WO2021078978A1 - Ciment osseux antibactérien et ses utilisations - Google Patents

Ciment osseux antibactérien et ses utilisations Download PDF

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Publication number
WO2021078978A1
WO2021078978A1 PCT/EP2020/079961 EP2020079961W WO2021078978A1 WO 2021078978 A1 WO2021078978 A1 WO 2021078978A1 EP 2020079961 W EP2020079961 W EP 2020079961W WO 2021078978 A1 WO2021078978 A1 WO 2021078978A1
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bone cement
seq
amino acid
oligopeptidic compound
peptide
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PCT/EP2020/079961
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English (en)
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Marit Otterlei
Synnøve BRANDT-RÆDER
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Norwegian University Of Science And Technology (Ntnu)
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Publication of WO2021078978A1 publication Critical patent/WO2021078978A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0015—Medicaments; Biocides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/108—Specific proteins or polypeptides not covered by groups A61L24/102 - A61L24/106
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
    • C12Y114/11033—DNA oxidative demethylase (1.14.11.33)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • the present invention relates to bone cements, particularly to kits for the production of antibacterial bone cements.
  • the bone cements provided herein contain bactericidal oligopeptidic compounds that also inhibit the development of antibiotic resistance in bacteria and the formation of bacterial biofilms.
  • the bone cements may also contain other therapeutically active agents, such as antibiotics.
  • the bone cements find utility in therapy and surgery, particularly in treating or preventing bacterial infections, such as orthopaedic infections. Methods and compositions comprising bactericidal oligopeptidic compounds for treating or preventing prosthetic joint infections are also provided.
  • Implant infections are considered to be a serious clinical problem for implant surgery and new solutions for anti-infective implant materials are needed.
  • Staphylococcus aureus and Staphylococcus epidermidis are known to be nosocomial bacterial species and the most common species isolated from orthopaedic implant infections. While S. epidermidis is a part of human skin flora and normally not associated with disease, S. aureus is known to be a virulent pathogen.
  • the introduction of S. epidermidis to surgical implants or to immunocompromised patients can cause chronic difficult-to-treat infections, in contrast to the more acute S. aureus infections.
  • Biofilms can provide additional resistance towards antibiotics by inhibiting the penetrance of the drug or containing slow or non-growing bacteria with reduced metabolism.
  • Biofilm-producing S. epidermidis from orthopaedic implant infections show a significantly higher resistance towards several antibiotics, including gentamicin, compared to non-biofilm forming strains, and it has also been suggested that these antibiotics (aminoglycosides) can induce biofilm formation.
  • APIM peptides are a group of peptides that interact with PCNA (proliferating cell nuclear antigen) via a novel PCNA interacting motif (Gilljam et al., 2009. Identification of a novel, widespread, and functionally important PCNA-binding motif, J. Cell Biol. 186(5), pp. 645-654).
  • the motif has been termed APIM (AlkB homologue 2 (hABH2) PCNA-interacting motif) since it was first identified as mediating the interaction between hABH2 and PCNA, but APIM sequences have now been identified in a wide range of proteins.
  • PCNA binding motif found in APIM peptides typically is defined using the consensus sequence, [R/K]-[F/W/Y]- [L/I/V/A]-[L/I/V/A]-[K/R] (SEQ ID NO: 19), and it has been determined that a more diverse motif, [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T/N/Q/C]-
  • [L/I/V/A/M/G/S/T/N/Q/R/H/K/C]-[K/R/H/P] (SEQ ID NO: 2), is present in various proteins that interact with PCNA (see WO2015/067713 incorporated herein by reference).
  • PCNA is a member of the sliding clamp family of proteins, which is known to be involved in both DNA replication and DNA repair.
  • the main function of PCNA is to provide replicative polymerases with the high processivity needed for duplication of the genome.
  • APIM peptides have been shown to be useful in therapy.
  • APIM peptides have been shown to have a direct effect on bacteria, i.e. APIM peptides have direct cytotoxic effects on a variety of bacterial cells and also potentiate or enhance the effect of cytotoxic and/or cytostatic agents on bacterial cells, i.e. sensitize bacterial cells to various cytotoxic and/or cytostatic agents, e.g. antibiotics, particularly agents that act intracellularly rather than agents that function at the cell membrane or cell wall, e.g. to permeabilize cells (see WO2015/067713 and WO20 16/177899).
  • APIM peptides In addition to interacting with the mammalian DNA sliding clamp, PCNA, APIM peptides also interact with the highly conserved bacterial sliding clamp, the b- clamp. APIM peptides therefore affect DNA replication and repair in bacterial cells, resulting in cytotoxicity and/or reduction in cell growth. APIM peptides have also been show to interfere with the interaction between TLS polymerases and sliding- clamp proteins (e.g. the b-clamp), thereby inhibiting essential cellular functions, such as translesion synthesis (TLS).
  • TLS pathway utilizes specialized TLS polymerases for the bypass and tolerance of toxic DNA lesions.
  • TLS often being an error-prone process itself, results in mutagenesis a key factor in the development of antibiotic resistance.
  • APIM peptides have a strong anti-mutagenic effect in bacteria.
  • APIM peptides can be used prophylactically to limit frequency of infections and the development of antibiotic resistance during prosthetic joint surgery.
  • the inventors have found that the addition of APIM peptides to bone cement does not alter the mechanical properties of the cement.
  • APIM peptides are efficiently eluted from bone cement.
  • the inventors have shown that bone cement containing a combination of APIM peptide and gentamicin reduced the growth of gentamicin- resistant (GR) S. epidermidis in bone grafts at a level that was beyond expectations.
  • GR gentamicin- resistant
  • APIM peptides inhibit the development of gentamicin resistance in a gentamicin-sensitive (GS) S. epidermidis, inhibit biofilm formation at sub-MIC (minimum inhibitory concentration) concentrations and eradicate existing biofilms.
  • the invention can be seen to provide the use of an oligopeptidic compound comprising a PCNA interacting motif in an antibacterial bone cement, e.g. in the preparation of an antibacterial bone cement.
  • the invention may be seen to provide a method of preparing an antibacterial bone cement comprising: (i) providing components for producing bone cement, wherein one of the components comprises an oligopeptidic compound comprising a PCNA interacting motif; and
  • the step of providing components used for producing bone may involve a step of combining an oligopeptidic compound comprising a PCNA interacting motif with one or more components used for producing bone cement.
  • the method of preparing an antibacterial bone cement comprises:
  • the invention provides a kit for producing an antibacterial bone cement comprising:
  • the oligopeptidic compound may be pre-mixed with one or more of the components for producing bone cement. In some embodiments, the oligopeptidic compound may be provided separately to the components for producing bone cement.
  • the invention provides an antibacterial bone cement comprising an oligopeptidic compound comprising a PCNA interacting motif.
  • the bone cement is obtained or obtainable from the method and kit described above.
  • the invention also provides an antibacterial bone cement described herein for use in therapy or surgery, e.g. for use in the treatment or prevention of a bacterial infection.
  • the invention provides a method of treating or preventing a bacterial infection in a subject in need thereof, said method comprising administering (e.g. applying) (particularly administering (e.g. applying) an effective amount of) the antibacterial bone cement described herein to the subject.
  • the invention provides the use of a kit of the invention in the preparation of an antibacterial bone cement for therapy or surgery, e.g. for the treatment or prevention of a bacterial infection.
  • the invention provides an oligopeptidic compound comprising a PCNA interacting motif (e.g. a pharmaceutical composition comprising said oligopeptidic compound) for use in the treatment or prevention of a prosthetic joint infection.
  • a PCNA interacting motif e.g. a pharmaceutical composition comprising said oligopeptidic compound
  • the invention provides a method of treating or preventing a prosthetic joint infection in a subject in need thereof, said method comprising administering (particularly administering an effective amount of) an oligopeptidic compound comprising a PCNA interacting motif (e.g. a pharmaceutical composition comprising said oligopeptidic compound).
  • oligopeptidic compound refers to a compound which is composed of amino acids or equivalent subunits, which are linked together by peptide or equivalent bonds.
  • oligopeptidic compound includes peptides and peptidomimetics.
  • the oligopeptidic compound consists of or comprises a peptide, i.e. consisting of amino acids linked by peptide bonds.
  • PCNA interacting motif refers to a sequence of consecutive subunits (e.g. amino acids) within an oligopeptidic compound (e.g. peptide) that functions to facilitate the interaction of the oligopeptidic compound (e.g. peptide) with PCNA.
  • a PCNA interacting motif also facilitates the interaction of the oligopeptidic compound with PCNA equivalent proteins, e.g. proteins that are functionally equivalent and/or structurally similar to PCNA, such as the b-clamp protein from bacteria, e.g. E.coli.
  • the oligopeptidic compounds of the invention may be characterised insofar as they must be capable of interacting with a PCNA and a b-clamp protein.
  • the oligopeptidic compounds of the invention and for use in the methods and uses of the invention must be competent and/or proficient PCNA and b-clamp interacting molecules.
  • the oligopeptidic compounds of the invention must be capable of interacting with at least a b-clamp protein, i.e. must be competent and/or proficient b-clamp interacting molecules.
  • the PCNA interacting motif may be viewed as a b-clamp interacting motif.
  • the PCNA protein used to determine the capacity and/or affinity of the oligopeptidic compound:PCNA interaction may be from any suitable source, e.g. a PCNA from any animal, particularly a mammal such as a human, rodent (e.g. mouse, rat) or any other non-human animal.
  • a PCNA from any animal, particularly a mammal such as a human, rodent (e.g. mouse, rat) or any other non-human animal.
  • the oligopeptidic compound: PCNA interaction is determined, characterised or assessed using human PCNA protein.
  • the b-clamp protein used to determine the capacity and/or affinity of the oligopeptidic compound ⁇ -clamp interaction may be from any suitable source, e.g. a b-clamp from any bacteria, e.g. gram positive or gram negative bacteria.
  • the oligopeptidic compound ⁇ -clamp interaction is determined, characterised or assessed using the b-clamp protein from E.coli or a bacteria from the genus Staphylococcus, e.g. Staphylococcus aureus or Staphylococcus epidermidis.
  • the interaction may be direct or indirect, and may involve direct binding of the motif to the PCNA or b-clamp protein, or the motif may bind indirectly, for example binding may be mediated by another molecule.
  • This reference to "PCNA- interacting", “PCNA-binding", “b-clamp-interacting” or “b-clamp-binding” can thus include any form of interaction, and both direct and indirect binding.
  • the interaction is direct binding.
  • PCNA-interacting motif refers to a sequence of 5 or 6 consecutive subunits (e.g. amino acids) having the functional activity defined above.
  • PCNA-interacting motif refers to a sequence X1X2X4X5X6 (SEQ ID NO: 1) or X 1 X 2 X 3 X4X5X6 (SEQ ID NO: 1225) as defined herein.
  • the motif consists of 5 consecutive subunits having a sequence X1X2X4X5X6 (SEQ ID NO: 1) as defined herein.
  • Xi is preferably selected from lysine (K), arginine (R), histidine (H), ornithine (Orn), methyllysine (MeK), diaminobutyric acid (Dbu), citrulline (Cit), acetyllysine (AcK), and any suitable basic amino acid selected from the non-conventional amino acids in Table 2. Whilst the standard or conventional basic amino acids are preferred, e.g. K, R and H, particularly K and R, these may be substituted by any functionally equivalent non-conventional basic amino acid.
  • X2 is preferably selected from phenylalanine (F), tryptophan (W), tyrosine (Y), te/t-butylglycine, cyclohexylalanine, te/t-butylphenylalanine, biphenylalanine and tri te/t-butyltryptophan (in certain embodiments this list may exclude W).
  • the standard or conventional aromatic amino acids are preferred, e.g. F, W and Y, these may be substituted by any functionally equivalent non-conventional aromatic amino acid, e.g. from Table 2.
  • X2 may be selected from W and Y, F and Y, or F and W or in specific embodiments X2 may be F or W or Y, or a functionally equivalent non-conventional aromatic amino acid.
  • the binding of the motif to PCNA may in certain embodiments be improved when X 2 is W or Y.
  • X 2 is not F. However, as indicated above, in other embodiments it may be F.
  • X3 is absent.
  • subunits X2andX4 are directly connected via a peptide bond or equivalent.
  • X4 is the third amino acid in the motif, i.e. position 3 shown in the sequence listing.
  • X3 is present and is preferably selected from phenylalanine (F), tryptophan (W), tyrosine (Y), te/t-butylglycine, cyclohexylalanine, te/t-butylphenylalanine, biphenylalanine and tri te/t-butyltryptophan, leucine (L), isoleucine (I), valine (V), methionine (M), norleucine (Nor) (in certain embodiments this list may exclude W or L).
  • X3 is preferably an aromatic amino acid selected from phenylalanine (F), tryptophan (W), tyrosine (Y), te/t-butylglycine, cyclohexylalanine, te/t-butylphenylalanine, biphenylalanine and tri te/t- butyltryptophan (in certain embodiments this list may exclude W).
  • the standard or conventional aromatic amino acids are preferred, e.g. F, W and Y, these may be substituted by any functionally equivalent non-conventional aromatic amino acid, e.g. from Table 2.
  • X3 may be selected from W and Y, F and Y, or F and W or in specific embodiments X3 may be F, or W or Y, or functionally equivalent non-conventional aromatic amino acids.
  • X3 is preferably a hydrophobic amino acid with an R group containing at least 3 carbon atoms, particularly an aliphatic amino acid with an R group containing at least 3 carbon atoms.
  • X3 may be selected from leucine (L), isoleucine (I), valine (V), methionine (M) or any suitable hydrophobic amino acid with an R group containing at least 3 carbon atoms selected from the non-conventional amino acids in Table 2. More particularly, X3 may be selected from L, I, V, M or Nor and any suitable hydrophobic (preferably aliphatic) amino acid with an R group containing at least 3 carbon atoms selected from the non-conventional amino acids in Table 2.
  • Xs may be selected from L, I, V and M and preferably from L, I and V or I, V and M and optionally non-conventional functional equivalents thereof.
  • X 4 IS preferably a hydrophobic or polar amino acid, particularly an aliphatic amino acid or polar amino acid.
  • X 4 may be selected from leucine (L), isoleucine (I), valine (V), alanine (A) methionine (M), norleucine (Nor), serine (S), threonine (T), glutamine (Q), asparagine (N) or cysteine (C) or any suitable hydrophobic or polar amino acid selected from the non-conventional amino acids in Table 2.
  • X 4 may be selected from L, I, V, A, M, Nor, S or T and any suitable hydrophobic (preferably aliphatic) or polar (preferably a polar amino acid that does not contain an amine group (NH 2 ) in the R-group) amino acid selected from the non-conventional amino acids in Table 2.
  • X 4 is not N or Q or a non-conventional functional equivalent thereof and/or in certain embodiments X 4 is not M, S and/or T or a non-conventional functional equivalent thereof.
  • X 4 may not be glycine (G) or proline (P) and this limitation is also intended to exclude non-conventional functional equivalents thereof.
  • X 4 may be selected from L, I, A, V, M, S and T, and preferably from L, I, A, V, S and T and optionally non-conventional functional equivalents thereof.
  • X 4 may be a hydrophobic, and more preferably an aliphatic amino acid.
  • X 4 may be selected from L, I, A, V, M, and preferably from L, I, V and A, and optionally non-conventional functional equivalents thereof.
  • X 5 IS preferably a hydrophobic, polar, basic or thiol-containing amino acid or proline.
  • X 5 an aliphatic amino acid or a polar amino acid.
  • the polar amino acid does not contain an amine group (NH 2 ) in the R-group.
  • X 5 preferably may be selected from L, I, V, A, M, Nor, S, T, Q, N, H, K, R, G, C or P and any suitable hydrophobic (preferably aliphatic) or polar (preferably a polar amino acid that does not contain an amine group (NH 2 ) in the R-group), basic or thiol-containing amino acid selected from the non-conventional amino acids in Table 2.
  • X 5 is an uncharged amino acid other than an aromatic amino acid, as defined in X 4 .
  • the basic amino acid may be selected from the amino acids as defined in Xi although in some embodiments, e.g. where X 3 is not an aromatic amino acid, e.g. when X 3 is L, X 5 is not K.
  • X 5 is not C or a non-conventional functional equivalent thereof and/or N or Q or a non-conventional functional equivalent thereof.
  • X 5 is not H and preferably X 5 is not R, K or H or a non-conventional functional equivalent thereof.
  • X 5 is not S or T or a non-conventional functional equivalent thereof.
  • X 5 is not P.
  • X 5 may not be an aromatic amino acid (as defined in X 2 ) or acidic amino acid, e.g. aspartic acid (D) or glutamic acid (E), and this limitation is also intended to exclude non-conventional functional equivalents thereof.
  • Xs may be selected from L, V, I, A, M, S, T and G, and preferably from L, V, A, I, S and T and optionally non-conventional functional equivalents thereof.
  • X 5 may be a hydrophobic amino acid, and more preferably an aliphatic amino acid or G.
  • Xs may be selected from L, I, A, V, M, and G and preferably from L, V, I and A, and optionally non-conventional functional equivalents thereof.
  • Xe may be a hydrophobic, polar, basic or thiol-containing amino acid or proline as defined above with respect to X 5 .
  • Xe is not a hydrophobic, polar or thiol-containing amino acid when X 3 is not an aromatic amino acid.
  • Xe may be a hydrophobic, polar or thiol-containing amino acid only when X 3 is an aromatic amino acid.
  • Xe is not a hydrophobic, polar or thiol-containing amino acid when X 3 is not an aromatic amino acid or when X 3 is absent (i.e. in a motif consisting of 5 amino acids).
  • Xe is preferably a basic amino acid or proline and may be selected from K, R, H, Orn, MeK, Dbu, Cit, AcK, P and any functionally equivalent amino acid selected from the non-conventional amino acids in Table 2. Whilst the standard or conventional amino acids are preferred, e.g. K, R, H and P, particularly K, R and H, e.g. K and R, these may be substituted by any functionally equivalent non-conventional basic amino acid.
  • Xe is a basic amino acid, preferably selected from K, R and H and optionally non-conventional functional equivalents thereof.
  • X 4 and/or X 5 is a polar amino acid. Accordingly, in certain embodiments only one of X 4 and X 5 is a polar amino acid.
  • X 5 and/or Xe is a basic amino acid. Accordingly, in certain embodiments Xe is a basic amino acid. In some embodiments, X4 and X5 are both uncharged amino acids other than an aromatic amino acid.
  • X2 and X3 are both aromatic amino acids, but preferably are not both W, as described further below.
  • a functionally equivalent amino acid may be defined as an amino acid that may be used as a substitute in a peptide or protein for a conventional amino acid without affecting significantly the function of the peptide or protein (or an amino acid that would not be expected to affect or alter significantly the function of the peptide or protein), e.g. an amino acid that has similar structural and/or chemical properties to the conventional amino acid.
  • a functionally equivalent amino acid may be viewed as having the base structure of a standard amino acid, with a non-standard or non-conventional R-group that is structurally and/or chemically similar to the standard R-group.
  • the R-group is structurally similar to the standard R- group of the amino acid being substituted.
  • a conventional or standard amino acid is an amino acid that is used in vivo to produce a polypeptide or protein molecule, i.e. a proteinogenic amino acid.
  • the oligopeptidic compound comprises a 5 subunit (e.g. amino acid) motif defined as [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T/N/Q/C]- [L/IA//A/M/G/S/T/N/Q/R/H/K/C]-[K/R/H/P] (SEQ ID NO: 2), wherein said oligopeptidic compound is capable of interacting with PCNA (and/or a b-clamp protein as defined above).
  • a 5 subunit e.g. amino acid motif defined as [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T/N/Q/C]- [L/IA//A/M/G/S/T/N/Q/R/H/K/C]-[K/R/H/P] (SEQ ID NO: 2), wherein said oligopeptidic compound is
  • the motif may be defined as: [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G/S/T/R/K]-[K/R/H] (SEQ ID NO:
  • the motif may be defined as: [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G/S/T]-[K/R/H] (SEQ ID NO: 7).
  • the motif may be defined as: [R/K]-[W/F/Y]-[L/IA//A/M/S/T]-[L/IA//A/M/G/S/T]-[K/R] (SEQ ID NO: 8).
  • the motif may be defined as: [R/K]-[W/F]-[L/IA//A/M/S/T]-[L/I/V/A/M/G/S/T]-[K/R] (SEQ ID NO: 9).
  • the motif may be defined as: [R/K]-[W/F]-[L/I/V/A/M/T]-[L/IA//A/M/G/S/T]-[K/R] (SEQ ID NO: 10).
  • the motif may be defined as: [R/K]-[W/F]-[L/I/V/A/M/T]-[L/I/V/A/M/S/T]-[K/R] (SEQ ID NO: 11).
  • the motif may be defined as: [R/K]-[W/F]-[L/IA//A/M/S/T]-[L/I/V/A/M/G]-[K/R] (SEQ ID NO: 12).
  • the motif may be defined as: [R/K]-[W/F]-[L/I/AA//M/T]-[L/I/V/A/M/S/T]-[K/R] (SEQ ID NO: 13).
  • the motif may be defined as: [R/K]-[W/F]-[L/IA//A/M/S/T]-[L/V/A/I/S/T]-[K/R] (SEQ ID NO: 14).
  • the motif may be defined as: [R/K]-[W/F]-[L/V/I/A/T]-[L/V/A/I/S/T]-[K/R] (SEQ ID NO: 15).
  • the motif may be defined as: [R]-[W/F/Y]-[L/V/I/A]-[L/V/A/S/T/M]-[K/R] (SEQ ID NO: 16).
  • the motif may be defined as: [R]-[W/F/Y]-[L/V/I/A/T]-[LA//A/S/T/M]-[K] (SEQ ID NO: 17).
  • the motif may be defined as: [R/K]-[F/Y]-[L/V/I/A]-[L/V/A/I/M]-[K/R] (SEQ ID NO: 18).
  • the motif may be defined as: [R/K]-[F/W/Y]-[L/I/V/A]-[L/I/V/A]-[K/R] (SEQ ID NO: 19).
  • the motif may be defined as: [R/K]-[W/Y]-[L/V/I/A/S/T]-[L/V/A/S/T/M]-[K/R] (SEQ ID NO: 20).
  • the motif may be defined as: [K]-[F/Y/W]-[L/V/I/A/T]-[L/V/A/I/S/T/M]-[K] (SEQ ID NO: 21).
  • the oligopeptidic compound comprises a 6 subunit (e.g. amino acid) motif defined as [R/K/H]-[W/F/Y]-[W/F/Y/L/IA///M]- [L/I/V/A/M/S/T/N/Q/C]-[L/IA//A/M/G/S/T/N/Q/R/H/K/C/P]- [K/R/H/P/L/l/V/A/M/G/S/T/N/Q/C] (SEQ ID NO: 1224), wherein said oligopeptidic compound is capable of interacting with PCNA (and/or a b-clamp protein as defined above).
  • 6 subunit e.g. amino acid motif defined as [R/K/H]-[W/F/Y]-[W/F/Y/L/IA///M]- [L/I/V/A/M/S/T/N/Q/C]-
  • the third amino acid (X 3 ) is L (e.g. X 3 is not an aromatic amino acid)
  • the fifth amino acid (X5) is not K and/or when X 3 is not an aromatic amino acid
  • the sixth amino acid (C d ) is K, R, H or P, wherein said oligopeptidic compound is capable of interacting with PCNA.
  • the motif may be defined as: [R/K/H]-[W/F/Y]-[L/IA//M]-[L/I/V/A/M/S/T/N/Q/C]- [L/IA//A/M/G/S/T/N/Q/R/H/C/P]-[K/R/H/P] (SEQ ID NO: 1226).
  • the motif may be defined as: [R/K/H]-[W/F/Y]-[L/IA//M]-[L/I/V/A/M/S/T]-[L/IA//A/M/G/S/T/N/Q/R/H]- [K/R/H/P] (SEQ ID NO: 1227).
  • the motif may be defined as: [R/K/H]-[W/F/Y]-[L/IA//M]-[L/I/V/A/M/S/T]-[L/IA//A/M/G/S/T/N/Q/R/H]-[K/R/H] (SEQ ID NO: 1228).
  • the motif may be defined as: [R/K/H]-[W/F/Y]-[L/IA//M]-[L/I/V/A/M/S/T]-[L/IA//A/M/G/S/T/R]-[K/R/H] (SEQ ID NO: 1229).
  • the motif may be defined as: [R/K]-[W/F/Y]-[L/I/V/M]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G/S/T]-[K/R] (SEQ ID NO:
  • the motif may be defined as: [R/K]-[W/F]-[L/I/V/M]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G/S/T]-[K/R] (SEQ ID NO:
  • the motif may be defined as: [R/K]-[W/F]-[L/I/V/M]-[L/IA//A/M/T]-[L/I/V/A/M/G/S/T]-[K/R] (SEQ ID NO:
  • the motif may be defined as: [R/K]-[W/F]-[L/I/V/M]-[L/IA//A/M/T]-[L/I/V/A/M/S/T]-[K/R] (SEQ ID NO: 1234). In another embodiment the motif may be defined as: [R/K]-[W/F]-[L/I/V/M]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G]-[K/R] (SEQ ID NO: 1235).
  • the motif may be defined as: [R/K]-[W/F]-[L/I/V/M]-[L/I/A/V/M/T]-[L/I/V/A/M/S/T]-[K/R] (SEQ ID NO: 1236). In another embodiment the motif may be defined as: [R/K]-[W/F]-[L/I/V/M]-[L/I/V/A/M/S/T]-[L/V/A/I/S/T]-[K/R] (SEQ ID NO: 1237).
  • the motif may be defined as: [R/K]-[W/F]-[L/I/V/M]-[L/V/I/A/T]-[L/V/A/I/S/T]-[K/R] (SEQ ID NO: 1238).
  • the motif may be defined as: [R]-[W/F/Y]-[L/IA//M]-[L/V/I/A]-[L/V/A/S/T/M]-[K/R] (SEQ ID NO: 1239).
  • the motif may be defined as: [R]-[W/F/Y]-[L/IA//M]-[L/V/I/A/T]-[L/V/A/S/T/M]-[K] (SEQ ID NO: 1240).
  • the motif may be defined as: [R/K]-[F/Y]-[L/I/V/M]-[L/V/I/A]-[L/V/A/I/M]-[K/R] (SEQ ID NO: 1241).
  • the motif may be defined as: [R/K]-[F/W/Y]-[L/I/V/M]-[L/I/V/A]-[L/IA//A]-[K/R] (SEQ ID NO: 1242).
  • the motif may be defined as: [R/K]-[W/Y]-[L/I/V/M]-[L/V/I/A/S/T]-[L/V/A/S/T/M]-[K/R] (SEQ ID NO: 1243). In yet another embodiment the motif may be defined as: [K]-[F/Y/W]-[l_/l/V/M]-[l_/V/l/A/T]-[L/V/A/l/S/T/M]-[K] (SEQ ID NO: 1244).
  • the motif may be defined as: [R/K]-[W/F]-[L/IA/]-[L/V/l/A/T]-[L/V/A/l/S/T]-[K/R] (SEQ ID NO: 1245).
  • the motif may be defined as: [R]-[W/F/Y]-[L/I/V]-[L/V/I/A]-[L/V/A/S/T/M]-[K/R] (SEQ ID NO: 1246).
  • the motif may be defined as: [R]-[W/F/Y]-[L/I/V]-[L/V/I/A/T]-[LA//A/S/T/M]-[K] (SEQ ID NO: 1247).
  • the motif may be defined as: [R/K]-[F/Y]-[L/IA/]-[L/V/l/A]-[L/V/A/l/M]-[K/R] (SEQ ID NO: 1248).
  • the motif may be defined as: [R/K]-[F/W/Y]-[L/I/V]-[L/I/V/A]-[L/I/V/A]-[K/R] (SEQ ID NO: 1249).
  • the motif may be defined as: [R/K]-[W/Y]-[L/IA/]-[L/V/l/A/S/T]-[L/V/A/S/T/M]-[K/R] (SEQ ID NO: 1250).
  • the motif may be defined as: [K]-[F/Y/W]-[L/IA/]-[L/V/l/A/T]-[L/V/A/l/S/T/M]-[K] (SEQ ID NO: 1251).
  • the motif may be defined as: [R/K/H]-[W/F/Y]-[W/F/Y]-[L/I/V/A/M/S/T/N/Q]-[L/IA//A/M/G/S/T/N/Q/R/H/K]- [K/R/H/P/L/l/V/A/M/G/S/T/N/Q/C] (SEQ ID NO: 1252).
  • the motif may be defined as: [R/K/H]-[W/F/Y]-[W/F/Y]-[L/I/V/A/M/S/T]-[L/IA//A/M/G/S/T/N/Q/R/H/K]- [K/R/H/P/L/l/V/A/M/G/S/T/N/Q/C] (SEQ ID NO: 1253).
  • the motif may be defined as: [R/K/H]-[W/F/Y]-[W/F/Y]-[L/I/V/A/M/S/T]-[L/IA//A/M/G/S/T/N/Q/R/H/K]-
  • the motif may be defined as: [R/K/H]-[W/F/Y]-[W/F/Y]-[L/I/V/A/M/S/T]-[L/IA//A/M/G/S/T/N/Q/R/H/K]- [K/R/H/P/L/l/V/A/M] (SEQ ID NO: 1255).
  • the motif may be defined as:
  • the motif may be defined as: [R/K/H]-[W/F/Y]-[W/F/Y]-[L/I/V/A/M/S/T]-[L/IA//A/M/G/S/T/R/K]-[K/R/H] (SEQ ID NO: 1257).
  • the motif may be defined as: [R/K/H]-[W/F/Y]-[W/F/Y/L/I/V/M]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G/S/T]-[K/R/H] (SEQ ID NO: 1258).
  • the motif may be defined as: [R/K]-[W/F/Y]-[W/F/Y/L/I/V/M]-[L/I/V/A/M/S/T]-[L/IA//A/M/G/S/T]-[K/R] (SEQ ID NO:
  • the motif may be defined as: [R/K]-[W/F]-[W/F/Y/L/I/V/M]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G/S/T]-[K/R] (SEQ I D NO: 1260). In another embodiment the motif may be defined as:
  • the motif may be defined as: [R/K]-[W/F]-[W/F/Y/L/I/V/M]-[L/IA///A/M/T]-[L/I/V/A/M/S/T]-[K/R] (SEQ ID NO: 1262).
  • the motif may be defined as: [R/K]-[W/F]-[W/F/Y/L/I/V/M]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G]-[K/R] (SEQ ID NO:
  • the motif may be defined as: [R/K]-[W/F]-[W/F/Y/L/I/V/M]-[L/I/A/V/M/T]-[L/I/V/A/M/S/T]-[K/R] (SEQ ID NO:
  • the motif may be defined as: [R/K]-[W/F]-[W/F/Y/L/I/V/M]-[L/I/V/A/M/S/T]-[LA//A/I/S/T]-[K/R] (SEQ ID NO:
  • the motif may be defined as: [R/K]-[W/F]-[W/F/Y/L/I/V/M]-[L/V/I/A/T]-[L/V/A/I/S/T]-[K/R] (SEQ ID NO:
  • the motif may be defined as: [R]-[W/F/Y]-[W/F/Y/L/I/V/M]-[L/V/I/A]-[LA//A/S/T/M]-[K/R] (SEQ ID NO:
  • the motif may be defined as: [R]-[W/F/Y]-[W/F/Y/L/I/V/M]-[L/V/I/A/T]-[LA//A/S/T/M]-[K] (SEQ ID NO:
  • the motif may be defined as: [R/K]-[F/Y]-[W/F/Y/L/I/V/M]-[L/V/I/A]-[L/V/A/I/M]-[K/R] (SEQ ID NO: 1269).
  • the motif may be defined as: [R/K]-[F/W/Y]-[W/F/Y/L/I/V/M]-[L/I/V/A]-[L/IA//A]-[K/R] (SEQ ID NO: 1270).
  • the motif may be defined as: [R/K]-[W/Y]-[W/F/Y/L/I/V/M]-[L/V/I/A/S/T]-[L/V/A/S/T/M]-[K/R] (SEQ ID NO:
  • the motif may be defined as: [K]-[F/Y/W]-[W/F/Y/L/I/V/M]-[L/V/I/A/T]-[L/V/A/I/S/T/M]-[K] (SEQ ID NO:
  • the motif may be defined as: [R/K]-[W/F]-[W/F/Y/L/IA/]-[L/IA//A/M/S/T]-[L/V/A/l/S/T]-[K/R] (SEQ ID NO:
  • the motif may be defined as: [R/K]-[W/F/Y]-[F/Y/L/I/V/M]-[L/I/V/A/M/S/T]-[L/V/A/I/S/T]-[K/R] (SEQ ID NO:
  • the motif may be defined as: [R/K]-[W/F]-[W/F/Y/L/IA/]-[L/V/l/A/T]-[L/V/A/l/S/T]-[K/R] (SEQ ID NO: 1275). In another embodiment the motif may be defined as: [R]-[W/F/Y]-[W/F/Y/L/IA/]-[L/V/l/A]-[L/V/A/S/T/M]-[K/R] (SEQ ID NO: 1276).
  • the motif may be defined as: [R]-[W/F/Y]-[W/F/Y/L/I/V]-[L/V/I/A/T]-[L/V/A/S/T/M]-[K] (SEQ ID NO: 1277).
  • Xi and X2 are RW, RF, KF, KW, RY or KY.
  • X 2 and X 3 are not both W.
  • the second and third amino acids in the sequences above consisting of 6 subunits i.e. X 2 and X 3 ) are selected from the group consisting of WF, WY,
  • X 4 and X 5 are LL, LA, LV, AL, VL, VI, LI, IL, VV, VA,
  • X 4 and X 5 are LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA, Al, AM, LM, LS, LT, IS, MV, TV, AA, IM, LN, LQ or VM.
  • X 4 and X 5 are LV, IV, SV, LS, AV, LG, LA, IR, LR, VR, AR, IK, LK, VK or AK.
  • X 4 and X 5 are LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA, Al, AM, LM, LS or LT, preferably LL, LA, LV, AL, VL, VI, LI, I L, VV, VA, IV,
  • X 4 and X 5 are not any one or more of AG, AC, CC, NN, QQ, NQ, QN, TS, SS, ST or TT.
  • X 4 and X 5 are SL, LS, SV, LT or AV.
  • X 3 and X 4 are not FS, FT, WA or WS. In some embodiments X 3 and X 4 are FS or FT.
  • Cb ⁇ e K In some embodiments Cb ⁇ e P.
  • the oligopeptidic compound has or comprises (i.e. the motif consists of) the sequence selected from any one of more of SEQ ID NOs: 22-297, 1206 and 1207.
  • the oligopeptidic compound has or comprises (i.e. the motif consists of) the sequence RWLVK (SEQ ID NO: 28) or KFIVK (SEQ ID NO: 119). In still further embodiments, the oligopeptidic compound has or comprises (i.e.
  • the motif consists of) the sequence RWLTK (SEQ ID NO: 76), RFLSK (SEQ ID NO: 41), RFSLK (SEQ ID NO: 1206), RWLSK (SEQ ID NO: 40), RWSVK (SEQ ID NO: 52) RWAVK (SEQ ID NO: 58) or RWLVP (SEQ ID NO: 1207).
  • the oligopeptidic compound has or comprises (i.e. the motif consists of) the sequence RWLVK (SEQ ID NO: 28) or RFSLK (SEQ ID NO: 1206).
  • the oligopeptidic compound has or comprises the sequence selected from any one of more of SEQ ID NOs: 1282-1559, wherein X 3 (the third residue in the motif) is selected from the group consisting of W, F, Y, L, I,
  • V and M preferably W, F and Y.
  • the motifs consisting of 6 amino acids do not contain two adjacent tryptophan residues, i.e. the sequences do not contain WW.
  • the second amino acid (X 2 ) is W
  • X (X 3 ) is selected from the group consisting of F, Y, L, I, V and M.
  • the second and third amino acids in the motifs consisting of 6 amino acids above i.e.
  • X 2 and X 3 are selected from the group consisting of WF, WY, WL, Wl, WV, WM, FW, FF, FY, FL, FI, FV, FM, YW, YF, YY, YL, Yl, YV and YM.
  • X 2 and X 3 are selected from WF, WY, FF, FY, FW, YF, YY and YW.
  • the oligopeptidic compound has or comprises the sequence RWXLVK (SEQ ID NO: 1288) or RFXLVK (SEQ ID NO: 1289) or RWXVIK (SEQ ID NO: 1498) or RFXVIK (SEQ ID NO: 1499).
  • the oligopeptidic compound has or comprises the sequence selected from any one of more of SEQ ID NOs: 1560-1593.
  • the oligopeptidic compound has or comprises the sequence RWXLTK (SEQ ID NO: 1336), RFXLSK (SEQ ID NO: 1301),
  • RWXLSK (SEQ ID NO: 1300), RWXSVK (SEQ ID NO: 1312) RWXAVK (SEQ ID NO: 1318) or RWXLVP (SEQ ID NO: 1559), wherein X (X 3 ) is selected from the group consisting of W, F, Y, L, I, V and M, preferably W, F and Y, as defined above.
  • the PCNA interacting motifs listed above are preferred motifs of the invention, in some embodiments any one or more of these motifs may be excluded, e.g. any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more motifs may be excluded, such as any 25, 30, 40, 50 or more motifs or any integer in this range.
  • the oligopeptidic compound does not have or comprise a sequence selected from any one or more of SEQ ID NOs: 22-297, 1206, 1207 and 1282-1593.
  • the oligopeptidic compound is preferably an isolated compound, e.g. an isolated peptide and most preferably the oligopeptidic compound is a synthetic compound, e.g. a synthetic peptide. In other words, the oligopeptidic compound is a non-native, i.e. non-naturally occurring, molecule.
  • the oligopeptidic compound In order that the oligopeptidic compound may function as an antibacterial compound, the compound must be capable of entering the bacterial cells, i.e. crossing the cell membrane and cell wall, if present, into the cytosol (cytoplasm).
  • the uptake of the oligopeptidic compound may be achieved by associating the oligopeptidic compound with one or more molecules that are known to be capable of facilitating the uptake of molecules into bacterial cells, e.g. an import peptide.
  • an oligopeptidic compound that comprises a domain that assists the transit of the compound across the cell membrane, i.e. to generate a fusion peptide or chimeric peptide (a peptide formed from two or more domains that are not normally found together in nature).
  • a peptide comprising a cell membrane permeable motif e.g. a cell penetrating peptide (an uptake or import peptide, or a peptide transduction domain).
  • the oligopeptidic compound comprises a PCNA interacting motif and a domain that facilitates its uptake.
  • the oligopeptidic compound comprises at least 5 or 6 residues (depending on the size of the motif) and the final size of the compound will be dependent on the size and number of the domains that make up said compound, i.e. the PCNA interacting motif and uptake (import) peptide may be viewed as domains of the oligopeptidic compound.
  • a domain may be viewed as a distinct portion (i.e. a sequence within the full-length peptidic sequence) of the oligopeptidic compound that can be assigned or ascribed a particular function or property.
  • the oligopeptidic compound of the invention comprises at least 2 domains, i.e. the PCNA interacting motif domain and the domain that facilitates the cellular uptake of said compound, e.g. uptake (import) peptide sequence domain.
  • the oligopeptidic compound may comprise additional domains that may facilitate its function and/or stability, e.g. the capacity of the peptide to interact with its target, i.e. PCNA or b-clamp protein.
  • the oligopeptidic compound may comprise at least 2, 3, 4 or 5 domains, e.g. 6, 7, 8, 9, 10, 12, 15 or more domains.
  • the oligopeptidic compound may comprise one or more linker domains, i.e. a domain that interspaces between two other domains, i.e. occupies the space in between and connects two domains of the oligopeptidic compound.
  • the linker domain may be inert, i.e. it may have no physiological function in the bacterial cell in which the oligopeptidic compound is active and simply functions to physically separate the other domains in the oligopeptidic compound.
  • the linker domain may have an additional function.
  • the linker domain may also function as a cleavage domain, i.e. the linker domain may contain a peptide bond that is susceptible to cleavage under physiological conditions, e.g. inside a bacterial cell, such that the peptide is cleaved following its uptake.
  • the linker may conveniently be a signal peptide, e.g. a NLS, even though it has no known function in a bacterial cell.
  • a signal peptide domain may function as a linker domain in some embodiments, e.g. an NLS sequence may be used as a linker domain.
  • the oligopeptidic compound may comprise a PCNA interacting motif domain, a domain that facilitates its cellular uptake (e.g. an uptake (import) peptide sequence domain) and a linker domain.
  • a PCNA interacting motif domain e.g. an uptake (import) peptide sequence domain
  • a linker domain e.g. an uptake (import) peptide sequence domain
  • the oligopeptidic compound of the invention may take the form of a construct containing (i.e. comprising) an oligopeptidic compound which comprises a PCNA interacting motif as defined herein, together with a domain that facilitates its cellular uptake (e.g. an uptake peptide sequence) and optionally additional domains.
  • the invention may accordingly be seen to provide a construct comprising an oligopeptidic compound which is capable of interacting with a PCNA ⁇ -clamp protein as described above.
  • the domain that facilitates the uptake of the oligopeptidic compound may be an uptake (import) peptide sequence, which may be a sequence which acts to transport the oligopeptidic compound into a cell, or across a cell membrane (i.e. into the interior of a cell). It may thus be a so-called “cell penetrating” sequence (or more particularly “cell penetrating peptide”) also known in the art as a protein transduction domain (PTD) or protein transduction sequence.
  • uptake (import) peptide sequence which may be a sequence which acts to transport the oligopeptidic compound into a cell, or across a cell membrane (i.e. into the interior of a cell). It may thus be a so-called “cell penetrating” sequence (or more particularly "cell penetrating peptide”) also known in the art as a protein transduction domain (PTD) or protein transduction sequence.
  • PTD protein transduction domain
  • the invention may provide a construct comprising (i) an oligopeptidic compound comprising an APIM motif (i.e. a PCNA- interacting motif) as defined herein, and (ii) a cell penetrating sequence (more particularly a cell penetrating peptide).
  • an APIM motif i.e. a PCNA- interacting motif
  • a cell penetrating sequence more particularly a cell penetrating peptide
  • CPP Cell penetrating peptide
  • CPPs are not characterized by a single structural or functional motif
  • tools to identify CPPs are available and the skilled person can readily determine whether a peptide sequence may function to facilitate the uptake of the peptide of which it forms a domain, i.e. whether a peptide sequence may function as an uptake (import) peptide, e.g. a CPP.
  • a peptide sequence may function as an uptake (import) peptide, e.g. a CPP.
  • Hansen etal Predicting cell- penetrating peptides, Advanced Drug Delivery Reviews, 2008, 60, pp.
  • CPPs may be derived from naturally-occurring proteins which are able to translocate across cell membranes such as the Drosophila homeobox protein Antennapedia (a transcriptional factor), viral proteins such as the HIV-1 transcriptional factor TAT and the capsid protein VP22 from HSV-1, and/or they may be synthetically-derived, e.g. from chimeric proteins or synthetic polypeptides such as polyarginine.
  • Drosophila homeobox protein Antennapedia a transcriptional factor
  • viral proteins such as the HIV-1 transcriptional factor TAT and the capsid protein VP22 from HSV-1
  • synthetically-derived e.g. from chimeric proteins or synthetic polypeptides such as polyarginine.
  • RRMKWKK (SEQ ID NO: 305) WO FQN RRMKWKK (SEQ ID NO: 306) 97/12912 RREKWKK (SEQ ID NO: 307) RRQKWKK (SEQ ID NO: 308) KRMKWKK (SEQ ID NO: 309) RKMKWKK (SEQ ID NO: 310) RROKWKK (SEQ ID NO: 311) RRMKQKK (SEQ ID NO: 312) RRMKWFK (SEQ ID NO: 313) RORKWKK (SEQ ID NO: 314) RRMWKKK SE ID NO 315 Pep-1 KETWWETWWTEWSQ P KKKR KV Wyman (SEQ ID NO: 326) Biochemistry
  • Vectocell VKRGLKLRHVRPRVTRMDV (SEQ ID NO: 329) Coupade peptides SRRARRSPRHLGSG * (SEQ ID NO: 330) (2005) LRRERQSRLRRERQSR * (SEQ ID NO: 331) Biochem. J. GAYDLRRRERQSRLRRRERQSR 407 (SEQ ID NO: 332) indicates addition of cys for conjugation to cargo
  • Antennapedia-derived CPPs represent a class of CPPs based around the 16 amino acid Penetratin sequence as shown in Table 1 , which corresponds to the third loop of antennapedia protein and was shown to be responsible for translocation of the protein.
  • Penetratin has been extensively developed as a delivery vehicle, including particularly for pharmaceutical use, and a wide range of Penetratin derivatives and modified sequences have been proposed and described. Reference may be made in particular to WO 91/1891, WO 00/1417, WO 00/29427, WO 2004/069279 and US 6,080,724 (herein incorporated by reference).
  • the 16 amino acid sequence of Penetratin may be modified and/or truncated, or the peptide may be chemically-modified or retro-, inverso- or retro- inverso analogues may be made whilst retaining cell-penetrating activity.
  • HIV-TAT and fragments thereof represent a preferred class of CPPs for use according to the present invention.
  • TAT-based CPPs are described in US 5,656,122 (herein incorporated by reference).
  • An exemplary HIV-TAT peptide as used in the Examples below is RKKRRQRRR (SEQ ID NO: 335) but it will readily be appreciated that longer or shorter TAT fragments may be used.
  • CPPs may be identified by particular features, such as for example peptides which are amphipathic and net positively charged.
  • Other groups of CPPs may have a structure exhibiting high a- helical content.
  • Another group may be peptides characterised by a high content of basic amino acids.
  • CPPs may thus be or may comprise oligomers of basic amino acids such as arginine e.g. 5 to 20, 6 to 15 or 6 to 12 R residues e.g. R 7 (SEQ ID NO: 334) , R 8 (SEQ ID NO: 336), R 10 (SEQ ID NO: 1623) or Rn (SEQ ID NO: 337) or QSR 8 (SEQ ID NO: 338).
  • the domain that facilitates the uptake of the oligopeptidic compound may be defined as a peptide of 4-30 amino acids (e.g. 5-29, 6-28, 7-27, 8-26, 9-25 etc. amino acids), wherein at least 4 amino acids, optionally at least 4 consecutive amino acids, (e.g. at least 5, 6, 7, 8, 9, 10 or 11 amino acids, e.g. 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, 11-13 amino acids) are positively charged amino acids, preferably selected from K, R or H.
  • 4-30 amino acids e.g. 5-29, 6-28, 7-27, 8-26, 9-25 etc. amino acids
  • at least 4 amino acids optionally at least 4 consecutive amino acids, (e.g. at least 5, 6, 7, 8, 9, 10 or 11 amino acids, e.g. 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, 11-13 amino acids) are positively charged amino acids, preferably selected from K, R or H.
  • Proline-rich amphipathic peptides are another class of CPP and such peptides characterised by the presence of pyrrolidine rings from prolines are described in Pujals et al. 2008 Advanced Drug Delivery Reviews 60, pages 473-484 (herein incorporated by reference).
  • CPPs include pVEC (Elmquist etal. 2003 Biol. Chem 384, pages 387-393; Holm etal. 2005 Febs Lett. 579, pages 5217- 5222, all herein incorporated by reference) and calcitonin-derived peptides (Krauss etal. 2004 Bioorg. Med. Chem. Lett., 14, pages 51-54 herein incorporated by reference).
  • CPPs include Chariot, based on the Pep-1 peptide (Active Motif, France), the Syn-B vectors based on the protegrin peptide PG-1 (Syntem, France), and Express-si Delivery based on the MPG peptide from Genospectra, USA.
  • CPPs include the R41, R8, M918 and YTA-4 peptides (SEQ ID NOs: 1213-1216, respectively) disclosed in Eriksson et al. 2013, Antimicrobial Agents and Chemotherapy, vol. 57(8), pp. 3704-3712 (incorporated herein by reference). ln some embodiments the CPPs may be cyclic peptides, such as those disclosed in Oh et ai, 2014, Mol. Pharmaceutics, vol. 11, pp. 3528-3536 (incorporated herein by reference). In particular, the CPPs may be amphiphilic cyclic CPPs, particularly containing tryptophan and arginine residues.
  • the CPPs may be cyclic polyarginine peptides and may be modified by the addition of a fatty acyl moiety, e.g. octanoyl, dodecanoyl, hexadecanoyl, N- acetyl-L-tryptophanyl-12-aminododecanoyl etc.
  • a fatty acyl moiety e.g. octanoyl, dodecanoyl, hexadecanoyl, N- acetyl-L-tryptophanyl-12-aminododecanoyl etc.
  • Suitable cyclic CPPs for use in the invention are presented in SEC ID NOs: 1217-1223.
  • novel or derivative CPP peptides may be designed and synthesized based on known or reported criteria (e.g. known CPP sequences or features such as basic amino acid content, a-helical content etc. as discussed above). Additionally, randomly-designed or other peptides may be screened for CPP activity, for example by coupling or attaching such a peptide containing a reporter molecule, e.g. a detectable label or tag such as a fluorescent tag to the desired cargo (e.g. an oligopeptidic compound as described herein) and testing to see if the construct is translocated across the cell membrane, for example by adding these peptides to live cells followed by examination of cellular import e.g. using confocal microscopy.
  • a reporter molecule e.g. a detectable label or tag such as a fluorescent tag
  • desired cargo e.g. an oligopeptidic compound as described herein
  • CPPs also facilitate the uptake of peptides into prokaryotic cells. It is thought that the capacity of CPPs to function in prokaryotic cells is a result of their structural similarity to anti-bacterial peptides, e.g. short, cationic peptides with amphipathic properties.
  • the skilled person will be aware of suitable cell penetrating peptide sequences that may facilitate the uptake of the oligopeptidic compound, but by way of example the sequences may include Penetratin TM, a 16- amino acid peptide corresponding to the third helix of the homeodomain of Antennapedia protein, R rich tags such as R6-Penetratin (in which arginine- residues were added to the N-terminus of Penetratin) and derivatives of the HIV Tat protein such as GRKKRRQRRRPPQQ (SEQ ID NO: 339).
  • Penetratin TM a 16- amino acid peptide corresponding to the third helix of the homeodomain of Antennapedia protein
  • R rich tags such as R6-Penetratin (in which arginine- residues were added to the N-terminus of Penetratin)
  • derivatives of the HIV Tat protein such as GRKKRRQRRRPPQQ (SEQ ID NO: 339).
  • the domain that facilitates the cellular uptake of the oligopeptidic compound is a CPP and may be selected from any one of:
  • an amphipathic class peptide selected from an amphipathic and net positively charged peptide, a proline-rich amphipathic peptide, a peptide based on the Pep-1 peptide and a peptide based on the MPG peptide;
  • the domain that facilitates the cellular uptake of the oligopeptidic compound is a CPP and may be selected from a sequence selected from any one of SEC ID NOs: 302-1162 and 1213-1223 or a fragment and/or derivative thereof.
  • the details and properties of the CPPs identified in SEC ID NOs: 340-1162 can be found at http://crdd.osdd.net/raghava/cppsite/index.php, CPPSite: A database of cell penetrating peptides (herein incorporated by reference).
  • the domain that facilitates the cellular uptake of the oligopeptidic compound is a CPP which is or comprises an arginine oligomer, e.g. comprising 5-20, 7-15 or 9-12 consecutive arginine residues, such as 9, 10 or 11 consecutive arginine residues.
  • the CPP is selected from SEC ID NOs: 334, 336, 337 or 1623.
  • the domains (which may be viewed as components, elements or separate parts) of an oligopeptidic compound or construct of the invention as described herein may be attached or linked to one another in any desired or convenient way according to techniques well known in the art.
  • the domains may be linked or conjugated chemically, e.g. using known chemical coupling technologies or the compound or constructs may be formed as a single whole using genetic engineering techniques e.g. techniques for forming fusion proteins, or they may simply be synthesized as a whole, e.g. using peptide synthesis techniques.
  • the domains may be linked directly to each other or they may be linked indirectly by means of one or more linker (or spacer) sequences.
  • a linker sequence may interspace or separate two or more individual domains (i.e. parts, e.g. or separate motif elements) in an oligopeptidic construct or compound.
  • the precise nature of the linker sequence is not critical and it may be of variable length and/or sequence, for example it may have 0-40, more particularly 0-20, 0-15, 0-12, 0-10, 0-8, 0-7, 0-6, 0-5, 0-4 or 0-3 residues e.g. 1 , 2 or 3 or more residues.
  • the linker sequence if present, may have 1-15, 1-12, 1- 10, 1-8, 1-7, 1-6, 1-5 or 1-4 residues etc.
  • residues may for example be any amino acid, e.g. a neutral amino acid, or an aliphatic amino acid, or alternatively they may be hydrophobic, or polar or charged or structure-forming e.g. proline.
  • linker sequences have been shown to be of use, including short (e.g. 1-7) sequences of neutral and/or aliphatic amino acids.
  • Exemplary linker sequences thus include any single amino acid residue, e.g. A, I, L, V, G, R, Q, T, or W, or a di-, tri- tetra- penta- or hexa-peptide composed of such residues.
  • linkers may be mentioned I, II, IL, R, W, WW, WWW, RIL, RIW, GAQ, GAW, VAT, IILVI (SEQ ID NO: 1180), IILVIII (SEQ ID NO: 1181), GILQ (SEQ ID NO: 1621), GILQWRK (SEQ ID NO: 1622), GILQWRKI (SEQ ID NO:
  • the linker domain comprises a sequence selected from any one of SEQ ID NOs: 1163-1179 or 1621, 1622 or 1624 or a fragment and/or derivative thereof, preferably wherein said fragment and/or derivative comprises at least 4 positively charged amino acids, preferably selected from any of K, R or H.
  • the linker domain comprises a sequence selected from any one of SEQ ID NOs: 1621, 1622 or 1624.
  • an oligopeptidic compound or construct according to the present invention may comprise at least three domains, including (i) an APIM motif domain as defined herein, (ii) a linker domain as defined herein, and (iii) a peptide sequence domain that facilitates the cellular uptake of said compound or construct (i.e. an uptake/import peptide sequence domain, e.g. cell penetrating signal sequence domain), as defined herein.
  • the separate elements or components (domains) of a construct according to the present invention may be contained or presented in any order, but preferably in the orders indicated above (e.g. motif-CPP or motif-linker-CPP).
  • the motif is located at or towards the N-terminus of the peptide.
  • the APIM motif may be described as being N-terminal to the peptide sequence domain that facilitates the cellular uptake of said compound (e.g. the CPP) and optionally N-terminal to the linker sequence, if present.
  • the oligopeptidic compound comprises a PCNA interacting motif as set forth in SEQ ID NO: 28 or 1206 and a cell penetrating signal sequence as set forth in SEQ ID NO: 334, 336, 337 or 1623.
  • the oligopeptidic compound comprises a PCNA interacting motif as set forth in SEQ ID NO: 28 or 1206, a linker domain as set forth in SEQ ID NO: 1176, 1621 , 1622 or 1624 and a cell penetrating signal sequence as set forth in SEQ ID NO: 334, 336, 337 or 1623, preferably SEQ ID NO: 337 or 1623.
  • the oligopeptidic compound comprises a PCNA interacting motif as set forth in SEQ ID NO: 28, a linker domain as set forth in SEQ ID NO: 1176 and a cell penetrating signal sequence as set forth in SEQ ID NO: 337, e.g. a sequence as set forth in SEQ ID NO: 1198.
  • the oligopeptidic compound comprises a PCNA interacting motif as set forth in SEQ ID NO: 28, a linker domain as set forth in SEQ ID NO: 1624 and a cell penetrating signal sequence as set forth in SEQ ID NO: 337.
  • the oligopeptidic compound comprises a PCNA interacting motif as set forth in SEQ ID NO: 28, a linker domain as set forth in SEQ ID NO: 1624 and a cell penetrating signal sequence as set forth in SEQ ID NO: 1623.
  • the oligopeptidic compound comprises a PCNA interacting motif as set forth in SEQ ID NO: 1206, a linker domain as set forth in SEQ ID NO: 1624 and a cell penetrating signal sequence as set forth in SEQ ID NO: 337.
  • the oligopeptidic compound comprises a PCNA interacting motif as set forth in SEQ ID NO: 1206, a linker domain as set forth in SEQ ID NO: 1624 and a cell penetrating signal sequence as set forth in SEQ ID NO: 1623.
  • an oligopeptidic compound or construct according to the invention may contain more than one PCNA-interacting motif.
  • a construct or oligopeptidic compound may for example contain 1-10, e.g. 1-6, or 1-4 or 1-3 or one or two motifs.
  • motifs may be spaced or located according to choice, e.g. they may be grouped together, or they may be separated by other domains, e.g. motif-motif- CPP, motif-linker-motif-CPP; or motif-linker-motif-motif-CPP; or motif-motif-linker- CPP etc.
  • a “fragment” may comprise at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% of the amino acids of the sequence from which it is derived.
  • Said fragment may be obtained from a central or N-terminal or C- terminal portions of the sequence. Whilst the size of the fragment will depend on the size of the original sequence, in some embodiments the fragments may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid residues shorter than the sequence from which it is derived, e.g. 1-10, 2-9, 3-8, 4-7 amino acid residues shorter than the sequence from which it is derived.
  • a "derivative" of a sequence is at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the sequence to which it is compared.
  • Sequence identity may be determined by, e.g. using the SWISS-PROT protein sequence databank using FASTA pep-cmp with a variable pamfactor, and gap creation penalty set at 12.0 and gap extension penalty set at 4.0, and a window of 2 amino acids. Preferably said comparison is made over the full length of the sequence, but may be made over a smaller window of comparison, e.g. less than 200, 100, 50, 20 or 10 contiguous amino acids.
  • sequence identity related polypeptides i.e. derivatives
  • sequence identity related polypeptides are functionally equivalent to the peptides which are set forth in the recited SEQ ID NOs.
  • the peptides with sequences as set forth in the SEQ ID NOs. may be modified without affecting the sequence of the polypeptide as described below.
  • fragments as described herein may be functional equivalents. Preferably these fragments satisfy the identity (relative to a comparable region) conditions mentioned herein.
  • the peptide may show some reduced efficacy in performing the function relative to the parent molecule (i.e. the molecule from which it was derived, e.g. by amino acid substitution), but preferably is as efficient or is more efficient.
  • functional equivalence may relate to a peptide which is effective in localizing or directing the oligopeptidic compound into the cell, e.g. to facilitate to the uptake of the peptide as described above. This may be tested by comparison of the effects of the derivative peptide relative to the peptide from which it is derived in a qualitative or quantitative manner, e.g. by performing the in vitro analyses described above. Where quantitative results are possible, the derivative is at least 30, 50, 70 or 90% as effective as the parent peptide.
  • Functionally-equivalent peptides which are related to or derived from the parent peptide, may be obtained by modifying the parent amino acid sequence by single or multiple amino acid substitution, addition and/or deletion (providing they satisfy the above-mentioned sequence identity requirements), but without destroying the molecule's function.
  • the parent sequence has less than 20 substitutions, additions or deletions, e.g. less than 10, 5, 4, 3 or 2 such modifications.
  • Such peptides may be encoded by "functionally-equivalent nucleic acid molecules" which may be generated by appropriate substitution, addition and/or deletion of one or more bases.
  • Representative compounds (or more particularly constructs) containing a PCNA interacting motif consisting of 5 amino acids include:
  • MDRWLVKRILKKKRKVATKG (SEQ ID NO: 1183), MDRWLVKGAQPKKKRKVLRQIKIWFQNRRMKWKK (SEQ ID NO: 1184), MDRWLVKGAWKKKRVKIIRKKRRQRRRK (SEQ ID NO: 1185), MDRWLVKGAWKKKRKIIRKKRRQRRRG (SEQ ID NO: 1186), MDRWLVKGAWKKKRKIIRKKRRQRRRK (SEQ ID NO: 1187), MDRWLVKRIWKKKRKIIRKKRRQRRRK (SEQ ID NO: 1188), MDRWLVKWWWKKKRKIIRKKRRQRRRK (SEQ ID NO: 1189), MDRWLVKWWRKRHIIKKRKKRRQRRRK (SEQ ID NO: 1190), MDRWLVKRIWKKKRKIIRRRRRRRRRK (SEQ ID NO: 1191), MDRWLVKRIWKKKRKIIRRRRRRRRR
  • the oligopeptidic compounds shown above comprise N-terminal amino acids that do not form part of the domains that are essential for the compounds to have activity in the methods and uses of the invention, i.e. an "MD" sequence. Some of the peptides may also comprise N-terminal modification, e.g. acetyl groups. These additional amino acids and modifications may facilitate the production of the oligopeptidic compounds, e.g. in vitro or in vivo, and/or help to protect the compounds from degradation in vivo.
  • the oligopeptidic compounds do not require these additional amino acids or modifications for their activity. Accordingly, further representative sequences according to the invention include any of SEQ ID NOs: 1182 to 1204, 1208-1212, 1594-1620, 1625 or 1626 omitting the N-terminal "MD". Furthermore, the presence of additional amino acids or modifications at either terminus would not be expected to disrupt or inhibit the function of the oligopeptidic compounds described herein.
  • the oligopeptidic compound may comprise an N- terminal sequence, e.g. a sequence at the N-terminus that does not comprise a domain defined above, e.g. a so-called N-terminal flanking sequence.
  • the oligopeptidic compound may comprise a C-terminal sequence, e.g. a sequence at the C-terminus that does not comprise a domain defined above, e.g. a so-called C-terminal flanking sequence.
  • the oligopeptidic compound may comprise an N-terminal and C-terminal flanking sequence.
  • the oligopeptidic compound may also comprise a C-terminal modification, e.g. an amide group.
  • the C-terminal residue may be amidated.
  • the oligopeptidic compound comprises an amidated C-terminal arginine residue.
  • the oligopeptidic compound of the invention may be in the form of a salt.
  • the oligopeptidic compound may be in the form of an acidic or basic salt.
  • the oligopeptidic compound is in a neutral salt form.
  • the oligopeptidic compound may be in the form of an acetate salt or derivative thereof, e.g. trichloroacetate (TCA), trifluoroacetate (TFA) etc.
  • TCA trichloroacetate
  • TFA trifluoroacetate
  • the oligopeptidic compound may be stabilized by preparing it in the form of a salt, e.g. an acetate salt.
  • a flanking sequence may comprise from about 1-150 amino acids, such as 1-120, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-35, 1-30 etc.
  • a flanking sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
  • amino acids e.g. 1-40, 2-39, 3-38, 4-37, 5-36, 6-35, 7- 34, 8-33, 9-32, 10-31 , 11-30, 12-29, 13-28, 14-27, 15-26 amino acids or any combination thereof.
  • K stands for lysine (Lys)
  • I stands for isoleucine (lie) and so on.
  • the oligopeptidic compound may comprise non-conventional or non-standard amino acids. Other domains in the oligopeptidic compound may also incorporate non-standard amino acids.
  • the oligopeptidic compound may comprise one or more, e.g. at least 1 , 2, 3, 4 or 5 non-conventional amino acids, i.e. amino acids which possess a side chain that is not coded for by the standard genetic code, termed herein "non-coded amino acids" (see e.g. Table 2).
  • amino acids which are formed through metabolic processes such as ornithine or taurine, and/or artificially modified amino acids such as 9/-/-fluoren-9- ylmethoxycarbonyl (Fmoc), (tert)-(B)utyl (o)xy (c)arbonyl (Boc), 2, 2, 5,7,8- pentamethylchroman-6-sulphonyl (Pmc) protected amino acids, or amino acids having the benzyloxy-carbonyl (Z) group.
  • non-coded amino acids are present, they are not located within the motif, but in some embodiments one or more non-coded amino acids are present within the motif.
  • non-coded amino acids are present in more than one domain of the oligopeptidic compound.
  • ln vitro and/or in vivo stability of the oligopeptidic compound may be improved or enhanced through the use of stabilising or protecting means known in the art, for example the addition of protecting or stabilising groups, incorporation of amino acid derivatives or analogues or chemical modification of amino acids, Such protecting or stabilising groups may for example be added at the N and/or C-terminus.
  • An example of such a group is an acetyl group and other protecting groups or groups which might stabilise a peptide are known in the art.
  • the oligopeptidic compounds of the invention will typically comprise only amino acids having the L-configuration, but one or more amino acids having the D configuration may be present.
  • the oligopeptidic compound contains at least 1, 2, 3, 4 or 5 D-amino acids and they are preferably found in the motif, but in another embodiment, D-amino acids are present only outside of the motif. In a still further embodiment, D-amino acids may be found in more than one domain of the oligopeptidic compound.
  • the oligopeptidic compound may be linear or cyclic, preferably linear.
  • Retro-inverso oligopeptidic compounds of the oligopeptidic compounds of the invention comprise D-amino acids in reverse (opposite) order to the parental or reference compound sequence.
  • a retro-inverso analogue thus has reversed termini and reversed order of e.g. peptide bonds, while approximately maintaining the topology of the side chains as in the parental or reference sequence.
  • the oligopeptidic compound may include partial retro-inverso sequences, i.e. a domain or part of a domain may comprise a retro-inverso sequence.
  • equivalent subunit is meant a subunit which is structurally and functionally similar to an amino acid.
  • the backbone moiety of the subunit may differ from a standard amino acid, e.g. it may incorporate one or more nitrogen atoms instead of one or more carbon atoms.
  • the subunit comprises a standard amino acid backbone, i.e. the backbone of a standard or coded amino acid.
  • the subunit is an amino acid.
  • the amino acid subunit may comprise a non-standard (non-coded) R-group.
  • peptidomimetic is meant a compound which is functionally equivalent or similar to a peptide and which can adopt a three-dimensional structure similar to its peptide counterparts, but which is not solely composed of amino acids linked by peptide bonds.
  • a preferred class of peptidomimetics are peptoids, i.e. /V-substituted glycines. Peptoids are closely related to their natural peptide counterparts, but they differ chemically in that their side chains are appended to nitrogen atoms along the molecule's backbone, rather than to the a-carbons as they are in amino acids.
  • Peptidomimetics particularly non-peptidic molecules may be generated through various processes, including conformational-based drug design, screening, focused library design and classical medicinal chemistry. Not only may oligomers of unnatural amino acids or other organic building blocks be used, but also carbohydrates, heterocyclic or macrocyclic compounds or any organic molecule that comprises structural elements and conformation that provides a molecular electrostatic surface that mimics the same properties of the 3-dimensional conformation of the peptide may be used by methods known in the art.
  • peptidomimetics may bear little or no resemblance to a peptide backbone.
  • Peptidomimetics may comprise an entirely synthetic non-peptide form (e.g. based on a carbohydrate backbone with appropriate substituents) or may retain one or more elements of the peptide on which it is based, e.g. by derivatizing one or more amino acids or replacing one or more amino acids with alternative non-peptide components.
  • Peptide-like templates include pseudopeptides and cyclic peptides. Structural elements considered redundant for the function of the peptide may be minimized to retain a scaffold function only or removed where appropriate.
  • peptidomimetics retain one or more peptide elements, i.e. more than one amino acid, although such amino acids may be replaced with a non-standard or structural analogue thereof.
  • Amino acids retained in the sequences may also be derivatised or modified (e.g. labelled, glycosylated or methylated) as long as the functional properties of the oligopeptidic compound are retained.
  • the peptidomimetics are referred to as being "derivable from" a certain polypeptide sequence. By this it is meant that the peptidomimetic is designed with reference to the peptide sequence defined above, such that it retains the structural features of the peptide which are essential for its function.
  • This may be the particular side chains of the peptide, or hydrogen bonding potential of the structure.
  • Such features may be provided by non-peptide components or one or more of the amino acid residues or the bonds linking said amino acid residues of the polypeptide may be modified so as to improve certain functions of the peptide such as stability or protease resistance, while retaining the structural features of the peptide which are essential for its function.
  • non-standard or structural analogue amino acids which may be used are D amino acids, amide isosteres (such as N-methyl amide, retro-inverse amide, thioamide, thioester, phosphonate, ketomethylene, hydroxymethylene, fluorovinyl, (E)-vinyl, methyleneamino, methylenethio or alkane), L-N methylamino acids, D-a methylamino acids, D-N-methylamino acids. Examples of non- conventional, i.e. non-coded, amino acids are listed in Table 2.
  • Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyric acid Abu L-N-methylalanine Nmala a-amino-a-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile
  • the oligopeptidic compound is a peptide.
  • the oligopeptidic compound is a peptide consisting of L-amino acids.
  • the oligopeptidic compound is a peptide consisting of standard or coded L-amino acids.
  • the oligopeptidic compound may comprise non standard amino acids.
  • the oligopeptidic compound may incorporate di-amino acids and/or b-amino acids.
  • at least the APIM motif domain consists of a-amino acids.
  • the oligopeptidic compound i.e. all domains and optionally all flanking sequences, consists of a-amino acids.
  • the oligopeptidic compound defined herein comprises at least 5 or 6 subunits.
  • the oligopeptidic compound comprises more than 5 or 6 subunits, but the length of the construct/compound will depend on the size of the uptake peptide sequence and on the number and size of other domains, e.g. linker domains, flanking sequences etc., if present.
  • the prefix "oligo” is used to designate a relatively small number of subunits such as amino acids, i.e. less than 200, preferably less than 150, 100, 90, 80, 70, 60 or 50 subunits.
  • the oligopeptidic compound of the invention may thus comprise more than 5 but no more than 200 subunits.
  • it comprises at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 subunits.
  • it comprises no more than 50, 45, 40, 35, 34, 33, 32, 31 or 30 subunits.
  • Representative subunit ranges thus include 12-50, 12-45, 12-40, 12-35, 12-30, 12-
  • the nature of the subunits of the oligopeptidic compound outside of the APIM motif domain and the uptake peptide sequence is not critical, so the subunits outside of the motif may for example be alanine residues or any other suitable residues.
  • Peptidomimetics typically have a longer half-life within a patient's body, so they may be preferred in embodiments where a longer lasting effect is desired. However, for bio-safety reasons a shorter half-life may be preferred in other embodiments; in those embodiments peptides are preferred.
  • bone cement refers to substances that are used in orthopaedic and trauma surgery to join or connect bone to other surfaces, such as bone or prosthetics, e.g. implants, such as artificial joints. Thus, bone cement is commonly used for implant fixation. However, bone cements typically do not have intrinsic adhesive properties and instead function as a space-fillers to generate a close mechanical interlock between the irregular bone surfaces and/or bone surface and a prosthesis.
  • bone cement typically refers to polymethylmethacrylate (PMMA) bone cement.
  • PMMA based bone cements are well-known in the art and were first used in joint replacement surgery in the 1960s in the anchorage of a femoral stem prosthesis to the shaft of a femur (J. Bone Joint Surg. 42 (1960) 28-30).
  • PMMA bone cements comprise a liquid monomer component and a powder component.
  • the monomer component generally contains the monomer, methylmethacrylate, and an accelerator dissolved therein.
  • the powder component also referred to as bone cement powder, typically comprises one or more polymers, such as PMMA, a radio-opacifier, and an initiator.
  • PMMA polymers
  • the radical polymerisation of the methylmethacrylate is initiated by radicals that are formed during the mixing process. As polymerisation of the methylmethacrylate advances the viscosity of the cement dough increases until the cement dough solidifies.
  • PMMA may be categorised as a polymerisable bone cement.
  • CPCs calcium phosphate cements
  • GPCs Glass polyalkenoate (ionomer) cements
  • the oligopeptidic compound defined herein may be used in the preparation of any type of bone cement.
  • the bone cement of the present invention is a polymerisable bone cement.
  • the bone cement is a PMMA bone cement, i.e. the bone cement comprises PMMA.
  • components for producing bone cement and “means for producing bone cement” refer to the ingredients that are required to produce a bone cement when combined (i.e. mixed) under suitable conditions.
  • the components or ingredients that, when mixed in the appropriate ratios under suitable conditions, react to produce a material that can be used as bone cement, i.e. a material or substance (e.g. a dough) that can be shaped plastically and subsequently hardens to a provide a substance that mimics the physical properties of natural bone.
  • the components for producing bone cement may be provided in two-part preparations comprising a solid (dry) component and a liquid component. When the solid and liquid components are combined they react (e.g. polymerize) to form a hardened substance mimicking many of the physical properties of natural bone.
  • the solid component may be in the form of a powder comprising a polymeric material.
  • the powder contains an acrylate polymer.
  • the powder contains PMMA, such as styrene methyl methacrylate copolymer.
  • the particle size of the powder may depend on the utility of the bone cement.
  • the particle size of the powder may be in the micron range, e.g. 1- 100 microns.
  • the particle size of the powder may be in the submicron range, i.e. less than 1.0 microns, such as 0.9 microns or less.
  • the use of particles in the submicron range may function to fill any voids left in the bone cement composition to produce stronger bone cement.
  • the liquid component of the bone cement includes a reactive monomer, such as methylmethacrylate.
  • a PMMA comprising bone cement
  • Other components that may be used for producing bone cement include an initiator, an accelerator, a stabilizer and a radio-opacifer.
  • the term “initiator” refers to a component (chemical species) in the bone cement mixture that reacts with the reactive monomer to form an intermediate compound capable of linking successively with a large number of other monomers into a polymeric compound.
  • the initiator is provided as part of the solid (dry) component of the bone cement (e.g. the powder containing the polymeric material, e.g. acrylate polymer).
  • the solid (dry) part of the bone cement components comprises a polymeric material and an initiator. Any suitable initiator may be used in the bone cement preparations of the invention.
  • the initiator comprises benzoyl peroxide.
  • the term “accelerator” refers to a component (chemical species) in the bone cement mixture that functions to encourage the polymer and monomer to polymerise.
  • An accelerator may be included in the components for the bone cement to promote the polymerisation to occur at room temperature, i.e. to provide a cold curing cement.
  • the accelerator is provided as part of the liquid part of the bone cement components.
  • the liquid component of the bone cement preparation comprises a reactive monomer and an accelerator.
  • the accelerator may be provided as a separate component, e.g. a separate liquid component, to be added when the polymeric material and reactive monomer are combined.
  • the liquid and solid components may be combined in any suitable order and determining the order is within the ability of the skilled person.
  • the accelerator may be combined with the liquid reactive monomer prior to combining the liquid and solid components.
  • Any suitable accelerator may be used in the bone cement preparations of the invention.
  • the accelerator comprises N, N-Dimethyl para-toluidine and/or dimethyl para-toluidine.
  • stabilizer refers to a component (chemical species) in the reactive monomer component that functions to prevent or inhibit polymerisation of the monomer before it is combined with the solid component comprising the polymer.
  • a stabilizer may alternatively be termed an “inhibitor”.
  • exposure of the monomer to light and/or heat may cause premature polymerisation of the monomer and the stabilizer inhibits the polymerisation reaction under these conditions.
  • the stabilizer is present, it is provided as part of the liquid part of the bone cement components.
  • the liquid part of the bone cement components comprises a reactive monomer, a stabilizer and optionally an accelerator. Any suitable stabilizer may be used in the bone cement components of the invention.
  • the stabilizer comprises hydroquinone.
  • radio-opacifier refers to a component (chemical species) in the bone cement mixture that functions to make the bone cement visible under radio (e.g. x-ray) imaging, i.e. to make the bone cement radioopaque.
  • a radio- opacifier may also be referred to as a “contrast agent” and is any physiologically acceptable chemical species with high absorption of x-rays, e.g. particles or compounds of barium (e.g. barium sulfate), tungsten, or other high atomic weight elements.
  • the radio-opacifier is provided as part of the solid part of the bone cement components.
  • the solid part (e.g. powder) of the bone cement component comprises a polymeric material, a radio-opacifier and optionally an initiator.
  • the radio-opacifier comprises barium sulfate and/or zirconium dioxide.
  • various components for producing the bone cement may be provided separately, e.g. as separate solid (powder) components and/or separate liquid components.
  • Providing the ingredients for each component separately may provide the end user with flexibility to omit ingredients that are not required for particular uses, e.g. additional therapeutically active agents as described below.
  • the components for producing bone cement according to the invention comprise:
  • a solid component e.g. a powder
  • a polymeric material as defined above
  • the solid component further comprises an initiator and/or radio-opacifier as defined above, preferably both.
  • the liquid component further comprises a stabilizer and/or accelerator, preferably both.
  • the solid component may contain further components (e.g. ingredients) depending on the utility of the bone cement.
  • the solid component may comprise one or more fluoride salts.
  • the addition of fluoride salts to the bone cement functions to produce a stronger bond between the bone cement and the bone of the patient.
  • the solid component may comprise one or more calcium salts, e.g. calcium hydroxide or calcium acetate.
  • the addition of calcium salts to the bone cement functions to improve the biocompatibility of the bone cement and promote the formation of hydroxyapatite, thereby promoting the formation of a bond between the bone and the bone cement.
  • these additional components may be provided as part of the solid component (i.e. mixed with the polymeric material), in some embodiments, these additional components may be provided as separate solid components that can be mixed with the polymeric material by the end user.
  • the oligopeptidic compound may be provided in any suitable form for use in the production of a bone cement.
  • the oligopeptidic compound may be provided as a solid, e.g. powder, such as in the form of a salt as defined herein. This solid form of the oligopeptidic compound may be provided as part of the bone cement powder, e.g. combined with the polymeric material.
  • the oligopeptidic compound may be provided separately to the other components for producing the bone cement. When the oligopeptidic compound is provided in the kit separately to the other components for producing the bone cement (particularly separately to the bone cement powder), it may be mixed with the other solid components prior to combining the solid and liquid components.
  • the oligopeptidic compound may be dissolved, e.g. in water, and added to the liquid component of the bone cement.
  • the oligopeptidic compound may be provided in the kit as part of the liquid component, i.e. it may be dissolved in the liquid component of the bone cement, e.g. combined with the reactive monomer.
  • the oligopeptidic compound may be provided as a separate liquid component, e.g. a solution, which may be combined with the other components of the bone cement in any suitable order.
  • the oligopeptidic compound solution provided in the kit is combined with the other liquid component(s) of the bone cement prior to combining the solid and liquid components of the bone cement.
  • the components for producing the bone cement further comprise one or more additional therapeutically active agents, e.g. in order to enhance or complement the effect of oligopeptidic compound defined herein, i.e. to improve the antibacterial properties of the bone cement.
  • additional agents may be provided as part of the solid or liquid components of the bone cement or separately thereof and will depend on the nature of the agent.
  • the additional therapeutically active agent may be used to prevent a bacterial infection or treat an existing bacterial infection.
  • the additional therapeutically active agent may be used to treat symptoms of the bacterial infection, e.g. secondary symptoms, and such agents may be, e.g. an anti-inflammatory compound, steroid (e.g. a corticosteroid) etc.
  • the additional therapeutically active agent may be used to treat or ameliorate symptoms or side effects associated with the surgery involving the use of the bone cement.
  • the oligopeptidic compound as defined herein may the only therapeutically active agent in the bone cement.
  • the additional therapeutically active agent is an agent that has been indicated for an anti-bacterial application.
  • the additional therapeutically active agent is an antibiotic.
  • the bone cement of the invention may be viewed as an antibiotic-loaded bone cement (ALBC).
  • ALBC antibiotic-loaded bone cement
  • the kit, use and method of the invention may be for the preparation of an ALBC.
  • Suitable antibiotic agents include but are not limited to any one or more of Aminocoumarins (such as Novobiocin, Albamycin, Coumermycin and Clorobiocin), Aminoglycosides (such as Amikacin, Apramycin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin and Spectinomycin), Ansamycins (such as Geldanamycin, Herbimycin, Rifaximin and Streptomycin), Carbapenems (such as Ertapenem, Doripenem, Cilastatin ('Imipenem') and Meropenem), Cephalosporins (such as Cefadroxil, Cefazolin, Cefalothin ('Cefalotin'), Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefoperazone, Cefotaxime
  • Doxycycline, Minocycline, Oxytetracycline and Tetracycline) and Others such as Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin ('Rifampin'), Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Platensimycin, Guinupristin (Dalfopristin), Thiamphenicol, Tigecycline, Tinidazole and Trimethoprim).
  • Others such as Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin ('Rifampin'), Rifabutin, Rifapentine, Strepto
  • the antibiotic is an aminoglycoside, preferably gentamicin.
  • the antibiotic is provided as part of the solid component of the bone cement, e.g. the bone cement powder comprises the polymeric material and the antibiotic.
  • the invention provides kit for producing an antibacterial bone cement comprising:
  • an oligopeptidic compound comprising a PCNA interacting motif consisting of an amino acid sequence as set forth in SEC ID NO:28 or 1206; and optionally
  • an antibiotic preferably an aminoglycoside, such as gentamicin.
  • the invention provides the use of an oligopeptidic compound comprising a PCNA interacting motif consisting of an amino acid sequence as set forth in SEC ID NO:28 or 1206 in the preparation of an antibacterial PMMA bone cement.
  • the PMMA bone cement is an ALBC comprising an antibiotic, preferably an aminoglycoside, such as gentamicin.
  • the invention provides a method of preparing an antibacterial bone cement comprising:
  • components e.g. separate solid and liquid components
  • one of the components comprises an oligopeptidic compound comprising a PCNA interacting motif consisting of an amino acid sequence as set forth in SEC ID NO:28 or 1206;
  • the invention provides a method of preparing an antibacterial bone cement comprising:
  • the PMMA bone cement is an ALBC comprising an antibiotic, preferably an aminoglycoside, such as gentamicin.
  • the one or more components for producing a PMMA bone cement includes an antibiotic, preferably an aminoglycoside, such as gentamicin.
  • a further embodiment of the invention is an antibacterial PMMA bone cement comprising an oligopeptidic compound comprising a PCNA interacting motif consisting of an amino acid sequence as set forth in SEQ ID NO:28 or 1206.
  • PMMA bone cement is an ALBC comprising an antibiotic, preferably an aminoglycoside, such as gentamicin.
  • the oligopeptidic compound comprising a PCNA interacting motif consisting of an amino acid sequence as set forth in SEQ ID NO:28 or 1206 is selected from the oligopeptidic compounds defined above that comprise SEQ ID NO: 28 or 1206, e.g. oligopeptidic compounds also containing a domain that facilitates the cellular uptake of the compound, such as a CPP (e.g. a CPP comprising an amino acid sequence as set forth in SEQ ID NO: 334, 336, 337 or 1623).
  • a CPP e.g. a CPP comprising an amino acid sequence as set forth in SEQ ID NO: 334, 336, 337 or 1623.
  • the oligopeptidic compound may be combined with the other components for the production (i.e. preparation) of the bone cement in any suitable form or order.
  • the method of preparing an antibacterial bone cement comprises:
  • components e.g. separate solid and liquid components
  • one of the components comprises an oligopeptidic compound comprising a PCNA interacting motif
  • the method of preparing an antibacterial bone cement comprises:
  • an oligopeptidic compound comprising a PCNA interacting motif with one or more components for producing bone cement (e.g. the solid or liquid component, preferably the liquid component, such as the reactive monomer); and
  • the oligopeptidic compound may be provided as a solid, e.g. powder, such as in the form of a salt as defined herein.
  • the step of combining an oligopeptidic compound with one or more components for producing bone cement comprises mixing the oligopeptidic compound with the bone cement powder, i.e. the powder comprising the polymeric material.
  • the step of combining an oligopeptidic compound with one or more components for producing bone cement comprises dissolving the oligopeptidic compound in a solvent (e.g. water) to provide a solution and mixing the solution containing the oligopeptidic compound with the liquid component of the bone cement, i.e. the liquid comprising the reactive monomer.
  • a solvent e.g. water
  • the step of dissolving the oligopeptidic compound in a solvent (e.g. water) to provide a solution is omitted.
  • the step of combining the oligopeptidic compound with one or more components for producing bone cement may utilise any means suitable to mix the oligopeptidic compound with the one or more components.
  • the step may use any means suitable to distribute the oligopeptidic compound within the one or more components, e.g. uniformly mix the components.
  • the combining step results in a homogeneous mixture of the components.
  • the step of combining the components to produce a bone cement may utilise any means suitable to produce a bone cement in which the oligopeptidic compound is distributed throughout the bone cement, e.g. uniformly distributed.
  • the combining step results in a homogeneous bone cement.
  • the components of the bone cement may combined (mixed) in any suitable order.
  • two or more of the solid and/or liquid components are provided separately (e.g. in the kit)
  • it is preferred that all of the solid components are combined to produce a single solid component, e.g. bone cement powder, and all of the liquid components are combined to produce a single liquid component, prior to combining the solid and liquid components to form the bone cement.
  • Bone cement components are typically mixed at room temperature, e.g. 18-25°C, preferably about 23°C.
  • the process of combining the components of bone cement to produce the bone cement may be termed “curing”.
  • the curing process may be split into four stages: a) mixing, b) sticky/waiting, c) working, and d) hardening.
  • the mixing step of the curing process can be done by hand or with the aid of centrifugation or vacuum technologies.
  • any deviation from the recommended temperature for mixing the bone cement components will affect the handling characteristics and setting time of the cement. For instance, manual handling and body temperature reduces the final setting time. Variations in humidity also affect the cement handling characteristics and setting time. Vacuum mixing of the cement components can also accelerate the setting time of the cement.
  • pre-chill the components e.g. below room temperature
  • This will also increase the setting time.
  • the ratio of each of the components of the bone cement may be adjusted according to the utility of the bone cement, e.g. to produce a bone cement of the appropriate strength and/or consistency. Such adjustments are within the purview of the skilled person.
  • the oligopeptidic compound may be combined with the other bone cement components to provide a bone cement with a final concentration of oligopeptidic compound of about 0.1-10% w/w, such as about 0.2- 9.5% w/w, about 0.3-9.0% w/w, about 0.4-8.5% w/w, about 0.5-8.0% w/w, about 1.0-7.5% w/w, about 12-7.0% w/w or about 15-6.5% w/w.
  • the final concentration of oligopeptidic compound in the bone cement is about 1.5- 5.0% w/w, e.g. about 1.5, 2.0, 2.5, 3.0, 3.5 or 4.5 w/w.
  • the bone cement (or bone cement components) also comprises an additional therapeutically active agent, e.g. an antibiotic such as gentamicin
  • the agent may be combined with the other bone cement components to provide a bone cement with a final concentration of agent as defined above.
  • the combined concentration of the oligopeptidic compound and agent in the bone cement is within the range defined above.
  • all of the components of the bone cement are sterile.
  • the inventors have shown that the bone cement of the invention is able to inhibit bacterial growth on the bone cement and on the surrounding bone.
  • the inventors have also shown that the bone cement reduces biofilm formation and inhibits the development of antibiotic resistance in infectious bacteria.
  • the bone cement of the invention finds utility in preventing bacterial infections, e.g. preventing orthopaedic infections following a surgical procedure or intervention, and in treating bacterial infections, e.g. treating an existing orthopaedic infection that has necessitated the replacement of a prosthetic (i.e. a revision).
  • the invention provides an antibacterial bone cement described above for use in therapy.
  • the invention provides an antibacterial bone cement described above for use in surgery, e.g. bone repair surgery, e.g. bone graft surgery or prosthetic joint surgery.
  • Bone repair surgery may involve applying bone cement to a bone fracture.
  • bone cement may be applied, e.g. injected, to a fractured vertebrae to fill gaps in the bone caused by the fracture, which may stabilise and/or facilitate the healing of the vertebrae.
  • Bone repair surgery may also refer to bone grafts in which a missing piece of bone is replaced in order to repair a damaged bone, e.g. a bone fracture.
  • the bone graft may be autologous (i.e. using bone harvested from the patient’s own body, e.g. from the iliac crest), an allograft (e.g. using a cadaveric bone), or synthetic (e.g. using a biocompatible substance, such as hydroxyapatite).
  • the bone cement may be applied to the bone graft material and/or bone, which are subsequently contacted together.
  • Prosthetic joint surgery or joint replacement surgery refers to a procedure of orthopaedic surgery in which joint surface is replaced with an orthopaedic prosthesis.
  • the joint replacement is used to replace an arthritic (e.g. caused by osteoarthritis or rheumatoid arthritis) or dysfunctional joint that causes severe joint pain or dysfunction and is not alleviated by less-invasive therapies.
  • Prosthetic joint surgery typically involves the replacement of a hip or knee joint with a prosthetic joint (implant).
  • other joints that may be replaced with a prosthetic include elbow, wrist, ankle, shoulder, finger and toe joints.
  • the antibacterial bone cement of the invention is used in hip, knee, elbow, wrist, ankle, shoulder, finger or toe prosthetic joint surgery.
  • the prosthetic joint used in the methods of the invention may be a hip, knee, elbow, wrist, ankle, shoulder, finger or toe prosthetic joint.
  • the antibacterial bone cement of the invention may be used to prevent a bacterial infection following surgery, e.g. bone repair surgery, such as bone graft surgery or prosthetic joint surgery.
  • the antibacterial bone cement of the invention may be used to prevent a bone graft or prosthetic joint bacterial infection.
  • the antibacterial bone cement of the invention may be used to prevent or inhibit the formation of a bacterial biofilm, e.g. a biofilm on the bone graft or prosthetic joint.
  • Implant infections are a serious clinical problem and, in some circumstances, necessitate revision surgery, i.e. replacement of the implant.
  • the antibacterial bone cement of the invention may find particular utility in revision surgery, particularly where the surgery is needed due to a prosthetic joint infection, e.g. a chronic infection of the prosthetic.
  • the antibacterial bone cement of the invention may be used to treat a bacterial infection, e.g. bone graft infection or prosthetic joint infection.
  • the antibacterial bone cement of the invention may be used to treat a chronic bacterial infection.
  • the invention provides a method of bone repair surgery comprising using an antibacterial bone cement of the invention to repair a bone in a subject.
  • Using the antibacterial bone cement may involve applying the bone cement to a fractured bone (e.g. to fill a gap in the bone), applying the bone cement to a bone graft and/or bone in the subject (e.g. a damaged or fractured bone) that are subsequently contacted together and/or applying the bone cement to a prosthetic (e.g. a prosthetic joint) and/or bone (e.g. a damaged or fractured bone) in the subject that are subsequently contacted together.
  • a prosthetic e.g. a prosthetic joint
  • bone e.g. a damaged or fractured bone
  • the invention provides a method of preventing a bacterial infection (e.g. an orthopaedic infection) in a subject (e.g. following surgery, e.g. bone repair surgery, such bone graft surgery or prosthetic joint surgery), comprising applying or administering an antibacterial bone cement of the invention to a subject in an effective amount to prevent a bacterial infection (e.g. a bone graft or prosthetic joint bacterial infection).
  • a bacterial infection e.g. a bone graft or prosthetic joint bacterial infection
  • the method involves preventing or inhibiting the formation of a bacterial biofilm, e.g. a biofilm on the bone graft or prosthetic joint.
  • applying or administering the bone cement may involve applying the bone cement to a fractured bone (e.g.
  • a prosthetic e.g. a prosthetic joint
  • a bone e.g. a damaged or fractured bone
  • a prosthetic joint e.g. a prosthetic joint
  • the invention provides a method of treating a bacterial infection (e.g. an orthopaedic infection) in a subject (e.g. following surgery, e.g. bone repair surgery, such bone graft surgery or prosthetic joint surgery), comprising applying or administering an antibacterial bone cement of the invention to a subject in an effective amount to treat the bacterial infection (e.g. the bone graft or prosthetic joint bacterial infection).
  • applying or administering the bone cement may involve applying the bone cement to a fractured bone (e.g. to fill a gap in the bone), applying the bone cement to a bone graft and/or bone in the subject (e.g.
  • a damaged or fractured bone that are subsequently contacted together and/or applying the bone cement to a prosthetic (e.g. a prosthetic joint) and/or bone (e.g. a damaged or fractured bone) in the subject that are subsequently contacted together.
  • a prosthetic e.g. a prosthetic joint
  • bone e.g. a damaged or fractured bone
  • the bone cement may be applied or administered using any suitable means.
  • the bone cement may be applied to the appropriate surface (e.g. bone, bone graft or prosthetic) digitally or with a device, e.g. a cement gun.
  • a device e.g. a cement gun.
  • it may be advantageous to pressurize the bone cement to facilitate its application to the appropriate surface, e.g. to reduce porosity.
  • the oligopeptidic compounds defined herein are capable of eradicating (i.e. eliminating, removing, combating etc.) an existing or established bacterial biofilm, e.g. such as is found in a prosthetic joint infection. This has led the inventors to contemplate a further utility of the oligopeptidic compounds, i.e. the treatment of prosthetic joint infections, such as chronic prosthetic joint infections.
  • the present invention provides a method of treating or preventing a prosthetic joint infection, said method comprising administering (particularly administering an effective amount of) an oligopeptidic compound comprising a PCNA interacting motif as defined herein or a composition (e.g. a pharmaceutical composition comprising the oligopeptidic compound) to a subject in need thereof.
  • the invention provides an oligopeptidic compound comprising a PCNA interacting motif as defined herein or a composition (e.g. a pharmaceutical composition comprising the oligopeptidic compound, particularly comprising an effective amount thereof), for use in treating or preventing a prosthetic joint infection.
  • a composition e.g. a pharmaceutical composition comprising the oligopeptidic compound, particularly comprising an effective amount thereof, for use in treating or preventing a prosthetic joint infection.
  • an oligopeptidic compound comprising a PCNA interacting motif as defined herein in the manufacture of a medicament for the treatment or prevention of a prosthetic joint infection.
  • the oligopeptidic compound may be used in combination with one or more additional active (e.g. therapeutic) agents, e.g. an antibiotic as defined above.
  • additional active e.g. therapeutic
  • the composition containing the oligopeptidic compound may further comprise one or more additional active agents.
  • the combination therapy may be in the form of a single composition comprising both the oligopeptidic compound as defined herein and the one or more additional active agents, e.g. an antibiotic, or it may be in the form of a kit or product containing them for separate (e.g. simultaneous or sequential) administration.
  • additional active agents e.g. an antibiotic
  • the invention provides a method of treating or preventing a prosthetic joint infection, said method comprising administering an oligopeptidic compound or composition as defined herein, and separately, simultaneously or sequentially administering of one or more additional active agents, e.g. an antibiotic, to a subject in need thereof.
  • additional active agents e.g. an antibiotic
  • the invention provides an oligopeptidic compound or composition as defined herein and one or more additional active agents, e.g. an antibiotic, as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of a prosthetic joint infection.
  • the invention provides the use of an oligopeptidic compound or composition as defined herein in the manufacture of a medicament for the treatment or prevention of a prosthetic joint infection, wherein the medicament is administered separately, simultaneously or sequentially with one or more additional active agents, e.g. an antibiotic.
  • additional active agents e.g. an antibiotic.
  • prosthetic joint infection refers to any bacterial infection affecting the site of an orthopaedic implant and/or the adjacent tissue. This includes bacterial infections which are developed following any orthopaedic surgery, e.g. bone repair surgery, bone graft surgery or prosthetic joint surgery, such as bone graft infections or orthopaedic infections.
  • osteopaedic surgery e.g. bone repair surgery, bone graft surgery or prosthetic joint surgery, such as bone graft infections or orthopaedic infections.
  • chronic prosthetic joint infections represent as serious problem due to the difficulty in eradicating biofilms.
  • the oligopeptidic compound or composition of the invention finds utility in the treatment of chronic prosthetic joint infections.
  • composition refers to a composition comprising the oligopeptidic compound together with at least one pharmacologically (or pharmaceutically) acceptable carrier or excipient.
  • the excipient may include any excipients known in the art, for example any carrier or diluent or any other ingredient or agent such as buffer, antioxidant, chelator, binder, coating, disintegrant, filler, flavour, colour, glidant, lubricant, preservative, sorbent and/or sweetener etc.
  • any carrier or diluent or any other ingredient or agent such as buffer, antioxidant, chelator, binder, coating, disintegrant, filler, flavour, colour, glidant, lubricant, preservative, sorbent and/or sweetener etc.
  • the excipient may be selected from, for example, lactic acid, dextrose, sodium metabisulfate, benzyl alcohol, polyethylene glycol, propylene glycol, microcrystalline cellulose, lactose, starch, chitosan, pregelatinized starch, calcium carbonate, calcium sulfate, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, powdered cellulose, sodium chloride, sorbitol and/or talc.
  • the composition may be provided in any suitable form known in the art, for example as a tablet, capsule, coated tablet, liquid, suspension, solution, tab, sachet, implant, inhalant, powder, pellet, emulsion, lyophilisate, effervescent, spray, salve, emulsion, balm, plaster or any mixtures thereof. It may be provided e.g. as a gastric fluid-resistant preparation and/or in sustained action form.
  • the composition may be administered to the subject using any suitable means, and the route of administration will depend on formulation of the pharmaceutical composition. In some embodiments, the composition may be administered locally.
  • “Local administration” includes any form of administration in which the composition is administered directly to, or to the area in the local vicinity of or adjacent to, the site of the prosthetic joint infection.
  • the agent may be administered by topical, subcutaneous, transdermal, intramuscular and/or intra-articular administration.
  • the composition is administered by intra-articular administration, e.g. by injection.
  • composition may be administered systemically.
  • Systemic administration includes any form of non-local administration in which the agent is administered to the body at a site other than directly adjacent to, or in the local vicinity of the prosthetic joint infection, resulting in the whole body receiving the administered composition.
  • systemic administration is by parenteral delivery (e.g. intravenous, intraperitoneal, intramuscular, or subcutaneous).
  • systemic administration is by enteral delivery, e.g. orally.
  • the effective amount of the oligopeptidic compound may depend on the method of administration, the size and nature of the prosthetic joint infection to be treated, and on the age and weight of the patient (i.e. subject). It will be seen that the amount of the oligopeptidic compound which is administered may be varied or adjusted according to choice. For example, where the oligopeptidic compound is administered systemically it may be necessary to use an increased amount of the oligopeptidic compound, relative to the amount that would be used for local administration.
  • an effective amount of the oligopeptidic compound is an amount which results in the effective treatment or prevention of the prosthetic joint infection (e.g. eradication or elimination of the infection), and will be capable of calculating such an effective amount.
  • the amount of the oligopeptidic compound which is administered may be defined based on the minimal inhibitory concentration (MIC), e.g. as determined in the Examples.
  • MIC minimal inhibitory concentration
  • an effective amount of the oligopeptidic compound may be at least 1x, 2x, 3x, 4x, 5x, or 10x the MIC.
  • treatment refers broadly to any effect or step (or intervention) beneficial in the management of a clinical condition or disorder. Treatment may include reducing, alleviating, ameliorating, slowing the development of, or eliminating the condition or one or more symptoms thereof, which is being treated, relative to the condition or symptom prior to the treatment, or in any way improving the clinical status of the subject.
  • a treatment may include any clinical step or intervention which contributes to, or is a part of, a treatment programme or regimen.
  • preventing is used to refer to a prophylactic treatment, which may include delaying, limiting, reducing or preventing the condition or the onset of the condition, or one or more symptoms thereof, for example relative to the condition or symptom prior to the prophylactic treatment.
  • Prophylaxis thus explicitly includes both absolute prevention of occurrence or development of the condition, or symptom thereof, and any delay in the onset or development of the condition or symptom, or reduction or limitation on the development or progression of the condition or symptom.
  • treatment includes killing, inhibiting or slowing the growth of bacterial cells, or the increase in size of a body or population of bacterial cells, reducing bacterial cell number or preventing the spread of bacterial cells (e.g. to another anatomic site), reducing the size of a bacterial cell colony or infection site etc.
  • treatment does not necessarily imply the cure or complete abolition or elimination of bacterial cell growth, or growth of bacterial cells.
  • treatment results in the eradication or elimination of a prosthetic joint infection. Eradication or elimination refers to killing the majority of bacterial cells at the infection site, e.g. reducing the number of bacterial cells (e.g. in a sample (e.g. tissue, blood sample) taken from the site of the infection) to undetectable levels, e.g. using detection means well known in the art, e.g. PCR.
  • inhibitor is used broadly to include any reduction or decrease in bacterial cell growth as well as the prevention or abolition of bacterial cell growth. “Inhibition” thus includes the reduction or prevention of bacterial cell growth, e.g. including reducing the rate of cell growth. This may be determined by any appropriate or convenient means, such as determining or assessing cell number, cell viability and/or cell death etc., as may be determined by techniques well known in the art.
  • “Growth” of bacterial cells as referred to herein is also used broadly to include any aspect of bacterial cell growth, including in particular the proliferation (i.e. increase in number) of bacterial cells.
  • the antibacterial bone cement of the invention may thus be used in the treatment or prevention of any bacterial infection, particularly an orthopaedic infection, which may be a disease or condition (used broadly herein to include any disorder or any clinical situation) which is responsive to reduction of bacterial cell growth (particularly bacterial cell proliferation).
  • the bone cement accordingly finds utility in any therapy (or treatment, e.g. prophylactic treatment) which targets bacterial cell growth (or proliferation).
  • the bone cement may be used in any therapeutic application in which it desirable or advantageous to inhibit bacterial cell proliferation.
  • oligopeptidic compound defined herein may be used in the treatment or prevention of any prosthetic joint bacterial infection as defined herein.
  • a "bacterial infection” may be defined as any atypical, unwanted, undesirable, excessive and/or harmful infection and may be defined as a disease, condition or disorder caused by the invasion of a subject, e.g. one or more organs or tissues (e.g. joints) of said subject, by one or more bacteria and their subsequent multiplication.
  • an infection may be characterised by the reaction of the subject (e.g. organ or tissues of said subject) to said organisms and, in some cases, to the toxins produced by said organisms.
  • a bacterial infection may be local or systemic.
  • the bone cement may be used to treat or prevent a local bacterial infection, e.g. a prosthetic joint infection.
  • any proliferating bacterial cell may be targeted in the therapies and utilities disclosed and encompassed herein.
  • the bacteria may be a gram positive or gram negative, or gram test non-responsive. They may be aerobic or anaerobic bacteria.
  • the bacteria may be from the genus Acinetobacter, Bacillus, Burkholderia, Chlamydia, Clostridium, Escherichia, Enterococcus, Helicobacter, Staphylococcus, Streptococcus, Pseudomonas, Legionella, Listeria, Micrococcus, Mycobacterium, Proteus, Klebsiella, Fusobacterium or other enteric or coliform bacteria.
  • the bacterium is from the genus Staphylococcus.
  • the bacterial infection consists of or comprises a Staphylococcus infection.
  • the bacterial infection is caused by Staphylococcus aureus or Staphylococcus epidermidis.
  • the bacterium is a Multidrug resistant (MDR) bacterium.
  • Multidrug resistance in bacteria describes the situation where a bacterium is resistant to at least three classes of drugs, specifically in the context of bacteria, at least three classes of anti-bacterial agents.
  • Antibiotics in one class are functionally unrelated, structurally unrelated, or both, to antibiotics in a different class.
  • MDR in bacteria is thus often termed multiple anti-bacterial drug resistance or multiple antibiotic resistance.
  • Bacteria displaying multidrug resistance phenotypes are referred to as MDR bacteria (or sometimes MAR bacteria). Again, these terms are used interchangeably in the art and herein.
  • the MDR bacterium is a Methicillin-resistant Staphylococcus aureus (MRSA) bacterium or a Methicillin-resistant Staphylococcus epidermidis (MRSE) bacterium.
  • MRSA Methicillin-resistant Staphylococcus aureus
  • MRSE Methicillin-resistant Staphylococcus epidermidis
  • the MDR bacterial infection is an MRSA or MRSE infection.
  • the concentration of the oligopeptidic compound in the bone cement may be defined as a concentration that is sufficient, either alone or in combination with an additional therapeutically active agent (e.g. an antibiotic), to inhibit bacterial cell growth (in vivo or in vitro).
  • a suitable concentration may be defined as a concentration that is sufficient, either alone or in combination with an additional therapeutically active agent to kill the majority of the bacterial cells causing, or associated with, the infection.
  • a suitable concentration may be defined as a concentration that is sufficient to sensitize a bacterium to an antibiotic (e.g. gentamicin), wherein contacting the bacterium with (e.g. treatment with or administration of) a bone cement of the invention further comprising an antibiotic is sufficient to inhibit bacterial cell growth (e.g. in vivo or in vitro) and/or sufficient to kill the majority of the bacterial cells causing, or associated with, the infection.
  • a suitable concentration may be defined as a concentration that does not induce apoptosis in animal cells significantly, i.e. an apoptosis non-inducing concentration for animal cells, particularly human cells.
  • the "majority of cells” may be defined as at least 50% of the bacterial cells, e.g. at least 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of the cells, e.g. cells causing, or associated with, the infection.
  • a concentration that does not induce apoptosis in animal cells significantly may be viewed as a concentration that causes or induces apoptosis of less than 20% of the animal cells in the target area, e.g. less than 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% of the animal cells in the target area.
  • the target area is area to which the bone cement is administered (i.e. in the local vicinity of the bone cement).
  • the effective amount of bone cement may depend on the characteristics of the oligopeptidic compound, e.g. the strength of the interaction between the PCNA interacting motif and the binding domain of the target protein(s), how efficiently it is eluted from the bone cement. Furthermore, the effective amount may depend upon the age and weight of the patient (i.e. subject), the bacterial infection or the body area to be treated and may be varied or adjusted according to choice.
  • the subject is an animal (i.e. any human or non-human animal), preferably a mammal, most preferably a human.
  • antibacterial refers to the ability to kill bacteria or suppress their growth or their ability to reproduce.
  • the antibacterial bone cement of the invention may be used to prevent a bacterial infection, e.g. an orthopaedic infection. Accordingly, in some embodiments, the invention may be seen to provide an anti-infective bone cement.
  • prosthetic refers to an artificial body part, particularly an artificial body part that is implanted in the body, e.g. a prosthetic joint.
  • a prosthetic may be seen as referring to any prosthetic (implant) that requires the use of bone cement to secure it within the body, e.g. to adhere the prosthetic to bone.
  • prosthetic and implant may be used interchangeably herein.
  • Figure 1 shows the results of a time killing assay showing rapid killing with addition of APIM-peptide on MRSE cultures.
  • Antibiotic treatments APIM- peptide/gentamicin/vancomycin
  • Figure 3 demonstrates that APIM-peptide inhibits gentamicin resistance development of a gentamicin sensitive S. epidermidis.
  • Figure 4 shows that APIM-peptide is eluted from bone cement and inhibits bacteria growth in vitro.
  • A To examine if APIM-peptide is eluted from bone cement, cement pellets containing no treatment (control) or APIM-peptide were incubated in 100 pl_ water for 15 minutes or 1 hour and then the water was added to MRSE cultures containing. MRSE cultures were also added cement pellet directly (pellet in culture). MRSE cultures (2ml_) were grown for 24 hours.
  • B-C MRSE bacterial load in infected bone grafts filled with bone cement containing APIM-peptide, gentamicin or a combination. Control bone grafts contain bone cement without treatment. 24 hours after infection the bone grafts (B) and the cement inside (C) were separated and bacterial load was quantified. Student two tailed paired t-test, **p>0.01. Detection limit: 33.3 CFU/mL.
  • Figure 5 shows the antibacterial effect of APIM-peptide and gentamicin in bone cement on MRSE in bone grafts.
  • Control bone grafts contain cement without antibiotics.
  • the bone cement (B) inside the bone graft was removed and bacterial load was quantified separately in bone and cement after sonication in saline (0.9% NaCI).
  • Figure 7 shows the results of time killing assays with MRSE cultures treated with APIM-peptide, vancomycin or gentamicin. Addition of APIM-peptide, vancomycin or gentamicin was conducted at time point 0. The control was added the corresponding amount of water. The dotted horizontal line shows the detection limit of CFU/mL.
  • Figure 8 shows CFU determination of biofilms established by growing ATCC 35984 for 5 days on steel rods 1 , 6, and 24 hours after APIM-peptide (or R11 peptide) addition, plotted as an average from three independent experiments. Two- tailed, unpaired student t-test at 24-hour timepoint, **p ⁇ 0.01, ***p ⁇ 0.001,
  • Example 1 The combination of APIM-peptide and gentamicin shows an additive effect on the growth of both gentamicin resistant and susceptible strains of S. epidermidis
  • Table 1 shows minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) for APIM-peptide and gentamicin alone and in combination, in addition to fractional inhibitory concentration (FIC) for combined values.
  • MIC minimal inhibitory concentration
  • MBC minimal bactericidal concentration
  • FIC fractional inhibitory concentration
  • the fractional inhibitory concentration (FIC) index was calculated to 0.83 and 0.73 for the GS S. epidermidis and the MRSE, respectively, which means there is an additive effect of the combination of APIM-peptide and gentamicin. This effect could be explained by the different mode of action APIM-peptide and gentamicin exhibit, targeting DNA synthesis and protein synthesis respectively.
  • the FIC-index for the MRSE strain was slightly lower than the GS S. epidermidis strain, but still fits the definition for an additive effect.
  • the GS S. epidermidis strain showed a 3-fold reduction in gentamicin MIC when combined with APIM-peptide, while APIM- peptide MIC is reduced 2-fold.
  • the MRSE strain show a much larger reduction in the gentamicin MIC when combined with APIM-peptide than the GS S. epidermidis strain, reducing the gentamicin MIC 8-fold in the combination.
  • the reduction in APIM-peptide MIC in the combination is however more similar in between the strains ( ⁇ 2-fold). This indicates that although the additive effect of the combination of APIM-peptide and gentamicin is found in both gentamicin resistant and susceptible strains, APIM-peptide has a larger effect on sensitizing resistant bacteria towards gentamicin.
  • Table 1 S. epidermidis sensitivity towards APIM-peptide and gentamicin MIC and FIC values for two S. epidermidis strains, one GS and one GR MRSE, for APIM-peptide and gentamicin alone and in combination (checkerboard assay).
  • APIM-peptide is bactericidal
  • CFU colony forming units
  • APIM-peptide at 1/2x MIC
  • gentamicin at 1x MIC
  • Example 3 APIM-peptide inhibits gentamicin resistance development of S. epidermidis
  • Example 4 APIM-peptide is released from bone cement and has a strong antibacterial effect in an in vivo bone infection model in combination with gentamicin
  • ALBC inside bone grafts functions as a local distributor of the antibiotic and the addition of bacterial inoculum to the grafts mimics a setting where bacteria is introduced at the time of surgery, colonizing the grafts’ surface.
  • APIM-peptide was added to bone-cement (1.6% w/w) and it was found that it was efficiently eluted from the cement, both in water and in growth media, as growth of S. epidermidis was inhibited (Figure 4A).
  • Bone grafts treated with APIM- peptide or gentamicin alone also showed a significant reduction of bacterial growth compared to control, however less than the combination treatment.
  • a large fraction, 77% (10/13), of the bone grafts with combination treatment had no detectable bacterial growth.
  • the fraction of bone grafts with no detectable growth was lower in bone grafts containing only APIM-peptide or gentamicin, 4/12 and 5/13 (33 and 38%), respectively.
  • Bone grafts with cement without any additions, control had only one bone graft without detectable growth.
  • the bacterial load on the bone cement correlates with the bacterial load on found in the bone, except for the very low bacterial load found on cement containing only APIM-peptide (Figure 5B). All the treatments significantly reduced bacterial load compared to control, but the cement containing APIM-peptide (alone and in combination) had significantly lower bacterial load than cement with only gentamicin. This indicates that APIM-peptide inhibits adhesion of bacteria to the cement.
  • APIM-peptide and gentamicin inhibits bacterial growth on both the bone cement and in the bone graft in vivo, which indicates that APIM-peptide in bone cement may find utility in inhibiting orthopaedic infections, such as prosthetic joint infections (PJIs).
  • PJIs prosthetic joint infections
  • S. epidermidis pathogenicity factor is biofilm formation, which often leads to a poorer outcome for the patients and low penetrance of antibiotics.
  • Gentamicin has previously shown to increase Pseudomonas aeruginosa biofilm formation. Thus, the effect of APIM-peptide on biofilm formation was examined directly.
  • APIM-peptide was also found to be capable of eradicating existing biofilms on C-wire steel rods (Figure 8).
  • 25 mM of APIM-peptides completely eradicated the biofilm without re-growth after the rods were transferred into fresh media.
  • Lower doses of the peptide also significantly reduced the biofilm; 62% and 95% reduction was observed after 24 hours at 5 pM (MIC) and 10 pM respectively.
  • MIC 5 pM
  • R11 was also included as a control.
  • APIM-peptides show an overall antibacterial effect of APIM-peptides on S. epidermidis. While a bactericidal effect is seen in vitro, the reduced bacterial load seen in vivo in a bone infection model combining APIM-peptide and gentamicin in bone cement was better than expected. In addition, APIM-peptide inhibits the development of gentamicin resistance and inhibits biofilm formation in concentrations lower than the MIC. APIM-peptide is also capable of eradicating existing biofilms.
  • S. epidermidis strains Two clinical isolates S. epidermidis strains were used (Department of Microbiology, St. Olavs University Hospital, Trondheim, Norway); one GS S. epidermidis and one MRSE strain.
  • the MRSE strain is resistant to gentamicin, erythromycin, cl oxaci 11 i n/d i cloxaci 11 i n/pen ici Min.
  • S. epidermidis strain ATCC35984 and ATCC12228 were used as a strong and poor biofilm forming strains, respectively.
  • S. epidermidis strains were normally cultured in tryptic soy broth (TSB, 1 % glucose) or Mueller-Hinton Broth (MHB) for MIC assays. Blood agar plates were utilized for plating, incubated at 37 °C with 5% CO2.
  • Minimal inhibitory concentration (MIC) and Minimal bactericidal concentration (MBC) was determined following the protocol recommended by National Committee of Laboratory Safety and Standards (NCCLS) for microtiter broth dilution assay, conducted with modifications suggested by R.E.W. Hancock Lab for cationic peptides. Briefly, bacterial suspension in MHB was adjusted to 0.5 McFarland and diluted to 5x10 5 CFU/mL. Suspension (0.1 mL) was seeded in polypropylene microtiter plates (Greiner), 5x10 4 CFU/well in each well. APIM- peptide and gentamicin were added as 10% of the total volume.
  • NCCLS National Committee of Laboratory Safety and Standards
  • the checkerboard assay was performed in microtiter plates and performed as MIC assay, with series of concentrations of each drug, alone and in combination.
  • Time-kill curves were conducted according to NCCLS standards by inoculating the MRSE strain in 10 mL MHB, 5x 10 5 CFU/mL.
  • the cultures were grown in a shaking incubator (250 rpm, 37 °C and 5% CO2) over 24 hours. Samples were harvested at the time points 0, 2, 4, 8, 12 and 24 hours, serial diluted in normal saline and plated (duplicates) on blood agar plates. CFU were counted after 48 hours of incubation and plotted as logio CFU/mL. The detection limit is 50 CFU/mL (1.72 log 10 CFU/mL).
  • 1 ⁇ 2x MIC of APIM-peptide was also combined with 1x MIC of gentamicin.
  • the cultures were grown in a shaking incubator (250 rpm, 37 °C, and 5% C02.).
  • Samples were harvested at the time points from minimum 5 minutes up to maximum 48 hours, serial diluted in normal saline, and plated (duplicates) on blood agar plates. CFUs were counted after 24 hours of incubation. The detection limit is calculated to 50 CFU/mL (1.72 log10CFU/mL). For some cultures, the culture density was also determined after the exchange of fresh media after 48 hours to quantify CFUs after the removal of the antibacterial agent. In this case, 5 mL of the culture was centrifuged (2 minutes, 14000 rpm), and the pellet re suspended in fresh MHB before serial dilution and plating.
  • Corticospongeous bone grafts were harvested from Norwegian white sheep, delivered from Nortura Malvik, Norway. Rib bones were cut to approximately 1x1 cm in size, height -0.5 cm, hollowed from end to end with a 3 mm drill through the bone marrow, creating a cylindrical hole through the bone. In addition to this, a 2 mm groove was drilled on the top of the bone grafts, perpendicular to the cylindrical hole in the bone marrow. Periosteum from the bone was removed and sharp edges of the bone grafts were rounded, and the bone grafts were sterilized by autoclaving.
  • the cylindrical hole of the bone grafts was filled with bone cement (Biomet® Bone Cement R) with and without addition of APIM-peptide, or gentamicin containing bone cement (PALACOS®R+G) with and without addition APIM-peptide.
  • APIM- peptide (Innovagen, Sweden) was dissolved in water (160 pg/pL) before mixing it with the cement liquid component, enabling a homogenous cement containing 2.5% APIM-peptide (25 mg APIM-peptide/g cement, corresponding to 1.6% active ingredient). The corresponding amount of water was added to the control cement without APIM-peptide.
  • the groove on top of the bone grafts were kept hollow, to allow inoculation of bacteria to reach the cement inside the bone.
  • the bone grafts containing cement filled with APIM-peptide, gentamicin or a combination of these, were each added an inoculum of 50 pl_ TSB in the groove on the top, containing 1000 CFU of MRSE. After 24-hour incubation (37°C, 5% CO2) the bone grafts were harvested for quantification of bacterial load. The bone grafts were “opened” with a sterile scalpel to separate the cement inside from the bone. The bone and cement pieces were added in 3 ml_ normal saline each (0.9% NaCI) in sterile glass tubes.
  • Samples were sonicated for 5 minutes in a water bath (40 kHz, 200W, BactoSonic®, Bandelin GmbH, Berlin) in addition to being vortexed 30 seconds (2500 rpm) before and after sonication. Samples were serial diluted in normal saline (1:10) and plated in triplicates per concentration on blood agar plates.
  • the animals were acclimatized for a minimum of a week after arrival to the Comparative Medicine Core Facility (NTNU, St. Olav’s Hospital). The animals were housed in solitude during the experiment and had access to environmental enrichment and standard food/water ad libitum in a 12-hour-night-day cycle. The rats were weighed on the first and the last day of the experiment, and only minor weight loss were observed.
  • the exposed area was washed with 70% alcohol and a ⁇ 4 cm long incision was made in the midline of the upper part of the back.
  • One intramuscular cavity was made on each side of the back midline with a scissor.
  • Two bone grafts were each instilled with 5 mI_ saline, containing 1000 CFU of S. epidermidis, in the groove of the bone graft, before being placed sub-fascial in the intramuscular pockets, one bone graft in each side.
  • the incision was closed with sutures and the rats were given analgesia (Buprenorphine; Temgesic, Reckitt & Colman), 0.05 mg/kg) at the end of the surgery and postoperatively (8-12 hours after the surgery).
  • the bone grafts were retrieved after four days. The sutures on the back of the rats were removed and the incision made on day one was re-opened. The bone grafts were harvested with a sterile tweezer and a muscle biopsy (approximately 5 mm) was taken from the surrounding tissue of the bone graft. The bacterial load was quantified as described in the in vitro bone infection model. The rats were euthanized after the experiment with pentobarbital (Mebumal 10%, 0.1ml_/100g), by intracardiac injection or intravenously injection in tail. Biopsies from to spleen (5-10 mm) was also harvested to check for systemic infection. No signs of systemic infection were found, i.e.
  • S. epidermidis strain ATCC35984 and ATCC12228 were seeded in suspension (200 pL/well) adjusted to 0.5 McFarland turbidity standard in TSB in microtiter plates.
  • APIM-peptide was added directly to the culture in the wells, 10% of the total volume, to minimum 3 wells per treatment. After 24 hours incubation (37°C) the wells were washed and vortexed (3x1 min, 600 rpm) with normal saline before fixation with 99% methanol for 15 minutes. The wells were emptied and dried followed by staining (5 min) with crystal violet (0.5-2%). Wells were washed with water and dried before stain was re-solubilized with 160 pl_ glacial acetic acid (33%). Finally, absorbance was measured at 570 nm.
  • the gentamicin sensitive S. epidermidis strain was inoculated (1:1000) from an overnight culture into 4 ml_ TSB with addition of 50% MIC of gentamicin, APIM- peptide, a combination of these or a corresponding amount of water as control.
  • the cultures were grown in a shaking incubator (37°C) and were re-inoculated (1:100) every 24 hours in fresh TSB and supplements.
  • the cultures were plated on blood agar with and without gentamicin (10 mg/L) every day for 4 days to count the frequency of resistant bacteria (CFU on gentamicin plates/total CFU).
  • Stainless steel wire (C-WIRE, 0.89mm, ConMed) was cut to 6 mm rods and autoclaved.
  • the rods were placed in 200 pl_ bacterial suspension with S. epidermidis ATCC35984 (106 CFU/mL in MHB) in 96-well polypropylene microtiter plates and grown for 5 days to allow for biofilm formation. Fresh media was exchanged every 24 hours. After 5 days the rods were placed into fresh media and treated with APIM-peptides (1x, 2x or 5x MIC), water (control), or a peptide containing only the cell penetrating arginine tail of the APIM-peptide (R11 , x5 MIC).
  • the rods were harvested 1, 6, and 24 hours after the addition of peptides, washed briefly in normal saline before sonication in 0.5 ml_ normal saline in glass tubes for 6 minutes (40 kHz, 200W, BactoSonic®). The rods were vortexed 20 seconds (2500 rpm) before and after sonication. Samples were serial diluted in normal saline (1:10) and plated in duplicates per concentration on blood agar plates. CFU counts were conducted after 24 hours incubation at 37 °C and 5% CO2. Lastly, after sonication the rods were put into 3 mL fresh media and incubated 24 hours (37 °C, shaking) to check for visible re-growth of bacteria from the rods.

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Abstract

La présente invention concerne des ciments osseux contenant des composés oligopeptidiques bactéricides qui inhibent le développement de la résistance aux antibiotiques dans les bactéries et la formation de biofilms bactériens. L'invention concerne un kit de production d'un ciment osseux antibactérien comprenant : (i) des composants pour la production de ciment osseux ; et (ii) un composé oligopeptidique comprenant un motif d'interaction PCNA.
PCT/EP2020/079961 2019-10-24 2020-10-23 Ciment osseux antibactérien et ses utilisations WO2021078978A1 (fr)

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001891A1 (fr) 1989-08-02 1991-02-21 Kallos Verlag Und Versand Gmbh Carte de felicitations
US5656122A (en) 1993-07-30 1997-08-12 Lam Research Corporation Shadow clamp
WO2000001417A1 (fr) 1998-07-03 2000-01-13 Cyclacel Limited Systeme d'administration
WO2000029427A2 (fr) 1998-11-13 2000-05-25 Cyclacel Limited Vecteurs de transport
US6080724A (en) 1995-10-05 2000-06-27 Centre National De La Recherche Scientifique (Cnrs) Peptides which can be used as vectors for the intracellular addressing of active molecules
US6645501B2 (en) 1997-12-10 2003-11-11 Washington University Anti-pathogen system and methods of use thereof
WO2004069279A1 (fr) 2003-01-07 2004-08-19 Centre National De La Recherche Scientifique Composition pour le transport intracellulaire de macromolecules ou particules biologiques
WO2015067713A1 (fr) 2013-11-06 2015-05-14 Norwegian University Of Science And Technology (Ntnu) Agents antimicrobiens et leur utilisation en thérapie
WO2016177899A1 (fr) 2015-05-06 2016-11-10 Norwegian University Of Science And Technology (Ntnu) Agents antibactériens et leur utilisation en thérapie

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001891A1 (fr) 1989-08-02 1991-02-21 Kallos Verlag Und Versand Gmbh Carte de felicitations
US5656122A (en) 1993-07-30 1997-08-12 Lam Research Corporation Shadow clamp
US6080724A (en) 1995-10-05 2000-06-27 Centre National De La Recherche Scientifique (Cnrs) Peptides which can be used as vectors for the intracellular addressing of active molecules
US6645501B2 (en) 1997-12-10 2003-11-11 Washington University Anti-pathogen system and methods of use thereof
WO2000001417A1 (fr) 1998-07-03 2000-01-13 Cyclacel Limited Systeme d'administration
WO2000029427A2 (fr) 1998-11-13 2000-05-25 Cyclacel Limited Vecteurs de transport
WO2004069279A1 (fr) 2003-01-07 2004-08-19 Centre National De La Recherche Scientifique Composition pour le transport intracellulaire de macromolecules ou particules biologiques
WO2015067713A1 (fr) 2013-11-06 2015-05-14 Norwegian University Of Science And Technology (Ntnu) Agents antimicrobiens et leur utilisation en thérapie
WO2016177899A1 (fr) 2015-05-06 2016-11-10 Norwegian University Of Science And Technology (Ntnu) Agents antibactériens et leur utilisation en thérapie

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
ELMQUIST ET AL., BIOL. CHEM, vol. 384, 2003, pages 387 - 393
ERIKSSON ET AL., ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 57, no. 8, 2013, pages 3704 - 3712
GAUTAM ET AL.: "CPPSite: a curated database of cell penetrating peptides", DATABASE, 2012, Retrieved from the Internet <URL:http://crdd.osdd.net/raghava/cppsite/index.php>
GILLJAM ET AL.: "Identification of a novel, widespread, and functionally important PCNA-binding motif", J. CELL BIOL., vol. 186, no. 5, 2009, pages 645 - 654, XP055459102, DOI: 10.1083/jcb.200903138
HALLBRINK ET AL.: "Prediction of Cell-Penetrating Peptides", INTERNATIONAL JOURNAL OF PEPTIDE RESEARCH AND THERAPEUTICS, vol. 11, no. 4, 2005, pages 249 - 259, XP019287639, DOI: 10.1007/s10989-005-9393-1
HANSEN ET AL.: "Predicting cell-penetrating peptides", ADVANCED DRUG DELIVERY REVIEWS, vol. 60, 2008, pages 572 - 579, XP022476847, DOI: 10.1016/j.addr.2007.09.003
HOLM ET AL., FEBS LETT., vol. 579, 2005, pages 5217 - 5222
J. BONE JOINT SURG, vol. 42, 1960, pages 28 - 30
JARVER ET AL., BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1758, 2006, pages 260 - 263
KANG TING ET AL: "Synergistic reinforcement of polydopamine-coated hydroxyapatite and BMP2 biomimetic peptide on the bioactivity of PMMA-based cement", COMPOSITES SCIENCE AND TECHNOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 123, 5 January 2016 (2016-01-05), pages 232 - 240, XP029395251, ISSN: 0266-3538, DOI: 10.1016/J.COMPSCITECH.2016.01.002 *
KRAUSS ET AL., BIOORG. MED. CHEM. LETT., vol. 14, 2004, pages 51 - 54
LIU ZHENZHEN ET AL: "Synergistic effect of HA and BMP-2 mimicking peptide on the bioactivity of HA/PMMA bone cement", COLLOIDS AND SURFACES B: BIOINTERFACES, ELSEVIER AMSTERDAM, NL, vol. 131, 21 April 2015 (2015-04-21), pages 39 - 46, XP029179053, ISSN: 0927-7765, DOI: 10.1016/J.COLSURFB.2015.04.032 *
OH ET AL., MOL. PHARMACEUTICS, vol. 11, 2014, pages 3528 - 3536
PUJALS, ADVANCED DRUG DELIVERY REVIEWS, vol. 60, 2008, pages 473 - 484
SANDERS ET AL.: "Prediction of Cell Penetrating Peptides by Support Vector Machines", PLOS COMPUTATIONAL BIOLOGY, vol. 7, no. 7, 2011, pages 1 - 12

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