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WO2012113037A1 - Procédé d'inhibition de protéines - Google Patents

Procédé d'inhibition de protéines Download PDF

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Publication number
WO2012113037A1
WO2012113037A1 PCT/AU2012/000188 AU2012000188W WO2012113037A1 WO 2012113037 A1 WO2012113037 A1 WO 2012113037A1 AU 2012000188 W AU2012000188 W AU 2012000188W WO 2012113037 A1 WO2012113037 A1 WO 2012113037A1
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Prior art keywords
lactoferrin
composition
gingivalis
activity
cpi
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English (en)
Inventor
Eric C. Reynolds
Stuart G. DASHPER
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University of Melbourne
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University of Melbourne
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Priority claimed from AU2011900664A external-priority patent/AU2011900664A0/en
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Publication of WO2012113037A1 publication Critical patent/WO2012113037A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
    • A61K9/0058—Chewing gums
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0063—Periodont
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis

Definitions

  • the invention relates to methods and compositions for inhibiting bacterial proteinases, specifically cysteine proteinases from pathogenic bacteria such as Porphyromonas gingivalis.
  • the invention also relates to methods and compositions for inhibiting gingipains and to methods for preventing cell and tissue invasion mediated by the gingipains and for treating diseases associated with gingipains.
  • Arg- and Lys- gingipains are proteins produced by the bacterium Porphyromonas gingivalis. The gingipains are thought to be critical virulence factors since deficient mutants are less pathogenic in animal models.
  • Gingipains R (RgpA and RgpB) and K (Kgp) are cysteine endopeptidases in class 3.4.22 of NC-RJBMB with specificity for arginyl and lysyl peptide bonds, respectively. Other members of this class include calpain, the cathepsins, the caspsases, papain, and sortases A and B. All require reducing conditions for activity. Gingipains R and K are included in peptidase family 25.
  • the crystal structure of gingipain R (RgpB) shows a 435-residue, single-polypeptide chain organized into a catalytic domain and an immunoglobulin-like, hemagglutinin domain.
  • the catalytic domain is divided into two subdomains comprising four- and six-stranded beta-sheets sandwiched between alpha- helices. Each subdomain has topological similarities to the p20-pl0 heterodimer of caspase-1, the type example for clan CD.
  • the second subdomain contains the Cys/His catalytic dyad and a nearby Glu.
  • the SI specificity pocket contains an Asp residue that is believed to be responsible for the specificity preference for Arg in PI.
  • P. gingivalis gingipains have been shown to hydrolyse a range of growth regulatory proteins and peptides and are involved directly in tissue destruction and evasion and modulation of host immune defences.
  • gingipains may contribute to the development and maintenance of an inflammatory state, possibly by activation of the kallikrein/kinin- generating cascade, which results in increased vascular permeability or by activation of blood coagulation factor X to generate activated factor X which can modulate the production of proinflammatory cytokines.
  • gingipains R and K can activate different cell types, including macrophages and monocytes leading to the secretion of proinflammatory cytokines including TNF a and interleukin 8. While P.
  • gingivalis gingipains have been relatively well studied as more genomes are being sequenced it is becoming apparent that gingipains are produced by many other bacteria, archaea, protozoa, fungi, animals, viruses and plants and may be implicated in many biological activities. While not all gingipains have cysteine proteinase activity, according to the MEROPS peptidase database all have the same basic 3-D structure which is typical of clan CD members.
  • the clan CD contains five families of endopeptidases, CI 1 typified by clostripain, C13 typified by legumain (plant beta form), C14 typified by caspase-1, C25 typified by gingipain R and C50 typified by separase (yeast type). Gingipains belong to the endopeptidase family C25 and can be distinguished from other endopeptidases in clan CD.
  • Identification of an inhibitor of gingipains may have implications in the treatment of a variety of diseases, disorders or biological activities possessed by bacteria, archaea, protozoa, fungi, or viruses which produce gingipains.
  • Periodontal diseases range from simple gum inflammation to serious disease that results in major damage to the soft tissue and bone that support the teeth. Periodontal diseases include gingivitis and periodontitis. Bacteria, such as P. gingivalis causes inflammation of the gums known as "gingivitis". In gingivitis, the gums become red, swollen and can bleed easily. When gingivitis is not treated, it can advance to periodontitis (which means "inflammation around the tooth.”). In periodontitis, gums pull away from the teeth and form pockets that are infected. The body's immune system fights the bacteria as the plaque spreads and grows below the gum line. If not treated, the bones, gums, and connective tissue that support the teeth are destroyed.
  • the teeth may eventually become loose and may exfoliate or have to be removed.
  • Chronic periodontitis is an inflammatory condition involving a host response to bacterial components that have entered the gingival tissue of the periodontal pocket and these compounds can be released from planktonic pathogenic bacteria associated with the epithelium.
  • Some commensal oral bacterial species colonise the hard, non-shedding surface of the tooth root however the pathogenic bacteria associated with disease initiation and progression are late colonizers of the periodontal pocket and may not necessarily be strongly associated with a biofilm.
  • planktonic cells can invade the host tissue and cells and spread the infection to another site of the body. Later stages of periodontitis are characterized by host tissue (intercellular) and cell (intracellular) invasion by pathogenic bacteria and the inability of the host immune system to remove pathogenic components which results in continual external stimulation, leading to a chronic inflammatory state.
  • Wakabayasbi et al. 2009. Antimicrob. Agents and Chemo. Vol. 53(8): 3308 to 3316 describes the inhibition of a single species biofilm (P. gingivalis or P. intermedia) in vitro by bovine lactoferrin where the biofilm is grown on a microliter plate.
  • Wakabayasbi does not describe any experiments which recreate or model growth of a biofilm that occurs in a disease state in vivo.
  • Biofilms found in the oral cavity in vivo are polymicrobial and are exposed to a completely different environment than the in vitro biofilm was exposed to in Wakabayasbi.
  • in vivo environments include temperature fluctuations and variations in nutrient, carbon and nitrogen availability.
  • Wakabayasbi The substratum used for attachment and biofilm formation by Wakabayasbi has no relevance to the oral cavity. Wakabayasbi does not describe the colonisation of an existing polymicrobial biofilm by P. gingivalis or methods for its inhibition. Wakabayasbi is not concerned with nor describes any experimental data relating to bacterial tissue or cell invasion.
  • lactofemn is an inhibitor of P. gingivalis proteinases that require metal ions for activity.
  • lactoferrin and various forms thereof can inhibit the proteolytic activity of P. gingivalis cysteine proteinases including the gingipains Rgp and Kgp.
  • the invention provides use of a cysteine protease inhibitor (CPI) in an individual for inhibiting the cysteine protease activity of a pathogen of periodontal disease in the individual, wherein the CPI is a protein capable of binding to an ion comprised in an active site of a cysteine protease, thereby inhibiting the activity of the cysteine protease.
  • CPI cysteine protease inhibitor
  • cysteine protease activity of the pathogen inhibits the pathogen from invading tissues and/or cells of an individual.
  • the inhibition of the cysteine protease activity of the pathogen prevents the individual from acquiring periodontitis or periodontal disease.
  • the CPI is lactoferrin, preferably the lactoferrin comprises a metal ion.
  • the CPI is provided by oral administration.
  • the CPI is provided in the form of a food, a drink, a supplement, a medicament or a pharmaceutical.
  • the pathogen is P. gingivalis.
  • the form of P. gingivalis may be planktonic.
  • the invention provides a composition for inhibiting the cysteine protease activity of a pathogen of periodontal disease including:
  • the carrier adapts the composition to form a gum, gel or the like.
  • An aspect of the invention provides a method for inhibiting a P. gingivalis proteinase, the method comprising admimstering lactoferrin to a subject.
  • the proteinase is a cysteine proteinase, preferably the cysteine proteinase is a gingipain, even more preferably RgpA, RgpB or Kgp.
  • lactoferrin for inhibiting a P.gingivalis proteinase and the use of lactoferrin in the manufacture of a medicament for inhibiting a P.gingivalis proteinase.
  • a method of treating a disease, disorder or biological effect that is associated with host tissue or cell invasion by P. gingivalis including administering lactoferrin.
  • a method of treating a disease, disorder or biological effect that is associated with host tissue or cell invasion by P. gingivalis including the steps of
  • the formulation including lactoferrin.
  • the formulation including a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a gel suitable for oral administration.
  • the disease is a disease of the oral cavity. More preferably, the disease is periodontitis and the subject is identified as exhibiting one or more symptoms of periodontitis. Even more preferably, the subject exhibits one or more symptoms of chronic periodontitis.
  • lactoferrin for use in treating a disease, disorder or biological effect that is associated with host tissue or cell invasion by P. gingivalis.
  • a method of treating a disease, disorder or biological effect modulated by a gingipain including administering lactoferrin.
  • lactoferrin for treating a disease, disorder or biological effect modulated by a gingipain and the use of lactoferrin in the manufacture of a medicament for treating a disease, disorder or biological effect modulated by a gingipain.
  • a method of preventing, reducing or inhibiting host tissue or cell invasion by P. gingivalis including the steps of
  • lactoferrin in the manufacture of a preparation for the treatment or prevention of a disease, disorder or biological effect modulated by a gingipain.
  • the disease is periodontitis.
  • Particularly useful preparations include tablets, aqueous or oily suspensions, lozenges, troches, powders, granules, emulsions, gels, capsules, syrups, chewing gums, toothpastes, toothpowders, and dentifrices, mouth washes, dental pastes, gargle tablets, dairy products, elixirs or other foodstuffs.
  • the preparation is formulated as a gel suitable for oral administration.
  • a composition including lactoferrin and one or more one or more pharmaceutically acceptable carriers, excipients or diluents for the treatment or prevention of a disease, disorder or biological effect modulated by a gingipain.
  • the disease is periodontitis.
  • Particularly useful forms of the composition include tablets, aqueous or oily suspensions, lozenges, troches, powders, granules, emulsions, gels, capsules, syrups, chewing gums, toothpastes, toothpowders, and dentifrices, mouth washes, dental pastes, gargle tablets, dairy products, elixirs or other foodstuffs.
  • the composition is formulated as a gel suitable for oral administration.
  • a composition for use in the treatment or prevention of a disease, disorder or biological effect modulated by a gingipain the composition having lactoferrin as an active ingredient.
  • the composition is formulated as a gel suitable for oral administration. Any biologically active lactoferrin may be employed in the aspects of the invention.
  • lactoferrin is administered orally.
  • An alternative embodiment provides orally ingestible or orally administrable lactoferrin for inhibiting a P. gingivalis proteinase or for treating a disease, disorder or biological effect modulated by P. gingivalis proteinase.
  • the oral ingestible or orally administrable lactoferrin is formulated as a gel.
  • a further aspect of the invention provides a method of inhibiting gingipain-associated pathogen invasion of host cells or tissues by administering lactoferrin.
  • kits for use in a method of the invention including a composition including lactoferrin and one or more pharmaceutically acceptable carriers, excipients or diluents.
  • the kit further includes written instructions for use.
  • lactoferrin is formulated as a gel suitable for oral administration.
  • kit for use in a method of the invention mentioned above including:
  • the lactoferrin in the kit is formulated as a gel.
  • the kit may contain one or more further components for treatment or prevention of a disease, disorder or biological effect modulated by a gingipain, for example, antibiotics or antibiofilm agents.
  • Figure 1 shows the time-dependent inhibition of RgpA activity by lactoferrin.
  • the figure specifically shows the effect of lactoferrin at different concentrations ( ⁇ ) on the activity (Ln%) of RgpA over time.
  • the slope of these lines provided the apparent inactivation rate constants (kapp).
  • FIG. 2 An orthographic view of the modeled lactoferrin-RgpB complex.
  • the solvent accessible surface of RgpB is shown as a space filling model.
  • the arrow (A) indicates the zinc ion, depicted a dark sphere, bound to the catalytic histidine (His 244 ) of RgpB.
  • Lactoferrin is shown as a ribbon structure and the side-chains of residues that moved to within 3 A of RgpB during the dynamics simulation are shown as 'capped sticks'.
  • the location of the atoms of the RgpB-inhibitor, DFFR-chloromethylketone, is shown by the arrow (B) pointing to the dark space-filling atoms in the left panel.
  • Figure 3 A SDS-PAGE of LF incubated with P. gingiva!is.
  • Lane 1 LF (1 ⁇ g); lanes 2-7: LF and P. gingivalis.
  • lane 2 1 min incubation;
  • lane 3 10 min incubation;
  • lane 4 3 h incubation;
  • lane 5 6 h incubation;
  • lane 6 18 h incubation;
  • lane 7 3 day incubation.
  • the arrow indicates the sample fractionated by gel filtration (6 h incubation).
  • Major fragments of LF are labelled with Roman numerals, and the gel bands have been analysed by MS.
  • B LF was treated with different concentrations of trypsin at 37°C for 18 h.
  • Lane 1: LF/trypsin 2000/1;
  • FIG. 4 The primary sequence of LF, underlined sequences denote the peptides identified by peptide mass fingerprint (PMF) analysis of Fragments I to V.
  • PMF peptide mass fingerprint
  • the cleavage site between Fragment I and IV was determined by In Source Decay (LSD) MALDI-TOF MS. All arginine and lysine residues are shown in bold.
  • Figure 5 A: RP-HPLC analysis of LF after 6 h hydrolysis by P. gingivalis whole cells.
  • B-D MS spectra of RP-HPLC peaks 1-3, respectively.
  • FIG. 6 In Source Decay MS spectrum of Fragment I of LF-Pg.
  • the fragment ions labelled correspond to N-terminal (c-type) fragments of lactoferrin starting from c-12, and extending to c-38.
  • the mass difference between each peak corresponds to the amino acid residue shown. Extrapolation of these data indicated that the N-terminal sequence of this polypeptide was 285 SFOLFGSPPGORDLLFKDSALGFLRIPSKVDSALYLGS. with the underlined portion evident in the spectrum.
  • the fractions were collected at an interval of 1 mL and two fractions (marked as Fl and F2) were collected from the peak at the elution volume between 9 and 11 mL.
  • B LF-Pg and the SEC fractions from LF-Pg were analysed by SDS-PAGE. Lane 1: MW marker; lanes 2 and 3: Fl and F2 from the peak eiuted from SEC; lane 4: LF-Pg without SEC.
  • Figure 8 shows the effect of bovine LF on P. gingivalis biofilm formation, expressed as % inhibition.
  • P. gingivalis was incubated with LF for 18 h ( ), 24 h (* ), and 48 h ( ⁇ ). Each data point represents the mean and standard deviation of three replicates. Note categorical scale on the x axis.
  • Figure 9 shows the effect of LF on planktonic growth of P. gingivalis in batch culture,
  • LF concentrations (mg/mL):- 0 (X), 0.5 ( ⁇ ), 2.5 ( ⁇ ), 5.0 ( ⁇ ), 10 ( ⁇ ), uninoculated growth medium ( ⁇ ).
  • Figure 10 shows a comparison of the biofilm inhibitory activity of native LF, LF incubated with P. gingivalis (LF-Pg) and fractions obtained from RP-HPLC separation of LF-Pg.
  • LF-Pg P. gingivalis
  • LF-Pg fractions obtained from RP-HPLC separation of LF-Pg.
  • Peaks 1 to 3 correspond to the three RP-HPLC fractions in Fig. 5.
  • LFfract was native LF eluted under the same condition as that for separation of LF-Pg sample. The number in each treatment represents the protein concentration at mg/mL.
  • lactoferrin is an inhibitor of bacterial proteinases requiring metal ions for proteolytic activity.
  • lactoferrin is an inhibitor of the gingipains secreted by various pathogenic bacteria including P. gingivalis.
  • the gingipains are involved in various stages of pathogenic disease progression and in the case of periodontal diseases such as periodontitis, gingipains are involved in late stage disease, for example, host tissue and cell invasion by pathogenic bacteria.
  • Bacterial strains can express varying levels of gingipains but cells that express high levels of gingipains are more cytotoxic and likely to invade host tissues and/or cells.
  • Gingipains can penetrate the host cell while also activating other enzymes which are involved in host tissue and cell invasion.
  • treatment of a subject in late stage periodontal disease with lactoferrin in accordance with the invention can also stop the spread of infection in vivo.
  • lactoferrin has the capacity to interact with a metal ion in a proteinase, and/or the general conformation of the active site of the proteinase associated with metal ion binding for activity thereby reducing or inhibiting proteolytic activity.
  • reduction or inhibition of proteolytic activity is proposed to be via obstruction of the substrate binding site on the proteinase.
  • lactoferrin in the inhibition of proteinases is based directly on the unexpected observation that as well as being resistant to hydrolysis by P. gingivalis gingipains Rgp and Kgp, lactoferrin directly inhibits the proteinase inhibitory activity of P. gingivalis gingipains Rgp and Kgp.
  • the inventors have shown that lactoferrin inhibits gingipains Rgp and Kgp in a time dependent manner.
  • P. gingivalis gingipains are involved in systemic disease, enabling the bacterium to enter the blood stream, invade the endothelial cell lining and persist and replicate in host cells.
  • Inhibition of gingipains by lactoferrin is proposed to inhibit gingipain-associated pathogen invasion of host cells or tissues and consequently allows treatment of any disease, disorder or biological effect modulated by a gingipain.
  • Reducing or inhibiting tissue and cell invasion, treatment of a subject in late stage periodontal disease with lactoferrin in accordance with the invention can also stop the spread of infection in vivo and in combination with treatment with other active agents, such as antibiotics, or treatment methods, such as root scaling, can provide an effective therapy.
  • a "gingipain” as referred to herein is any protein having a tertiary structure having a number of features that relate to the well characterised Porphyromonas gingivalis gingipain proteinases. It must have a tertiary structure that has significant similarity to that of P. gingivalis gingipains, this can be determined using computer programs such as FUGUETM that recognises distant structural homologs of a target sequence by sequence-structure comparison. It assesses the compatibility between a target sequence and structural profiles of all known protein structural families. A Z-score of greater than 6 is recognised as being predictive of structural homology.
  • the primary sequence of a gingipain must include a histidine, cysteine catalytic dyad.
  • Tryptophan and tyrosine are acceptable substitutions for histidine if the organism exists in the appropriate environment for proton transfer to take place during catalysis in the active site.
  • the catalytic "histidine” is usually in the motif GHG; exceptions being HG, GH, GY, GWG.
  • the catalytic cysteine is located within 33-42 residues, C-terminal of the catalytic "histidine”.
  • the sequence must contain two conserved structural glycines; one 8 to 10 residues N-terminal of the catalytic "histidine” and one 25-30 residues C-terminal of the catalytic cysteine.
  • the sequence must match the consensus sequence: [GSDA]-x(0,31)-[GAHDT]-[HWY]-[GSIAYL]-x(26,42)- C-x(18,30)-G.
  • the protein primary sequence must have a propeptide in its N-terminal region.
  • the sequence must contain additional conserved amino acids in the interface between the N- terminal region and the active site domain.
  • the sequences should have the following salt bridge and calcium binding residues: G 77 , D 78 , R 112 , E 258 (P. gingivalis RgpB numbering).
  • a gingipain includes a His, Cys catalytic dyad.
  • the catalytic His occurs in a His-Gly motif and is preceded by a block of hydrophobic residues; the catalytic Cys occurs in the motif Ala-Cys and is preceded by a second block of hydrophobic residues.
  • the gingipain tertiary structure includes alpha/beta proteins with a fold that consists of an alpha/beta/alpha sandwich.
  • the beta sheet contains six strands (preferably in the order 213456) and strand 6 is anti-parallel to the rest.
  • the gingipain is gingipain R or gingipain K from P. gingivalis.
  • gingipain is calpain, cathepsin, caspase, sortase, clostripain, legumain, separase or RTX self-cleaving toxin.
  • a gingipain is intended to relate to either one gingipain or a combination of two or more gingipains from the same or different source.
  • the term "inhibition” is intended to include any reduction in gingipain activity compared to the activity of the gingipain in the absence of lactoferrin.
  • the reduction may be 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99% or any integer therebetween or may be total inhibition of gingipain activity.
  • gingipain activity defines the activity of the gingipain to be inhibited.
  • gingipain is gingipain R (RgpA or B) or gingipain K (Kgp)
  • the gingipain activity is proteinase activity.
  • the gingipain activity is cysteine proteinase activity.
  • a cysteine protease inhibitor as used herein is a compound that can inhibit the protease activity of a cysteine protease. Inhibition of cysteine protease activity by the inhibitor may be by steric hindrance of the substrate to the active site of the cysteine protease. As is explained in detail in the Examples below, a cysteine protease inhibitor may bind to the cysteine protease via the metal ion in the active site of the proteinase. For example, where the cysteine protease is a gingipain, such as a Kgp or Rgp from P.
  • cysteine protease inhibitor may bind to the gingipain via the zinc-ion in the proteinase active site. Binding to the metal ion in the active site of the proteinase would then inhibit access of substrates to the active site.
  • cysteine protease inhibitors include members of the transferrin family.
  • transferrin family include blood serotransferrin (or siderophilin, usually called transferrin); lactotransferrin (lactoferrin); milk transferrin; egg white ovotransferrin (conalbumin); and membrane-associated melanotransferrin.
  • cysteine protease inhibitors may be naturally occurring polypeptides, e.g. isolated or purified from milk or other fluid, recombinant polypeptides, or synthetic polypeptides.
  • Particularly useful forms of members of the transferrin family include any peptide than exhibits metal ion binding activity. Any of these inhibitors could be manufactured, modified, extracted or formulated as described for lactoferrin below.
  • a particularly useful cysteine protease inhibitor is lactoferrin.
  • the lactoferrin to be used in the methods of the invention includes, but is not limited to, lactoferrin, mutant lactoferrins, truncated lactoferrins, lactoferrin lobes or fusions of any of the above to other peptides, polypeptides, proteins or hydrolysates of lactoferrin.
  • Lactoferrin is an 80 kD iron-binding glycoprotein present in most exocrine fluids, including tears, bile, bronchial mucus, gastrointestinal fluids, cervico-vaginal mucus, seminal fluid, and milk. It is a major constituent of the secondary specific granules of circulating polymorphonuclear neutrophils. The richest source of lactoferrin is mammalian milk and colostrum.
  • Lactoferrin circulates at a concentration of 2 to 7 ⁇ g/mL. It has multiple postulated biological roles, including regulation of iron metabolism, immune function, and embryonic development. Lactoferrin has anti-microbial activity against a range of pathogens including Gram positive and Gram negative bacteria and fungi, including yeasts. The anti-microbial effect of lactoferrin is thought to be based on its capability of binding iron, which is essential for the growth of the pathogens. Lactoferrin also inhibits the replication of several viruses and increases the susceptibility of some bacteria to antibiotics and lysozyme by binding to lipid A component of lipopolysaccharides on bacterial membranes.
  • lactoferrin refers to pure lactoferrin, naturally derived, recombinant or synthetic lactoferrin, fragments of lactoferrin, variants of lactoferrin, hydrolysates of lactoferrin, or any mixture or combination thereof.
  • the lactoferrin may be a pure lactoferrin polypeptide containing no more than two (i.e., 0, 1 , or 2) metal ions per molecule.
  • the lactoferrin may be isolated or purified.
  • isolated or purified lactoferrin is substantially free of at least one agent or compound with which it is naturally associated.
  • an isolated protein is substantially free of at least some cellular material or contaminating protein from the cell or tissue source from which it is derived.
  • substantially free of cellular material refers to preparations where the lactoferrin is at least SO to 59% (w/w) pure, at least 60 to 69% (w/w) pure, at least 70 to 79% (w/w) pure, at least 80-89% (w/w) pure, at least 90 to 95% pure, or at least 96%, 97%, 98%, 99% or 100% (w/w) pure.
  • the purity of a polypeptide can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Practically, the measurement of the ion/lactoferrin ratio for a preparation of lactoferrin can be in the range of 0-2.5.
  • the lactoferrin can be a naturally occurring polypeptide, e.g. isolated from milk, a recombinant polypeptide, or a synthetic polypeptide.
  • Recombinant lactoferrin may be produced by expression in cell-free expression systems or in transgenic animals, plants, fungi or bacteria, or other useful species.
  • Recombinant human lactoferrin is available from ProSpec Protein Specialists.
  • lactoferrin may be produced using known organic synthetic methods.
  • the lactoferrin may be isolated from milk by cation exchange chromatography followed by ultrafiltration and diafiltration.
  • Useful lactoferrin fragments include individual components of hydrolysates of lactoferrin, fragments that include either or both the N and C lobe, fragments of the N- or C- lobes, lactoferricin and fragments generated (by artificial or natural processes) and identified by known techniques as discussed below.
  • lactoferrin can be of a mammalian origin. Verified sequences of bovine and human lactotransferrins (lactoferrin precursors), lactoferrins and peptides therein can be found in Swiss-Prot (http://au.expasy.org/cgi-bin/sprot- search-ful).
  • the lactoferrin may include, for example bovine lactotransferrin precursor accession number P24627 or its fragment bovine Lactoferricin B, or human lactotransferrin precursor accession number P02788 or its fragments Kaliocin-1, Lactoferroxin A, Lactoferroxin B, or Lactoferroxin.
  • lactoferrin amino acid and mRNA sequences that have been reported and are useful in any one of the first to fourth aspects include, but are not limited to: the amino acid (Accession Numbers AAW71443 and NP 002334) and mRNA (Accession Number NM 002343) sequences of human lactoferrin; the amino acid (Accession Numbers NP 851341 and CAA38572) and mRNA (Accession Numbers X54801 and NM_180998) sequences of bovine lactoferrin; the amino acid (Accession Numbers JC2323, CAA55517 and AAA97958) and mRNA (Accession Number U53857) sequences of goat lactoferrin; the amino acid (Accession Number CAA09407) and mRNA (Accession Number AJ010930) sequences of horse lactoferrin; the amino acid (Accession Number NP_001020033) and mRNA (Accession Number NM_001024862)
  • the lactoferrin is sheep, goat, pig, mouse, water buffalo, camel, yak, horse, donkey, llama, bovine or human lactoferrin.
  • the lactoferrin is buffalo or deer lactoferrin.
  • An animal from which lactoferrin may be produced may be a transgenic animal designed to over-express lactoferrin in its milk.
  • Variants of a wild-type lactoferrin polypeptide e.g., a fragment of the wild-type lactoferrin polypeptide containing at least 2 (e.g., 4, 6, 8, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700) amino acids, or a recombinant protein containing a lactoferrin polypeptide sequence
  • a recombinant protein containing a lactoferrin polypeptide sequence that maintain the biological activity of a wild-type lactoferrin polypeptide may be employed.
  • the lactoferrin can be produced using genetic engineering or chemical synthesis techniques well-known in the art.
  • variant refers to a naturally occurring (an allelic variant, for example) or non-naturally occurring (an artificially generated mutant, for example) lactoferrin that varies from the predominant wild-type amino acid sequence of a lactoferrin of a given species by the addition, deletion or substitution of one or more amino acids. Methods for generating such variants are known in the art. Useful recombinant lactoferrins and lactoferrin fragments and methods of producing them are reported in U.S. patent specifications U.S. Pat. No. 5,571,691, U.S. Pat. No. 5,571,697, U.S. Pat. No. 5,571,896, U.S. Pat. No.
  • lactoferrin may contain any number of conservative changes its amino acid sequence without altering its biological properties to produce a "variant". Such conservative amino acid modifications are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary conservative substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Also included within the meaning of the term "variant" are homologues of lactoferrin.
  • a homologue is typically a polypeptide from a different species but sharing substantially the same biological function or activity as the corresponding polypeptide. Variants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • Variant lactoferrin may be generated by techniques including but not limited to techniques for mutating wild type proteins such as, but not limited to: site-directed mutagenesis of a wild-type nucleotide sequence encoding lactoferrin and expression of the resulting polynucleotide; techniques for generating expressible polynucleotide fragments such as PCR using a pool of random or selected primers; techniques for full or partial proteolysis or hydrolysis of wild type or variant lactoferrin polypeptides; and techniques for chemical synthesis of polypeptides.
  • Variants or fragments of lactoferrin may be prepared by expression as recombinant molecules from lactoferrin DNA or RNA, or variants or fragments thereof.
  • Nucleic acid sequences encoding variants or fragments of lactoferrin may be inserted into a suitable vector for expression in a cell, including eukaryotic cells such as, but not limited to, Aspergillus or bacterial cells such as but not limited to E. coli.
  • Lactoferrin variants or fragments may be prepared using known PCR techniques including but not limited to error-prone PCR and DNA shuffling. Error-prone PCR is a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product.
  • DNA shuffling refers to forced homologous recombination between DNA molecules of different but highly related DNA sequence in vitro, caused by random fragmentation of the DNA molecule based on sequence homology, followed by fixation of the crossover by primer extension in a PCR reaction. Variants or fragments of lactoferrin may also be generated by known organic synthetic methods.
  • the lactoferrin used in the present invention can contain an iron ion (as in a naturally occurring lactoferrin polypeptide) or a non-iron metal ion (e.g., a copper ion, a chromium ion, a cobalt ion, a manganese ion, a zinc ion, or a magnesium ion).
  • an iron ion as in a naturally occurring lactoferrin polypeptide
  • a non-iron metal ion e.g., a copper ion, a chromium ion, a cobalt ion, a manganese ion, a zinc ion, or a magnesium ion.
  • lactoferrin isolated from bovine milk can be depleted of iron and then loaded with another type of metal ion.
  • copper loading can be achieved according to the same method for iron loading described above. Methods for loading lactoferrin with other metal ions are known
  • a preparation of lactoferrin can contain polypeptides of a single species, e.g., every molecule binding two iron ions. It can also contain polypeptides of different species, e.g., some molecules binding no ion and others each binding one or two ions; some molecules each binding an iron ion and others each binding a copper ion; some molecules each being a biological active lactoferrin polypeptide (full-length or shorter than full-length) that contains 0, 1, or 2 metal ions and others each being a fragment (same or different) of the polypeptide; or all molecules each being a fragment (same or different) of a full- length lactoferrin polypeptide that contains 0, 1, or 2 metal ions.
  • Metal ion-binding fragments of lactoferrin may be obtained by known techniques for isolating metal-binding polypeptides including, but not limited to, metal affinity chromatography. Fragments of lactoferrin may be contacted with free or immobilised metal ions, such as Fe 3+ and purified in a suitable fashion. For example, fragments may be contacted at neutral pH with a metal ion immobilised by chelation to a chromatography matrix comprising iminodiacetic acid or tris(carboxymethyl)-ethylenediamine ligands. Bound fragments may be eluted from the supporting matrix and collected by reducing the pH and ionic strength of the buffer employed. Metal-bound fragments may be prepared according to methods known in the art.
  • a mixture of full-length lactoferrin polypeptides and various fragments of full-length lactoferrin polypeptides can be prepared from a hydrolysate, e.g., a partial digest such as a proteinase digest, of full-length lactoferrin polypeptides.
  • a mixture of various fragments of full- length lactoferrin polypeptides can be prepared, for example, by complete digestion (i.e., no full-length polypeptides remain after digestion) of full-length lactoferrin polypeptides, or by mixing different fragments of full-length lactoferrin polypeptides.
  • the degree of digestion can be controlled according to methods well known in the art, e.g., by manipulating the amount of proteinase or the time of incubation. Otherwise, a mixture of full-length lactoferrin polypeptides and various fragments of full-length lactoferrin polypeptides can be obtained by mixing full- length lactoferrin polypeptides with various fragments of full-length lactoferrin polypeptides (e.g., synthetic fragments).
  • the lactoferrin comprises a full or partial enzyme hydrolysate (including but not limited to a proteinase, trypsin, chymotrypsin, chymosin, plasmin, pepsin, papain, peptidase, or aminopeptidase hydrolysates), a full or partial microorganism hydrolysate (including but not limited to hydrolysis by a bacterium from the genera Bacillus, Bifidus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Propionbacter, Pseudomonas or Streptococcus or a mixture thereof), a full or partial acid hydrolysate (including but not limited to trifluoro acetate and hydrochloric acid hydrolysates), a cyanogen bromide hydrolysate, or a mixture thereof.
  • a full or partial enzyme hydrolysate including but not limited to a proteinase, tryps
  • the lactoferrin hydrolysate may be a hydrolysate of a natural, recombinant or synthetic lactoferrin polypeptide or a mixture thereof.
  • the lactoferrin hydrolysate may be a human lactoferrin hydrolysate or a bovine lactoferrin hydrolysate or mixtures thereof.
  • the lactoferrin may be non-glycosylated or glycosylated.
  • the lactoferrin may be fully or partially glycosylated with naturally occurring or non-naturally occurring glycosyl groups.
  • the lactoferrin may be modified, for example by conjugation to a polymer to increase its circulating half-life such as by pegylation or other chemical modification. It may also be desirable to introduce a modification to lactoferrin to improve storage stability. Such modified lactoferrin is also envisaged for use according to the invention.
  • the lactoferrin may comprise about 50 to 100% by weight, or at least about 50, 55, 60,
  • lactoferrin 65, 70, 75, 80, 85, 90, 95 or 99% by weight, of lactoferrin.
  • Fresh skim milk (7 L, pH 6.5) is passed through a 300 ml column of S Sepharose Fast Flow equilibrated in milli Q water, at a flow rate of 5 ml/min and at 4° C. Unbound protein is washed through with 2.5 bed volumes of water and bound protein eluted stepwise with approximately 2.5 bed volumes each of 0.1 M, 0.35 M, and 1.0 M sodium chloride. Lactoferrin eluting as a discreet pink band in 1 M sodium chloride is collected as a single fraction and dialysed against milli Q water followed by freeze-drying.
  • the freeze-dried powder is dissolved in 25 mM sodium phosphate buffer, pH 6.5 and subjected to rechromatography on S Sepharose Fast Flow with a sodium chloride gradient to 1 M in the above buffer and at a flow rate of 3 ml/min.
  • Fractions containing lactoferrin of sufficient purity as determined by gel electrophoresis and reversed phase HPLC are combined, dialysed and freeze-dried.
  • Final purification of lactoferrin is accomplished by gel filtration on Sephacryl 300 in 80 mM dipotassium phosphate, pH 8.6, containing 0.15 M potassium chloride. Selected fractions are combined, dialysed against milli Q water, and freeze-dried.
  • the purity of this preparation is greater than 95% as indicated by HPLC analysis and by the spectral ratio values (280 nm/465 nm) of " 19 or less for the iron- saturated form of lactoferrin.
  • Iron saturation is achieved by addition of a 2:1 molar excess of 5 mM ferric nitrilotriacetate to a 1% solution of the purified lactoferrin in 50 mM Tris, pH 7.8 containing 10 mM sodium bicarbonate. Excess ferric nitrilotriacetate is removed by dialysis against 100 volumes of milli Q water (twice renewed) for a total of 20 hours at 4° C. The iron-loaded (holo-) lactoferrin is then freeze-dried.
  • Iron-depleted (apo-) lactoferrin is prepared by dialysis of a 1% solution of the highly purified lactoferrin sample in water against 30 volumes of 0.1 M citric acid, pH 2.3, containing 500 mg/L disodium EDTA, for 30 h at 4° C. Citrate and EDTA are then removed by dialysis against 30 volumes of milli Q water (once renewed) and the resulting colourless solution freeze- dried.
  • the lactoferrin may be encapsulated, microencapsulated or nanoencapsulated, for example for oral administration.
  • the lactoferrin may be used to inhibit a gingipain or to treat any disease, disorder or biological activity modulated by a gingipain.
  • treating and “treatment” as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms (prophylaxis) and/or their underlying cause, and improvement or remediation of damage.
  • the present method of "treating" diseases modulated by gingipains encompasses both prevention of the condition or disorder in a predisposed individual and treatment of the condition or disorder in a clinically symptomatic individual.
  • Treating covers any treatment of, or prevention of a condition or disorder in a vertebrate, a mammal, particularly a human, and includes: inhibiting the condition or disorder, i.e., arresting its development; or relieving or ameliorating the effects of the condition or disorder, i.e., cause regression of the effects of the condition or disorder.
  • symptom refers to a phenomenon which arises from and accompanies a particular condition or disorder, i.e. underlying cause, and serves as an indication of that condition or disorder.
  • a "symptom” may be directly observable in a subject, or may be indirectly observable, for example by use of a laboratory test or assay.
  • treatment of a symptom includes treatment of the underlying cause and treatment of the underlying cause includes treatment of the symptom.
  • “Prophylaxis” or “prophylactic” or “preventative” therapy as used herein includes preventing the condition from occurring or ameliorating the subsequent progression of the condition in a subject that may be predisposed to the condition, but has not yet been diagnosed as having it.
  • Reference to a disease, disorder or biological effect being "modulated" by a gingipain is intended to mean that the gingipain plays a role in the disease, disorder or biological effect to the extent that reduction in the activity of the gingipain is desirable.
  • the gingipain need not be the sole causative agent.
  • Gingipains play a number of physiological roles in bacteria, more particularly in controlling the expression of other virulence factors as well as in the stability and/or processing of extracellular or cell surface proteins.
  • Grenier and Tanabe show that P. gingivalis gingipains K and R induce an inflammatory response in macrophages, through activation of intracellular kinases. They showed that gingipains K and R induce the production of TNF a and IL-8 by macrophages.
  • Other studies show that gingipains R and K stimulate the secretion of proinflammatory cytokines by monocytes through PAR -1, -2 and 3.
  • Gingipains R and K are found in the mouth, in the blood and on mucosal surfaces, such as in the gastrointestinal tract.
  • the present invention provides means to inhibit these gingipains and to reduce the risk of cell and tissue invasion by gingipain-producing pathogens thereby accordingly treat diseases modulated by gingipains.
  • Lactoferrin can therefore be used to reduce inflammation, particularly of mucosal surfaces such as in the GI tract. It may also be used to lower the risk of developing cancers and cardiovascular disease, associated with gingipain- producing pathogens.
  • a disease, disorder or biological effect are known to be modulated by a gingipain as defined herein, it will be evident that the disease, disorder or biological effect can be treated by administration of lactoferrin.
  • a subject at risk of oral cell or tissue invasion by P. gingivalis may be determined by the presence of planktonic P. gingivalis cells in the subgingiva.
  • a subject may also exhibit elevated gingipain activity in the subgingival pocket or associated with the epithelium of the subgingiva.
  • the lactoferrin is for treating a patient for a disorder modulated by a gingipain, which patient also has periodontitis.
  • a subject with periodontitis may be identified by any known clinical means.
  • Clinical manifestations of periodontitis include acute or chronic inflammation of the gingiva.
  • the hallmarks of acute inflammation may be present including an increased movement of plasma and leukocytes from the blood into the injured tissues.
  • Clinical signs of acute infection of the gingiva may also be present including rubor (redness), calor (increased heat), tumor (swelling), dolor (pain), and function laesa (loss of function).
  • Chronic inflammation may be characterised by leukocyte cell (monocytes, macrophages, lymphocytes, plasma cells) infiltration. Tissue and bone loss may be observed.
  • Periodontitis may also be characterised by an increased level of Porphyromonas bacteria, in particular P. gingivalis, above a normal range observed in individuals without periodontitis.
  • the lactoferrin used in any one of the aspects may be provided as a nutraceutical or as a pharmaceutical or veterinary formulation.
  • the term "nutraceutical” as used herein refers to an edible product that may be isolated or purified from food, e.g. a milk product, which is demonstrated to have a physiological benefit or to provide protection or attenuation of an acute or chronic disease or injury when orally administered.
  • the nutraceutical may thus be presented in the form of a dietary preparation or supplement, either alone or admixed with edible foods or clrinks.
  • “Nutraceuticals” are also referred to as "functional foods”.
  • Nutraceuticals can be produced by various methods and processes known in the art including, but not limited to, synthesis (chemical or microbial), extraction from a biological material, mixing functional ingredient or component to a regular food product, fermentation or using a biotechnological process.
  • a nutraceutical may exert its effects directly in the body or it may function e.g. through intestinal bacterial flora.
  • nutraceuticals will contain lactoferrin purified to some degree, or at the very least, all components of the nutraceutical will be verifiable.
  • suitable foods, drinks or edible consumer products include soluble powders, milk powders, confectionary, reconstituted fruit products, breakfast cereals, ready-to-eat bars, snack bars, muesli bars, spreads, dips, diary products including yoghurts and cheeses, a liquid or a ready-to-drink formulation including dairy and non-dairy based drinks (e.g.
  • milks, juices, teas, or soft drinks include food supplements, a dietary supplements (e.g., a hard or soft capsule, a mini-bag, or a tablet, a tea-bag), nutritional formulations, sports nutrition supplements including dairy and non-dairy based sports nutrition supplements, an infant formula, particularly a humanised milk formula for administration to infants, food additives such as protein sprinkles and dietary supplement products including daily supplement tablets.
  • a dietary supplements e.g., a hard or soft capsule, a mini-bag, or a tablet, a tea-bag
  • nutritional formulations sports nutrition supplements including dairy and non-dairy based sports nutrition supplements
  • sports nutrition supplements including dairy and non-dairy based sports nutrition supplements
  • an infant formula particularly a humanised milk formula for administration to infants
  • food additives such as protein sprinkles and dietary supplement products including daily supplement tablets.
  • the nutraceutical preferably has acceptable sensory properties (such as acceptable smell, taste and palatability).
  • the nutraceutical may be produced as is conventional; for example, the nutraceutical may be prepared by blending together the protein and other additives, for example, various flavours, fibres, sweeteners, and other additives may also be present. If used, an emulsifier may be included in the blend.
  • the nutraceutical may include other nutrients such as amino acids, a protein, or a carbohydrate. Additional vitamins and minerals may be added at this point but are usually added later to avoid thermal degradation. Further vitamins and/or minerals may be selected from at least one of vitamins A, Bl, B2, B3, B5, B6, Bl l, B12, biotin, C, D, E, H and calcium, magnesium, potassium, zinc and iron.
  • the lactoferrin may be admixed with additional components in powdered form.
  • the powder should have a moisture content of less than about 5% by weight.
  • Water preferably water which has been subjected to reverse osmosis, may then be mixed in to form a liquid mixture.
  • the nutraceutical is to be provided in a ready to consume liquid form, it may be heated in order to reduce the bacterial load. If it is desired to produce a liquid nutraceutical, the liquid mixture is preferably aseptically filled into suitable containers. Aseptic filling of the containers may be carried out using techniques commonly available in the art. Suitable apparatus for carrying out aseptic filling of this nature is commercially available.
  • nutraceutical also comprises one or more pharmaceutically acceptable carriers, diluents or excipients.
  • Nutraceuticals may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose, lactose, lactulose, or dextrans; mannitol or lactitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA; adjuvants and preservatives.
  • the lactoferrin used as a nutraceutical need not be totally pure. However, to reduce the amount of composition to be administered it is preferred that the lactoferrin is concentrated significantly with respect to its concentration in milk. Preferably the lactoferrin is administered in at a concentration of at least 10 times its concentration in milk and more preferably 20, 30, 40, or 50 times its concentration in milk.
  • a pharmaceutical formulation is one which is suitable for administration to humans.
  • a veterinary formulation is one that is suitable for administration to animals. Generally such formulations will contain purified lactoferrin or compositions comprising lactoferrin in which all other components are identifiable.
  • the pharmaceutical or veterinary formulation may comprise lactoferrin formulated with one or more carriers, diluents, adjuvants and/or excipients and optionally other therapeutic agents.
  • Each carrier, diluent, adjuvant and/or excipient may be pharmaceutically "acceptable”.
  • a “pharmaceutically acceptable carrier” is meant a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected active agent without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • a “pharmaceutically acceptable” salt or ester of a novel compound as provided herein is a salt or ester which is not biologically or otherwise undesirable.
  • a “pharmaceutical carrier” is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering the agent to the subject.
  • the carrier may be liquid or solid and is selected with the planned manner of administration in mind.
  • Each carrier must be pharmaceutically “acceptable” in the sense of being not biologically or otherwise undesirable i.e. the carrier may be administered to a subject along with the agent without causing any or a substantial adverse reaction.
  • the formulations may be administered orally, topically, or parenterally in formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • the formulations are administered orally.
  • the formulations may be administered orally as tablets, aqueous or oily suspensions, lozenges, troches, powders, granules, emulsions, gels, capsules, syrups, chewing gums, toothpastes, toothpowders, and dentifrices, mouth washes, dental pastes, gargle tablets, dairy products, elixirs or other foodstuffs.
  • the formulation for oral use may contain one or more agents selected from the group of sweetening agents, flavouring agents, colouring agents and preserving agents in order to produce pharmaceutically elegant and palatable preparations. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin.
  • Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar.
  • Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring.
  • Suitable preservatives include sodium benzoate, vitamin E, alphatocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite.
  • Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc.
  • Suitable time delay agents include glyceryl monostearate or glyceryl distearate.
  • the tablets may contain the agent in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be, for example, (1) inert diluents, such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents, such as corn starch or alginic acid; (3) binding agents, such as starch, gelatin or acacia; and (4) lubricating agents, such as magnesium stearate, stearic acid or talc.
  • These tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • tablets can be formulated in accordance with conventional procedures by compressing mixtures of the lactoferrin with a solid carrier and a lubricant.
  • solid carriers include starch and sugar bentonite.
  • the lactoferrin can also be administered in the form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tableting agent.
  • parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, subconjunctival, intracavity, transdermal and subcutaneous injection, aerosol for administration to lungs or nasal cavity or administration by infusion by, for example, osmotic pump.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride
  • lactated Ringer's intravenous vehicles include fluid and nutrient replenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, anti-microbials, anti-oxidants, chelating agents, growth factors and inert gases and the like.
  • the formulations may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.
  • the formulations may also be included in a container, pack, or dispenser together with instructions for administration.
  • the pharmaceutical and veterinary formulations may be presented for use in the form of oral care formulations, which may be presented, for example, by methods that are convenient in the art.
  • oral care formulations include dentifrice (toothpastes), tooth cremes, tooth powders and mouth wash formulations for OTC or dental professional application.
  • An oral formulation for use in the methods of the invention which contains the above- mentioned gingipain inhibitor may be prepared and used in various forms applicable to the mouth such as dentifrice including toothpastes, toothpowders and liquid dentifrices, mouthwashes, saliva substitute, troches, chewing gums, dental pastes, gingival massage creams, gargle tablets, dairy products and other foodstuffs.
  • An oral formulation for use in the methods of the invention may further include additional well known ingredients depending on the type and form of a particular oral formulation.
  • the formulation may further include one or more antibiotics that are toxic to or inhibit the growth of Gram negative anaerobic bacteria.
  • antibiotics include amoxicillin, tetracycline, doxycycline or metronidazole.
  • the oral formulation may be substantially liquid in character, such as a mouthwash or rinse.
  • the vehicle is typically a water-alcohol mixture desirably including a humectant as described below.
  • the weight ratio of water to alcohol is in the range of from about 1:1 to about 20:1.
  • the total amount of water-alcohol mixture in this type of formulation is typically in the range of from about 70 to about 99.9% by weight of the formulation.
  • the alcohol is typically ethanol or isopropanol. Ethanol is preferred.
  • the pH of such liquid and other formulations used in the methods of the invention is generally in the range of from about 5 to about 9 and typically from about 5.0 to 7.0.
  • the pH can be controlled with acid (e.g. citric acid or benzoic acid) or base (e.g. sodium hydroxide) or buffered (as with sodium citrate, benzoate, carbonate, or bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, etc).
  • the formulation for use in the methods of the invention may be substantially solid or pasty in character, such as toothpowder, a dental tablet or a toothpaste (dental cream) or gel dentifrice.
  • the vehicle of such solid or pasty oral formulations generally contains dentally acceptable polishing material.
  • the liquid vehicle may comprise water and humectant typically in an amount ranging from about 10% to about 80% by weight of the formulation.
  • Glycerine, propylene glycol, sorbitol and polypropylene glycol exemplify suitable humectants/carriers.
  • liquid mixtures of water, glycerine and sorbitol are also advantageous. In clear gels where the refractive index is an important consideration, about 2.5 - 30% w/w of water, 0 to about 70% w/w of glycerine and about 20-80% w/w of sorbitol are preferably employed.
  • Toothpaste, creams and gels typically contain a natural or synthetic thickener or gelling agent in proportions of about 0.1 to about 10, preferably about 0.5 to about 5% w/w.
  • a suitable thickener is synthetic hectorite, a synthetic colloidal magnesium alkali metal silicate complex clay available for example as Laponite (e.g. CP, SP 2002, D) marketed by Laporte Industries Limited.
  • Laponite D is, approximately by weight 58.00% Si0 2 , 25.40% MgO, 3.05% Na 2 0, 0.98% Li 2 0, and some water and trace metals. Its true specific gravity is 2.53 and it has an apparent bulk density of 1.0 g/ml at 8% moisture.
  • thickeners include Irish moss, iota carrageenan, gum tragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose, hydroxybutyl methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose (e.g. available as Natrosol), sodium carboxymethyl cellulose, and colloidal silica such as finely ground Syloid (e.g. 244).
  • Irish moss iota carrageenan
  • gum tragacanth starch
  • polyvinylpyrrolidone hydroxyethylpropylcellulose
  • hydroxybutyl methyl cellulose hydroxypropyl methyl cellulose
  • sodium carboxymethyl cellulose hydroxyethyl cellulose
  • colloidal silica such as finely ground Syloid (e.g. 244).
  • Solubilizing agents may also be included such as humectant polyols such propylene glycol, dipropylene glycol and hexylene glycol, cellosolves such as methyl cellosolve and ethyl cellosolve, vegetable oils and waxes containing at least about 12 carbons in a straight chain such as olive oil, castor oil and petrolatum and esters such as amyl acetate, ethyl acetate and benzyl benzoate.
  • humectant polyols such propylene glycol, dipropylene glycol and hexylene glycol
  • cellosolves such as methyl cellosolve and ethyl cellosolve
  • vegetable oils and waxes containing at least about 12 carbons in a straight chain such as olive oil, castor oil and petrolatum and esters such as amyl acetate, ethyl acetate and benzyl benzoate.
  • a bottle of mouth rinse will have a label describing it, in substance, as a mouth rinse or mouthwash and having directions for its use; and a toothpaste, cream or gel will usually be in a collapsible tube, typically aluminium, lined lead or plastic, or other squeeze, pump or pressurized dispenser for metering out the contents, having a label describing it, in substance, as a toothpaste, gel or dental cream.
  • Organic surface-active agents may be used in the oral formulation to achieve increased therapeutic or prophylactic action, assist in achieving thorough and complete dispersion of the active agent throughout the oral cavity, and render the instant formulations more cosmetically acceptable.
  • the organic surface-active material is preferably anionic, non-ionic or ampholytic in nature and preferably does not interact with the lactoferrin. It is preferred to employ as the surface-active agent a detersive material which imparts to the formulation detersive and foaming properties.
  • anionic surfactants are water-soluble salts of higher fatty acid monoglyceride monosulfates, such as the sodium salt of the monosulfated monoglyceride of hydrogenated coconut oil fatty acids, higher alkyl sulfates such as sodium lauryl sulfate, alkyl aryl sulfonates such as sodium dodecyl benzene sulfonate, higher alkylsulfo-acetates, higher fatty acid esters of 1,2-dihydroxy propane sulfonate, and the substantially saturated higher aliphatic acyl amides of lower aliphatic amino carboxylic acid compounds, such as those having 12 to 16 carbons in the fatty acid, alkyl or acyl radicals, and the like.
  • Examples of the last mentioned amides are N-lauroyl sarcosine, and the sodium, potassium, and ethanolamine salts of N-lauroyl, N-myristoyl, or N-palmitoyl sarcosine which should be substantially free from soap or similar higher fatty acid material.
  • the use of these sarconite compounds in oral formulations is particularly advantageous since these materials exhibit a prolonged marked effect in the inhibition of acid formation in the oral cavity due to carbohydrate breakdown in addition to exerting some reduction in the solubility of tooth enamel in acid solutions.
  • Examples of water- soluble non-ionic surfactants suitable for use are condensation products of ethylene oxide with various reactive hydrogen-containing compounds reactive therewith having long hydrophobic chains (e.g.
  • condensation products contain hydrophilic polyoxyethylene moieties, such as condensation products of poly (ethylene oxide) with fatty acids, fatty alcohols, fatty amides, polyhydric alcohols (e.g. sorbitan monostearate) and polypropyleneoxide (e.g. Pluronic materials).
  • the surface active agent is typically present in amount of about 0.1-5% by weight. It is noteworthy, that the surface active agent may assist in the dissolving of lactoferrin and thereby diminish the amount of solubilizing humectant needed.
  • Various other materials may be incorporated in the oral formulation for use in the methods of the invention such as whitening agents, preservatives, silicones, chlorophyll compounds and/or ammoniated material such as urea, diammonium phosphate, and mixtures thereof.
  • whitening agents such as whitening agents, preservatives, silicones, chlorophyll compounds and/or ammoniated material such as urea, diammonium phosphate, and mixtures thereof.
  • flavouring or sweetening material may also be employed.
  • suitable flavouring constituents are flavouring oils, e.g. oil of spearmint, peppermint, wintergreen, sassafras, clove, sage, eucalyptus, marjoram, cinnamon, lemon, and orange, and methyl salicylate.
  • suitable sweetening agents include sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP (aspartyl phenylalanine, methyl ester), saccharine, and the like.
  • flavour and sweetening agents may each or together comprise from about 0.1% to 5% more of the preparation.
  • formulations for use in the methods of the invention can also be incorporated in lozenges, or in chewing gum or other products, e.g. by stirring into a warm gum base or coating the outer surface of a gum base, illustrative of which are jelutong, rubber latex, vinylite resins, etc., desirably with conventional plasticizers or softeners, sugar or other sweeteners or such as glucose, sorbitol and the like.
  • Formulations intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical formulations and such formulations may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monoole
  • the aqueous suspensions may also contain one or more preservatives, for example benzoates, such as ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example benzoates, such as ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide palatable oral preparations. These formulations may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • the formulations may be presented for use in the form of veterinary formulations, which may be prepared, for example, by methods that are conventional in the art. Examples of such veterinary formulations include those adapted for:
  • oral administration external application, for example drenches (e.g. aqueous or non-aqueous solutions or suspensions); tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue, particularly adapted for protection through the rumen if to be administered to ruminants;
  • drenches e.g. aqueous or non-aqueous solutions or suspensions
  • tablets or boluses e.g. aqueous or non-aqueous solutions or suspensions
  • powders, granules or pellets for admixture with feed stuffs
  • pastes for application to the tongue, particularly adapted for protection through the rumen if to be administered to ruminants;
  • parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g. as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced in the udder via the teat;
  • topical applications e.g. as a cream, ointment or spray applied to the skin; or
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the formulations are to be administered in therapeutically effective amounts.
  • an "effective amount" of lactoferrin is a dosage which is sufficient to inhibit a gingipain.
  • a therapeutical effective amount may vary with the subject's age, condition, and sex, as well as the severity of the medical condition in the subject.
  • the dosage may be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • Appropriate dosages for administering lactofemn may range from 5 mg to 100 mg, from 15 mg to 85 mg, from 30 mg to 70 mg, or from 40 mg to 60 mg, 5 mg to 500 mg, 10 mg to 400 mg, 20 mg to 300 mg, 25 mg to 250 mg, 40 mg to 200 mg, 50 mg to 100 mg.
  • doses may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 125, 150, 175, 200, 225, 230, or 250 mg.
  • the formulations can be administered in one dose, or at intervals such as once daily, twice daily, once weekly, and once monthly. Dosage schedules can be adjusted depending on the half life of lactoferrin, or the severity of the medical condition in the subject.
  • a composition including lactoferrin for inhibiting proteinase activity, especially gingipain activity does not include an active ingredient for preventing or treating periodontal disease other than lactoferrin.
  • the only active principle for inhibiting proteinase activity, or otherwise for preventing or treating periodontal disease, especially conditions mediated by P. gingivalis (such as periodontitis) is lactoferrin.
  • the composition does not substantially include protein other than lactoferrin.
  • the lactoferrin may be the only protein acting as a cysteine proteinase inhibitor, or otherwise, lactoferrin may be the only protein contained in the composition.
  • the composition may be substantially devoid of other components of dairy, or extract or whey stream thereof. In these embodiments, the composition may not contain casein, lactoalbumin, or other significant protein, lipid or carbohydrate components of dairy, other than lactoferrin. All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference.
  • LF Native Bovine lactoferrin
  • Apo-LF had an iron saturation of 0.8%, native LF had an iron saturation of 7.4% whilst holo-LF was 98.7% iron saturated as determined by Atomic Absorption Spectrometry (AA240, Varian, Inc. CA, USA) at a wavelength of 248.3 nm.
  • Proteinase activity of P. gingivalis whole cells The Arg- and Lys-specific proteinase activities of P. gingivalis whole cells were measured in a 96-well plate using the chromogenic substrates N-a-benzoyl-Arg-p-nitroanilide (L-BApNA) and N-(p-Tosyl)-Gly-Pro-Lys 4-nitroanilide acetate salt (GPK-NA) (Sigma), respectively, essentially as described previously by O'Brien-Simpson et al., 2001 Infect Immun 69(12): 7527-7534. P.
  • L-BApNA N-a-benzoyl-Arg-p-nitroanilide
  • GPK-NA N-(p-Tosyl)-Gly-Pro-Lys 4-nitroanilide acetate salt
  • gingivalis cells that were grown as described above were harvested by centrifugation (8000 xg, 4°C, 20 min), the cell pellet aseptically collected, washed with 20 mL and suspended in 2 mL of ice cold TCI 50 buffer (pH 8.0) comprising 2.8 mM cysteine, 50 mM Tris-HCl, 150 mM NaCl, and 5 mM CaCl 2 . The final cell density was 4.5 x 10 7 cell/mL.
  • the proteinase activity of P. gingivalis was measured by the change in colour intensity at 405 nm after L-BApNA or GPK-NA was incubated with P. gingivalis cells in the presence and absence of LF. All colour intensity curves were linear from 20 s to at least 4.4 min after the addition of substrate. Therefore, proteinase activity was determined by calculating the absorbance change rate over this time.
  • P. gingivalis cell suspensions (2.5 ⁇ ) were pre-incubated with LF preparations at concentrations between 0.01 - 10 mg/mL in TCI 50 buffer with a total volume of 100 ⁇ at 37°C for 15 - 90 min.
  • substrate preparation 100 yl that contained 2 mM BApNA or GPK-NA, 30% (v/v) isopropanol, 400 mM Tris-HCl (pH 8), 100 mM NaCl, and 2 mM cysteine was added, and the absorbance at a wavelength of 405 nm was determined from 0 to 10 min.
  • RgpA-Kgp proteinase-adhesin complexes were prepared as described previously Pathirana RD et al., 2006 (Microbiology 152:2381-2394) with minor modifications.
  • P. gingivalis strain W50 cells were harvested from 1 L of planktonic culture grown to late exponential phase, washed and resuspended in 30 mL of TC buffer (50 mM Tris-HCl, pH 7.4, 5 mM CaCl 2 ) containing 50 mM NaCl.
  • the cell suspension was ultrasonicated using a Branson Sonifier 250 as described previously in Bhogal PS et al., 1997 Microbiology 143: 2485-2495.
  • the resulting cell sonicate was clarified by centrifugation (40,000 x g, 30 min, 4°C), made up to 50 mL with TC buffer containing 50 mM NaCl and 2 mM MgCl 2 , then incubated with freshly added benzonase (140 U/mL) for 1 h on ice.
  • the benzonase-treated cell sonicate was loaded onto a 50-mL Arginine Sepharose 4B column at 1 mL/min and monitored at 280 nm using an AKTA explorer 100 automatic liquid chromatography system (GE Healthcare Bio-Science AB SE-751 84 Uppsala, Sweden).
  • the column After being stepped-washed with 150, 200 and 250 mM NaCl in TC buffer, the column was re-equilibrated in TC buffer containing 50 mM NaCl, then eluted with 500 mM arginine (Sigma) in the same buffer at 1.6 mL/min to recover the bound RgpA- Kgp proteinase-adhesin complexes.
  • the eluted protein peak fraction of 25 mL was concentrated using a Centriprep YM-10 centrifugal filter device (Millipore) and buffer-exchanged into TC buffer with 50 mM NaCl using a PD-10 desalting column (GE Healthcare). The final protein fraction prepared from 1 L of P.
  • gingivalis culture contained ⁇ 3 mg protein with 6.8 U/mg and 2.1 U/mg of Arg-X and Lys-X specific activities, respectively.
  • the protein concentration was determined using Bradford assays (Bio-Rad) with bovine serum albumin standard.
  • the Arg-X and Lys-X activities were measured in a Diode Array spectrophotometer using the chromogenic substrates Bz-L-Arg-/?NA (L-BAPNA) (Sigma) and Z-L-Lys-/?NA (Novabiochem), respectively as described previously by Pathirana RD et ah, 2006.
  • One unit (U) of the activity is equivalent to 1 mmol substrate hydrolyzed per min at 37°C, pH 8.0.
  • P. gingivalis (ATCC 33277) was cultured and harvested as described above. The cells were washed and resuspended to the initial volume with Pga buffer (pH 7.5), which was modified from Milner et al. (1996) FEMS Microbiol Lett 140(2-3): 125-130.
  • LF was dissolved in Pga buffer to a concentration of 2 mg/mL. Equal volumes of LF solution and cell suspension were mixed thoroughly and incubated at 37°C for between 10 min - 3 days. To terminate P. gingivalis proteolytic activity 0.46 M acetic acid was added to lower the pH to 4.4. The preparation was filtered (0.22 um) to remove the cells and this preparation was referred to as LF-Pg. The degree of hydrolysis was determined by SDS-PAGE.
  • Size exclusive chromatography was applied to separate the fragments in LF-Pg using a SuperdexTM 75 10/300 GL column (GE Healthcare) connected to an AKTA explorer 100 (GE Healthcare).
  • the elution buffer was 50 mM phosphate buffer pH 6.0 with 750 mM NaCl.
  • Flow rate was 0.5 mL/min with an injection volume of 250 ⁇ . of filtered (0.22 um) sample.
  • LF-Pg preparation at 6 h incubation was subjected to fractionation using an Aquapore OD-300 reverse-phase column (7 ⁇ , 4.6 x 250 mm, PerkinElmer Brownlee Columns, Shelton, CT, USA) connected to an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA).
  • the eluting solvents consisted of (A): 0.01% (v/v) trifluoroacetic acid (TFA) in milliQ water and (B): 0.01% (v/v) TFA in 80% acetonitrile and 20% milliQ water (HPLC grade).
  • the injection volume was 100 per sample, and the system was operated at a flow rate of 1 mL/min.
  • the protein content was detected by a diode array detector at wavelengths of 214 and 280 nm.
  • the column was initially equilibrated with 100% mobile phase A for 10 min, followed by elution with a linear gradient from 0% to 40% of mobile phase B for 40 min, from 40% to 50% for 30 min, from 50% to 60% for 10 min, from 60% to 100% for 5 min.
  • the elution peaks were collected, freeze dried, and reconstituted with milliQ water.
  • the protein concentrations were determined using Bradford micro-assay from Bio-Rad (Bradford, 1976 Anal Biochem 72: 248-254.).
  • LF fractions were mixed 1:1 with matrix B (sinapinic acid saturated in 0.1% TFA, 30% acetonitrile) and 1 deposited onto the thin layer and allowed to dry.
  • ISD spectra were acquired using a standard reflectron method optimised for peptides except for an increased PIE delay of 200 ns, and enhanced sensitivity settings on the digitiser and reflector detector. Laser power was increased until ISD peaks appeared, and then spectra from 1600 laser shots were acquired.
  • P. gingivalis was anaerobically grown in brain heart infusion (BHI) broth (37 g L, Oxoid Australia Pty Ltd, South Australia, Australia), supplemented with haemin (5 ⁇ g/mL), vitamin K3 (5 ⁇ g mL) and cysteine (0.5 mg/mL) at 37°C until a cell density of 2.8 x 10 10 cfu/mL was reached (OD of the culture reached 0.6 at a wavelength of 650 nm). The culture was then 10 times diluted with supplemented BHI and kept on ice.
  • BHI brain heart infusion
  • LF and other antimicrobials were prepared 10 times more concentrated than the final concentration, and sterilized by passing through a 0.22 um filter. All antimicrobial solutions (20 ⁇ ) were added to 96-well plate with 6 replicates while control samples contained 20 ⁇ L distilled water. The bacterial culture (180 iL) was added to each well to make the total volume of 200 ⁇ L ⁇ . The plate was incubated at 37°C for 24 h in an anaerobic chamber (MK3 Anaerobic work-station, Don Whitley Scientific Ltd., Sydney, NSW, Australia) with an atmosphere of 5% hydrogen, 10% carbon dioxide and 85% nitrogen. To determine the effect of haemin on biofilm formation haemin was not added to growth medium.
  • the plate was shaken at 100 rpm at 37°C for 15 min, and all media were removed. Each well was washed with milliQ water and blow-dried by air for at least 3 h. Crystal violet (0.1%) was used to stain the biofilm on each well surface at room temperature for 15 min. Unbound crystal violet was then removed by washing twice with milliQ water. The plate was blow-dried by air briefly and 100 ⁇ ⁇ ethanol containing 20% (v/v) acetone was used to dissolve the bound colour from the well surface. The mass of the biofilm on the well surface was expressed as the absorbance at 600 nm for each sample in the well, using a UV-Spectrometer (VICTOR3TM1420 Multilabel Counter, PerkinElmer, MA, USA).
  • P. gingivalis planktonic growth assay The effect of LF on P. gingivalis was determined in a 96 well plate assay using supplemented BHI growth medium under anaerobic conditions at 37°C as described previously (Malkoski et al., 2001 Antimicrob Agents Chemother 45(8): 2309-15).
  • Crystallographic models of RgpB and the C-lobe of lactoferrin (PDBrlcvr and 3taj respectively) were down-loaded from the Protein Data Bank. Structures were prepared for further modeling using the AMBER-99 force-field and the program Sybyl. Crystallographic water molecules were removed, and the resulting structures initially energy minimized to a maximum energy derivative of 0.5 kcal mol '1 A "1 .
  • the 3taj (C-lobe lactoferrin) structure was then manually docked against the lcvr (RgpB) structure so that Glu 659 of the C-lobe of lactoferrin that binds a zinc ion in the crystal structure could be constrained to the zinc ion bound to the catalytic histidine of RgpB, His 244 .
  • the atoms of the RgpB-inhibitor (DFFR- chloromethylketone) were removed for the dynamics simulation.
  • the docked structures were then solvated with 'TIP3P' waters using a 'droplet' solvation model and energy minimized to a maximum energy derivative of 0.5 kcal mol "1 A "1 .
  • LF inhibited both the Arg- and Lys-specific proteinase activities of P. gingivalis whole cells by approximately 40% at 1.0 mg mL (12.5 uM) and over 70% at 10 mg/mL (125 ⁇ ) (Table 1).
  • Bovine Serum Albumin (BSA) had no effect on P. gingivalis Arg- and Lys-specific proteinase activities at concentrations up to 1 mg/mL.
  • BSA Bovine Serum Albumin
  • BSA displayed a small effect on the hydrolysis of the chromogenic substrate, which was attributed to substrate competition (Table 1).
  • gingivalis RgpA/ gp proteinase-adhesin complexes by LF demonstrated time-dependent inhibition with a first-order inactivation rate constant (ki nact ) of 0.023 min " 1 and an inhibitor affinity constant (Ki) of 5.02 ⁇ (Fig. 1).
  • LF was incubated with purified RgpB which lacks the adhesin domains of RgA and Kgp.
  • LF inhibited RgpB activity by 77% at a concentration of 1.0 mg mL and by 95% at 10 mg mL confirming the inhibition was independent of adhesins.
  • LF was incubated anaerobically with P. gingivalis whole cells for 3 days in Pga buffer, which was formulated to ensure the survival of the bacterium and activity of the cell surface proteinases. Samples taken at specified time points were subjected to SDS-PAGE analysis and used in the P. gingivalis biofilm assay. There was a limited initial hydrolysis of LF by P. gingivalis resulting in two major products, Fragment I (53 kDa) and Fragment IV (33 kDa) and a minor product, Fragment III (40 kDa) (Lanes 2-4 in Fig 3A).
  • Fragment I was a C- terminal fragment of LF and that Fragment IV was an N-terminal fragment (Fig 4). It appears that Fragment IV was further hydrolysed by loss of the N-terrninal portion to yield Fragment V. Fragment II which was only generated in significant quantities after 6 h incubation with P. gingivalis corresponds to a C-terminal fragment of Fragment I (Fig 4).
  • the sequence DLLFKDSALGFLRI PSKVDSALYLGSRY was directly determined from the series of peaks, while the mass of each peak indicated that the N-terminus of Fragment I was S 285 .
  • the primary cleavage site of LF when exposed to P. gingivalis was therefore R -S (Fig 4).
  • Analysis of LF-Pg samples with size exclusion chromatography revealed a single peak (SEC, Fig 7A), and SDS-PAGE analysis of the material in this peak showed it consisted of the two fragments (33 and 53 kDa) as well as a much lower amount of native LF (Fig 7B).
  • bovine serum albumin BSA
  • ⁇ -lactoglobulin ⁇ -Lg
  • the major virulence factors of P. gingivalis are its cysteine proteinases RgpA and Kgp and associated adhesins that form large complexes on the cell surface and cleave C-terminal to arginine or lysine residues. These proteinase-adhesin complexes hydrolyse a range of host regulatory proteins, peptides and cell receptors leading to dysregulation of the host immune response and subsequent tissue damage.
  • the proteinases are essential for tissue invasion by P. gingivalis in an animal model that has been used extensively to determine the invasive characteristics of pathogenic bacteria.
  • LF is a slow binding inhibitor with a slow dissociation rate.
  • the molecular dynamics simulation of the interaction between LF and RgpB (Fig 2) provided a mechanism for the observed inhibition.
  • the zinc-ion binding C-lobe of LF can bind the zinc ion in the active site of the RgpA/B and Kgp proteinases ultimately forming a stable structure explaining the observed time-dependent inactivation of the proteinases.
  • Antimicrobial peptides and proteins in host secretions may have reduced efficacy against bacteria due to their susceptibility to hydrolysis by cell surface or secreted bacterial proteinases.
  • P. gingivalis proteinases have been shown to degrade a range of host proteins including human transferrin and hemoglobin. The high level of P. gingivalis cell surface and extracellular proteolytic activity has been shown to hydrolyse antibacterial proteins and peptides such as histatin, thereby reducing their efficacy.
  • Fig 4 despite an abundance of arginine and lysine residues (Fig 4) LF was relatively resistant to hydrolysis by P. gingivalis proteinases (Fig 3). After 3 h of incubation of LF with P.
  • LF gingivalis whole cells in a physiological buffer only two major polypeptides (33 and 53 kDa) were detected and these fragments resulted from cleavage at a single site.
  • LF has been reported to be relatively resistant to degradation by both trypsin and chymotrypsin and the N-linked glycosylation of LF has been shown to help protect the protein from trypsin hydrolysis.
  • LF was more extensively hydrolysed by trypsin than the P. gingivalis proteinases (Fig 3) which is most likely related to the ability of LF to inhibit the P. gingivalis proteinases.
  • Bovine LF contains five N-linked glycosylation sites (Asn-233, -281, -368, -476 and - 545) and the majority of glycans are located in the N-terminal region of LF (33 kDa fragment) consequently the variation in glycosylation could explain why this fragment eluted in two distinct peaks from RP-HPLC (Fig 5).
  • the glycosylation of both fragments made it impossible to use the measured masses of the peptide fragments to identify the cleavage site. ISD-MS was therefore used to analyse the primary cleavage site of LF when exposed to P. gingivalis (Figs 4 & 6).
  • the R -S cleavage site identified occurs on an exposed external hydrophilic loop of the LF molecule (2). Cleavage at this site is unlikely to cause dissociation of the two polypeptides such that the molecule would retain its tertiary structure. This was confirmed by Size Exclusion Chromatography (SEC) analysis of LF-Pg (6h incubation) that demonstrated that the two fragments (33 kDa and 53 kDa) eluted as a single peak with native LF (Fig 7).
  • SEC Size Exclusion Chromatography
  • Bovine native, apo- and holo-LF all had a strong P. gingivalis biofilm formation inhibitory activity, inhibiting biofilm formation by >84% at concentrations above 0.01 mg mL. At concentrations as low as 0.001 mg mL, native LF still significantly inhibited P. gingivalis biofilm formation by 50% (Fig. 10).
  • the P. gingivalis biofilm inhibitory activity of LF was not a general protein effect as BSA and ⁇ -Lg did not inhibit P. gingivalis biofilm formation and in fact enhanced it. This is consistent with recent data which showed that LF inhibited P. gingivalis biofilm formation by ⁇ 60% at a concentration of 0.008 mg/mL. Native LF reduced P. gingivalis planktonic growth at high concentrations, whilst significantly increasing the mean generation time indicating that LF was slowing growth rather than having a bactericidal action (Fig 9).
  • LF is resistant to hydrolysis by P. gingivalis proteinases and directly inhibits proteinase activity by a novel mechanism. Therefore LF is capable of inhibiting gingipains and may be useful in the treatment of periodontitis and other conditions involving gingipains.
  • composition of the invention includes embodiments of the invention discussed above including lactoferrin with or without a cation.
  • composition of the invention 1.0
  • composition of the invention 0.2 Water balance
  • composition of the invention 0.2
  • composition of the invention 0.3
  • composition of the invention 0.3
  • composition of the invention 0.3
  • composition of the invention 3.0
  • Colloidal silicon dioxide (such as Aerosil® 200TM) 1.0
  • composition of the invention 0.3

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Abstract

La présente invention concerne des procédés et des compositions destinés à inhiber les protéinases bactériennes, notamment les cystéine protéinases de bactéries pathogènes telles que Porphyromonas gingivalis.
PCT/AU2012/000188 2011-02-25 2012-02-24 Procédé d'inhibition de protéines Ceased WO2012113037A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004346020A (ja) * 2003-05-22 2004-12-09 Morinaga Milk Ind Co Ltd プロテアーゼ阻害剤
US20050148504A1 (en) * 2002-11-29 2005-07-07 Nobuhiko Katunuma Cysteine protease inhibitor
JP2005306890A (ja) * 2005-07-25 2005-11-04 Lion Corp 歯周病細菌の内毒素中和方法および付着抑制方法
JP2007016002A (ja) * 2005-07-11 2007-01-25 Niigata Univ 歯周病菌プロテアーゼ阻害剤、並びにこれを用いた口腔組成物及び食料品
JP2008150325A (ja) * 2006-12-18 2008-07-03 Lion Corp アルジンジパイン阻害剤及び口腔用組成物
WO2010024702A2 (fr) * 2008-08-28 2010-03-04 Akademia Medyczna Im. Piastow Slaskich We Wroclawiu Composition pharmaceutique pour le traitement des maladies parodontales

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050148504A1 (en) * 2002-11-29 2005-07-07 Nobuhiko Katunuma Cysteine protease inhibitor
JP2004346020A (ja) * 2003-05-22 2004-12-09 Morinaga Milk Ind Co Ltd プロテアーゼ阻害剤
JP2007016002A (ja) * 2005-07-11 2007-01-25 Niigata Univ 歯周病菌プロテアーゼ阻害剤、並びにこれを用いた口腔組成物及び食料品
JP2005306890A (ja) * 2005-07-25 2005-11-04 Lion Corp 歯周病細菌の内毒素中和方法および付着抑制方法
JP2008150325A (ja) * 2006-12-18 2008-07-03 Lion Corp アルジンジパイン阻害剤及び口腔用組成物
WO2010024702A2 (fr) * 2008-08-28 2010-03-04 Akademia Medyczna Im. Piastow Slaskich We Wroclawiu Composition pharmaceutique pour le traitement des maladies parodontales

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