic windows.
  • peptide or peptide agent in accordance with the invention include platelet-derived growth factor (PDGF), transforming growth factor- ⁇ (TGF- ⁇ ), platelet- derived wound healing factor, insulin growth factor (IGF), keratinocyte growth factor (KGF), anti-inflammatory agents and anti-microbial agents.
  • PDGF platelet-derived growth factor
  • TGF- ⁇ transforming growth factor- ⁇
  • IGF insulin growth factor
  • KGF keratinocyte growth factor
  • anti-inflammatory agents and anti-microbial agents.
  • IDO indoleamine 2,3-dioxygenase
  • tryptophan tryptophan
  • TDO dioxygenase
  • SIP spingosine-1 -phosphate
  • N-acylethanolamines grapefruit extract and other plant phytochemicals including ascein, green tea catechins, melatonin, arginine and other amino acids for support of blood vessel growth.
  • a peptide embodied by the invention or utilised in a method embodied by the invention will generally be formulated into a medicament (e.g., a pharmaceutical composition comprising the protein and a pharmaceutically acceptable carrier), and the use of peptides as described herein for the manufacture of a medicament for applications in accordance with the invention are expressly encompassed.
  • a medicament e.g., a pharmaceutical composition comprising the protein and a pharmaceutically acceptable carrier
  • the peptide according to the invention to be formulated into a medicament is an isolated or substantially purified peptide.
  • Suitable pharmaceutical compositions include topically acceptable formulations such as creams, lotions, ointments and gels for internal or external application. Topically acceptable compositions can be applied directly to the site of treatment including by way of dressings and the like impregnated with the preparation. Gels comprising precipitate of the peptide are particularly suitable. Gels and other pharmaceutical compositions as described herein may also include calcium ions and/or salts for enhancement of the wound healing process.
  • a pharmaceutical composition embodied by the invention can also incorporate one or more preservatives such as parabens, chlorobutanol, and sorbic acid, binders such as corn starch or gelatin, thickening agents, emulsifiers, surfactants, gelling agents, and other components typically used in such compositions.
  • Pharmaceutically acceptable carriers include any suitable conventionally known topically and physiologically acceptable solvents, dispersion media, isotonic preparations and solutions. Use of such ingredients and media for pharmaceutically active substances is well known. Except insofar as any conventional media or agent is incompatible with the active peptide agent, use thereof is expressly encompassed.
  • compositions embodied by the invention include therapeutically active agents, derivatives, and derivatives thereof.
  • compositions for human or veterinary use will have a pH suitable for application of the composition directly to a wound.
  • the pH will be above 3.8 and usually, about 4 or higher.
  • a pharmaceutical composition will generally contain at least about 0.001% by weight of the peptide or peptide agent up to about 80% w/w of the composition.
  • the composition can contain about 0.001%, 0.01%, 0.05%, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% by weight of the peptide or peptide agent.
  • the amount of the peptide or peptide agent in the composition will be such that a suitable effective dosage will be delivered to the subject taking into account the proposed mode of administration.
  • a peptide or peptide agent administered in accordance with an embodiment of the invention will depend on a number of factors including whether it is to be administered for prophylactic or therapeutic use, the disease or condition for which it is intended to be
  • the severity of the condition, the sex and age of the subject, and related factors including weight and general health of the subject can be determined in accordance with accepted medical principles. For instance, a low dosage can initially be given which is subsequently increased or decreased following evaluation of the subject's response.
  • the frequency of administration can be determined in the same way that is, by continuously monitoring the subject's response between each dosage and if desirable, increasing the frequency of administration or alternatively, reducing the frequency of administration.
  • the peptide or peptide agent will be administered in accordance with a method embodied by the invention at a dosage up to about 50 mg/kg body weight and preferably, in a range of about 5 ⁇ g/kg to about 100 ⁇ g/kg body weight.
  • the peptide or peptide agent will be administered to a tissue or wound site in a dose range of about 0.1 ⁇ g to 100 ⁇ g/cm "1 .
  • the dose administered topically can for instance be about 0.1, 0.5, 1.0, 5.0, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 ⁇ g/cm "I .
  • the peptide or peptide agent will be administered at a dosage in a range of from 1.0 to 10.0 ⁇ g/cm 2 of the tissue to be treated.
  • Routes of administration include but are not limited to topically, by respiration (e.g., by inhalation but not limited thereto), intravenously, orally, intraperitonealy, subcutaneously, intramuscularly, rectally, topically and by implant.
  • intravenous routes particularly suitable routes are via injection into blood vessels which supply the target tissue to be treated.
  • the peptide or peptide agent can also be delivered into cavities such for example the pleural or peritoneal cavity, or be injected directly into the tissues to be treated.
  • the peptide or peptide agent can be encapsulated or otherwise provided in an enteric form for passage through the stomach and release in the small intestine. Any suitable such enteric formulation or coating can be utilized.
  • penetration enhancers may be utilised for enhancing or facilitating penetration of the skin by the peptide or peptide agent in accordance with the invention.
  • Suitable penetration enhancers include pharmaceutically acceptable oils such as castor oils, ethoxylated oils, and sunflower oil, linoleic acid and polyethylene glycol (PEG).
  • transdermal delivery techniques can be utilised, examples of which include transdermal patches, iontophoresis, electroporation and sonophoresis, all of which are well known in the art.
  • Peptide or peptide agents as described herein can also be coated onto the surface of a stent or balloon of a catheter such as an angioplasty catheter, or other surgical instrument for application to the interior wall of a blood vessel during angioplasty or other surgical procedure.
  • the peptide or peptide agent can for instance be applied to the wall of the blood vessel in this manner in the form of a gel or any other appropriate formulation for contact with the tissue to be treated upon expansion of the stent or balloon catheter.
  • Slow release formulations are particularly suitable for deposition on the tissue contacting surfaces of catheters and the such like, as may be obtained by the formation of physiologically acceptable salt complexes of the active peptide or peptide agent as described herein and divalent cations (e.g., calcium salts).
  • dressings e.g., bandages, sticking plasters, cloth material, gauze, muslin, and film dressings etc.
  • gauze dressings can be coated or impregnated with peptide or peptide agents for topical application to tissues and skin wounds in accordance with embodiments of the invention.
  • gauze dressings can be coated with purified ⁇ 2 crystallin derived peptide as follows. Firstly, the gauze is placed into an aqueous salt solution (10 mM acetic acid pH 3.5 containing 2M calcium chloride although a number of different divalent salts can be used as well as ammonium sulphate), and is then removed and blotted dry on filter paper until essentially free of excess water.
  • the cloth is subsequently placed into an acidic solution of the purified ⁇ 2 crystallin peptide (10-50 mg/mL peptide in 10 mM acetic acid pH 3.5, although the pH can range from 2.0 to 5.0).
  • the surface of the cloth is coated with a white protein/peptide precipitate ( ⁇ 2 crystallin salt) as shown by electron microscopy.
  • the intact form of native ⁇ 2 crystallin can also be coated on the surface of previously soaked cloth under identical conditions.
  • the level of peptide coating may be controlled by altering the salt concentration, peptide concentration and the length of time the cloth, gauze or other substrate dressing material is in contact with the peptide solution.
  • the coated dressing can then be dried and sterilized (e.g., wiped with 70% alcohol, or gamma irradiated etc.) for subsequent use.
  • sponge material can also be coated with peptide (e.g., ovine ⁇ 2 crystallin derived peptide) using the above protocol although in this instance, the sponge can be squeezed to remove excess water prior to being placed in the peptide solution.
  • peptide e.g., ovine ⁇ 2 crystallin derived peptide
  • This type of wound dressing may be suitable for use in wounds that produce high levels of exudate.
  • the peptide or peptide agent may be provided on, or impregnated in, a dressing in a composition (e.g., in a gel or cream) including a suitable absorbent preparation to assist in the absorption of wound exudate and for slow release of the peptide or peptide agent to the wound to facilitate wound healing.
  • a composition e.g., in a gel or cream
  • suitable absorbent preparation to assist in the absorption of wound exudate and for slow release of the peptide or peptide agent to the wound to facilitate wound healing.
  • Alginate is particularly suitable for this purpose and a number of dressings coated with a hydrophilic alginate gel absorbent preparation are currently commercially available. Besides providing a high level of absorption, the alginate gel provides a barrier against bacterial access to the wound, promotes granulation of tissue and maintains a moist environment for wound healing.
  • alginate Whilst alginate is particularly suitable, any other physiologically suitable polysaccharide absorbent may be utilised and particularly, suitable anionic polysaccharides.
  • the calcium salt form of the peptide is slowly released from the alginate dressing as the alginate binds the calcium from the insoluble peptide salt form.
  • the peptide is retained within the dressing in this form until it is made soluble by removal of calcium. Also the peptide is not available for wound proteases to break down which improves its bioavailability and efficacy.
  • Suitable pharmaceutically acceptable carriers and formulations useful in compositions embodied by the invention can for instance be found in handbooks and texts well known to the skilled addressee such as Remington's Pharmaceutical Science, 15 th ed., Mack Publishing Company, Easton, Pa., United States, the contents of which is incorporated herein in its entirety by reference.
  • EXAMPLE 1 Modulation of cell communication by the peptide FHPSS (SEQ ID NO: 3)
  • Peptide release was monitored using RP HPLC and peptide was collected by ultrafiltration on a 5 kDa UF membrane.
  • the filtrate was acidified with acetic acid prior to the addition of 1M calcium chloride.
  • the pH of the resulting solution was increased to pH 11.8 with NaOH to cause precipitation of the peptide calcium salt.
  • the peptide salt was collected by filtration and neutralized with acetic acid. After drying under vacuum the peptide was analysed using FTIR, TGA, Karlfisher analysis and RP HPLC. The naturally derived peptide was compared to a synthetically produced peptide. Essentially an identical FTIR spectral profile was obtained for the natural and synthetic peptide.
  • a peptide alginate wound dressing was prepared by mixing the calcium peptide salt with alginate solution to provide even distribution of the peptide salt throughout the alginate solution. This solution was then poured into a sterile plastic dish containing muslin pre-soaked in calcium chloride in a laminar flow hood to maintain sterility. After 30 minutes the alginate had set and the gel could be peeled off the muslin backing to provide the dressing. More particularly, the peptide dressing was prepared as follows.
  • the peptide calcium salt (EL lbFp2 -batch #3) 1.42 g was mixed with 300 mL of 1.25 % alginic acid (Sigma A-2158 Lot 93H0342 sodium salt from Macrocystis pyrifera (Kelp) Low Viscosity) (sterile).
  • the peptide calcium salt was suspended into the alginate solution through the use of a stick blender.
  • the blender was made sterile by washing with 70% ethanol and then allowed to dry.
  • the mixture was then poured onto a sterile plastic tray 30 cm x 41 cm containing 2 layers of sterile baby muslin which had previously made damp by the addition of 70 mL of 5% CaCl 2 (final calcium concentration was 19.6 MI cm 2 ).
  • the dressing was then allowed to set at room temp in a laminar flow hood for 2 hours.
  • the use of the muslin enables a controlled release of calcium which provides for a consistent protein gel to be formed.
  • the dressing (Dressing 4 ELlbFp2-D4) was cut into 18 mm circular dressings by using a sterile 18 mm centre punch.
  • the circular dressings were sprayed with 70% ethanol and placed into Esselte (40 microns Code 480583) zip lock bags (10 per bag) 75 mm x 100 mm (Dressing 4 ELlbFp2-D4) and stored at 4 °C until use.
  • PBS
  • Synthetic peptides corresponding to the amino acid sequences FHPSS (SEQ ID NO: 3) 08B2 crystallin C-terminal derived peptide), SREEKPSSAPSS (SEQ ID NO: 1) (oA crystallin C- terminal derived peptide) and RPRPDDLEI (SEQ ID NO: 76) (ACT1 : C-terminal end of Cx43) were used as positive controls (1 mg/mL stock solution) and 1, 10 and 100 iL were added to wells to give final concentrations of 1, 10 or 100 ⁇ g of peptide per well. The results are shown in Fig. 1.
  • a pig animal model was employed to assess the efficacy of different of dressings in wound healing. Briefly, five rows (A-E) of 4 x 2cm diameter wounds were made along the side of a pig. The dressings (18mm diameter) were placed into or on top of the wound at various time points. One row of wounds was biopsied on each of Days 3 (row A), 9 (row B), 14 (row C), 24 (row D) and 42 (row E). Various parameters of the wounds were analysed, including wound area, closure rate, wound gape, blood vessel numbers, and scar area.
  • the pig was single housed in an animal facility in a raised cage pen with a perforated metal floor and wire mesh walls adjacent to other pigs to provide company, and was fed commercial pig grower feed twice daily. Water was provided ad lib. Pens were approximately 2m x 2m in size and were hosed out daily. Methadone was administered for pain relief during anaesthesia for the surgery to create the wounds, four hours later then again once on the following three mornings post operatively (the third morning was also the day of biopsy of row A wounds). Intermittent diarrhoea was observed on 6, 10, 11 and 15 days after the initial surgery. Faeces were soft and not watery. Food was withheld on Day 6, otherwise no treatment was given. It was believed unlikely that the observed diarrhoea was related to surgery or anaesthesia. A digital camera was used to image the wounds at the time of each biopsy, other than on Day 42.
  • Ketamine 100 mg/mL, approx. 1.3 mL/10 kg
  • midazolam 5 mg/mL, approx. 3 mL/10 kg
  • the pig was allowed to recover from surgery on Day 42 and provided as a companion for another pig in a different study.
  • Dressings were changed on Days 3, 6, 9, and 14,and the wounds were examined for inflammation, redness, weeping exudate level, and signs of infection at those times.
  • the dressings utilised in this study were as follows and are further described in Example 1.3 below.
  • Dressings were wetted with sterile saline before application if required depending on the level of wound exudate. Dressings from each dressing change were stored on wet ice at 4°C or frozen at approx. -20 °C prior to analysis. 1.3 Dressing preparation
  • Ovine eyes (150) were obtained from the local abattoir from sheep that were less than 2 years of age and stored by refrigeration prior to being processed within 1 hour of receipt.
  • a scalpel was used to slice a 1-2 cm incision across the cornea to remove the lens. The scalpel was rinsed in 70% ethanol prior to incision. Extraction of the lenses was performed in a laminar flow hood using sterile techniques. The weight of the lenses obtained was 116.3 g. Sterile water 1163 mL was added to the lenses in a sterile beaker. The mixture was then homogenised for 4 minutes and stored overnight at 4 °C. It was noted that the central core nucleus of the lens was white after overnight storage at 4 °C believed to be due to the presence of ⁇ crystallins. The following day the extract was centrifuged in sterile 300 mL centrifuge bottles. Centrifugation was performed in a GS3 rotor for 10 min at 8K rpm at 10 °C. The pellet formed by
  • centrifugation was weakly attached to the side of the centrifuge tube and the supernatant obtained was passed through 4 layers of sterile muslin to ensure that it was free of cellular debris.
  • the supernatant (ELI -batch #3) was separated from the pellet and placed in a sterile beaker and adjusted to pH 5.0 with glacial acetic acid. A white precipitate formed which was collected by centrifugation for 10 min at 8K rpm at 10 °C.
  • the pellet 38.92 g was separated from the supernatant and stored at 4 °C (EL la-batch #3), which was used to prepare the protein alginate dressing.
  • Ovine protein material 38.92 g of wet ELla-batch #3 was mixed with 300 mL of 1.25 % alginic acid (Sigma A-2158 Lot 93H0342 sodium salt from Macrocystis pyrifera (Kelp) - Low viscosity sterile), and the dressing prepared in a laminar flow hood using sterile techniques as per the dressing preparation protocol described in Example l .
  • the dressing (Dressing 3 ELla-D3) was cut with a sterile scalpel into manageable sections (10 cm x 8 cm), before being peeled off the muslin and placed into sterile zip lock bags (Glad zip slide bags 17 cm x 21 cm, No.
  • the ratio of wet protein precipitate to alginate was determined to provide a final alginate concentration of 1%.
  • the standard batch size of 100 ovine eye lenses provided sufficient protein to make 300 mL of dressing.
  • the initial concentration of alginate 1.25% was reduced to 1% after the addition of the protein precipitate extract.
  • a calcium salt of purified ovine FHPSS (SEQ ID NO: 3) peptide (ELlbFp2-batch #3) (1.42 g) was mixed with 300 mL of 1.25 % alginic acid by suspending the peptide calcium salt into the alginate solution with the use of a stick blender, prior to pouring the peptide alginate solution onto sterile muslin and allowed to set as described above.
  • the dressing (Dressing 4 ELlbFp-2-D4) was then prepared and stored as for the protein alginate dressing.
  • Fibracol PlusTM collagen wound dressing with alginate is a commercially available collagen and alginate preparation (Johnson & Johnson, New Brunswick, NJ, USA).
  • the dressing was cut under sterile conditions in a laminar flow hood into 18 mm circular dressings using a sterile 18 mm centre punch and stored dry in Esselte (40 microns Code 480583) zip lock bags (10 per bag) 75 mm x 100 mm, which were then stored at 4 °C.
  • Biopsies of wounds were performed as per the following steps:
  • the animal was given ketamine/midazolam mixture as an intramuscular injection as described above. When the animal was satisfactorily sedated it was weighed and relocated to a surgical table.
  • Anaesthesia was maintained with isoflurane, delivered via endotracheal tube or a mask if the animal could not be intubated.
  • the animal was positioned in a ventral recumbency with forelimbs extended forward and hindlimbs extended back.
  • a single dose of the analgesic Temgesic (1 ml) was given intramuscularly during biopsy of rows B to E. Pig behaviour and appetite were used to gauge health and well being.
  • the wound dressings were removed from all wounds if possible.
  • the Day 3 wounds had absorbed the dressings so they were unable to be removed.
  • the wounds were cleaned of debris to ensure an accurate measurement of the wound area.
  • Biopsy of each wound in the respective row was taken at each biopsy time point making sure a section of tissue across the entire wound was obtained as well as at least 0.2 cm of the wound margin.
  • the biopsies were rinsed quickly in sterile saline solution and cut in half. One half of each biopsy was placed into neutral 10% formalin buffered saline for histochemistry analysis. The other half was frozen on dry ice prior to being stored at -80 °C for biochemical analysis.
  • the surgical site was draped prior to biopsy such that only the row to be biopsied was exposed.
  • the animal was stabilized and monitored by the anaesthetist.
  • a scalpel was used to make an elliptical shaped incision to excise the wound. Care was taken to ensure removal of the entire wound margin whilst taking the least possible amount of excess skin.
  • the elliptical incision was made as narrow as possible with a 2 mm margin from the wound to ensure the wound edges remained opposed after suturing.
  • the wound was closed using Ethilon blue monofilament nylon suture, size 2/0 (3 metric). Additionally, a biopsy of normal skin was taken from the mid-line of each biopsied row using an 8 mm sterile biopsy punch.
  • the animal was extubated if required and returned to its pen when ready, where it was monitored.
  • the analysis of the wounds at the various time points provided a snapshot of the wound healing cascade during the inflammatory phase, proliferation phase (which includes
  • Photographic digital images of the wounds at the specified time points were taken with a Canon digital IXUS500 5.0 mega pixel camera.
  • the images were imported into Image J 1.42q and analysed for wound area by first measuring the circumference of the wound and calibrating the area by measuring the distance on a graduated ruler included in the photographic image.
  • the results are shown in Fig. 4.
  • the peptide alginate wound dressing promoted faster wound closure compared to alginate alone (negative control), Fibracol PlusTM (positive control) and the protein alginate wound dressing containing aA crystallin protein.
  • Trichrome Masson staining is routinely used to identify mature collagen in normal tissue and immature collagen in scar and healing wounds.
  • the boundary between bright blue staining (indicative of mature collagen) and light blue staining (immature collagen) defines the wound margins (e.g., Wilgus et al., 2008, the entire contents of both of which are incorporated herein by cross-reference).
  • the final biopsy was taken at Day 42 which is equivalent to fully closed partially remodelled wounds. Biopsied tissue samples were fixed in formalin in PBS, sectioned and stained with Trichrome Masson to identify mature and immature collagen. The slides were examined under a microscope and images taken that contained the healed wound plus
  • Mature collagen (3 sections of a 168 x 168 pixel square) was averaged to identify the collagen colour map. Histo grams from six sections of a 168 x 168 pixel square were averaged within the wound area for each of the treatments at Day 42.
  • the data obtained were plotted to identify shifts in the colour intensity that relates to the collagen maturity and degree of scar formation.
  • the area highlighted by the stain as scar tissue was determined and used to gauge the degree of scarring.
  • the number of cell nuclei in the scar tissue was determined at Day 42 to indicate the quality of healing.
  • Particle count analysis was performed in Image J to determine the number of cellular nuclei. The number of cells at Day 42 within the healed wound was greatest for the peptide alginate treated wound.
  • Mature collagen from unwounded tissue is shown by the peak furthest to the left in Fig. 5 followed by peaks for the respective wound treatments in the order peptide alginate dressing > Fibracol PlusTM >Alginate > Protein alginate dressing. This indicates that the peptide alginate dressing has the most mature collagen as staining is most closely resembling that of mature unwounded collagen.
  • the alginate negative control produced a profile shifted to the right. This suggests a wound with less mature collagen and may reflect its stage of healing within the cascade.
  • the protein alginate treated wound produced a considerably different profile to the other of the treatments which may be due to the high protein content present within the wound dressing.
  • the wounds treated with the protein alginate and peptide alginate dressings appeared to have smaller scar areas when compared to the alginate and Fibracol PlusTM Day 42 treated wounds (see Fig. 6). However, the protein alginate treated wound at Day 42 had a prominent long raised white scar whereas the peptide alginate treated wound did not have a readily visible scar.
  • VEGF vascular endothelial growth factor
  • TNFa levels were highest at Day 3 as expected due to the presence of high levels of inflammatory cells.
  • the TNFo; level was well above that found in control tissue (unwounded).
  • TNFo; levels were lowest for the Fibracol Plus treatment on Day 9 but then an additional spike of TNFa was observed at Day 14 for the wound treated with
  • TNFc level for the peptide alginate dressing treated wounds was lowest on day 14 and remained low when tested on Day 24 and Day 42.
  • the protein alginate and alginate samples had very similar TNFa profiles that reduced in a hyperbolic fashion from day 3 to Day 24 and remained low at Day 42.
  • the alginate treated wound exhibited a lower TNFo; level at day 42 similar to that observed for the peptide alginate treated wound at this time point.
  • the peptide alginate dressings showed an anti-inflammatory effect as reflected by TNFo; wound levels.
  • CD68 staining Cell surface expressed CD68 is a marker for monocytes and macrophages. Monocytes differentiate into macrophage upon entering the interstitial tissue. Macrophages can be destructive and lead to scarring when present in high numbers during the early stages of inflammation but are also thought to drive the wound healing cascade through the release of growth factors and cytokines. Wound tissue treated with the different dressings at Days 3, 9 and 14 were stained as follows. Briefly, sections were cut and placed onto polylysine coated slides which were in turn placed into a Bond automated immunohistological apparatus for blocking, staining and developing for CD68 antibody labelling. The analysis of the staining density was performed using Adobe Photoshop v5.0 using the selection tool (colour selection settings R 199, G 178, B 172; sample colour fuzziness 50 and histogram pixel count).
  • the peptide alginate dressing exhibited very little staining compared to the other wounds indicating that inflammation was reduced by this treatment.
  • the CD68 stained cells for the wound treated with the peptide alginate dressing at Day 9 appeared to be confined to the blood vessels. This was a surprising finding and indicates that monocytes are prevented from entering into the interstitial tissue by modulation of gap junction communication between the monocytes and endothelial cells, and were thereby unable to differentiate into macrophages in the interstitial tissue.
  • Smooth muscle cell actin is produced by differentiated fibroblasts during wound contraction which aids in wound closure but can also lead to increased scar deposition within the wound. A smaller area of staining for SMA would therefore be beneficial and indicate of a reduction in scarring potential. Briefly, biopsied tissue sections were cut and placed onto polylysine coated slides which were placed into a Bond automated immunohistological apparatus for blocking, staining and developing for antibody to smooth muscle actin (SMA).
  • the peptide alginate dressing SMA stained area was the smallest and a significant reduction was evident (see Fig. 9).
  • the protein alginate and alginate treatments resulted in very similar SMA stained areas.
  • the Fibracol PlusTM treated Day 24 wound had a smaller SMA stained area than that of the alginate treated wound but the peptide alginate treated wound had the smallest area stained using this antibody.
  • Analysis of the SMA stained areas revealed differences in staining intensity as shown in Fig. 10.
  • the density of staining as indicated by the peak heights of the graph indicates the amount of SMA produced by differentiated fibroblasts.
  • the level of SMA produced followed the trend Alginate > Protein alginate > Fibracol PlusTM > Peptide alginate treatment.
  • CD34 Wound blood vessel numbers were determined for pig wounds at Days 3, 9, 14, 24 and 42 by analysis of wound tissue by staining for CD34. CD34 is expressed by both mature and immature endothelial cells.
  • E cadherin staining identifies tight junctions and occurs between epithelial cells
  • keratinocytes and endothelial cells lining the blood vessels. It was postulated that modulation of gap junction communication by the FHPSS (SEQ ID NO: 3) peptide may also effect the expression of E-cadherin or the ability of keratinocytes and endothelial cells to form tight junctions. Understanding how the peptide affects various cell types may provide further insight in the timings used for dressing changes. Day 9, 14 and 24 wounds were therefore analysed using immuno Histochemistry (IHC) staining for E cadherin. An antibody specific for E- cadherin was used to detect tight junctions present in the epithelium which form upon
  • Fig. 12 shows E-cadherin immuno stained wound tissue at Day 14 for the various wound dressings treatments and Day 14 and 24 for the peptide alginate wound treatment.
  • the protein alginate treated wound appeared to have completed epithelialisation at Day 14 but contained a sero-cellular crust/scab on top of the wound.
  • the wound treated with the Fibracol PlusTM dressing was substantially closed by the migrating epithelial tongues.
  • the alginate negative control and the peptide alginate treated wounds still had a considerable wound gape at this stage.
  • the presence of E-cadherin was observed but the typical morphology of the stratified skin was not present (Fig. 12.
  • Day 14 was the last day the dressings were reapplied.
  • the presence of normal keratinocyte morphology of the skin was observed for the peptide alginate treated wounds.
  • the ovine protein alginate dressing was made by precipitation of crystallin soluble proteins at pH 5.0, providing a protein material that stimulated vascular endothelial growth factor levels in a sheep aortic ring model.
  • the protein precipitate was blended into an alginate solution and then made into a gel, using calcium chloride soaked muslin cloth. Once set, the gel was peeled off the muslin and the protein dressing cut into shape and treated with 70% ethanol for sterilization.
  • the peptide alginate dressing contained the C-terminal peptide FHPSS (SEQ ID NO: 3) derived from ovine ⁇ 2 crystallin.
  • the peptide was shown to down regulate cell to cell communication which is known to be beneficial in accelerating wound healing.
  • the peptide was released by elastase I cleavage from the soluble proteins remaining in the supernatant after protein precipitation. Hydrolysis by elastase was performed in the absence of calcium which provided greater specificity at releasing the FHPSS (SEQ ID NO: 3) peptide. This unexpected finding allowed greater control of the protease hydrolysis and led to greater peptide purity.
  • the aim of the present study was to determine the efficacy of the peptide alginate and the protein alginate dressings to determine which of these had the best utility in treating wounds.
  • the two dressings were applied to 18 mm full thickness dorsal excisional wounds in a pig.
  • the dressings were applied on Day 0, 3, 6, 9 and Day 14.
  • Comparison to alginate (negative control) and Fibracol Plus (positive control) was undertaken. Wound closure rates were determined based on photographic evidence.
  • the peptide alginate wound dressing treated wounds closed faster than the other wounds. At Day 9 all three wound treatments were shown to have faster wound closure than that of the alginate negative control. Some positional effects were observed.
  • Biochemical analysis of extractable protein, growth factors, vascular endothelial growth factor (VEGF) and transforming growth factor alpha (TNF a) were performed. Analysis of GAG and DNA content was also performed. The GAG content was highest on Day 14 for the wound treated with Fibracol PlusTM. Free DNA analysis on Day 3 was low for the peptide alginate treated wound but high for the protein alginate treated wound. On Day 9 the levels of free DNA had increased for the peptide alginate treated dressing. Analysis of growth factors revealed that the protein alginate treated wound on Day 3 had the highest VEGF levels, but at Day 42 the peptide alginate dressing treated wound had the highest levels of VEGF and the protein alginate treated wound had the lowest VEGF levels.
  • VEGF vascular endothelial growth factor
  • TNF a transforming growth factor alpha
  • the peptide alginate treated wounds had the highest number of blood vessels but it also had the lowest level of VEGF. This suggests that the initial angio genesis stimulation was completed by Day 9 for this dressing treatment but then it increased again at Days 14 and 42. Analysis of TNFc levels for the four dressings produced some interesting results. Firstly, at Day 9 the Fibracol PlusTM treated wound exhibited the lowest TNFo; concentration. By Day 14 the wound treated with this dressing had a spike in the concentration of TNF ⁇ x The other dressings having a lower TNFo; concentration at Day 14, with the peptide alginate treated wound having the lowest level suggesting that it has anti-inflammatory properties.
  • CD68 positive cells monocytes
  • extravassation miration through the blood vessel
  • the peptide alginate dressing showed the greatest efficacy in the present study and was found to accelerate wound healing, reduce inflammation, and decrease scarring as well as to stimulate angiogenesis in the pig full thick dorsal wound model.
  • the peptide alginate dressing exhibited considerably improved wound healing properties compared to the Fibracol PlusTM dressing.
  • the protein alginate wound dressing also appeared to enhance the rate of wound healing. At Day 42 it had a smaller scar than both the alginate treated and the Fibracol PlusTM treated wounds. The scar morphology was considerably different and it stained deeply in parts. The reason for this staining pattern is unknown.
  • the smooth muscle actin staining at Day 24 for the protein alginate treated wound was similar to that of the Fibracol Plus treated wound but smaller than that for the alginate treated wound.
  • the efficacy of the protein alginate dressing appeared to be on a similar par to the Fibracol PlusTM dressing based on the findings in this study.
  • the gap junction communication assay was performed substantially as described in Example 1 as follows. Whole sheep eye lenses were removed from the eye retaining the outer membrane but without the pigmented epithelium. In a 24 well plate one lens was added per well with 1 mL of sterile PBS and either 50 or 100 ⁇ , of test compound or a 5 x 5 mm section of dressing prepared as outlined in 1.3.2. After 30 minutes incubation of the samples with the eye lenses, 5 of 3 mg/mL fluorescein in DMSO was added to each well. The plates were then incubated over night at room temperature (RT). After approximately 16 hours the lenses were removed and placed into a clean 24 well plate and an image of the fluorescence present within the eye lens was captured and analysed using an Alpha Imager gel documentation system.
  • RT room temperature
  • FHPSS (SEQ ID NO: 3), FKPSS (SEQ ID NO: 6), FHPS (SEQ ID NO: 13), FLSS (SEQ ID NO: 26), FHPAS (SEQ ID NO: 23), AHPSS (SEQ ID NO: 5) and FHPIN (SEQ ID NO: 2) gave the best results at reducing/down regulating gap junction communication, whereas the peptides WHPSS (SEQ ID NO: 107), YHPSN (SEQ ID NO: 103), YHPSS (SEQ ID NO: 100), YPSS (SEQ ID NO: 105), FHPSA (SEQ ID NO: 108), Ac-YHPSN-NH 2 (SEQ ID NO: 109) and Ac- YHPSS-NH 2 (SEQ ID NO: 89) stimulated/up-regulated gap junction communication.
  • the N- and C- terminal modified FHPSS (SEQ ID NO: 3) sequence was also shown to be effective at reducing gap junction communication. Without wishing to be bound by theory, the inventor believes that the difference between FHPSS (SEQ ID NO: 3) (inhibitory) and FHPSA (SEQ ID NO: 108) (stimulatory) peptides may highlight a role for the C-terminal serine residue in modulation of gap junction communication.
  • the FHPSS (SEQ ID NO: 3) peptide can be cleaved from the C-terminal native ⁇ 2 crystallin.
  • the need for the release of the peptide may control its ability to modulate gap junction communication within the eye.
  • Additional synthetic peptides were prepared for evaluation of their effect on gap junction communication.
  • the additional peptides are listed in Table 3 along with those peptides previously discussed; e.g. as shown in Tables 1 and 2.
  • Ahx coupling used for attachment of FITC to the N-terminus of FHPSS (SEQ ID NO: 3)
  • P1016 (3S, 4/?)-4-hydroxypyrrollidine-3-carboxylic acid.
  • the gap junction communication assay was performed substantially as described in Examples 1 and 3. The results are shown in column 2 of Table 3. Numbers below 100 indicate a decrease in gap junction communication as compared to controls. Numbers above 100 indicate an increase in gap junction communication as compared to controls.
  • FHPIN SEQ ID NO: 2
  • FHPSS SEQ ID NO: 3
  • Ac-FHPSS-NH 2 SEQ ID NO: 4
  • AHPSS SEQ ID NO: 5
  • FKPSS SEQ ID NO: 6
  • FH(P1030)S SEQ ID NO: 7
  • KNMSS SEQ ID NO: 8
  • FH(P1030)SN SEQ ID NO: 9
  • ECEGSS SEQ ID NO: 10
  • SLFSS SEQ ID NO: 1 1
  • FH(P1030)AS SEQ ID NO: 12
  • FHPS SEQ ID NO: 13
  • NLVSS SEQ ID NO: 14
  • VSVSS SEQ ID NO: 15
  • FH(P1016)SA (SEQ ID NO: 16), APPSS (SEQ ID NO: 17), AHHS (SEQ ID NO: 18), FQESS (SEQ ID NO: 19), FH(P1016)SS (SEQ ID NO: 20), RCDSS (SEQ ID NO: 21), VTYSS (SEQ ID NO: 22), FHPAS (SEQ ID NO: 23), Ac-QIQSS (SEQ ID NO: 24), FH(P1030)SA (SEQ ID NO: 25), FLSS (SEQ ID NO: 26), FK(P1016)SS (SEQ ID NO: 27), Ac-WHPSS-NH 2 (SEQ ID NO: 29), VEGSS (SEQ ID NO: 30), WH(P1030)SS (SEQ ID NO: 31), IT ASS (SEQ ID NO: 32), VHHSS (SEQ ID NO: 33), FHPIS (SEQ ID NO: 34), RGVSS (SEQ ID NO: 35) and VTTSS (SEQ ID NO: 36) gave the best results at
  • the peptides DRMSS (SEQ ID NO: 54), GPPSS (SEQ ID NO: 55), APESS (SEQ ID NO: 56), YH(P1016)SN (SEQ ID NO: 57), Ac-FRPSS-NH 2 (SEQ ID NO: 58), KCDWSS (SEQ ID NO: 59), VTGSS (SEQ ID NO: 60), NSPSS (SEQ ID NO: 61), FKISI (SEQ ID NO: 62), DKMSS (SEQ ID NO: 63), GDMSS (SEQ ID NO: 64), GRLSS (SEQ ID NO: 65), FKPVN (SEQ ID NO: 66), YH(P1030)SN (SEQ ID NO: 67), FHASS (SEQ ID NO: 68), FRPSS (SEQ ID NO: 69), AcQNDNK (SEQ ID NO: 70), RENSS (SEQ ID NO: 71), SPLSS (SEQ ID NO: 72), WLPSSS (SEQ ID
  • AH(P1016)SS (SEQ ID NO: 80), ADQSS (SEQ ID NO: 81), TPLNSS (SEQ ID NO: 82), GVPSS (SEQ ID NO: 83), VTVSS (SEQ ID NO: 84), FH(P1016)AS (SEQ ID NO: 86),
  • GGGPSS (SEQ ID NO: 87), AHHSS (SEQ ID NO: 88), Ac-YHPSS-NH 2 (SEQ ID NO: 89), SPASS (SEQ ID NO: 90), APPSSS (SEQ ID NO: 91), IKISS (SEQ ID NO: 92), Ac-FHPTN- NH 2 (SEQ ID NO: 93), Ac-FHPSN-NH 2 (SEQ ID NO: 94), FAPSS (SEQ ID NO: 95), NTGSS (SEQ ID NO: 96), WPSS (SEQ ID NO: 97), DTQSS (SEQ ID NO: 98), FH(P1030)IN (SEQ ID NO: 99), YHPSS (SEQ ID NO: 100), SAPSS (SEQ ID NO: 101), Ac-FLSS-NH 2 (SEQ ID NO: 102), YHPSN (SEQ ID NO: 103), YPSS (SEQ ID NO: 105), FITC-(Ahx) FHPSS (S
  • peptides that stimulate gap junction communication.
  • the peptides were shown to be able to bind to Cx43 C-terminal tail near Ser368. Without wishing to be bound by theory, the inventor believes that this may potentially block phosphorylation of this residue thereby prevent disassembly of the connexin, which results in the stabilisation of gap junction communication.
  • the peptides are predicted to be substrates to a range of relevant kinases that are involved in phosphorylation of Cx43 and are involved in the formation and regulation of cell to cell communication.
  • lens fibre cells contain a high level of crystallin proteins and have become DNA free and cell organelle free during their differentiation process. Gap junction communication in skin is controlled by gene expression and the turnover of gap junction protein in cells occurs rapidly.
  • the peptides that up regulate gap junction communication may be useful for the prevention of diabetic retinopathy by preventing the migration of endothelial cells.
  • the peptides identified in this specification were also shown to be able to bind to divalent cations including calcium. Previously it has been shown that uncoupling of gap junction communication can occur through the addition of calcium and magnesium salts. As FHPSS (SEQ ID NO: 3) was formulated as a calcium salt in the form of an alginate gel the presence of calcium may have also enhanced the uncoupling of gap junctions. The fact that the alginate control, which was prepared in the presence of calcium, did not show this effect suggests that the level of free calcium was insufficient in the control alginate gel and that FHPSS (SEQ ID NO: 3) calcium salt was more effective at delivering calcium to the wound and thereby promote the uncoupling. This additional mode of uncoupling may further improve the efficacy observed for FHPSS (SEQ ID NO: 3). LITERATURE REFERENCES CITED

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Abstract

La présente invention concerne des procédés et des peptides permettant de moduler la communication par jonction communicante entre des cellules. Plus particulièrement, la présente invention concerne des procédés permettant de moduler la communication par jonction communicante entre des cellules de mammifère, le procédé comprenant le traitement des cellules avec une quantité efficace d'un peptide possédant la séquence d'acides aminés FHPSS (SEQ ID NO: 3), ou d'une forme variante de celui-ci présentant une identité de séquence d'au moins 60 % avec la séquence d'acides aminés et contenant au moins 4 acides aminés.
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WO2024023535A1 (fr) * 2022-07-28 2024-02-01 The University Of Birmingham Agoniste peptidique

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