AU1327299A - Antimicrobial peptides - Google Patents

Description

WO 99/26971 PCT/AU98/00972 1 Antimicrobial Peptides FIELD OF THE INVENTION 5 The present invention relates to novel antimicrobial peptides which can be obtained from the milk protein casein or chemically synthesised or produced by recombinant DNA technology. These peptides can be used in foods as antimicrobial preservatives, in oral care products (eg. toothpaste, mouthwash, dental floss) for the control of dental plaque and suppression of 10 pathogens associated with dental caries and periodontal diseases. The antimicrobial peptides may also be used in pharmaceutical preparations for topical or parenteral application or oral administration for the control of oro-pharangeal and gastro-intestinal pathogens as well as systemic or localised infections. 15 BACKGROUND OF THE INVENTION Periodontal diseases are bacterial-associated inflammatory diseases of the supporting tissues of the teeth and are a major public health problem. 20 Nearly all of the human population is affected by periodontal diseases to some degree. In a recent Melbourne survey (Spencer et al., 1985) only 20% of the adult dentate sample did not require periodontal treatment while 62% required intermediate treatment and 18% required complex treatment. Brown et al. (1989), from an extensive US Dental Health survey reported that 25 only 15% of the studied population was free of periodontal diseases. The major form of periodontal disease is gingivitis which is associated with the non-specific accumulation of dental plaque at the gingival margin. In contrast, the less prevalent, destructive form of periodontal disease (periodontitis) is associated with a subgingival infection of specific 30 Gram-negative bacteria. Periodontitis is a major cause of tooth loss in Australian adults. Although gingivitis may not be a necessary precondition for the development of periodontitis (Christersson et al., 1989) gingivitis is likely to predispose susceptible sites to more serious forms of periodontal disease 35 since the specific Gram-negative bacteria that predominate in periodontitis, but which are not detectable in the healthy periodontium, have been found WO 99/26971 PCT/AU98/00972 2 in low proportions in gingivitis (Moore et al., 1987). Further, the environmental conditions that develop during gingivitis are likely to favour the subsequent colonisation or growth of the species implicated in periodontitis. The control of supragingival plaque is therefore considered an 5 important part of a preventive strategy for the control of periodontal diseases and in fact various plaque control programs have proven to be successful in the prevention of periodontal diseases (Loesche, 1976). In the majority of individuals the customary oral hygiene method of toothbrushing is usually insufficient by itself over long periods to provide a level of plaque control 10 compatible with oral health. Consequently the incorporation of antimicrobial agents into dental products as an aid to controlling dental plaque and gingivitis has been advocated (Addy, 1988; Marsh, 1991) and is of considerable interest to toothpaste and mouthwash companies. A number of agents have been suggested as antiplaque toothpaste additives (eg. 15 bisbiguanides, phenols, metal ions, quartenary ammonium salts) but have either negligible intra-oral activity, undesirable side-effects (eg. mucosal irritation, tooth discolouration) and/or an incompatibility with toothpaste formulations. Triclosan (2,4,4'-trichloro-2'-hydroxy diphenyl ether) an antimicrobial agent used extensively in deodorants, soaps and other 20 dermatological preparations is currently being used as an anti-plaque toothpaste additive in some countries however there is considerable interest to find a clinically efficacious, safe and natural antiplaque agent. Antimicrobial peptides are widely distributed in nature and play a role in the host defence of plants and animals (Boman and Hultmark, 1987; 25 Bevins and Zasloff, 1990). They include amongst others, the amphipathic channel forming peptides, for example the cecropins isolated from the cecropia moth (Boman and Hultmark, 1987), the magainins isolated from skin secretions of the African clawed frog Xenopus laevis (Bevins and Zasloff, 1990), the dermaseptins isolated from the skin of the arboreal frog (Mor and 30 Nicolas, 1994) and the bombinins from the skin of Bombina variegata (Simmaco et al., 1991). Other antimicrobial peptides include the cyclic cationic peptides containing an intramolecular disulphide, for example ranalexin from bullfrog skin (Clark et al., 1994) and bactenecin from bovine neutrophils (Romeo et a]., 1988). Proline-containing antimicrobial peptides 35 also have been identified and these include the apidaecins from the lymph WO 99/26971 PCT/AU98/00972 3 fluid of the honeybee (Casteels et al., 1989) and the pig myeloid antimicrobial peptide PMAP-23 (Zanetti et al., 1994). It is now well established that the milk protein casein should be considered not only as a nutrient but also as a protecting agent against 5 bacterial infection of the neonate mucosa as specific peptides released by tryptic or in situ digestion have been shown to possess marked biological activity. These bioactive peptides are relatively resistant to further proteolytic breakdown and have been detected in the distal portion of the small intestine and blood of humans after ingestion of cow's milk (Svedberg 10 et al., 1985). Migliore-Samour et al. (1989) have shown that peptides 13-casein(63-68) PGPIPN and us,-casein(194-199) TTMPLW at concentrations as low as 0.1 pM exert a significant protective effect in mice against Klebsiella pneumoniae infection when injected intravenously at 0.3 mg/kg, before lethal infectious challenge. An antibacterial peptide from bovine 15 as 2 -casein [las 2 -casein (f172-203)] released by treatment of milk with glacial acetic acid has recently been characterised and shown to inhibit the growth of Escherichia coli and Staphylococcus carnosus (Zucht et al., 1995). Antimicrobial peptides having activity against a range of Gram-positive and Gram-negative bacteria have potential in the area of oral 20 care, functional foods, food preservatives and pharmaceuticals. Oral care products include toothpaste, mouthwash, dental floss and professionally applied materials. Functional foods include chewing gum, confectionery, breakfast cereals, infant formula, beverages, lozenges etc. Food preservatives application could include dairy products, soups, salad dressings, processed 25 meats, baked goods, sauces etc. Pharmaceutical use would include systemic and topically applied antibiotics and anti-infectives and medications for the treatment of ulcers and other gastro-intestinal tract diseases. For food applications, natural antimicrobials are typically used for the maintenance and extension of shelf-life in sauces, wet salads, baked goods 30 and pastries, processed meats, refrigerated dairy products, salad dressings and soaps. Nisin has limited application as a food preservative due to a relatively narrow spectrum of antimicrobial activity and high cost. Food manufacturers using casein antimicrobial peptides as a preservative may use "all natural" label claims which are not allowed when using artificial or 35 chemical preservatives. A major trend in the food industry is the increasing demand for low fat products which in general tend to have increased WO 99/26971 PCT/AU98/00972 4 moisture levels. This creates a demand for better food preservation systems such as the incorporation of natural antimicrobials. The global market for medications for wound healing, treatment of upper gut ulcers and inflammatory based disease represents a major 5 pharmaceutical market. Clinicians working in the area of duodenal and gastric ulcers currently focus on the bacterium Helicobacterpylori as the causative agent in upper gut ulcers. Channel forming antimicrobial peptides that allow H+ to enter the bacterial cell have the potential for treatment of H. pylori infections by enhancing the sensitivity of the bacterium to the acid 10 secretions of the stomach. SUMMARY OF THE INVENTION The present inventors have developed new peptides which have 15 antimicrobial activity. These peptides can be produced synthetically, however, they can most conveniently be derived from casein. Accordingly, in a first aspect the present invention consists in an antimicrobial peptide, the peptide being non-glycosylated, less than about 100 amino acids, preferably less than about 70 amino acids, and including an 20 amino acid sequence selected from the group consisting of: AVESTVATLEAEPEVIESPPE, AVESTVATLEDZPEVIESPPE, AVESTVATLEASPEVIESPPE, AVESTVATLEDSPEVIESPPE, 25 DMPIQAFLLYQQPVLGPVR, and conservative substitutions therein. In a preferred embodiment of the present invention the peptide includes an amino acid sequence selected from the group consisting of: AVESTVATLEALPEVIESPPE, AVESTVATLEDIPEVIESPPE, 30 AVESTVATLEASPEVIESPPE, AVESTVATLEDSPEVIESPPE, and DMPIQAFLLYQQPVLGPVR, preferably AVESTVATLEAZPEVIESPPE, AVESTVATLEDIPEVIESPPE, or DMPIQAFLLYQQPVLGPVR.

WO 99/26971 PCT/AU98/00972 5 In a further preferred embodiment of the present invention the peptide includes an amino acid sequence selected from the group consisting of: MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEALPEVIESPPEINT 5 VQVTSTAV; MAIPPKKNQDKTEIPTINTIAIGEPTSTPTIEAVESTVATLEALPEVIESPPEINT VQVTSTAV; MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDEPEVIESPPEINT VQVTSTAV; 10 MAIPPKKNQDKTEIPTINTIAIGEPTSTPTTEAVESTVATLEDEPEVIESPPEINT VQVTSTAV; TEIPTINTIASGEPTSTPTIEAVESTVATLEALPEVIESPPEINTVQVTSTAV; TEIPTINTIAEGEPTSTPTIEAVESTVATLEAZPEVIESPPEINTVQVTSTAV; TEIPTINTIASGEPTSTPTTEAVESTVATLEDIPEVIESPPEINTVQVTSTAV; 15 TEIPTINTIAEGEPTSTPTTEAVESTVATLEDZPEVIESPPEINTVQVTSTAV; MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINT VQVTSTAV; MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINT VQVTSTAV; 20 TEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV; TEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINTVQVTSTAV; and conservative substitutions therein. It is further preferred that the peptide includes an amino acid sequence selected from the group consisting of: 25 MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEALPEVIESPPEINT VQVTSTAV; MAIPPKKNQDKTEIPTINTIAZGEPTSTPTIEAVESTVATLEALPEVIESPPEINT VQVTSTAV; MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDIPEVIESPPEINT 30 VQVTSTAV; MAIPPKKNQDKTEIPTINTIAEGEPTSTPTTEAVESTVATLEDZPEVIESPPEINT VQVTSTAV; TEIPTINTIASGEPTSTPTIEAVESTVATLEALPEVIESPPEINTVQVTSTAV; TEIPTINTIAIGEPTSTPTIEAVESTVATLEALPEVIESPPEINTVQVTSTAV; 35 TEIPTINTIASGEPTSTPTTEAVESTVATLEDIPEVIESPPEINTVQVTSTAV;

TEIPTINTIALGEPTSTPTTEAVESTVATLEDIPEVIESPPEINTVQVTSTAV;

WO 99/26971 PCT/AU98/00972 6 MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINT VQVTSTAV; MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINT VQVTSTAV; 5 TEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV; and TEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINTVQVTSTAV. In yet a further preferred embodiment of the present invention the peptide is selected from the group consisting of: MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEALPEVIESPPEINT 10 VQVTSTAV; MAIPPKKNQDKTEIPTINTIAEGEPTSTPTIEAVESTVATLEAZPEVIESPPEINT VQVTSTAV; MLAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDZPEVIESPPEINT VQVTSTAV; 15 MAIPPKKNQDKTEIPTINTIAZGEPTSTPTTEAVESTVATLEDEPEVIESPPEINT VQVTSTAV; TEIPTINTIASGEPTSTPTIEAVESTVATLEAZPEVIESPPEINTVQVTSTAV; TEIPTINTIAEGEPTSTPTIEAVESTVATLEAEPEVIESPPEINTVQVTSTAV; TEIPTINTIASGEPTSTPTTEAVESTVATLEDIPEVIESPPEINTVQVTSTAV; 20 TEIPTINTIAEGEPTSTPTTEAVESTVATLEDIPEVIESPPEINTVQVTSTAV; AVESTVATLEALPEVIESPP; AVESTVATLEDIPEVIESPP; MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINT VQVTSTAV; 25 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINT VQVTSTAV; TEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV; TEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINTVQVTSTAV; AVESTVATLEASPEVIESPP; 30 AVESTVATLEDSPEVIESPP; and DMPIQAFLLYQQPVLGPVR. As will be understood by those skilled in this field the peptide of the present invention can be conjugated to other molecules, such as acyl derivatives, to alter the delivery profile or pharmacokinetics of the peptide. 35 Such conjugates are described in PCT/AU90/00599, the disclosure of which is incorporated herein by reference.

WO 99/26971 PCT/AU98/00972 7 In a second aspect the present invention consists in a chimeric compound, the compound including the peptide of the first aspect of the present invention conjugated to a non-peptide molecule. It is preferred that the non-peptide molecule includes acyl groups. 5 In a third aspect the invention provides an antimicrobial compositions including the peptide of the first aspect of the present invention together with a pharmaceutically-acceptable carrier. Such compositions may be dental, intra-oral compositions, therapeutic anti-infective compositions for topical and systemic application. Dental 10 compositions or therapeutic compositions may be in the form of a gel, liquid, solid, powder, cream or lozenge. Therapeutic compositions may also be in the form of tablets or capsules. In a fourth aspect, there is provided a method of treating or preventing dental caries or periodontal disease in a subject, the method 15 comprising the step of administering a peptide or composition of the present invention to the teeth or gums of a subject in need of such treatments. Topical administration of the peptide is preferred. Without wishing to be bound by scientific theory it is believed that the peptides of the present invention exert their antimicrobial activity by 20 virtue of their amphipathic nature. It is believed that the peptides are incorporated into the bacterial membrane where they form aggregates. These aggregates provide or form pores or channels through the membrane through which ions may pass. The uncontrolled passage of ions across the bacterial membrane results in the death of the bacterial cell. 25 As it is the physical nature of the peptides rather than the specific sequence of the peptide which results in their antimicrobial activity so called conservative substitutions may be made in the peptide sequence with no substantial loss of activity. It is intended that such conservative substitutions which do not result in a substantial loss of activity are 30 encompassed in the present invention.

WO 99/26971 PCT/AU98/00972 8 Whilst the concept of conservative substitution is well understood by the person skilled in the art, for the sake of clarity conservative substitutions are those set out below. G, A, V, I, L, M; 5 D, E, S; N, Q; S, T; K, R, H; F, Y, W, H; and 10 P, Nca-alkalamino acids. Where I is a phosphoseryl residue. The peptides of the present invention have a number of applications, for example, they can be used in foods as antimicrobial preservatives, in oral care products (toothpastes and mouthrinses) for the control of dental plaque 15 and suppression of pathogens associated with dental caries and periodontal diseases. The antimicrobial peptides of the present invention may also be used in pharmaceutical preparations (eg, topical and systemic anti-infective medicines). Throughout this specification the word "comprise", or variations such 20 as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. DETAILED DESCRIPTION OF THE INVENTION 25 The present invention relates to the novel antimicrobial peptides. These peptides were initially derived from casein, K-casein (106-169) and 3-casein (184-202) [Table 1]. These peptides have potential to be used for the following micro-organisms inter alia. 30 Streptococcus mutans Staphylococcus aureus Streptococcus sanguinis Escherichia coli Salmonella typhimurium 35 Pseudomonas aeruginosa Porphyromonas gingivalis WO 99/26971 PCT/AU98/00972 9 Campylobacter jejuni Listeria monocytogenes Helicobacter pylori Clostridium botulinum 5 Streptococcus pyogenes Streptococcus pneumoniae Ccuidida albicans The antimicrobial peptides Ser(P) 4 9 K-casein B (106-169) and 10 Ser(P) 1 4 9 K-casein B (117-169) both had a minimum inhibitory concentration (MIC) of 2.4 tM against the oral pathogens Streptococcus mutans and Streptococcus sobrinus and at a ten-fold lower concentration (0.24pM) inhibited growth of these bacteria by 41%. The antimicrobial peptides Ser(P) 1 49 K-casein (117-169) and Ser(P) 127 15 Ser(P) 149 K-casein (117-169) and -casein (184-202) can be purified from a tryptic digest of bovine casein using standard chromatographic procedures of anion exchange and reversed-phase chromatography (HPLC). Ser(P) 149 K-casein (106-169) and Ser(P) 1 27 , Ser(P) 1 49 K-casein (106-169) can also be prepared from cheese whey and rennet whey by removal of the whey 20 proteins by ultrafiltration, or acid precipitation followed by reversed-phase HPLC purification of the phosphopeptides. The peptides can be prepared from casein of other species, eg. goat, sheep etc.

WO 99/2697 1 PCT/AU98/00972 10 Table 1. Casein Antimicrobial Peptides Peptide Sequence' Ser(P) 1 4 9 i<-casein B (106-169) MIAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVA TLEAEPEVIESPPEINTVQVTSTAV Ser(Pl' 2 1, Ser(P) 1 4 9 K-Gasein B MAIPPKKNQDKTEIPTINTIAZGEPTSTPTIEAVESTVA (106-169) TLEAY-PEVIESPPEINTVQVTSTAV Ser(P) 1 4 9 , ic-casein A (106-169) IIAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVA TLEDZIPEVIESPPEINTVQVTSTAV Ser(P) 1 2 7 , Ser(P) 1 4 9 K-casein A MAIPPKKNQDKTEIPTINTIAZ-GEPTSTPTTEAVESTV (106-169) ATFLEDFPEVIESPPEINTVQVTSTAV Ser(P) 1 4 9 K-casein B (117-169) TEIPTIINTIIASGEPTSTPTIEAVESTVATLEAT-PEVIESPP EINTVQVTSTAV Ser(P) 12, Ser(P) 1 4 9 K-casein B TEIPTINTIAX-GEPTSTPTIEAVESTVATLEAY-PEVIESP (117-169) PEINTVQVTSTAV Ser(P) 1 4 9 K-casein A (117-169) TEIPTINTIASGEPTSTPTTEAVESTVATLEDZPEVIESP PEINTVQVTSTAV Ser(P) 1 1 7 , Ser(P) 1 4 9 ic-casein A TEIPTINTIAX-GEPTSTPTTEAVESTVATLEDEPEVIESP (117-169) PEINTVQVTSTAV Ser(P) 1 4 9 K-casein B (138-158) AVESTVATLEAY-PEVIESPP Ser(P) 1 4 9 ic-casein A (138-158) AVESTVATLEDY-PEVIESPP K-casein B (106-169) MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVA TLEASPEVIESPPEINTVQVTSTAV K-casein A (106-169) MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVA TLEDSPEVIESPPEINTVQVTSTAV K-casein B (117-169) TEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPP EINTVQVTSTAV K-casein A (117-169) TEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESP PEINTVQVTSTAV K-casein B (138-158) AVESTVATLEASPEVIESPP ic-c asein A (138-158) AVESTVATLEASPEVIESPP f-casein (184-202) DMPIQAFLLYQQPVLGPVR a. Sequence identified using the one letter amino acid code where Y- = Ser(P) WO 99/26971 PCT/AU98/00972 11 The peptide K-casein (106-169) is present in cheese whey or rennet whey in several different forms. The peptide has two major genetic variants (A and B) and is post-translationally modified by glycosylation and phosphorylation. The glycosylated forms, known as the 5 Kappa-caseino-glycopeptide or glycomacropeptide have been described by Neeser [US patent Nos. 4,992,420 and 4,994,441] as anti-plaque and anti-caries agents by virtue of the oligosaccharide chains linked to threonine residues of the peptide. Neeser claims that the oligosaccharide chains of the glycopeptide, by specifically binding to plaque-forming oral bacteria, block 10 the adherence of these bacteria onto salivary-coated tooth enamel. The glycosylated forms of K-casein (106-169) can be separated from the non-glycosylated forms by chromatography (eg. anion exchange and reversed-phase HPLC) or by selective precipitation or ultrafiltration. Only the non-glycosylated forms of K-casein (117-169) or K-casein (106-169) 15 showed antimicrobial activity. As glycosylation destroys antimicrobial activity it is desirable to separate the glyco- and aglyco-forms of K-casein (117-169) or K-casein (106-169) which can be achieved using chromatography, selective precipitation or ultrafiltration. Phosphorylation of Ser 149 and to a lesser extent Ser' 27 are important for antimicrobial activity 20 and the phosphorylated forms of the two major genetic variants (A and B) appear to possess equal activity [Table 1]. The Neeser patents do not disclose the antimicrobial activity of K-casein(106-169) nor the use of the non-glycosylated forms of the peptide for the suppression of bacterial pathogens. 25 In a particularly preferred embodiment of the invention, the antimicrobial peptide is incorporated into dentifrices such as toothpaste, mouth washes or formulations for the mouth to aid in the prevention and/or treatment of dental caries and periodontal diseases. The peptide may comprise 0.01-50% by weight of the composition, preferably 0.1-10%. For 30 oral compositions it is preferred that the amount of the peptide administered is 0.01 -50% by weight, preferably 0.1-10% by weight of the composition. The oral composition of this invention which contains the above-mentioned peptides may be prepared and used in various forms applicable to the mouth such as dentifrice including toothpastes, toothpowders and liquid dentifrices, 35 mouthwashes, troches, chewing gums, dental pastes, gingival massage creams, gargle tablets, lozenges, dairy products and other foodstuffs. The WO 99/26971 PCT/AU98/00972 12 oral composition according to this invention may further include additional well known ingredients depending on the type and form of a particular oral composition. In certain highly preferred forms of the invention the oral 5 composition 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 preparation is 10 typically in the range of from about 70 to about 99.9% by weight of the preparation. The alcohol is typically ethanol or isopropanol. Ethanol is preferred. The pH of such liquid and other preparations of the invention is generally in the range of from about 4.5 to about 9 and typically from about 15 5.5 to 8. The pH is preferably in the range of from about 6 to about 8.0, preferably 7.4. 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). 20 Other desirable forms of this invention, the oral composition may be substantially solid or pasty in character, such as toothpowder, a dental tablet or a dentifrice, that is a toothpaste (dental cream) or gel dentifrice. The vehicle of such solid or pasty oral preparations generally contains dentally acceptable polishing material. Examples of polishing materials are 25 water-insoluble sodium metaphosphate, potassium metaphosphate, tricalcium phosphate, dihydrated calcium phosphate, anhydrous dicalcium phosphate, calcium pyrophosphate, magnesium orthophosphate, trimagnesium phosphate, calcium carbonate, hydrated alumina, calcined alumina, aluminium silicate, zirconium silicate, silica, bentonite, and 30 mixtures thereof. Other suitable polishing material include the particulate thermosetting resins such as melamine-, phenolic, and urea-formaldehydes, and cross-linked polyepoxides and polyesters. Preferred polishing materials include crystalline silica having particle sized of up to about 5 microns, a mean particle size of up to about 1.1 microns, and a surface area of up to 35 about 50,000 cm 2 /gm., silica gel or colloidal silica, and complex amorphous alkali metal aluminosilicate.

WO 99/26971 PCT/AU98/00972 13 When visually clear gels are employed, a polishing agent of colloidal silica, such as those sold under the trademark SYLOID as Syloid 72 and Syloid 74 or under the trademark SANTOCEL as Santocel 100, alkali metal alumino-silicate complexes are particularly useful since they have refractive 5 indices close to the refractive indices of gelling agent-liquid (including water and/or humectant) systems commonly used in dentifrices. Many of the so-called "water insoluble" polishing materials are anionic in character and also include small amounts of soluble material. Thus, insoluble sodium metaphosphate may be formed in any suitable 10 manner as illustrated by Thorpe's Dictionary of Applied Chemistry, Volume 9, 4th Edition, pp. 510-511. The forms of insoluble sodium metaphosphate lknlown as Madrell's salt and Kurrol's salt are further examples of suitable materials. These metaphosphate salts exhibit only a minute solubility in water, and therefore are commonly referred to as insoluble metaphosphates 15 (IMP). There is present therein a minor amount of soluble phosphate material as impurities, usually a few percent such as up to 4% by weight. The amount of soluble phosphate material, which is believed to include a soluble sodium trimetaphosphate in the case of insoluble metaphosphate, may be reduced or eliminated by washing with water if desired. The 20 insoluble alkali metal metaphosphate is typically employed in powder form of a particle size such that no more than 1% of the material is larger than 37 microns. The polishing material is generally present in the solid or pasty compositions in weight concentrations of about 10% to about 99%. 25 Preferably, it is present in amounts from about 10% to about 75% in toothpaste, and from about 70% to about 99% in toothpowder. In toothpastes, when the polishing material is silicious in nature, it is generally present in amount of about 10-30% by weight. Other polishing materials are typically present in amount of about 30-75% by weight. 30 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 preparation. Glycerine, propylene glycol, sorbitol and polypropylene glycol exemplify suitable humectants/carriers. Also advantageous are liquid mixtures of water, glycerine and sorbitol. In clear 35 gels where the refractive index is an important consideration, about WO 99/26971 PCT/AU98/00972 14 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 5 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% SiO 2 , 25.40% MgO, 3.05% Na 2 0, 0.98% Li 2 0, and some water and trace metals. Its true specific 10 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 15 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 20 petrolatum and esters such as amyl acetate, ethyl acetate and benzyl benzoate. It will be understood that, as is conventional, the oral preparations are to be sold or otherwise distributed in suitable labelled packages. Thus, a jar of mouthrinse will have a label describing it, in substance, as a 25 mouthrinse 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. 30 Organic surface-active agents are used in the compositions of the present invention to achieve increased prophylactic action, assist in achieving thorough and complete dispersion of the active agent throughout the oral cavity, and render the instant compositions more cosmetically acceptable. The organic surface-active material is preferably anionic, 35 nonionic or ampholytic in nature which does not denature the antimicrobial peptide of the invention, and it is preferred to employ as the surface-active WO 99/26971 PCT/AU98/00972 15 agent a detersive material which imparts to the composition detersive and foaming properties while not denaturing the peptide. 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 5 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 10 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 15 the oral compositions of the present invention is particularly advantageous since these materials exhibit a prolonged marked effect in the inhibition of acid formation in the oral cavity due to carbohydrates breakdown in addition to exerting some reduction in the solubility of tooth enamel in acid solutions. Examples of water-soluble nonionic surfactants suitable for use with peptides 20 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 25 acids, fatty alcohols, fatty amides, polyhydric alcohols (e.g. sorbitan monostearate) and polypropyleneoxide (e.g. Pluronic materials). 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 the peptide of the invention and thereby diminish the amount 30 of solubilizing humectant needed. Various other materials may be incorporated in the oral preparations of this invention such as whitening agents, preservatives, silicones, chlorophyll compounds and/or ammoniated material such as urea, diammonium phosphate, and mixtures thereof. These adjuvants, where 35 present, are incorporated in the preparations in amounts which do not substantially adversely affect the properties and characteristics desired.

WO 99/26971 PCT/AU98/00972 16 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. 5 Suitable sweetening agents include sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP (aspartyl phenyl alanine, 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. In the preferred practice of this invention an oral composition 10 according to this invention such as mouthwash or dentifrice containing the composition of the present invention is preferably applied regularly to the gums and teeth, such as every day or every second or third day or preferably from 1 to 3 times daily, at a pH of about 4.5 to about 9, generally about 5.5 to about 8, preferably about 6 to 8, for at least 2 weeks up to 8 weeks or more up 15 to a lifetime. The compositions of this invention can 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 may be mentioned jelutong, rubber latex, vinylite resins, etc., desirably with 20 conventional plasticisers or softeners, sugar or other sweeteners or such as glucose, sorbitol and the like. In another embodiment, the peptide of the invention is formulated in foods to act as a preservative preferably comprising 0.01-10% w/w, more preferably 0.1-5% w/w, most preferably 1-5% and particularly 2% w/w. 25 The present invention provides compositions including pharmaceutical compositions comprising the antimicrobial peptide as described together with a pharmaceutically-acceptable carrier. Such compositions may be selected from the group consisting of dental, intra-oral compositions, therapeutic anti-infective compositions for topical and 30 systemic application. Dental compositions or therapeutic compositions may be in the form of a gel, liquid, solid, powder, cream or lozenge. Therapeutic compositions may also be in the form of tablets or capsules. The present invention also provides a method of treating or preventing dental caries or periodontal disease comprising the step of 35 administering a peptide or composition of the invention to the teeth or gums WO 99/26971 PCT/AU98/00972 17 of a subject in need of such treatments. Topical administration of the peptide is preferred. The invention also provides a method of producing the antimicrobial peptide from casein or whey. K-casein (106-169) can be obtained from cheese 5 or rennet whey by ultrafiltration or acid precipitation. Ultrafiltration of whey through a 10,000 - 30,000 nominal molecular weight cut off (NMCO) membrane filter at neutral or preferably acidic pH (3-5) retains the majority of whey proteins producing a permeate rich in casein peptides, lactose and minerals. Ultrafiltration and concentration of the permeate using a 1000 10 NMCO membrane filter produces a fraction rich in K-casein (106-169). This fraction is then incubated with trypsin and the resulting hydrosylate subjected to reversed-phase HPLC producing a relatively pure K-casein (117-169) peptide. Alternatively the peptides K-casein (117-169) and 3(184-202) can be obtained from a tryptic digest of casein using 15 reversed-phase HPLC. Peptide K-casein (138-158) can be obtained by a partial endo-Glu-C digest of K-casein (106-169) followed by purification using reversed-phase HPLC. It will be clearly understood that, although this specification refers specifically to applications in humans, the invention is also useful for 20 veterinary purposes. Thus in all aspects the invention is useful for domestic animals such as cattle, sheep, horses and poultry; for companion animals such as cats and dogs; and for zoo animals. In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described with reference to 25 the following non-limiting examples. FIGURE LEGENDS Figure 1. Reversed-phase HPLC of a tryptic digest of a Whey Protein 30 Concentrate (WPC). The WPC tryptic digest (8 mg) was applied to a Brownlee RP-300 C 8 column. The sample was eluted using a stepwise linear gradient of 0 - 20% B in 2 min followed by 20 - 45% B in 40 min at a flow rate of 1 ml/min. Eluant A was 0.1% (v/v) TFA in water and eluant B was 80% (v/v) acetonitrile in 0.1% (v/v) TFA in water. 35 WO 99/26971 PCT/AU98/00972 18 Figure 2. Anion exchange chromatography of peak 9 from RP-HPLC of the WPC tryptic digest. Peak 9 was applied to a Mono Q column attached to a SMART T M system and eluted using 0 - 75% elutant B in 40 min at a flow rate of 100 tl/min. Elutant A was 20 mM Tris-HCI pH 8.0, 10 mM KCl and 5 elutant B was 20 mM Tris-HCI pH 8.0, 500 mM KC1. Figure 3. Determination of the MIC of K-casein A Ser(P) 149 (f117-169) for Streptococcus mutans Ingbritt. The MIC was 2.4 pM. 10 Figure 4. Analytical reversed-phase HPLC elution profile of UF-whey. A sample of UF-whey was dissolved in solvent A and applied to an analytical column (C18) and then eluted using a linear gradient from 0-35% Solvent B in 5 min followed by 35-80% Solvent B in 40 min at a flow rate of 1.0 ml/min. Solvent A consisted of 0.1% TFA in water and Solvent B 15 contained 90% acetonitrile (v/v/0.1% TFA in water). Peaks were detected at 215 nm, collected manually at 215 nm and lyophilised. Figure 5. Purification of peak 4 (from RP-HPLC) using gel filtration. Peak 4 was applied to a gel filtration column connected to an ABI system. 20 Material was eluted using 30% acetonitrile (v/v)/0.1% TFA at a flow rate of 1 ml/min and monitored at 215 nm. Figure 6. Purification of peptides generated by the hydrolysis of TCA-soluble UF-whey by endopeptidase Glu-C. A sample was dissolved in 25 Solvent A (0.1% TFA v/v in water) and applied to an analytical column (C18). Peaks were eluted using a gradient of 0-20% Solvent B (90% acetonitrile v/v/0.1% TFA in water) in 4 min followed by 20%-40% Solvent B in 40 min. Peaks were monitored at 215 nm. 30 Specific examples of formulations containing the antimicrobial peptide of the invention are provided below.

WO 99/26971 PCT/AU98/00972 19 EXAMPLE 1 Preparation of antimicrobial peptides from a tryptic digest of whey protein concentrate 5 Whey protein concentrate (50 mg/ml) in water (pH 8.0) was hydrolysed using Novo trypsin (1 mg/ml) at 50 0 C for 2 h with the pH maintained at 8.00 + 0.01 by the addition of 1N NaOH. Hydrolysis was terminated by the addition of 1M HC to pH 4.6. The hydrolysate was centrifuged (11,600 g for 10 min) and then filtered through a 0.2 ptm PVDF 10 filter before being applied to a 7 ptm C 8 (Brownlee) reversed-phase column (4.6 x 220 mm). The sample was eluted using an Applied Biosystems 140 A Solvent Delivery System to generate a stepwise linear gradient from 0-20% B in 2 min followed by 20-45% B in 40 min at a flow rate of 1 ml/min. Eluant A was 0.1% (v/v) TFA in water and eluant B was 80% (v/v) acetronitrile, 15 0.1% (v/v) TFA in water. The eluant was monitored using an Applied Biosystems 1000S Diode Array detector at a primary wavelength of 215 nm. The chromatogram obtained is shown in Fig. 1. Peaks were collected and assayed for antimicrobial activity. Antimicrobial assays were carried out in liquid growth medium using sterile 96 well plates, each well having a 20 capacity of 300 pL. The growth medium consisted of Todd Hewit broth (36.4 g/L), Yeast Extract (5.0 g/L) with 100 mmol/L potassium phosphate buffer. Routinely the pH of the growth medium was adjusted to 6.3. An inoculum of approximately 1.5 X 102 cells (Streptococcus sanguis, Streptococcus mutans, Porphyromonas gingivalis) that had been harvested 25 during the exponential phase of growth, was added to each well. The ionophore gramicidin (40 pmol/L final concentration) was added to a series of wells as a negative control. Positive controls contained only the inoculum and the growth medium. Growth was determined over a 30 hour period after inoculation by measuring the optical density of the cell suspensions at a 30 wavelength of 650 nm (OD 65 0 ), using a microplate reader (Biorad, model 450). Growth was determined by subtracting the initial reading, taken immediately after inoculation from the final reading (maximum culture OD). Antimicrobial assays were also carried out on agar plates containing suitable growth media that had been inoculated with a lawn of the test 35 bacterial species. Filter paper discs (6 mm diameter), to which was added 50 p L of the peptide solution, were placed on the surface of the agar plate.

WO 99/26971 PCT/AU98/00972 20 The diameter of the zone of growth inhibition around each disc was determined after three days of incubation and compared to a control that only had buffer added. Growth conditions depended on the bacterial species being tested, however they were routinely cultured in an anaerobic work 5 station at 37 oC. Only peak 9 of Fig. 1 exhibited antimicrobial activity. Analysis of this peak using amino acid sequence analysis (Hewlett Packard automated protein sequencer) and mass analysis (Perseptive Voyager IVIALDI-TOF mass spectrometer) revealed that the peak was heterogenous and so the sample was subjected to anion exchange chromatography on a 10 Mono Q PC 1.6/5 (10 jtm) column attached to a SMIVART

TM

' (Pharmacia) system. The sample was eluted using a linear gradient from 0-75% B in 40 min at a flow rate of 100 jtl/min. Eluant A was 20 mM Tris-HCI pH 8.0, 10 mM KC1. Eluant B was 20 mM Tris-HCI pH 8.0, 500 mM KC1. The eluant was monitored at 215 and 280 nm using the t Peak monitor. The anion 15 exchange chromatogram for peak 9 from RP-HPLC is shown in Fig. 2. Peaks were collected and assayed for antimicrobial activity and only peaks 9, 10 and 11 exhibited activity. N-terminal sequence and mass analyses revealed that fraction 9 contained Ser(P) 149 K-casein A (113-169), fraction 10 contained Ser(P) 149 K-casein A (124-169) and fraction 11 contained Ser(P) 149 K-casein A 20 (117-169). Mass analysis revealed that none of the peptides were glycosylated. The minimum inhibitory concentration (MIC) of pure Ser(P) 149 K-casein A (117-169) was then determined for the bacterium Streptococcus mutans and is shown in Fig. 3. The MIC obtained was 2.4 jtM. 25 EXAMPLE 2 A. Preparation of antimicrobial peptides from cheese whey Cheese whey was ultrafiltered (UF) though a 20,000 molecular weight cut off membrane. The filtrate was collected and proteins were precipitated 30 by addition of trichloroacetic acid (TCA) to a final concentration of 11% w/v. Precipitated proteins were removed by centrifugation (10,000g, 5 min) and the neutralised supernatant was lyophilised. The dried TCA-soluble UF whey was dissolved in 0.1% TFA in water and subjected to RP-HPLC. The sample was applied to a Brownlee aquapore analytical (C18) reversed-phase 35 column (220 x 4.6 mm) or a Brownlee (C18) preparative column (25 cm x 10 mm). Solvent B consisted of 90% acetonitrile containing 0.1% v/v TFA and WO 99/26971 PCT/AU98/00972 21 solvent A consisted of 0.1% TFA in water. The eluant was monitored using an Applied Biosystems Incorporated (ABI; Melbourne, Vic., Australia) 1000S diode array detector at a wavelength of 215 nm. The sample was applied to the Brownlee analytical column and 5 eluted using a gradient from 0-35% solvent B in 2 min, 35% solvent B in 2 min followed by 35-80% solvent B in 40 min at a flow rate of 1.0 ml/min. Fractions collected were assayed for antibacterial activity. Fractions were tested with the Gram-positive bacteria Streptococcus mutans Ingbritt, Streptococcus sanguinis (formerly S. sanguis), Streptococcus sobrinus 6715 10 WT15, Staphylococcus aureus ATCC 25923 and the Gram-negative bacteria, Escherichia coli NCTC 10418, Salmonella typhimurium ATCC 13311, and Pseudomonas aeruginosa ATCC 25619. The bacteria were stored in 30% glycerol broths at -20 0 C. The antibacterial assay was conducted in sterile 96 well plates 15 (Becton Dickinson, Melbourne, Australia). The growth media for the Gram-positive bacteria consisted of Todd Hewitt broth (TH; 36.4 g/1), Yeast extract (YE; 5 g/l) and 100 mM potassium phosphate, pH 6.28 (TYPB). The media for Gram-negative bacteria consisted of Nutrient broth at pH 6.28 and for P. gingivalis, Brain Heart Infusion media (with 1tg/ml haeme and 0.5g/1 20 cysteine) at pH 7.0. An inoculum was prepared by diluting exponentially growing cells in growth media, such that the inoculum contained approximately 2.7 x 10 4 viable cells/ml. To each well was added 250 pl media containing the peptide in varying concentrations and 50 p1 of bacterial inoculum. Control assays contained all components except peptide. The 25 negative control wells each contained 250 p1 media, 50 p1 inoculum and 5 p1 of gramicidin D (2.5 mM). Growth of the bacterium was determined as the difference between the final and initial Optical Density (OD) 650 nm readings using a microplate reader (BIORAD, model 450, NSW, Australia). The final OD represented the maximum culture OD and was recorded 30 normally 20-30 h after inoculation, during which time the cells were incubated at 37 0 C in aerobic conditions except for P. gingivalis which was incubated in anaerobic conditions at the same temperature. The minimal inhibitory concentration (MIC) was determined as the lowest concentration of peptide required to inhibit the growth of the bacterium. The peptide 35 concentration varied between 0.05 pM-500 tM WO 99/26971 PCT/AU98/00972 22 The antimicrobial activity of the neutralised starting material (TCA-soluble UF whey) is shown in Table 2. Table 2. Growth inhibition of Gram-positive and Gram-negative bacteria by 5 TCA-soluble UF-whey. Microbial growth was determined by optical density at a wavelength of 650 nm after 30 h incubation at 37C. Species Growth Inhibition by TCA-soluble UF-whey % 3.7 mg/ml 1.9 mg/ml Gram-positive bacteria S. mutans 89 ± 6 a 42 ± 11 S. aureus 47 ± 18 18 ± 18 S. sanguinis NIb NI Gram-negative bacteria E. coli 14 ± 8 12 ± 15 S. typhimurium 8 ± 7 NI P. aeruginosa 9 ± 6 NI a-% mean inhibition of growth ± standard deviation (n= 3-6) b- no inhibition 10 Fig. 4 shows the RP-HPLC of the TCA-soluble UF whey. Five peaks were collected and analysed for antibacterial activity. Peak 4 exhibited the highest specific antimicrobial activity as shown in Table 3. Peak 4 (RP4) was further subjected to gel filtration chromatography using a gel filtration 15 column (Supelco 30 x 7.8 cm) and eluted using 30% acetonitrile v/v 0.1% TFA in water at a flow rate of 1 ml/min.

WO 99/26971 PCT/AU98/00972 23 Table 3: Growth inhibition of streptococcal species by RP-HPLC peaks of UF-whey. The peaks generated were tested in the antibacterial assay. Microbial growth was determined by optical density at a wavelength of 650 nm after 30 h incubation at 37 0 C. 5 Sample Amountt Assay % Growth Inhibition (mg) concentrations (mg/ml) S. mutans S. sobrinus S. sanguinis RP1 + 1.7 1.4 23 ± 16 a _ b Mc RP2 RP3 1.0 0.90 17 ± 16 23 ± 20 17 7 RP4 0.64 0.53 91 ± 3 81 ± 5 26 7 RP5 0.60 0.50 79 ± 7 79 ± 14 23 5 a - % mean inhibition of growth ± standard deviation (n= 3-6) b - not determined c - no inhibition t - Amount of each peak estimated by 215nm absorbance 10 o0 - Concentration of peak in antibacterial assay Fig. 5 shows the gel filtration chromatography of peak 4 (RP4) from RP-HPLC of the TCA-soluble UF whey. Four peaks from the chromatography were collected and assayed for antimicrobial activity against S. mutans as shown 15 in Table 4.

WO 99/26971 PCT/AU98/00972 24 Table 4. The inhibition of growth of S. mutans by gel filtration peaks of peak 4 from RP-HPLC of TCA-soluble whey. Peak Amountt Assay %Growth (mg) Concentration' inhibition (mg/ml) RP4GF1 3.12 2.6 46 ± 9 a RP4GF2 b cNI RP4GF3 19.2 16 26 ± 12 RP4GF4 2.88 2.4 41 ± 11 a - % mean inhibition of growth ± standard deviation (n= 3-6) 5 b - not determined c - no inhibition t - Amount of each peak estimated by 215nm absorbance oo - Concentration of peak in antibacterial assay 10 The four peaks were also analysed by amino acid sequence analysis and by mass spectrometry. Mass spectrometric analysis (MS) of peptides was performed using a Perspective Biosystems (Framingham, MA, USA) Voyager linear matrix 15 assisted laser desorption/ionisation Time of Flight (MALDI-TOF) mass spectrometer equipped with delayed extraction. Samples were mixed (1:1 v/v) on the sample analysis plate with a 5 mg/ml solution of 2-5, dihydroxybenzoic acid in 50% aqueous acetonitrile, containing 0.25% v/v TFA in water. All spectra were obtained in linear, positive mode with an 20 accelerating voltage of 20 kV, grid voltage of 92% and pulse delay time of 125 ns. Calibration was performed using bovine insulin (MW 5733.54 Da). For sequence analysis peptides were applied to a preconditioned Hewlett-Packard (HP; Blackburn, Vic, Aust.) sequencing column in 1 ml of sample loading solution (2% v/v TFA in water) and then analysed using a HP 25 G1005A Protein sequencer.

WO 99/26971 PCT/AU98/00972 25 Table 5. Comparison of the peaks from gel filtration chromatography of peak 4 (RP4) of TCA-soluble UF whey as determined by sequence and mass spectrometric analysis. Peak Measured Calculated Assignment t Mass (Da) Mass (Da) RP4GF1 6756 6755 Ser(P) 14K-casein-B-(106-169) 6788 6787 Ser(P) 149 K-casein-A-(106-169) 6736 6835 Ser(P) 1 27 , Ser(P) 149 K-casein-B-(106-169) 6869 6867 Ser(P) 127, Ser(P) 149K-casein-A-(106-169) RP4GF2 - - f3-lactoglobulin, minor traces of ot-lactalbumin and K-casein (106-169) RP4GF3 - - A-lactalbumin RP4GF4 6758 6755 Ser(P) 1 49 K-casein-B-(106-169) 6788 6787 Ser(P) 149K-casein-A-(106-169) 6738 6835 Ser(P) 12 7 , Ser(P)149K-casein-B-(106-169) 6869 6867 Ser(P) 127, Ser(P) 149K-casein-A-(106-169) 5 t - Assigned by N-terminal amino acid sequencing The two gel filtration peaks with the same specific antimicrobial activity RP4 GF1 and RP4 GF4 (Table 4) contained the same peptides, presumably the higher molecular weight fraction RP4 GF1, represented an 10 aggregated state of the phosphopeptides. The active peptides were identified as: Ser(P) 149K-casein-B-(106-169) Ser(P) 149K-casein-A-(106-169) Ser(P) 127 , Ser(P) 149 K-casein-B-(106-169) 15 Ser(P) 127 , Ser(P)1 49 K-casein-A-(106-169) The identification of antimicrobial activity with the phosphorylated, non-glycosylated form of K-casein (106-169) is consistent with the identification of the tryptic casein peptide Ser(P) 149 K-casein (117-169) as an 20 antimicrobial peptide in Example 1.

WO 99/26971 PCT/AU98/00972 26 B. Preparation of antimicrobial peptides from TCA-soluble UF whey treated with endopeptidase Glu-C Endopeptidase Glu-C (Sigma Chemical Co, St. Louis, MO, USA) was 5 added (5.0 ptg/ml) to a solution of TCA-soluble UF-whey (1.0 mg/ml), in ammonium acetate (0.05 M, pH 4.0) and incubated at 37C for 24 h. The reaction was stopped by lowering the pH to 3.0 by the addition of glacial acetic acid. Enzymatic digestion products were separated by RP-HPLC. Fig. 6 shows the RP-HPLC of the endo Glu-C digest of TCA-soluble 10 UF whey. Twelve peaks were collected and only peak 12 exhibited antimicrobial activity against S. mutans as shown in Table 6. Peak 12 contained three peptides as shown by sequence and mass spectrometric analyses (Table 7). These peaks were further purified by analytical RP-HPLC and only peptide Ser(P) 149 K-casein A (138-158) exhibited antimicrobial 15 activity with a 100 pM concentration giving close to 100% growth inhibition of S. mutans. Table 6. Antimicrobial activity against S. mutans of lyophilised peaks 9-12 from RP-HPLC of an endo Glu-C hydrolysate of TCA-soluble UF-whey. 20 Peak Amountt Assay % Growth (mg) Concentration' inhibition (mg/ml) 9 0.64 0.53 NIa 10 0.30 0.25 NI 11 0.32 0.27 NI 12 0.40 0.34 84±9b a - no inhibition b - % mean inhibition of growth ±+ standard deviation (n= 3) t - Amount of each peak estimated by 215nm absorbance oo - Concentration of peak in antibacterial assay 25 WO 99/26971 PCT/AU98/00972 27 Table7. Composition of peaks 9-12 from RP-HPLC of an endo Glu-C hydrolysate of TCA-soluble UF-whey. Peak Measured Calculated Assignment Molecular Molecular mass (Da)a mass (Da) 9 3056.5 3050.2 Ser (P) 149 K-casein-B-(141-151) 4080.9 4076.2 K-casein-A-1 GalNAc, 1 Gal-(106-140) 4373.7 4367.2 K-casein-A-1 GalNAc, 1Gal, 1NeuAc-(106-140) + methionine sulfoxide 4750.9 4748.0 K-casein-A-2 GalNAc, 2Gal, 1NeuAc-(106-140) 5040.8 5039.0 K-casein-A-2 GalNAc, 2Gal, 2NeuAc-(106-140) + methionine sulfoxide 10 2438.2 2432.4 K-casein-A-1 GalNAc-(119-140) 2481.9 2474.0 Ser (P) 1 4 9 K-casein-A-1 GalNAc, 1 NeuAc-(141-158) 3423.8 3423.0 K-casein-B-(106-137) 4092.5 4092.2 K-casein-A-1 GalNAc, 1 Gal-(106-140) + methionine sulfoxide 4383.7 4383.2 K-casein-A-1 GalNAc, 1 Gal, 1 NeuAc-(106-140) + methionine sulfoxide 11 1890.1 1884.1 K-casein-A-(152-169) and k-casein-B-(152-169) 2938.4 2931.2 K-casein-A-(119-147) 3427.4 3423.8 K-casein-B-(106-137) 4094.2 4088.0 K-casein-B-1 GalNAc, 1 Gal-(106-140) 4385.3 4379.0 K-casein-B-1 GalNAc, 1 Gal, 1 NeuAc-(106-140) 12 2285.6 2279.3 Ser (P) 149 K-casein-A-(138-158) 2356.8 2348.4 Ser (P) 149 K-casein-B-(148-169) 2398.3 2392.5 Ser (P)149 K-casein-A-(148-169) a - molecular mass determined by MS WO 99/26971 PCT/AU98/00972 28 These results showed that only a 20 residue fragment of Ser(P) 149 K-casein A (106-169), Ser(P) 149 K-casein A (138-158) displayed antimicrobial activity albeit less potent (100 p~M MIC) compared with the longer peptide (2.5 pM MIC). The twenty residue fragment of the two major 5 genetic variants A and B are shown: Ser(P) 149 K-casein A (138-158) AVESTVATLEDIPEVIESPP Ser(P) 149 K-casein B (138-158) AVESTVATLEAZPEVIESPP The twenty residue fragment is amphipathic and has the potential to form an amphipathic helix and therefore a channel in the bacterial 10 membrane. A molecular model of K-casein (130-158) as a hexamer forming a polar channel with a non-polar exterior that could allow the passage of cations (Na , K , H etc.) through a bacterial cell membrane thereby dissipating transmembrane electrochemical gradients was constructed. It was interesting to note that the molecular model of the glycosylated form of 15 K-casein (130-158), which has no antimicrobial activity, has the channel blocked by sugar residues perhaps thereby possibly explaining the lack of activity with the glycosylated peptides. C. Synthesis of Ser(P) 14 K-casein (138- 160) 20 To confirm antimicrobial activity of Ser(P) 149 K-casein (138-158) related peptides were synthesised with and without the phosphorylation and assayed for antimicrobial activity. A peptide corresponding to Ser(P) 14 9 K-casein A (138-160), containing 25 a phosphoryl group on Ser 149, and K-casein A (130-158) were synthesised manually by standard solid-phase peptide synthesis protocols for Fmoc chemistry. The peptides were assembled as the carboxyl form using Pac-Peg-PS resin (PerSeptive Biosystems). Subsequent additions of the remaining Fmoc amino acids including Fmoc-Ser(PO(OBzl)OH)-OH were 30 accomplished with HBTU/HOBt activation using 4 equiv of Fmoc-amino acid and 6 equiv of DIPEA. The Fmoc group was removed with a continuous flow of 2% v/v DBU in DMF containing 2% v/v piperidine for 5 min. Cleavage of the peptide from the resin support was performed using TFA:TIPS:water (95:2.5:2.5) cleavage cocktail for 2.5 h under N2, in darkness. After cleavage 35 the resin was removed by filtration and the filtrate concentrated under a stream of nitrogen. After the peptide products were precipitated in cold WO 99/26971 PCT/AU98/00972 29 ether, they were centrifuged and washed three times. The peptide precipitate was then dissolved in water containing 0.1% v/v TFA and insoluble residue removed by centrifugation. Purification of synthesised peptides was performed using a Brownlee 5 C18 preparative column. Chromatograms were developed at a flow rate of 4.0 ml/min and peptides were eluted using a gradient of 0-100 % solvent B in 43 min. Peptide fractions collected from the column were applied to the Brownlee C18 analytical column and eluted using a gradient of 0-100% solvent B in 40 min. 10 All collected peptide fractions were lyophilised and subjected to analysis by MS. Table 8 shows the antimicrobial activity of the two synthetic peptides. These results show that the phosphorylation of Ser1 49 is essential for full antimicrobial activity. The phosphoseryl residue Ser(P)' 49 may be 15 necessary for the formation of an ion channel in the bacterial membrane or maybe necessary for solubility. Further, the higher MIC (100-150 pM) for the Ser(P) 14 9 K-casein A (138-160) compared with the larger peptide Ser(P) 49 K-casein A (117-169) (2.5 pIM MIC) suggests that the flanking residues of Ser(P) 149 K-casein (138-158) may be necessary for solubility and/or 20 interaction with the bacterial cell and formation of the ion channel. Table 8. Inhibition of S. mutans growth by synthetic peptides K-casein-A-(130-158) (non-phosphorylated) and Ser (P) 149 K-casein-A-(138-160). 25 Peptide MIC % Growth inhibition Concentration of synthetic peptides (mM) 100 75 50 25 10 K-casein-A-(130-158) 1.2 mM 17± 13 a _b 14 ± 13 11 ± 7 NIC Ser (P) 149 150 pM - 69 ± 6 52 ± 5 17 ± 9 6 ± 10 K-casein-A-(138-160) a - % mean inhibition of growth ± standard deviation (n= 3-6) b - not determined c - no inhibition WO 99/26971 PCT/AU98/00972 30 EXAMPLE 3 A. Trypsin hydrolysis Sodium caseinate was dissolved in 150 mM NIH 4

HCO

3 pH 8.0 at 10% 5 (w/v) and hydrolysed using Novo trypsin (2 g/L) at 50 oC for 2 h. Hydrolysis was terminated by the addition of 1N HCI to pH 4.6 and the undigested protein removed by centrifugation. A sample of the hydrolysate was applied to a 7 tm Applied Biosystems C 8 column (4.6 x 220 mm) and eluted as described in Example 1. Peaks were collected and assayed for antimicrobial 10 activity against Streptococcus sanguinis. Two peptides showed activity, Ser(P) 14 9 K-casein (117-169) and 13-casein (184-702). B. Rennet hydrolysis 15 Casein HC1 (5 g) was dissolved in 100 ml of 100 mM ammonium bicarbonate pH 8.0. Once the casein had dissolved the pH was lowered to 6.3 with 1 M HC1 and 1 mg of rennet (chymosin, Sigma) was added and the mixture incubated for 1 h at 37 0 C. TCA (11% w/v) was added to the solution or the pH was lowered to 4.5 by the addition of 1 M HC1 and the precipitated 20 proteins were removed by centrifugation. The supernatant was collected, neutralised and lyophilised. The dried sample was dissolved in solvent A (0.1% TFA in water) and applied to a Brownlee C18 preparative RP-HPLC column. The column was eluted using a gradient of 15% solvent B for 5 min 15-60% solvent B in 225 min followed by 60-100% solvent B in 1 min at a 25 flow rate of 4.0 ml/min. The eluant was monitored at 215 nm. Four peaks were obtained two of which had antimicrobial activity and corresponded to the non-glycosylated, phosphorylated K-casein (106-169). The active peptides were identified as: Ser(P) 14 9 ' K-casein A (106-169). 30 Ser(P) 1 2 7 , Ser(P) 149 K-casein A (106-169) and Ser(P) 149 K-casein B (106-169). Ser(P) 127 , Ser(P) 149 K-casein B (106-169).

WO 99/26971 PCT/AU98/00972 31 EXAMPLE 4 - PROPOSED TOOTHPASTE FORMULATIONS Formulation 1 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 Ser(P) 149 K-casein(106-169) 1.0 Water balance Formulation 2 Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Sorbitol 10.0 Glycerol 10.0 Sodium carboxymethyl cellulose 1.0 Sodium lauryl sulphate 1.5 Sodium lauroyl sarconisate 0.5 Flavour 1.0 Sodium saccharin 0.1 Sodium monofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Dextranase 0.01 Ser(P) 149 K-casein(106-169) 2.0 Water balance WO 99/26971 PCT/AU98/00972 32 Formulation 3 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 Ser(P) 149 K-casein(106-169) 5.0 Water balance Formulation 4 Ingredient % w/w Sorbitol 10.0 Irish moss 1.0 Sodium Hydroxide (50%) 1.0 Gantrez 19.0 Water (deionised) 2.69 Sodium monofluorophosphate 0.76 Sodium saccharin 0.3 Pyrophosphate 2.0 Hydrated alumina 48.0 Flavour oil 0.95 Ser(P) 149 K-casein(106-169) 1.0 Water balance WO 99/26971 PCT/AU98/00972 33 Formulation 5 Ingredient % w/w Sodium polyacrylate 50.0 Sorbitol 10.0 Glycerol 20.0 Sodium saccharin 0.1 Sodium monofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Ethanol 3.0 Ser(P) 14 9 K-casein(106-169) 2.0 Linolic acid 0.05 Water balance EXAMPLE 5 - PROPOSED MOUTHWASH FORMULATIONS Formulation 1 Ingredient % w/w Ethanol 20.0 Flavour 1.0 Sodium saccharin 0.1 Sodium monofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Lauroyl diethanolamide 0.3 Ser(P) 149 K-casein(106-169) 2.0 Water balance WO 99/26971 PCT/AU98/00972 34 Formulation 2 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 Ser(P) 149 K-casein(106-169) 2.0 Water balance EXAMPLE 6 - PROPOSED LOZENGE FORMULATION Ingredient % w/w Sugar 75-80 Corn syrup 1-20 Flavour oil 1-2 NaF 0.01-0.05 Ser(P) 1 4 9 K-casein(106-169) 3.0 Mg stearate 1-5 Water balance EXAMPLE 7 - PROPOSED 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 Chlorohexidine gluconate 0.1 Ser(P) 149 K-casein(106-169) 3.0 Water balance WO 99/26971 PCT/AU98/00972 35 EXAMPLE 8 - PROPOSED 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 Ser(P) 149 K-casein(106-169) 2.0 Water balance It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the 5 invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

WO 99/26971 PCT/AU98/00972 36 REFERENCES Addy M (1988) Rationale for chemotherapy in the treatment of periodontal disease In: Periodontology Today Guggenheim B (ed) pp 281-289, Basel: Karger. Bevins CL, Zasloff M (1990) Peptides from frog skin. Ann Rev Biochem. 59: 5 395-414. Boman HG, Hultmark D (1987) Cell-free immunity in insects. Ann Rev Microbiol 41:103-126 Brown LJ, Oliver RC, Loe H (1989) Periodontal diseases in the US in 1981: Prevalence, severity, extent and role in tooth mortality. JPeriodontol 60: 10 363-370. Casteels P, Ampe C, Jacobs F, Vaeck M, Tempst P (1989) Apidaecins: antibacterial peptides from honeybees. EMBOJ 8: 2387-2391. Christersson LA, Zambon JJ, Dunford RG, Grossi SG, Genco RJ (1989) Specific subgingival bacteria and diagnosis of gingivitis and periodontitis. J 15 Dent Res 68: 1633-1639. Clark DP, Durell S, Maloy WL, Zasloff M (1994) Ranalexin. A novel antimicrobial peptide from bullfrog (Rana catesbeiana) skin, structurally related to the bacterial antibiotic, polymyxin. J Biol Chem 269: 10849-10855. Loesch WJ (1976) The Gingival Index, the plaque index and the retention 20 index systems JPeriodontol 38: 610-616. Marsh PD (1991) Dentifrices containing new agents for the control of plaque and gingivitis: microbiological aspects. J Clin Periodontol 18: 462-467. Migliore-Samour D, Floch F, Jolles P (1989) Biologically active casein peptides implicated in immunomodulation. JDairy Res 56: 357-362. 25 Moore LVH, Moore WEC, Cato EP, Smibert RM, Burmeister JA, Best AM, Ranney RR (1987) Bacteriology of human gingivitis JDent Res 66: 989-995. Mor A, Nicolas P (1994) Isolation and structure of novel defensive peptides from frog skin. EurJBiochem 219: 145-154. Rogers AH, Reynolds EC (1990) The utilisation of casein and amino acids by 30 Streptococcus sanguis P 4

A

7 in continuous culture. J. Gen. Microbiol. 136: 2545-2550. Romeo D, Skerlavaj B, Bolognesi M, Gennaro R (1988) Structure and bactericidal activity of an antibiotic dodecapeptide purified from bovine neutrophils. J Biol Chem 263: 9573-9575.

WO 99/26971 PCT/AU98/00972 37 Simmaco M, Barra D, Chiarini F, Noviello L, Melchiorri P, Kreil G, Richter K (1991) A family of bombinin-related peptides from the skin of Bombina variegata. EurJBiochem 199: 217-222. Spencer AJ, Wright FAC, Brown DF, Hammond RH, Lewis JM (1985) A 5 socio-dental study of adult periodontal health: Melbourne, 1985 Community Dental Health Monograph No 5, Melbourne University Press. Svedberg J, de Haas J, Leimenstoll G, Paul F, Teschemacher H (1985) Demonstration of P-casomorphin immunoreactive materials in in vivo digests of bovine milk and in small intestine contents after bovine milk ingestion in 10 adult humans Peptides 6: 825-830. Zanetti M, Storici P, Tossi A, Scocchi M, Gennaro R (1994) Molecular cloning and chemical synthesis of a novel antibacterial peptide derived from pig myeloid cells. JBiol Chem 269: 7855-7858. Zucht HD, Raida M, Adermann K, Magert HJ, Forssmann WG (1995) 15 Casocidin-I: a casein-alpha s2 derived peptide exhibits antibacterial activity. FEBS Lett. 25, 372(2-3): 185-188.

WO 99/26971 PCT/AU98/00972 38 SEQUENCE LISTING <110> University of Melbourne and Victorian Dairy Industry Authority 5 <120> Antimicrobial peptides <130> 91383 10 <160> 17 <170> PatentIn Ver. 2.0 15 SEQ ID NO: 1 <211> 21 <212> PRT <213> Bovine 20 <400> 1 Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala Y Pro Glu Val Ile Glu 1 5 10 15 Ser Pro Pro Glu 25 20 SEQ ID NO: 2 <211> 21 30 <212> PRT <213> Bovine <400> 2 Ala Val Glu Ser Thr Val Ala Thr Leu Glu Asp E Pro Glu Val Ile Glu 35 1 5 10 15 Ser Pro Pro Glu 20 40 SEQ ID NO: 3 <211> 21 <212> PRT <213> Bovine 45 <400> 3 Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala Ser Pro Glu Val Ile 1 5 10 15 50 Glu Ser Pro Pro Glu 20 SEQ ID NO: 4 55 <211> 21 <212> PRT <213> Bovine <400> 4 60 Ala Val Glu Ser Thr Val Ala Thr Leu Glu Asp Ser Pro Glu Val Ile 1 5 10 15 Glu Ser Pro Pro Glu 20 65 WO 99/26971 PCT/AU98/00972 39 SEQ ID NO: 5 <211> 19 <212> PRT <213> Bovine 5 <400> 5 Asp Met Pro Ile Gln Ala Phe Leu Leu Tyr Gln Gln Pro Val Leu Gly 1 5 10 15 10 Pro Val Arg SEQ ID NO: 6 <211> 64 15 <212> PRT <213> Bovine <400> 6 Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr 20 1 5 10 15 Ile Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Ile Glu 20 25 30 25 Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala Z Pro Glu Val Ile Glu 35 40 45 Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val 50 55 60 30 SEQ ID NO: 7 <211> 63 <212> PRT 35 <213> Bovine <400> 7 Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr 1 5 10 15 40 Ile Asn Thr Ile Ala Gly Glu Pro Thr Ser Thr Pro Thr Ile Glu Ala 20 25 30 Val Glu Ser Thr Val Ala Thr Leu Glu Ala E Pro Glu Val Ile Glu Ser 45 35 40 45 Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val 50 55 60 50 SEQ ID NO: 8 <211> 64 <212> PRT <213> Bovine 55 <400> 8 Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr 1 5 10 15 60 Ile Asn Thr lie Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu 20 25 30 Ala Val Glu Ser Thr Val Ala Thr Leu Glu Asp E Pro Glu Val Ile Glu 35 40 45 65 Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val 50 55 60 WO 99/26971 PCT/AU98/00972 40 SEQ ID NO: 9 <211> 63 5 <212> PRT <213> Bovine <400> 9 Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr 10 1 5 10 15 Ile Asn Thr Ile Ala Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala 20 25 30 15 Val Glu Ser Thr Val Ala Thr Leu Glu Asp Z Pro Glu Val Ile Glu Ser 35 40 45 Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val 50 55 60 20 SEQ ID NO: 10 <211> 53 <212> PRT 25 <213> Bovine <400> 10 Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser 1 5 10 15 30 Thr Pro Thr Ile Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala 20 25 30 E Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr 35 35 40 45 Ser Thr Ala Val 50 40 SEQ ID NO: 11 <211> 52 <212> PRT <213> Bovine 45 <400> 11ii Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala Gly Glu Pro Thr Ser Thr 1 5 10 15 50 Pro Thr Ile Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala Y Pro 20 25 30 Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser 35 40 45 55 Thr Ala Val 50 WO 99/26971 PCT/AU98/00972 41 SEQ ID NO: 12 <211> 53 <212> PRT <213> Bovine 5 <400> 12 Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser 1 5 10 15 10 Thr Pro Thr Thr Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Asp 20 25 30 Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr 35 40 45 15 Ser Thr Ala Val 50 20 SEQ ID NO: 13 <211> 52 <212> PRT <213> Bovine 25 <400> 13 Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala Gly Glu Pro Thr Ser Thr 1 5 10 15 Pro Thr Thr Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Asp E Pro 30 20 25 30 Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser 35 40 45 35 Thr Ala Val 50 SEQ ID NO: 14 40 <211> 64 <212> PRT <213> Bovine <400> 14 45 Met Ala Ile Pro Pro Lys Lys Asn Gin Asp Lys Thr Glu Ile Pro Thr 1 5 10 15 Ile Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Ile Glu 20 25 30 50 Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala Ser Pro Glu Val Ile 35 40 45 Glu Ser Pro Pro Glu Ile Asn Thr Val Gin Val Thr Ser Thr Ala Val 55 50 55 60 WO 99/26971 PCT/AU98/00972 42 SEQ ID NO: 15 <211> 64 <212> PRT <213> Bovine 5 <400> 15 Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr 1 5 10 15 10 Ile Asn Thr lie Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu 20 25 30 Ala Val Glu Ser Thr Val Ala Thr Leu Glu Asp Ser Pro Glu Val Ile 35 40 45 15 Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val 50 55 60 20 SEQ ID NO: 16 <211> 53 <212> PRT <213> Bovine 25 <400> 16 Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser 1 5 10 15 Thr Pro Thr Ile Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala 30 20 25 30 Ser Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val 35 40 45 35 Thr Ser Thr Ala Val 50 SEQ ID NO: 17 40 <211> 53 <212> PRT <213> Bovine <400> 17 45 Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser 1 5 10 15 Thr Pro Thr Thr Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Asp 20 25 30 50 Ser Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val 35 40 45 Thr Ser Thr Ala Val 55 50 In each of the above sequences Y represent phosphoseryl.

Claims (10)

1. An antimicrobial peptide, the peptide being non-glycosylated, less than about 100 amino acids and including an amino acid sequence selected 5 from the group consisting of: AVESTVATLEAZPEVIESPPE, (SEQ. ID. NO. 1) AVESTVATLEDEPEVIESPPE, (SEQ. ID. NO. 2) AVESTVATLEASPEVIESPPE, (SEQ. ID. NO. 3) AVESTVATLEDSPEVIESPPE, (SEQ. ID. NO. 4) 10 DMPIQAFLLYQQPVLGPVR. (SEQ. ID. NO. 5) and conservative substitutions therein.

2. An antimicrobial peptide as claimed in claim 1 in which the peptide includes an amino acid sequence selected from the group consisting of: AVESTVATLEALPEVIESPPE, (SEQ. ID. NO. 1) 15 AVESTVATLEDIPEVIESPPE, (SEQ. ID. NO. 2) AVESTVATLEASPEVIESPPE, (SEQ. ID. NO. 3) AVESTVATLEDSPEVIESPPE, and (SEQ. ID. NO. 4) DMPIQAFLLYQQPVLGPVR. (SEQ. ID. NO. 5)

3. An antimicrobial peptide as claimed in claim 1 or claim 2 in which 20 the peptide is less than about 70 amino acids.

4. An microbial peptide as claimed in any one of claims 1 to 3 in which the peptide includes an amino acid sequence selected from the group consisting of: MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEALPEVIESPPEINT 25 VQVTSTAV; (SEQ. ID. NO. 6) MAIPPKKNQDKTEIPTINTIAZGEPTSTPTIEAVESTVATLEALPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO. 7) MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDZPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO. 8) 30 MAIPPKKNQDKTEIPTINTIAIGEPTSTPTTEAVESTVATLEDEPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO. 9) TEIPTINTIASGEPTSTPTIEAVESTVATLEAZPEVIESPPEINTVQVTSTAV; (SEQ. ID. NO. 10) TEIPTINTIAIGEPTSTPTIEAVESTVATLEALPEVIESPPEINTVQVTSTAV; 35 (SEQ. ID. NO. 11) WO 99/26971 PCT/AU98/00972 44 TEIPTINTIASGEPTSTPTTEAVESTVATLEDEPEVIESPPEINTVQVTSTAV; (SEQ. ID. NO. 12) TEIPTINTIAIGEPTSTPTTEAVESTVATLEDEPEVIESPPEINTVQVTSTAV; (SEQ. ID. NO. 13) 5 MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO. 14) MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO. 15) TEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV; 10 (SEQ. ID. NO. 16) TEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINTVQVTSTAV; (SEQ. ID. NO. 17) and conservative substitutions therein.

5. An microbial peptide as claimed in any one of claims 1 to 4 in which 15 the peptide includes an amino acid sequence selected from the group consisting of: MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEALPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO.

6) MAIPPKKNQDKTEIPTINTIAZGEPTSTPTIEAVESTVATLEAZPEVIESPPEINT 20 VQVTSTAV; (SEQ. ID. NO. 7) MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDIPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO. 8) MAIPPKKNQDKTEIPTINTIAZGEPTSTPTTEAVESTVATLEDIPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO. 9) 25 TEIPTINTIASGEPTSTPTIEAVESTVATLEAEPEVIESPPEINTVQVTSTAV; (SEQ. ID. NO. 10) TEIPTINTIALGEPTSTPTIEAVESTVATLEALPEVIESPPEINTVQVTSTAV; (SEQ. ID. NO. 11) TEIPTINTIASGEPTSTPTTEAVESTVATLEDEPEVIESPPEINTVQVTSTAV; 30 (SEQ. ID. NO. 12) TEIPTINTIAZGEPTSTPTTEAVESTVATLEDEPEVIESPPEINTVQVTSTAV; (SEQ. ID. NO. 13) MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO. 14) 35 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO. 15) WO 99/26971 PCT/AU98/00972 45 TEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV; and (SEQ. ID. NO. 16) TEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINTVQVTSTAV; (SEQ. ID. NO. 17) 5 6. An antimicrobial peptide as claimed in any one of claims 1 to 5 in which the peptide is selected from the group consisting of: AVESTVATLEAZPEVIESPPE, (SEQ. ID. NO. 1) AVESTVATLEDEPEVIESPPE, (SEQ. ID. NO. 2) AVESTVATLEASPEVIESPPE, (SEQ. ID. NO. 3) 10 AVESTVATLEDSPEVIESPPE, (SEQ. ID. NO. 4) DMPIQAFLLYQQPVLGPVR. (SEQ. ID. NO. 5) MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEAEPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO. 6) MAIPPKKNQDKTEIPTINTIAZGEPTSTPTIEAVESTVATLEALPEVIESPPEINT 15 VQVTSTAV; (SEQ. ID. NO. 7) MvAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDIPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO. 8) MAIPPKKNQDKTEIPTINTIAIGEPTSTPTTEAVESTVATLEDIPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO. 9) 20 TEIPTINTIASGEPTSTPTIEAVESTVATLEALPEVIESPPEINTVQVTSTAV; (SEQ. ID. NO. 10) TEIPTINTIAEGEPTSTPTIEAVESTVATLEALPEVIESPPEINTVQVTSTAV; (SEQ. ID. NO. 11) TEIPTINTIASGEPTSTPTTEAVESTVATLEDZPEVIESPPEINTVQVTSTAV; 25 (SEQ. ID. NO. 12) TEIPTINTIAZGEPTSTPTTEAVESTVATLEDEPEVIESPPEINTVQVTSTAV; (SEQ. ID. NO. 13) MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO. 14) 30 MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINT VQVTSTAV; (SEQ. ID. NO. 15) TEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV; and (SEQ. ID. NO. 16) TEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINTVQVTSTAV; 35 (SEQ. ID. NO. 17) WO 99/26971 PCT/AU98/00972 46

7. A chimeric compound, the compound including the peptide as claimed in any one of claims 1 to 6 conjugated to a non-peptide molecule.

8. A chimeric compound as claimed in claim 7 in which the non-peptide portion of the molecule includes acyl groups. 5

9. An antimicrobial composition, the composition including the peptide as claimed in any one of claims 1 to 6 and an acceptable carrier.

10. A method of treating or preventing dental caries or periodontal disease in a subject, the method comprising the step of administering a peptide as claimed in any one of claims 1 to 6 or the composition as claimed 10 in claim 9 to the teeth or gums of the subject in need of such treatments.