WO2012113037A1 - Method for inhibiting proteins - Google Patents

Abstract

The invention relates to methods and compositions for inhibiting bacterial proteinases, specifically cysteine proteinases from pathogenic bacteria such as Porphyromonas gingivalis.

Description

METHOD FOR INHIBITING PROTEINS

Field of the invention

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.

Background of the invention

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

Arg- and Lys- gingipains (otherwise known as gingipains R and K) 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. It is also proposed that 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. Recently it has been reported that 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. These 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. For example, in vivo environments include temperature fluctuations and variations in nutrient, carbon and nitrogen availability. 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.

Ultimately, what is required for successful treatment is to stop host tissue and cell invasion, rather than to stop biofilm formation. This is because host tissue and cell invasion by pathogenic bacteria allows the bacteria to persist and replicate as they are less likely to be removed by scaling and root planning and are more resistant to antibiotics. In addition, it is thought that an intracellular or intercellular population of pathogenic bacteria could repopulate a treated subgingival site. Further, P. gingivalis strains isolated from diseased sites in the oral cavity posses greater invasion capability than strains from non-diseased sites. There is a need for agents capable of inhibiting periodontitis and other diseases of the oral cavity involving host tissue and or cell invasion.

Summary of the invention

The present invention seeks to address one or more of the problems of the prior art. The present invention is based on the identification that lactofemn is an inhibitor of P. gingivalis proteinases that require metal ions for activity. Specifically, lactoferrin and various forms thereof can inhibit the proteolytic activity of P. gingivalis cysteine proteinases including the gingipains Rgp and Kgp.

In one aspect, 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. Preferably, cysteine protease activity of the pathogen inhibits the pathogen from invading tissues and/or cells of an individual. In a particularly useful embodiment of the invention, the inhibition of the cysteine protease activity of the pathogen prevents the individual from acquiring periodontitis or periodontal disease.

Typically, the CPI is lactoferrin, preferably the lactoferrin comprises a metal ion.

In one embodiment the CPI is provided by oral administration. Preferably, the CPI is provided in the form of a food, a drink, a supplement, a medicament or a pharmaceutical.

Typically the pathogen is P. gingivalis. The form of P. gingivalis may be planktonic.

In another aspect, the invention provides a composition for inhibiting the cysteine protease activity of a pathogen of periodontal disease including:

- a CPI; - a carrier for adapting the composition to a form that is suitable for oral administration of the composition. Preferably, 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. In one embodiment of this and each aspect of the invention, the proteinase is a cysteine proteinase, preferably the cysteine proteinase is a gingipain, even more preferably RgpA, RgpB or Kgp.

In another aspect there is provided lactoferrin for inhibiting a P.gingivalis proteinase and the use of lactoferrin in the manufacture of a medicament for inhibiting a P.gingivalis proteinase.

In another aspect there is provided 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.

In one embodiment there is provided 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

- identifying a subject that exhibits oral tissue or cell invasion by P. gingivalis; and

- administering to the subject a formulation including lactoferrin. Preferably, the formulation including a pharmaceutically acceptable carrier, diluent or excipient. Preferably, the formulation is a gel suitable for oral administration. Preferably, 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. In another aspect there is provided lactoferrin for use in treating a disease, disorder or biological effect that is associated with host tissue or cell invasion by P. gingivalis.

In one aspect there is provided a method of treatment of an individual for an oral disease characterised by host tissue or cell invasion including the step of

- providing lactoferrin to an individual requiring said treatment in an amount effective for therapy, thereby treating the individual for an oral disease.

In another aspect there is provided a method of treating a disease, disorder or biological effect modulated by a gingipain including administering lactoferrin.

In a further aspect there is provided 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.

In one embodiment there is provided a method of preventing, reducing or inhibiting host tissue or cell invasion by P. gingivalis including the steps of

- identifying a subject that is at risk of oral tissue or cell invasion by P. gingivalis; and - administering to the subject a formulation including lactoferrin thereby preventing host tissue or cell invasion by P. gingivalis.

In a further aspect there is provided use of lactoferrin in the manufacture of a preparation for the treatment or prevention of a disease, disorder or biological effect modulated by a gingipain. Preferably, 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. Even more preferably the preparation is formulated as a gel suitable for oral administration.

In one embodiment there is provided 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. Preferably, 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. Preferably the composition is formulated as a gel suitable for oral administration. In another embodiment there is provided 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. Preferably the composition is formulated as a gel suitable for oral administration. Any biologically active lactoferrin may be employed in the aspects of the invention. In a preferred embodiment 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. Preferably, 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.

Alternatives of the this aspect provide lactoferrin for inhibiting gingipain-associated pathogen invasion of host cells or tissues and the use of lactoferrin in the manufacture of a medicament for inhibiting gingipain-associated pathogen invasion of host cells or tissues. In one aspect there is provided a kit for use in a method of the invention including a composition including lactoferrin and one or more pharmaceutically acceptable carriers, excipients or diluents. Preferably, the kit further includes written instructions for use. Preferably, lactoferrin is formulated as a gel suitable for oral administration.

In another embodiment there is provided a kit for use in a method of the invention mentioned above, the kit including:

• a container holding lactoferrin or composition of the invention; and

• a label or package insert with instructions for use.

In a further embodiment there is provided a kit when used in a method of the invention mentioned above.

In a further embodiment, the lactoferrin in the kit is formulated as a gel. In certain embodiments 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.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings. Brief description of the drawings

Figure 1 shows the time-dependent inhibition of RgpA activity by lactoferrin. The figure specifically shows the effect of lactoferrin at different concentrations (μΜ) on the activity (Ln%) of RgpA over time. The slope of these lines provided the apparent inactivation rate constants (kapp). Plotting 1 ka p versus 1/[I] where I is the lactoferrin concentration yielded a straight line (r = 0.9995) with an intercept of 1 kinact and a slope of Ki/kinact. This produced a first order inactivation rate constant kjnaa of 0.023 min^"1 and a lactoferrin binding affinity (Ki) of 5.02 μΜ.

Figure 2. An orthographic view of the modeled lactoferrin-RgpB complex. The solvent accessible surface of RgpB is shown as a space filling model. In the left panel the arrow (A) indicates the zinc ion, depicted a dark sphere, bound to the catalytic histidine (His²⁴⁴) 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 μg); 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; lane 2: LF/trypsin = 5000/1.

Figure 4. The primary sequence of LF, underlined sequences denote the peptides identified by peptide mass fingerprint (PMF) analysis of Fragments I to V. The cleavage site between Fragment I and IV (the major polypeptides in LF-Pg sample) 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.

Figure 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 ²⁸⁵SFOLFGSPPGORDLLFKDSALGFLRIPSKVDSALYLGS. with the underlined portion evident in the spectrum. Figure 7 A: LF-Pg was fractionated by size exclusion chromatography (SEC) while native LF was eiuted as a control (indicated by an asterisk). 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 (^Δ ), 10 (▲), uninoculated growth medium (·). Each data point represents the mean of six biological replicates. 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. Each data point represents the mean of three biological replicates. 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.

Detailed description of the embodiments

The present inventors have identified that lactoferrin is an inhibitor of bacterial proteinases requiring metal ions for proteolytic activity. In particular, the inventors have shown that 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. By inhibiting gingipain activity thereby 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. Without being bound by any theory or mode of action, it is believed that 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. In some embodiments, reduction or inhibition of proteolytic activity is proposed to be via obstruction of the substrate binding site on the proteinase. Use of 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. These observations are surprising since one of the major reasons that antimicrobial peptides and proteins are ineffective against bacteria is that the bacteria either secrete or have cell surface proteinases which hydrolyse the antimicrobial peptides or proteins. Indeed it has previously been shown that P. gingivalis cell surface and extracellular proteins hydrolyse antibacterial proteins and peptides such as histatin, thereby reducing the efficiency of antimicrobial proteins and peptides against this bacterium.

It is proposed that 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 FUGUE™ 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. In one embodiment 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: G⁷⁷, D⁷⁸, R¹¹², E²⁵⁸ (P. gingivalis RgpB numbering). The sequence must match the consensus sequence: -G-[DNEIK]-x(0,31)-R-x(75,85)-[GSDA]-x(10,31)- [GAHDT]-[HWY]-[GSIAYL]-x(26,42)-C-x(l 0,15)-E-x(l 3,17)-G-x(60,70)-G-D-[PGAC]. In one embodiment a gingipain includes a His, Cys catalytic dyad. In one embodiment 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. In one embodiment the gingipain tertiary structure includes alpha/beta proteins with a fold that consists of an alpha/beta/alpha sandwich. In one embodiment the beta sheet contains six strands (preferably in the order 213456) and strand 6 is anti-parallel to the rest. In one embodiment the gingipain is gingipain R or gingipain K from P. gingivalis. Other family members of clan 25 are also gingipains because of the conservation of motifs around the catalytic residues. In one embodiment the gingipain is calpain, cathepsin, caspase, sortase, clostripain, legumain, separase or RTX self-cleaving toxin.

Any reference herein to "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.

The term "gingipain activity" defines the activity of the gingipain to be inhibited. For example if the gingipain is gingipain R (RgpA or B) or gingipain K (Kgp) the gingipain activity is proteinase activity. In one embodiment the gingipain activity is cysteine proteinase activity.

A cysteine protease inhibitor (CPI) 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. gingivalis, the 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. Examples of cysteine protease inhibitors include members of the transferrin family.

Members of the transferrin family include blood serotransferrin (or siderophilin, usually called transferrin); lactotransferrin (lactoferrin); milk transferrin; egg white ovotransferrin (conalbumin); and membrane-associated melanotransferrin. These 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 μg/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. As used herein, "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. For instance, 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. The phrase "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. Alternatively, 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.

The 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. Other examples of 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) sequences of sheep lactoferrin; the amino acid (Accession Numbers NP_999527, AAL40161 and AAP70487) and mRNA (Accession Number NM_214362) sequences of pig lactoferrin; the amino acid (Accession Numbers NP_032548 and A28438) and mRNA (Accession Number NM 008522) sequences of mouse lactoferrin; the amino acid (Accession Number C AA06441 ) and mRNA (Accession Number AJ005203) sequences of water buffalo lactoferrin; and the amino acid (Accession Number CAB53387) and mRNA (Accession Number AJ131674) sequences of camel lactoferrin. These sequences may be used in wild type or variant form.

In one embodiment the lactoferrin is sheep, goat, pig, mouse, water buffalo, camel, yak, horse, donkey, llama, bovine or human lactoferrin. In another embodiment, 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) that maintain the biological activity of a wild-type lactoferrin polypeptide may be employed. Alternatively, the lactoferrin can be produced using genetic engineering or chemical synthesis techniques well-known in the art.

As used herein, the term "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. 5,766,939, U.S. Pat. No. 5,849,881, U.S. Pat. No. 5,849,885, U.S. Pat. No. 5,861,491, U.S. Pat. No. 5,919,913, U.S. Pat. No. 5,955,316, U.S. Pat. No. 6,066,469, U.S. Pat. No. 6,080,599, U.S. Pat. No. 6,100,054, U.S. Pat. No. 6,111,081, U.S. Pat. No. 6,228,614, U.S. Pat. No. 6,277,817, U.S. Pat. No. 6,333,311, U.S. Pat. No. 6,455,687, U.S. Pat. No. 6,569,831, U.S. Pat. No. 6,635,447, US 2005-0064546 and US 2005-0114911. Useful variants also include bovine lactoferrin variants bLf-a and bLf-b.

It is understood by one of ordinary skill in the art that certain amino acids may be substituted for other amino acids in a protein structure without adversely affecting the activity of lactoferrin. It is thus contemplated by the inventors that various changes may be made in the amino acid sequences of lactoferrin without appreciable loss of their biological utility or activity. Such changes may include deletions, insertions, truncations, substitutions, fusions, shuffling of motif sequences, and the like.

One of skill in the art will recognise that 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.

Persons skilled in the art would readily appreciate the numerous software packages to enable them to design or identify homologues of the lactoferrin nucleotide and amino acid sequences, for example the "BLAST" program or other suitable packages.

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). For instance, lactoferrin isolated from bovine milk can be depleted of iron and then loaded with another type of metal ion. For example, 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 in the art.

A preparation of lactoferrin (e.g., lactoferrin isolated from bovine milk) 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 Fe³⁺ 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).

In one embodiment 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.

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. In addition 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,

65, 70, 75, 80, 85, 90, 95 or 99% by weight, of lactoferrin.

The following is an exemplary procedure for isolating lactoferrin from bovine milk.

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.

The terms "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. Thus, for example, 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" as used herein 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. As used herein, "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. Furthermore, as used herein, 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 (Toxins 2010, 2, 341-352) 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.

Once 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. The subject may or may not exhibit a subgingival biofilm. Identifying a subject at risk of oral cell or tissue invasion may include determining whether P. gingivalis cells are associated with the epithelium of the subgingiva or determining whether gingipains are associated with the epithelium of the subgingiva. A subject may also exhibit elevated gingipain activity in the subgingival pocket or associated with the epithelium of the subgingiva.

In one embodiment 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 functio 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.

Generally, although not entirely necessary, such nutraceuticals will contain lactoferrin purified to some degree, or at the very least, all components of the nutraceutical will be verifiable. Examples of 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), 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.

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 and calcium, magnesium, potassium, zinc and iron. If it is desired to produce a powdered nutraceutical, 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.

If 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.

Preferably the 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.

As bovine milk is a natural product that has been in food chain for hundreds of years, 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".

By "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. Similarly, 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. As used herein, 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. Preferably, 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. These 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. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

In one example, tablets can be formulated in accordance with conventional procedures by compressing mixtures of the lactoferrin with a solid carrier and a lubricant. Examples of 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.

The term 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. Examples of 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. Examples of such 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.

Optionally, the formulation may further include one or more antibiotics that are toxic to or inhibit the growth of Gram negative anaerobic bacteria. Potentially any bacteriostatic or bactericidal antibiotic may be used in a formulation for use in the invention. Preferably, suitable antibiotics include amoxicillin, tetracycline, doxycycline or metronidazole.

In certain preferred forms of the invention the oral formulation may be substantially liquid in character, such as a mouthwash or rinse. In such a preparation the vehicle is typically a water-alcohol mixture desirably including a humectant as described below. Generally, 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).

In other desirable forms, 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. In a toothpaste, 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. Also advantageous are liquid mixtures of water, glycerine and sorbitol. 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% Si0₂, 25.40% MgO, 3.05% Na₂0, 0.98% Li₂0, 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.

Other suitable 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). 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.

It will be understood that, as is conventional, the oral preparations for use in the methods of the invention will usually be sold or otherwise distributed in suitable labeled packages. Thus, 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. Suitable examples of 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. aliphatic chains of about 12 to 20 carbon atoms), which condensation products ("ethoxamers") 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. These adjuvants, where present, are incorporated in the formulations in amounts which do not substantially adversely affect the properties and characteristics desired.

Any suitable flavouring or sweetening material may also be employed. Examples of 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. Suitably, flavour and sweetening agents may each or together comprise from about 0.1% to 5% more of the preparation. The 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. These 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. For example, 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. Such 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 monooleate. 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.

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:

(a) 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;

(b) 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; (c) topical applications, e.g. as a cream, ointment or spray applied to the skin; or

(d) intravaginally, e.g. as a pessary, cream or foam.

It is especially advantageous to formulate the formulations in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein 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. As used herein, an "effective amount" of lactoferrin is a dosage which is sufficient to inhibit a gingipain. Generally, 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. For example, 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.

Generally, the formulations are administered as a bolus dose, to maximise the circulating levels of lactoferrin for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose. In certain embodiments, 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. As an example in these embodiments, 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. Here there may be trace or residual amounts of compounds that arise from composition preparation, rather than from an intention to supplement, add or modify the affect of lactoferrin on periodontal disease.

In other embodiments, the composition does not substantially include protein other than lactoferrin. For example, 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.

In those embodiments where lactoferrin is provided as a cysteine proteinase inhibitor, 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.

It must also be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

The invention will now be described with reference to the following, non-limiting examples. EXAMPLE 1

Native Bovine lactoferrin (LF) was provided by MG Nutritionals, Murray Goulburn Cooperative Limited, Australia. The purity of the LF was more than 95%, as determined by SDS- PAGE and in-gel digestion of protein bands with trypsin and MALDI-TOF mass spectrometric analysis (see below). Apo-LF and holo-LF were produced according to the methods of Shimazaki 2000 Animal Sci. J. 71: 329-347 and Brissona, Brittenb et al. 2007 International Dairy Journal 17: 617-624, respectively. 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. 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 CaCl₂. The final cell density was 4.5 x 10⁷ 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 μί) were pre-incubated with LF preparations at concentrations between 0.01 - 10 mg/mL in TCI 50 buffer with a total volume of 100 ί at 37°C for 15 - 90 min. To each well, 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.

Purification of P. gingivalis RgpA/Kgp proteinase/adhesin complexes

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 CaCl₂) containing 50 mM NaCl. Following the addition of 60 U/ml of benzonase (Sigma) and 2 mM MgCl₂, 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 MgCl₂, then incubated with freshly added benzonase (140 U/mL) for 1 h on ice. After filtering (0.22 mm), 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). 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.

LF hydrolysis by P. gingivalis

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. It contained 2.8 mM cysteine, 10 mM NaH₂P0₄, 10 mM KC1, 2 mM citric acid, 1.25 mM MgCl₂, 20 uM CaCl₂, 25 μΜ ZnCl₂, 50 μΜ MnCl₂, 5 μΜ CuCl₂, 10 uM CoCl₂, 5 μΜ H₃B0₃ and 1 μΜ Na₂Mo0₄ with the pH adjusted to 7.5 using 5 M NaOH at 37°C.

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.

SDS-polyacrylamide gel electrophoresis For denaturing polyacrylamide gel electrophoresis in the presence of ionic detergent

(SDS-PAGE), all LF and LF-Pg samples were analysed on NuPAGE^® (Invitrogen, Carlsbad, CA, USA) 4-12% Bis-Tris precast gels used with 3-( -morpholino) propane sulfonic acid (MOPS) SDS running buffer at pH 7.7. Protein samples (1 mg/mL) were mixed with NuPAGE^® lithium dodecyl sulphate (LDS) sample buffer and reducing agent, heated at 90°C for 5 min, and pulse-centrifuged at 6000 x g prior to loading on gels. The gels were stained overnight with 0.1% w/v Coomassie Blue G-250 in 17% w/v ammonium sulphate, 34% v/v methanol and 3% v/v o- phosphoric acid (Molloy et al, 1999 Electrophoresis 20(4-5): 701-4). Size exclusive chromatography

Size exclusive chromatography (SEC) was applied to separate the fragments in LF-Pg using a Superdex™ 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 μΐ. of filtered (0.22 um) sample. Fractionation of LF-Pg by RP-HPLC

LF-Pg preparation at 6 h incubation was subjected to fractionation using an Aquapore OD-300 reverse-phase column (7μ, 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.).

In-gel digestion of Lactoferrin and mass spectrometry. Protein bands from SDS-PAGE were excised, subjected to in-gel tryptic digestion and mass spectrometry analysis as published previously (O'Brien-Simpson et al., 2005 J Immunol 175(6): 3980-3989). Briefly, gel pieces were washed in 50 mM NH₄HC0₃/ethanol 1:1 vol/vol, reduced and alkylated with dithiothreitol (DTT) and iodoacetamide, respectively and digested with sequencing grade modified trypsin (10 ng/μΐ,) (Promega, NSW, Australia) overnight at 37 DC. Peptides were analysed on an Ultraflex TOF/TOF III (Bruker Daltonics, Bremen, Germany) using 4-hydroxy-a-cyanocinnamic acid as matrix (O'Brien-Simpson et al., 2005). Peaks were assigned to LF by using an in-house Mascot search engine (Matrix Science, London, UK) with bovine LF as the sequence database. Sample preparation in sinapinic acid and spectra acquisition for In Source Decay (ISD) were performed according to the FlexControl 3.0 user manual (Bruker Daltonics). Briefly, 1 uL of matrix A (sinapinic acid saturated in ethanol) was applied to a steel target to create a thin layer. 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 biofilm assay The biofilm assay was modified from the method of Wen and Burne (2002) Appl

Environ Microbiol 68(3): 1196-1203. P. gingivalis (ATCC 33277) was anaerobically grown in brain heart infusion (BHI) broth (37 g L, Oxoid Australia Pty Ltd, South Australia, Australia), supplemented with haemin (5 μg/mL), vitamin K3 (5 μg mL) and cysteine (0.5 mg/mL) at 37°C until a cell density of 2.8 x 10¹⁰ 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. 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 μί) were added to 96-well plate with 6 replicates while control samples contained 20 μL distilled water. The bacterial culture (180 iL) was added to each well to make the total volume of 200 μL·. 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. Following incubation, 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 μΐ_^ 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 (VICTOR3™1420 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).

Molecular dynamics simulation of the LF-RgpB interaction.

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 Glu⁶⁵⁹ 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, His²⁴⁴. 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. A 10-ns, molecular dynamics simulation was then performed for all atoms within a 20-A sphere centered on the Zn ion, with atoms more than 15 A from the Zn ion and all atoms in the RgpB structure being frozen at their initial coordinates. The NTV dynamics simulation used the AMBER-99 force-field, a constant dielectric of 4.0, a van der Waal's scale factor of 0.7827, a non-bonded cut-off of 12 A , and a set temperature of 300 K. Effect of LFon P. gingivalis proteinase activity.

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 μΜ) (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. At 10 mg/mL BSA displayed a small effect on the hydrolysis of the chromogenic substrate, which was attributed to substrate competition (Table 1).

Table 1. Arg- and Lys-specific proteinase activity of P. gingivalis whole cells presence of LF and BSA.

a - Units of proteinase activity/10 cells b - not determined

LF inhibited both the Arg-specific and Lys-specific activities of purified P. gingivalis RgpA/ gp proteinase-adhesin complexes by >96% at a concentration of 5 mg/mL. As the levels of inhibition were similar and there is a very high degree of similarity between RgpA and Kgp we then focused on the characterisation of the Arg-specific inhibitory activity. A kinetic analysis of the inhibition of Arg-specific proteolytic activity of purified P. 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 μΜ (Fig. 1). To confirm that LF was interacting with RgpA and Kgp by binding to the catalytic domain of the proteinases, 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.

Molecular dynamics simulation of the LF-RgpB interaction. As LF inhibited both the Arg- and Lys-specific proteinases (RgpA/B and Kgp) of P. gingivalis then this suggested a common inhibitory mechanism. The crystal structure of RgpB shows a zinc ion binding site involving residues Glu¹⁵² and the catalytic histidine, His²¹¹. Modelling of Kgp based on the known structure of RgpB indicates that residues Asp³⁸⁸ and His⁴⁴⁴ of Kgp would similarly bind a zinc ion in the active site of the proteinase. As lactoferrin is known to bind zinc ions then the interaction between LF and the zinc ion in the active site of RgpA/B and Kgp would explain the inactivation of the proteinases. The molecular modelling of the interaction between LF and RgpB (Fig 2) demonstrates that these two proteins could readily be cross-linked by sharing the zinc-ion bound by Glu¹⁵² and His²¹¹ of the RgpB active site. Energy minimization followed by molecular dynamics indicated that the unfavourable van der Waal's contacts observed between the proteins were readily relaxed and that additional favourable interactions between the two proteins developed on a relatively short-time scale being consistent with the time-dependent manner of the observed inactivation. As modelled there would be significant unfavourable van der Waal's interactions between Glu⁶⁵⁸ of LF and the terminal D-phenylalanyl residue of DFFR-chloromethylketone the substrate-analogue inhibitor of RgpB crystalised with the proteinase. This indicates that the interaction of LF with the zinc ion in the RgpB active site would sterically hinder substrate access to the active site (Fig 2).

Effect of P. gingivalis proteinase activity on LF.

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). This was followed by a slower more extensive hydrolysis (Lanes 5-7 in Fig 3 A) that after 3 days resulted in the almost complete disappearance of the intact LF band (85 kDa), and the production of Fragment Π (48 kDa). When LF was digested by trypsin a more extensive hydrolysis was seen with two major bands (23 and 46 kDa) detected in addition to the 33 and 53 kDa bands seen in LF digested by P. gingivalis (Fig 3B). Identification of LF cleavage site.

In order to identify the protein fragments that resulted from incubation of LF with P. gingivalis cells the gel bands shown in Fig 3A were subjected to in-gel digestion and analysis by mass spectrometry (MS). The protein bands were grouped as Fragments I, II, III, IV and V with relative molecular masses of 53, 48, 40, 33, and 22 kDa, respectively, while intact LF had a relative molecular mass of 85 kDa (Fig 3 A). Fragments I and IV were the main products during the first 6 h of incubation, and their predicted masses suggested that they resulted from cleavage at a single site in LF. Peptide mass fingerprint analysis indicated that 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).

RP-HPLC was used to obtain further information about these LF fragments and the fractions of LF-Pg (6h incubation) were analysed by MS (Fig 5). Peak 1 and 2 consisted of a polypeptide with a mass of 33 kDa whereas peak 3 consisted mainly of a polypeptide with a mass of 49 kDa (Fig 5). These peaks correspond to Fragment IV (33 kDa) and Fragment I (53 kDa) in SDS-PAGE analysis (Fig 3), respectively. The masses of these fragments as measured by MS total between 82.66 and 82.71 kDa which is close to the measured mass of 82.8 kDa for whole LF. This confirms that these two fragments are the products of cleavage at a single site in LF. However, due to potential glycosylation of both Fragments I and IV it was not possible to use these measured masses to identify the cleavage site. To determine the exact cleavage site In Source Decay - Mass Spectrometry (ISD-MS) which enables extensive N-terminal (and sometimes C-terminal) protein sequence to be elucidated was employed on the fractions. ISD-MS of Fragment IV was unsuccessful, however Fragment I was successfully fragmented yielding a strong series of peaks that corresponded to N-terminal fragment ions (Fig 6). 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 S²⁸⁵. 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).

LF growth inhibitory activity

When cultured in the presence of 5 μg mL of haemin P. gingivalis produced significant biofilms with an optical density of 0.315 ± 0.054 after 24 h of incubation, however in the absence of added haemin in the growth medium there was a significant reduction in the biofilm mass (0.168 ± 0.048; p<0.005). When LF was added to haemin-excess medium at concentrations above 0.05 mg mL it effectively abolished P. gingivalis biofilm formation and there was a significant inhibitory effect at concentrations as low as 0.001 mg mL (Fig 8). The activity was incubation time dependent as it was reduced when the incubation time was prolonged to 48 h. In contrast, bovine serum albumin (BSA) and β-lactoglobulin (β-Lg) did not inhibit P. gingivalis biofilm formation at concentrations up to 10.0 mg/mL, and in fact significantly enhanced biofilm formation (data not shown). Therefore, the biofilm inhibitory activity against P. gingivalis was LF specific. Native, apo- and holo-LF all had similar inhibitory effects on P. gingivalis biofilm formation at a concentration of 0.01 mg/mL, inhibiting biofilm formation by 87.2 ± 1.7, 88.1 ±. 1.6 and 84.2 ± 5.5%, respectively.

LF demonstrated a weak, dose-dependent inhibitory effect on the planktonic growth of the bacterium (Fig 9). At concentrations below 0.5 mg mL there was little effect on P. gingivalis planktonic growth whilst at 10 mg/mL planktonic growth was reduced by 43% after 30 h of incubation (Fig 9). Biofilm inhibitory activity of P. gingivalis treated LF

Native LF that had been pre-incubated with P. gingivalis for 6 h (LF-Pg) at a concentration of 0.1 mg/mL inhibited P. gingivalis biofilm formation by 83%, whilst LF-Pg at 0.01 mg mL inhibited biofilm formation by 56%. Individual RP-HPLC fractions of LF-Pg had no biofilm inhibitory activity (Fig 10). Native LF that was applied to RP-HPLC and eluted under the same conditions as that for LF-Pg (LF&act) still retained some activity, inhibiting P. gingivalis biofilm formation by 43% at a concentration of 0.04 mg/mL. Hydrolysis of LF by trypsin resulted in almost complete loss of P. gingivalis biofilm inhibitory activity (data not shown).

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.

In this study we have shown that LF inhibited the proteolytic activity of both RgpA and Kgp (Table 1). Shi et al. (2000; J Biol Chem 275:30002-8). have previously shown that human LF and bovine lactoferricin, the N-terminal domain of bovine LF, were able to bind to the P. gingivalis RgpA/Kgp proteinase-adhesin complexes and disrupt their quaternary structure, leading to the release of the haemoglobin-binding domain (Hbr). In our study LF inhibition of the proteolytic activity of RgpB, that does not contain adhesin domains, indicated that the protein was interacting with the catalytic domain of the proteinases. Kinetic analyses demonstrated that LF inhibited the proteinases of P. gingivalis in a time-dependent manner confirming that LF was interacting with the catalytic domain of the proteinases. The Ki of LF for RgpA activity was 5.02 uM. This inhibition is consistent with the work of Ohashi et al. (2003. Biochem. Biophys. Res. Commun. 306:98-103) who reported that LF inhibited the proteinases Cathepsin and Papain, although they did not examine the time-dependent nature nor the mechanism of inhibition. The time-dependency of the inhibition suggests that 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. In this study we showed that 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. 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. In our study 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).

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 β-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).

Importantly, even after incubation with P. gingivalis proteinases LF still retained biofilm inhibitory activity. This may indicate that LF retains its quaternary structure after cleavage at this site. The close association between these two fragments was confirmed by SEC where they co- eluted with native LF. When purified using RP-HPLC the two major fragments of LF-Pg preparations had no biofilm inhibitory activ{Shimazaki, 2000 #69}ity, however this may be due to the loss of conformation associated with the process.

This study is the first to report that 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.

Example 2

Formulations To help illustrate formulations for use in the invention, the following sample formulations are provided. As used in the following formulations, the phrase "composition of the invention" includes embodiments of the invention discussed above including lactoferrin with or without a cation.

The following is an example of a toothpaste formulation.

Ingredient % w/w

Dicalcium phosphate dihydrate 50.0

Glycerol 20.0 Sodium carboxymethyl cellulose 1.0 Sodium lauryl sulphate 1.5

Sodium lauroyl sarconisate 0.5 Flavour 1.0 Sodium saccharin 0.1

Chlorhexidine gluconate 0.01

Dextranase 0.01

Composition of the invention 1.0

Water balance

The following is an example of a further toothpaste formulation.

Ingredient % w/w

Dicalcium phosphate dihydrate 50.0

Sorbitol 10.0

Glycerol 10.0

Sodium carboxymethyl cellulose 1.0 Lauroyl diethanolamide 1.0 Sucrose monolaurate 2.0 Flavour 1.0

Sodium saccharin 0.1 Sodium monofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Dextranase 0.01

Composition of the invention 0.2 Water balance

The following is an example of a further toothpaste formulation.

Ingredient % w/w Sorbitol 22.0

Irish moss 1.0

Sodium Hydroxide (50%) 1.0 Gantrez 19.0 Water (deionised) 2.69 Sodium Mohofluorophosphate 0.76 Sodium saccharine 0.3 Pyrophosphate 2.0

Hydrated alumina 48.0 Flavour oil 0.95 Composition of the invention 0.1 sodium lauryl sulphate 2.00 The following is an example of a liquid toothpaste formulation.

Ingredient % w/w Sodium polyacrylate 50.0 Sorbitol 10.0 Glycerol 20.0 Flavour 1.0

Sodium saccharin 0.1 Sodium monofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Ethanol 3.0 Composition of the invention 0.2

Linolic acid 0.05 Water balance

The following is an example of a mouthwash formulation.

Ingredient % w/w Ethanol 10.0 Flavour 1.0

Sodium saccharin 0.1 Sodium monofluorophosphate 0.3

Chlorhexidine gluconate 0.01 Lauroyl diethanolamide 0.3

Composition of the invention 0.2

Water balance

The following is an example of a further mouthwash formulation useful as an oral lubricant, saliva substitute or as artificial saliva.

Ingredient % w/w

Gantrez® S-97 2.5

Glycerine 10.0

Flavour oil 0.4

Sodium monofluorophosphate 0.05

Chlorhexidine gluconate 0.01

Lauroyl diethanolamide 0.2

Composition of the invention 0.3

Water balance

The following is an example of a lozenge formulation.

Ingredient % w/w

Sugar 75-80

Corn syrup 1-20

Flavour oil 1-2

NaF 0.01-0.05

Composition of the invention 0.3

Mg stearate 1-5

Water balance

The following is an example of a gingival massage cream formulation.

Ingredient % w/w

White petrolatum 8.0

Propylene glycol 4.0

Stearyl alcohol 8.0 Polyethylene Glycol 4000 25.0

Polyethylene Glycol 400 37.0

Sucrose monostearate 0.5

Chlorhexidine gluconate 0.1

Composition of the invention 0.3

Water balance

The following is an example of a periodontal gel formulation.

Ingredient % w/w

Pluronic F127 (from BASF) 20.0

Stearyl alcohol 8.0

Composition of the invention 3.0

Colloidal silicon dioxide (such as Aerosil® 200™) 1.0

Chlorhexidine gluconate 0.1

Water balance The following is an example of a chewing gum formulation.

Ingredient % w/w

Gum base 30.0

Calcium carbonate 2.0

Crystalline sorbitol 53.0

Glycerine 0.5

Flavour oil 0.1

Composition of the invention 0.3

Water balance

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims

1. A 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.

2. The use of claim 1 wherein the inhibition of the cysteine protease activity of the pathogen inhibits the pathogen from invading tissues and/or cells of the individual.

3. The use of claim 1 wherein the inhibition of the cysteine protease activity of the pathogen prevents the individual from acquiring periodontitis or periodontal disease.

4. The use of any one of the preceding claims wherein the CPI is lactoferrin.

5. The use of claim 4 wherein the lactoferrin comprises a metal ion.

6. The use of any one of the preceding claims wherein the CPI is provided by oral administration.

7. The use of claim 6 wherein the CPI is provided in the form of a food, a drink, a supplement, a medicament or a pharmaceutical.

8. The use of any one of the preceding claims wherein the pathogen is P. gingivalis.

9. The use of any of the preceding claims wherein the pathogen is an a planktonic form.

10. A composition for preventing or treating periodontal disease including:

- a CPI for preventing or treating periodontal disease;

- a carrier for adapting the composition to a form that is suitable for oral administration of the composition.

11. The composition of claim 10 wherein the CPI is capable of binding to an ion comprised in an active site of a gingipain.

The composition of any one of the preceding claims wherein the CPI is lactoferrin.

The composition of any one of the preceding claims wherein the composition does not include an active ingredient for preventing or treating periodontal disease other than lactoferrin.

The composition of any one of the preceding claims wherein the composition does not substantially include protein other than lactoferrin.

The composition of any one of the preceding claims wherein the composition does not substantially include a dairy extract or component thereof other than lactoferrin.

The composition of any one of the preceding claims wherein in use, the composition provides for slow or extended release of the CPI.

The composition of any one of the preceding claims wherein the carrier is a gelling agent for adapting the composition to form a gel or the like.

18. The composition of any one of the preceding claims wherein in use, the composition forms a gum.