NZ533595A

NZ533595A - Methods and products for oral care using metal binding peptides

Info

Publication number: NZ533595A
Application number: NZ533595A
Authority: NZ
Inventors: David Bar-Or
Original assignee: Dmi Biosciences Inc
Priority date: 2001-11-20
Filing date: 2002-11-19
Publication date: 2009-07-31
Also published as: AU2008202759A1; NZ563229A

Abstract

Disclosed is an oral care product comprising one or more dosages of a peptide present in an amount effective for oral care, wherein a single dosage is less than 100mg of the peptide, and wherein the peptide has the formula: P1 - P2, wherein: P1 is: Xaa1Xaa2His: or Xaa1Xaa2HisXaa3; P2 is (Xaa4)n; Xaa1is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, p-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or hydroxymethylserine; Xaa3is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof. The peptides are metal-binding and can reduce inflammation of tissues of the mouth and can reduce the damage done by reactive oxygen species to such tissues.

Description

<div class="application article clearfix" id="description"> *10056947610* WO 03/043518 PCT/US02/37136 1 METHODS AND PRODUCTS FOR ORAL CARE FTRT J) OF TPF. INVENTION The invention relates to methods, oral care products and kits for treating mouth 5 tissues. In particular, the invention relates to methods, oral care products and kits which comprise certain metal-binding peptides, peptide derivatives and peptide dimers that can reduce inflammation of the tissues of the mouth and can reduce the damage done by reactive oxygen species (ROS) to such tissues. 10 BACKGROUND Reactive oxygen species (ROS) include free radicals (e.g., superoxide anion and hydroxyl, peroxyl, and alkoxyl radicals) and non-radical species {e.g., singlet oxygen and hydrogen peroxide). ROS are capable of causing extensive molecular, cellular and tissue damage, and they have been reported to play a major role in a variety of diseases and 15 conditions. Indeed, ROS have been implicated in over 100 diseases and pathogenic conditions, and it has been speculated that ROS may constitute a common pathogenic mechanism involved in all human diseases. Stohs, J. Basic Clin. Physiol. Pharmacol., 6, 205-228 (1995). For reviews describing ROS, their formation, the mechanisms by which they cause molecular, cellular andtissue damage, and their involvement in numerous diseases 20 and disorders, see, e.g., Man®), Rev. Port. Cardiol, 11,997-999 (1992); Florence, Aust. N Z J. Opthalmol., 23, 3-7 (1992); Stohs, J. Basic Clin. Physiol Pharmacol., 6, 205-228 (1995); Knight,/to. Clin. Lab. Sti., 25,111-121 (1995); Kerr et al., Heart & Lung, 25,200-209 (1996); Roth, Acta Chir. Hung., 36,302-305 (1997). Metal ions, primarily transition metal ions, can cause the production and 25 accumulation of ROS. In particular, copper and iron ions released from storage sites are one ofthe main causes oftheproduction of ROS following injury, including ischemia/reperfusion injury and injury due to heat, cold, trauma, excess exercise, toxins, radiation, and infection. Roth,j4eta Chir. Hung., 36,302-305 (1997). Copper and iron ions, as well as other transition metal ions (eg., vanadium, and chromium ions), have been reported to catalyze the 30 production of ROS. See, e.g., Stohs, J. Basic Clin. Physiol. Pharmacol, 6,205-228 (1995); Halliwell et al., Free Radicals In Biology And Medicine, pages 1-19 (Oxford University 1989); Marx et al., Biochem. J., 236,397-400 (1985); Quinlan et al., /. Pharmaceutical Sci., 81,611-614 (1992). Other transition metal ions (e.g, cadmium, mercury, and nickel ions) WO 03/043518 PCT/US02/37136 and other metal ions (eg., arsenic and lead ions) have been reported to deplete some of the molecules of the natural antioxidant defense system, thereby causing an increased accumulation of ROS. See, e.g., Stohs, J. Basic Clin. Physiol. Pharmacol, 6, 205-228 (1995). Although it has been reported that free copper ions bind nonspecifically to the amino 5 groups of essentially any protein (Gutteridge et al., Biochim. Biophys. Acta, 759, 38-41 (1983)), copper ions bound to proteins can still cause the production of ROS which damage at least the protein to which the copper ions are bound. See, e.g., Gutteridge et al., Biochim. Biophys. Acta, 759,38-41 (1983); Marx et al., Biochem. J236,397-400 (1985); Quinlan et al., J. Pharmaceutical Set, 81,611-614 (1992). 10 ROS may be present in the mouth for a variety of reasons. For instance, ROS are present in the mouth as a result of the use of tobacco products, exposure to environmental agents, exposure to radiation, and the use of oral care products comprising tooth whitening agents that liberate active oxygen or hydrogen peroxide. See, e.g,, U.S. Patents Nos. 5,906,811,6,228,347, and 6,270,781. ROS may also be present in the mouth as a result of 15 diseases and conditions that involve inflammation and/or infection, including gingivitis, periodontitis, injuries, surgeries, tooth extractions, cold sores, canker sores and ulcers. See, e.g„ U.S. Patents Nos. 6,228,347 and 6,270,781. Finally, although the normal pH of saliva is 7.2, acidic conditions often are present in the mouth, e.g., as a result of the breakdown of foods, especially carbohydrates. See, e.g., U.S. Patent No. 6,177,097. Acidic conditions 20 promote the release of copper ions from proteins to which they are bound and, as discussed above, free copper ions can cause the production of ROS. The ROS present in the mouth can cause damage to the tissues of the mouth. For instance, in inflammatory periodontal diseases, ROS and elevated levels of free iron and copper ions have been found in periodontal pockets, suggesting a significant role for ROS in periodontal tissue destruction. 25 See, e.g., Waddington et al., Oral Dis6:138-151 (2000). Albumin has been characterized as an extracellular antioxidant See, e.g., Halliwell and Gutteridge, Arch. Biochem. Biophys., 280,1-8 (1990); Das et al., Methods EnzymoL, 233,601-610 (1994); Stohs, J. Basic Clin. Physiol Pharmacol6,205-228 (1995); Dunphy et al., Am. J. Physiol. t276, HI 591 -HI598 (1999)). The antioxidant character of albumin has 30 been attributed to several of albumin's many physiological functions, including albumin's ability to bind metals (particularly copper ions), to bind fatty acids, to bind and transport WO 03/043518 PCT/DS02/37136 steroids, to bind and transport bilirubin, to scavenge HOC1, and others. See, e.g., Halliwell and Gutteridge, Arch. Biochem. Biophys280,1-8 (1990); Halliwell and Gutteridge, Arch. Biochem. Biophys., 246,501-514 (1986); Stohs, J. Basic Clin. Physiol. Pharmacol, 6,205-228 (1995); Dunphy et al., Am. J. Physiol, 276, H1591-H1598 (1999)). Albumin contains 5 several metal binding sites, including one at the N-terminus. Hie N-tenninal metal-binding sites of several albumins, including human, rat and bovine serum albumins, exhibit high-affinity fox Cu(D) and Ni(0), and the amino acids involved in the high-affinity binding of these metal ions have been identified. See Laussac et al., Biochem., 23,2832-2838 (1984); PredM et al.s Biochem. J., 287,211-215 (1992); Masuoka et al., J Biol Chem., 268,21533-10 21537(1993). It has been reported that copper bound to albumin at metal binding sites other than the high-affinity N-tenninal site produce free radicals which causes extensive damage to albumin at sites dictated by the location of the "loose" metal binding sites, resulting in the characterization of albumin as a "sacrificial antioxidant" See Maix et al., Biochem. J., 236, 397-400 (1985); Halliwell etal., Free RadicalsInBiologyAndMedicine,pages 1-19 (Oxford 15 University 1989); Halliwell and Gutteridge, Arch. Biochem. Biophys., 280, 1-8 (1990); Quinlan et al., J. Pharmaceutical Sci., 81,611-614 (1992). Despite the foregoing, attempts to use albumin as a treatment for cerebral ischemia have shown mixed results. It has been reported that albumin is, and is not, neuroprotective in animal models of cerebral ischemia. Compare Huh et al., Brain Res., 804,105-113 (1998) 20 and Retainers et al., Brain Res., 827,237-242 (1999), withLittle et al., Neurosurgery, 9,552-558 (1981) and Beaulieu et al., J. Cereb. Blood Flow. Metab., 18,1022-1031 (1998). Mixedresults have also been obtained using albumin in cardioplegia solutions for the preservation of excised hearts. As reported in Dunphy et al., Am. J. Physiol, 276, H1591-H1598 (1999), the addition of albumin to a standard cardioplegia solution for the 25 preservation of excised hearts did not improve the functioning of hearts perfused with the solution for twenty-four hours. Hearts did demonstrate improved functioning when perfused with a cardioplegia solution containing albumin and several enhancers (insulin, ATP, corticosterone, and pyruvic acid). This was a synergistic effect, since the enhancers alone, as well as the albumin alone, did not significantly improve heart function. An earlier report 30 of improved heart function using cardioplegia solutions containing albumin was also attributed to synergism between enhancers and albumin. Seethe final paragraph of Dunphy WO 03/043518 PCT/US02/37136 4 et al., Am. J. Physiol, 276, H1591-H1598 (1999) and Hisatarai et al., Transplantation, 52, 754-755 (1991), cited therein. In another study, hearts perfused with a cardioplegia solution containing albumin increased reperfusion injury in a dose-related manner, as compared to a solution not containing albumin. Suzer et al., Pharmacol. Res., 37,97-101 (1998). Based 5 on their study and the studies of others, Suzer et al. concluded that albumin had not been shown to be effective for cardioprotection. They further noted that the use of albumin in cardioplegia solutions could be unsafe due to possible allergic reactions and the risks associated with the use of blood products. Finally, although, albumin has been characterized as an antioxidant, it has also been 10 reportedtoenhancesuperoxideanionproductionbymicroglia(Sietal., 21,413-418 (1997)). This result led the authors to speculate that albumin leaking through the disrupted blood brain barrier in certain disorders potentiates the production of superoxide anion by microglia, and that this increased production of superoxide anion is responsible for the pathogenesis of neuronal damage in cerebral ischemia/reperfiision and some 15 neurodegenerative diseases. As noted above, theN-terminal metal-binding sites of several albumins exhibit high-affinity for Cu(JI) andNi(n). These sites have been studied extensively, and a general amino terminal Cu(H)- and Ni(IT)-binding (ATCUN) motif has been identified. See, e.g., Harford and Sarkar, Acc. Chem. Res., 30,123-130 (1997). The ATCUN motif can be defined as 20 being present in a protein or peptide which has a free -NH2 at the N-terminus, a histidine residue in the third position, and two intervening peptide nitrogens. See, e.g., Harford and Sarkar, Acc. Chem. Res., 30,123-130 (1997). Thus, the ATCUN motif is provided by the peptide sequence Xaa Xaa His, where Xaa is any amino acid except proline. See, e.g., Harford and Sarkar, Acc. Chem. Res., 30,123-130 (1997). The Cu(H) andNi(H) are bound 25 by four nitrogens provided by the three amino acids of the ATCUN motif (the nitrogen of the free -NHj, the two peptide nitrogens, and an imidazole nitrogen of histidine) in a slightly distorted square planar configuration. See, e.g., Harford and Sarkar, Acc. Chem. Res., 30, 123-130 (1997). Side-chain groups of the three amino acids of which the ATCUN motif consists can be involved in the binding of the Cu(D) and Ni(H), and amino acids near these 30 three N-terminal amino acids may also have an influence on the binding of these metal ions. See, e.g., Harford and Sarkar, Acc. Chem. Res., 30,123-130 (1997); Bal et al., Chem. Res. WO 03/043518 PCT/US02/37136 5 Toxicol., 10,906-914(1997). For instance, the sequence of the N-tenninal metal-binding site of human serum albumin is Asp Ala His Lys [SEQ ID NO:l], and the free side-chain carboxyl of the N-terminal Asp and the Lys residue have been reported to be involved in the binding of Cu(II) and Ni(D), in addition to the four nitrogens provided by Asp Ala His. See 5 Harford and Sarkar, Acc. Chem. Res., 30,123-130 (1997); Laussac et al., Biochem., 23,2832-2838 (1984); and Sadler et al., Eur. J. Biochem., 220,193-200 (1994). The ATCUN motif has been found in other naturally-occurring proteins besides albumins, and non-naturally-occurring peptides and proteins comprising the ATCUN motif have been synthesized. See, e.g., Harford and Sarkar, Acc. Chem. Res., 30,123-130(1997); 10 Baletal., Chem. Res. Toxicol., 10,906-914(1997); Mlynarz, etal.,Speciation98: Abstracts, http://www.iate.u-szeged.Irn/~spec98/abstr/mlvnar.html. Cu(D) and Ni(E) complexes of ATCUN-containing peptides and proteins have been reported to exhibit superoxide dismutase (SOD) activity. See Cotelle et al., J. Inorg. Biochem., 46,7-15 (1992); Ueda et al., J. Inorg. Biochem., 55, 123-130 (1994). Despite their reported SOD activity, these 15 complexes still produce free radicals which damage DNA, proteins and other biomolecules. See Harford and Sarkar, Acc. Chem. Res., 30, 123-130 (1997); Bal et al., Chem. Res. Toxicol, 10,915-21 (1997); Ueda et al., Free Radical Biol Med., 18,929-933 (1995); Ueda et al., J. Inorg. Biochem., 55,123-130 (1994); Cotelle et al., J. Inorg. Biochem., 46, 7-15 (1992). As a consequence, it has been hypothesized that at least some of the adverse effects 20 of copper and nickel in vivo (e.g., causing cancer and birth defects) are attributable to the binding of CufH) and Ni(D) to ATCUN-containing proteins which causes the production of damaging free radicals. See Harford and Sarkar,/lee. Chem. Res., 30,123-130 (1997); Bal et al., Chem. Res. Toxicol, 10,915-921 (1997); Cotelle et al., J. Inorg. Biochem., 46,7-15 (1992). Cf. Koch et al., Chem. & Biol, 4,549-60 (1997). The damaging effects produced 25 by a Cu(IQ complex of an ATCUN-containing peptide in combination with ascorbate have been exploited to kill cancer cells in vitro and to produce anti-tumor effects in vivo. See Harford and Sarkar, Acc. Chem. Res., 30,123-130 (1997). WO 03/043518 PCT/US02/37136 SUMMARY OP THE INVENTION The invention provides a method of treating a tissue of an animal's mouth. The method comprises contacting the tissue with an effective amount of a metal-binding peptide having the formula P] - P2 or a physiologically-acceptable salt thereof. The invention also 5 provides an oral care product comprising the peptide P, - or a physiologically-acceptable salt thereof, and a kit comprising the oral care product Oral care products include oral care devices and oral care compositions. In the formula P, - P2: P, is Xaaj XaajHis or Xaa, Xaaj His Xa%; and 10 P2 is (Xaa4)n. Xaat is glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (He), serine (Ser), threonine (Thr), aspartic acid (Asp), isoaspartic acid (i.e., Asp attached to Xaaj through its 7-carboxyl, hereinafter "isoAsp"), asparagine (Asn), glutamic acid (Glu), isoglutamic acid (i.e., Glu attached to Xa^ through its y-carboxyl, hereinafter "isoGlu"), 15 glutamine (Gin), lysine (Lys), hydroxylysine (Hylys), histidine (His), arginine (Arg), ornithine (Orn), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Tip), cysteine (Cys), methionine (Met) or a-hydroxymethylserine (HMS). In addition, Xaat can be an amino acid which comprises a 5-amino group (e.g., Orn, Lys) having another amino acid or a peptide attached to it (e.g., Gly (8)-Orn). Xaaj is preferably Asp, Glu, Arg, Thr, or HMS. More 20 preferably, Xaat is Asp or Glu. Most preferably Xaaj is Asp. Xaaj is Gly, Ala, (3-Ala, Val, Leu, He, Ser, Thr, Asp, Asn, Glu, Gin, Lys, Hylys, His, Arg, Orn, Phe, Tyr, Tip, Cys, Met or HMS. Xaaj is preferably Gly, Ala, Val, Leu, He, Thr, Ser, Asn, Met, His or HMS. More preferably Xaaj is Ala, Val, Thr, Ser, Leu, or HMS. Even more preferably Xaa^ is Ala, Thr, Leu, or HMS. Most preferably Xaaj is Ala. 25 Xaa3 is Gly, Ala, Val, Lys, Arg, Qm, Asp, Glu, Asn, Gin, or Trp, preferably Lys. Xaa4 is any amino acid. Finally, n is 0-100, preferably 0-10, more preferably 0-5, Mid most preferably 0. One or more of the amino acids of P, and/or P2 can be substituted with (a) a substituent that increases the lipophilicity of the peptide without altering the ability of P, to 30 bind metal ions, (b) a substituent that protects the peptide from proteolytic enzymes without altering the ability of P, to bind metal ions, or (c) a substituent which is a non-peptide, metal-binding functional group that improves the ability of the peptide to bind metal ions. The invention provides another method of treating a tissue of an animal's mouth. The method comprises contacting the tissue with an effective amount of a metal-binding peptide 5 (MBP) having attached thereto a non-peptide, metal-binding functional group. The metal-binding peptide MBP may be any metal-binding peptide; not just P, - P2. The invention also provides an oral care product comprising the metal-binding peptide MBP having attached thereto a non-peptide, metal-binding functional group and a kit comprising the oral care product. 10 The invention provides yet another method of treating a tissue of an animal's mouth. The method comprises contacting the tissue with an effective amount of a metal-binding peptide dimer of the formula P3 - L - P3, wherein each P3 may be the same or different and is a peptide which is capable of binding a metal ion, and L is a chemical group which connects the two P3 peptides through their C-terminal amino acids. In a preferred 15 embodiment, one or both of the two P3 peptides is P,. The invention also provides an oral care product comprising the metal-binding peptide dimer of the formula P3 - L - P3 and a kit comprising the oral care product. Unless the context clearly requires otherwise, throughout the description and 20 the claims, the words "comprise", "comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of "including, but not limited to". The applicant also refers to its related divisional application. '25 . BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A:D: Formulas of tetrapeptide Asp Ala His Lys [SEQ ID NO: 1] showing points of possible substitution. Figures 2A-B: Schematic diagrams of the synthesis of derivatives of the tetrapeptide 30 ^ ^ Lys [SEQ ID NO:l] coming within the formula of Figure 1C (Figure 2A) and Figure IB (Figure 2B). INTELLECTUAL PROPERTY OFFICE OF NZ. 2 0 DEC 2006 RECEIVED (Followed by page 7a) 7a Figure 3A-B: Formulas of cyclohexane diamine derivatives. Figures 3C-D: Schematic diagrams of syntheses of cyclohexane diamine derivatives of the tetrapeptide Asp Ala His Lys [SEQ ID NO:l]. Figure 4: Formula of a tetraacetic acid derivative of the tetrapeptide Asp AlaHis Lys [SEQIDNOrl]. Figure 5: Formula ofabispyridylethylamine derivative of the tetrapeptide Asp Ala His Lys [SEQ ID NO: 1]. (Followed by page 8) INTELLECTUAL PROPERTY OFFICE OF N± 20 DEC 2006 RECEIVED WO 03/043518 PCT7TJS02/37136 8 Figaro 6A-B: Formulas of mesopoiphyrin IX with (Figure 6B) and without (Figure 6A) a bound metal ion M. Figure 6C: Formula of mesopoiphyrin IX derivative of the tetrapeptide Asp Ala His Lys [SEQ ID NO: 1]. Figure 7: Formulas of monosaccharides. Figure 8A-B: Graphs of absorbance at 532 nm (A532) versus incubation time in mi assay for the production of hydroxyl radicals. In Figure 8 A, ■ - ascorbate only, ♦=copper and ascorbate, A= tetrapeptide (L-Asp L-Ala L-His L-Lys [SEQ ID NO:l]), copper and ascorbate (tetrapeptide/copper ratio of 1:1), X = tetrapeptide, copper and ascorbate (tetrapeptide/copper ratio of 2:1). In Figure 8B, ♦ = copper and ascorbate and ■ = tetrapeptide, copper and ascorbate (tetrapeptide/copper ratio of 2:1). Figure 9: Graph of % inhibition versus concentration tetrapeptide (L-Asp L-Ala L-His L-Lys [SEQ ID NO: l])-copper contplex at a tetrapeptide/copper ratio of 1:1 in the xanthine oxidase assay for superoxide dismutase activity. Figure 10: Graph of absorbance at 560 nm (A560) versus time in an assay for superoxide radical production. In Figure 10, ■=ascorbate only, ♦ = copper and ascorbate, A » tetrapeptide (L-Asp L-Ala L-His L-Lys [SEQ ID NO:l]), copper and ascorbate (tetrapeptide/copper ratio of 1: l),X=tetrapeptide, copper and ascorbate (tetrapeptide/copper ratio of 2:1). Figure 11: Gel after electrophoresis of DNA treated in various ways. Lane 1-17 (ig/ml plasmid DNA (untreated control); Lane 2 -17 fig/ml plasmid DNA and 50 |iM CuCl2; lane 3-17 (ig/ml plasmid DNA and 2.5 mM ascorbate; Lane 4-17 ng/ml plasmid DNA, 2.5 mM ascorbate, 50 pM CuCl^ and 200 >xM tetrapeptide (L-Asp L-Ala L-His L-Lys [SEQ ID NO:l]) (4:1 ratio tetrapeptide/copper); Lane 5-17 (ig/ml plasmid DNA, 2.5 mM ascorbate, 50 jiMCuCl^ and 100 jiM tetrapeptide (2:1 ratio tetrapeptide/copper); lane 6 -17 fig/ml plasmid DNA, 2.5 mM ascorbate, 50 fiM CuCl2, and 50 pM tetrapeptide (1:1 ratio tetrapeptide/copper); Lane 7-17 (ig/ml plasmidDNA, 2.5 mM ascorbate, 50 pM CuCl2, and 25 n-M tetrapeptide (1:2 ratio tetrapeptide/copper); Lane 8-17 jig/ml plasmid DNA, 2.5 mM ascorbate, 50 |u.M CuCl2, and 12.5 jiM tetrapeptide (1:4 ratio tetrapeptide/copper); Lane 9 -17 (ig/ml plasmid DNA, 2.5 mM ascorbate, and 50 fxM CuCl2 (positive control); and Lane 10-DNA ladder. WO 03/043518 PCT/US02/37136 9 Figure 12A: Formulas of peptide dimers according to the invention. Figures 12B-C: Diagrams illustrating the synthesis of peptide dimers according to the invention. Figure 13: TAE (tris acetic acidEDTA (ethylenediamine tetracetic acid)) agarose gel 5 visualized with ethidium bromide showing attenuation of ROS-induced DNA double strand breaks in genomic DNA by D-Asp Ala His Lys. Lane 1 - no treatment; Lane 2 - CuCl^ 50 |iM; Lane 3 - ascorbic acid, 100 (iM; Lane 4 - D-Asp Ala His Lys, 200 (iM; Lane 5 - CuCl^ 10 fiM + ascorbic acid, 50 |iM; Lane 6 - CuCl2,25 fiM + ascorbic acid, 50 }xM; Lane 7 -CuCLj, 50 jliM+ascorbic acid, 50 (iM; Lane 8 - CuCl2,50 (iM+ascorbic acid, 25 jjM; Lane 10 9 - CuCl2,50 jiM+ascorbic acid, 100 (iM; Lane 10 - CuCl2) 50 jiM+ascorbic acid, 100 (iM +D-Asp Ala His Lys, 50 (iM; Lane 11 - CuCl2,50 (iM+ascorbic acid, 100 (iM+D-Asp Ala His Lys, 100 (iM; Lane 12 - CuCl2,50 |xM + ascorbic acid, 100 fiM + D-Asp Ala IBs Lys, 200 jaM. Figure 14: TAE agarose gel visualized with ethidium bromide showing attenuation 15 of ROS-induced DNA double strand breaks in genomic DNA by D-Asp Ala His Lys. Lane 1 - no treatment; Lane 2 - CuCl^ 50 jiM; Lane 3 - ascorbic acid, 500 }iM; Lane 4 - D-Asp Ala His Lys, 200 (iM; Lane 5 - CuClg, 10 jiM + ascorbic acid, 500 pM; Lane 6 - CuCl^ 25 (iM + ascorbic acid, 500 fiM; Lane 7 - CuClj, 50 jiM + ascorbic acid, 500 (iM; Lane 8 -CuClj, 50 jiM + ascorbic acid, 100 jtM; Lane 9 - CuCI2,50 (iM + ascorbic acid, 250 jiM; 20 Lane 10 - CuCl?J 50 (iM + ascorbic add, 500 [iM + D-Asp Ala His Lys, 50 |iM; Lane 11-CuClj, 50 fiM+ascorbic acid, 500 (iM+D-Asp Ala His Lys, 100 [iM; Lane 12 - CuCI2, 50 (iM + ascorbic acid, 500 |iM + D-Asp Ala His Lys, 200 jiM. Figure 15: Southern Blot showing attenuation of ROS-induced DNA double strand breaks in telomere DNA by D-Asp Ala His Lys, Lane 1 - no treatment; Lane 2 - CuCl2s 50 25 (iM; Lane 3 - ascorbic acid, 100 jiM, Lane 4 - D-Asp Ala His Lys, 200 .uM; Lane 5 - CuCl2, 50 jiM + ascorbic acid, 100 (iM; Lane 6 - CuClj, 50 (iM + ascorbic acid, 100 jiM + D-Asp Ala His Lys, 200 (iM. Figure 16: Southern Blot showing attenuation of ROS-induced DNA double strand breaks in telomere DNA by D-Asp Ala His Lys. Lane 1 - no treatment; Lane 2 - CuCl2,50 30 (iM; Lane 3 - ascorbic acid, 500 (iM; Lane 4 - D-Asp Ala His Lys, 200 (iM; Lane 5 - CuCl2, 50 jiM+ascorbic add, 100 jiM; Lane 6 - CuCI2,50 jiM+ascorbic acid, 250 |iM; Lane 7 - WO 03/043518 PCT/US02/37136 10 CuCl2J 50 fiM + ascorbic acid, 500 |iM; Lane 8 - CuCl2,50 fiM+ascorbic acid, 500 fiM + D-Asp Ala His Lys, 50 p.M; Lane 9 - CuCl2J 50 jiM 4- ascorbic acid, 500 fiM + D-Asp Ala His Lys, 100 fiM. Figures 17A-C: Graphs of interleukin-8 (IL-8) concentration versus various 5 treatments of Jurkat cells (all treatments, except nil and copper-only treatments, contained ascorbic acid in addition to the other additives listed on the graphs). Figure 18: Graph showing the effect of copper and D-Asp D-Ala D-His D-Lys (d-DAHK), each alone or in combination, on interleukin 8 (IL-8) secretion from human umbilical vein endothelial cells (HUVEC). CTL = control. Values are mean ± standard 10 error. Figure 19A: Bar graph showing IL-8 concentrations for normal controls and gingivitis and peridontitis patients. Figure 19B: Bar graph showing tumor necrosis factor-a (TNFa) concentrations for normal controls and gingivitis and peridontitis patients. 15 Figure 19C: Bar graph showing soluble tumor necrosis factor-a receptor (sTNFR75) concentrations for normal controls and gingivitis and peridontitis patients. Figure 19D: Bar graph showing IL-8 concentrations before (open bars), and at 0.5 hours (gray bars), 6 hours (black bars), 24 hours (horizontal lines in bars), and 72 hours (cross-hatched bars) after, application of Crest Whitestrips™ to the teeth of five normal 20 controls (Patients D, O, H, K and Y). Figure 19E: Bar graph showing IL-8 concentrations before (open bars), and at 0.5 hours (gray bars) and 6 hours (black bars) after, application to the teeth of three normal controls (Patients R, BM and W) of Crest Whitestrips™ to which the tetrapeptide L-Asp L-Ala L-His L-Lys [SEQ ID NO:l] (also referred to as DAHK-1199) had been applied. 25 DETAILED DESCRIPTION OF THE PRESENTLY-PREFERRED EMBODIMENTS In the formula P, - P2, P, is Xaa, Xaa^ His or is Xaa, Xa&j His Xaa3, wherein Xaa„ Xaaj, and Xaa3 are defined above. P, is a metal-binding peptide sequence that binds 30 transition metal ions of Groups lb-7b or 8 of the Periodic Table of elements (including V, Co, Cr, Mo, Mn, Ba, Zn, Hg, Cd, Au, Ag, Co, Fe, Ni, and Cu) and other metal ions (including As, Sb andPb). The binding ofmetal ions by P, inhibits (i.e., reduces or prevents) WO 03/043518 PCT/US02/37136 11 the production of ROS and/or the accumulation of ROS by these metal ions and/or targets the damage done by ROS that may still be produced by the bound metal ions to the peptide itself. As a result, the damage that can be caused by ROS in the absence of die binding of the metal ions to P, is reduced. Id particular, Pj binds Cu(D), Ni(H), Co(H), and Mn(0) with 5 highaffinity. It should, therefore, be particularly effective in reducing the damage caused by the production and accumulation of ROS by copper and nickel. In addition, the binding of metal ions by P, inhibits inflammation (see Examples 10,12 and 13) and protects against inhibition of metabolism and energy utilization by cells (see Example 11). In Pj, Xaa, is most preferably Asp, Xaaj is most preferably Ala, and Xaa3 is most 10 preferably Lys (see above). Thus, the preferred sequences of P, are Asp Ala His and Asp Ala His Lys [SEQ ID NO: 1 J. Most preferably the sequence of P, is Asp Ala His Lys [SEQ ID NO: 1]. Asp Ala His is the minimum sequence of the N-terminal metal-binding site ofhuman serum albumin necessary for the high-affinity binding of Gu(D) and Ni(H), and Lys has been reported to contribute to the binding of these metal ions to this site. Also, Asp Ala His Lys 15 [SEQ ID NO:l] has been found by mass spectometry to bind Fe(H).. Other preferred sequences for Pt include Thr Leu His (the N-terminal sequence of human a-fetoprotein), Arg Thr His (the N-terminal sequence ofhuman sperm protamin HP2) and HMS HMS His (a synthetic peptide reported to form extremely stable complexes with copper; see Mlynarz et al., Spedation 98: Abstracts, http://www.tate.u-szeged.hu/spec98/abstr/tn1vnar.htm1. 20 4/21/98). P2 is (Xaa^,,, wherein Xaa4 is any amino acid and n is 0-100. When n is large (n > about 20), the peptides will reduce the damage done by ROS extracellularly. Smaller peptides are better able to enter cells, and smaller peptides can, therefore, be used to reduce the damage done by ROS both intracellularly and extracellularly. Smaller peptides are also 25 less subject to proteolysis. Therefore, in P2, preferably n is 0-10, more preferably n is 0-5, and most preferably n is 0. Although P2 may have any sequence, P2 preferably comprises a sequence which (1) binds a transition metal, (2) enhances the ability of the peptide to penetrate cell membranes, or (3) otherwise stabilizes or enhances the performance of the peptide. P2 together with P, may also be the N-terminal sapience of a protein having an N-30 terminal metal-binding site with high affinity for copper and nickel, such as human, rat or WO 03/043518 PCT/US02/37136 12 bovine serum albumin. In the case where n= 100, the peptide would have the sequence of approximately domain. 1 of these albumins. The sequences of many peptides which comprise a binding site for transition metal ions are known. See, e.g., U.S. Patents Nos. 4,022,888,4,461,724,4,665,054,4,760,051, 5 4,767,753,4,810,693,4,877,770, 5,023,237, 5,059,588,5,102,990, 5,118,665, 5,120,831, 5,135,913, 5,145,838, 5,164,367, 5,591,711, 5,177,061, 5,214,032, 5,252,559, 5,348,943, 5,443,816, 5,538,945,5,550,183, 5,591,711,5,690,905,5,759,515,5,861,139,5,891,418, 5,928,955, and 6,017,888, PCT applications WO 94/26295, WO 99/57262 and WO 99/67284, European Patent application 327263, Lappin et al., Inorg. Chem., 17, 1630-34 10 (1978), Bossu et al., Inorg. Chem., 17,1634-40 (1978), Chakrabarti, Protein Eng., 4,57-63 (1990), Adman, Advances In Protein Chemistry, 42,145-97 (1991), Cotelle et al., J". Inorg. Biochem46, 7-15 (1992), Canters et al., FEBS, 325, 39-48 (1993), Regan, Anna. Rev. Biophys. Biomol. Struct., 22,257-281 (1993), Ueda et al., J. Inorg. Biochem., 55, 123-30 (1994), Ueda et al., Free Radical Biol. Med., 18,929-33 (1995), Regan, TIBS, 20,280-85 15 (1995), Ueda et aL, Chem. Pharm. Bull., 43,359-61 (1995), Bal et al., Chem. Res. Toxicol., 10,906-914 (1997), Bal et aL, Chem. Res. Toxicol, 10,915-21 (1997), Koch et al., Chem. Biol, 4,549-60 (1997), Kowalik-Jankowska et al., J. Inorg. Biochem., 66,193-96 (1997), Harford and Sarkar, Acc. Chem. Res., 30,123-130 (1997), Prince et al., TIBS, 23,197-98 (1998), Mlynarz, et al., Spedation 98: Abstracts, http://www.jate.u-20 szeged.hu/~snec98/abstr/m1vnar.htm1- and Aitken, Molec. Biotechnol, 12,241-53 (1999), Whittal et al., Protein Science, 9,332-343 (2000). P2 may comprise the sequence of one or more of the metal-binding sites of these peptides. When P2 comprises a metal-binding site, it preferably has a sequence which includes a short spacer sequence between P, and the metal binding site of P^ so that the metal-binding 25 sites of Pj and P2 may potentially cooperatively bind metal ions (similar to a 2:1 peptide:metal complex; see Example 8). Preferably, the spacer sequence is composed of 1-5, preferably 1-3, neutral amino acids. Thus, the spacer sequence maybe Gly, Gly Gly, Gly Ala Gly, Pro, Gly Pro Gly, etc. In particular, when P2 comprises a metal-binding site, it preferably comprises one of 30 the following sequences: (Xaa4)m Xaaj Xafy His Xaa3 or (Xaa4)m Xa% Xaaj His. Xaaj, Xaaj andXaa4 are defined above, and mis 0-5, preferably 1-3. The Xaa4 amino acid(s), if present, WO 03/043518 PCT/US02/37136 13 form(s) a short spacer sequence between P, and the metal binding site of P2 so that the metal-binding sites of Pj and P2 may cooperatively bind metal ions, andXaa4 is preferably a neutral amino acid (see the previous paragraph). Xaaj is an amino acid which comprises a 8-amino group (preferably Orn or Lys, more preferably Orn) having the Xaa4 amino acid(s), if present, orP, attachedto it bymeansoftheS-amino group. See Harford and Saikar, Acc. Chem.Res., 30,123-130 (1997) and ShuUenberger et al., J. Am. Chem. Soc., 115,11038-11039 (1993) (as a result of this means of attachment, the a-amino group of Xaa$ can still participate in binding metals by means of the ATCUN motif). Thus, for instance, P,-P2 could be Asp Ala His Gly Gly (8)-Qm Ala His [SEQ ID NO:2], in addition, P2 may comprise one of the following sequences: [(Xaa4)m Xaa,- Xaa^, His Xaa3]n [(Xaa4)m Xaa3 Xaa, His]„ [(Xaa4)ra Xaaj Xaa, His (Xaa4)m Xm, Xaa, His],, and [(XaaJ,,, Xa% Xaaj His(Xaa4)m Xaa$ Xa% His XaaJ,, wherein Xaaj, Xaa^ Xaa4, Xaa^ and m are defined and described above, and r is 2-100. In this manner metal-binding polymers may be formed. In another preferred embodiment, P2 comprises a peptide sequence that can bind Cu(I). As discussed in more detail below, Cu(D) is converted to CuQ) in the presence of ascorbic acid or other reducing agents, and the Cu(I) reacts with oxygen to produce ROS (see equations in Examples 8 and 9). Pj can bind Cu(D) tightly and is very effective by itself in inhibiting the production of ROS by copper (see Examples). However, as can be seen from the equations in Examples 8 and 9, it would be desirable to also employ a P2 which could bind Cu(T>. Peptide sequences which can bind Cu(I) are known in the art. See, e.g, Pickering et al., J. Am. Chem. Soc., 115,9498-9505 (1993); Winge et aL, in Bioinorganic Chemistry Of Copper, pages 110-123 (Karlin and Tyeklar, eds., Chapman & Hall, New York, NY, 1993); Koch et al., Chem & Biol., 4,549-560 (1997); Cobine et al., in Copper Transport And Its Disorders, pages 153-164 (Leone and Mercer eds., Kluwer Academic/Plenum Publishers, New York, NY, 1999). These sequences include: Met Xaa4 Met, Met Xaa4 Xaa4 Met, Cys Cys, CysXaa^Cys, WO 03/043518 PCT/US02/37136 14 Cys Xaa4 Xaa4 Cys, Met Xaa4 Cys Xaa4 Xaa4 Cys, Gly Met Xaa4 Cys Xaa4 Xaa4 Cys [SEQ ID N0:7], Gly Met Thr Cys Xaa4 Xaa4 Cys [SEQ ID NO: 8], and 5 Gly Met Thr Cys Ala Asn Cys [SEQ ID N0:9], wherein Xaa4 is defined above. Glutathione (y-Glu Cys Gly) is also known to bind Cu(I). Additional Cu(T)-binding peptide sequences can be identified using a metallopeptide combinatorial library as described in, e.g., PCT application WO 00/36136. Preferably, the Cu(T)-binding peptide comprises the sequence Cys Xaa4 Xaa4 Cys (e.g., Gly Met Xaa4 Cys 10 Xaa4 Xaa4 Cys [SEQ ID NO:7], more preferably Gly Met Thr Cys Xaa4 Xaa* Cys [SEQ ID NO :8], most preferably Gly Met Thr Cys Ala Asn Cys [SEQ ID NO:9]). To enhance the ability of the P, - P2 peptide to penetrate cell membranes, P2 is preferably hydrophobic or an arginme oligomer (see Rouhi, Chem. & Eng. News, 49-50 (January 15,2001)). When P2 is hydrophobic, it preferably contains 1-3 hydrophobic amino 15 acids (e.g., Gly Gly), preferably D-amino acids. The arginine oligomer preferably contains 6-9 Arg residues, most preferably 6-9 D-Arg residues (see Rouhi, Chem. &Eng. News,49-50 (January 15, 2001). The use of a P2 which is an arginine oligomer may be particularly desirable when Pt - P2 is to be administered topically. The amino acids of the peptide may be L-amino acids, D-amino acids, or a 20 combination thereof. Preferably, at least one of the amino acids of P, is a D-amino acid (preferably Xaa] and/or His), except for (3-AIa, when present Most preferably, all of the amino acids of P„ other than (J-Ala, when present, are D-amino acids. Also, preferably about 50% of the amino acids of P2 are D-amino acids, and most preferably all of the amino acids of P2 are D-amino acids. D-amino acids are preferred because peptides containing D-amino 25 acids are resistant to proteolytic enzymes, such as those that would be encountered in the mouths of animals (including humans). Also, the use of D-amino acids would not alter the ability of the peptide to bind metal ions, including the ability of the peptide to bind copper with high affinity. The peptides of the invention may be made by methods well known in the art For 30 instance, the peptides, whether containing L-amino acids, D-amino acids, or a combination of L- and D-amino acids, may be synthesized by standard solid-phase peptide synthesis WO 03/043518 PCT/US02/37136 15 methods. Suitable techniques are well known in the art, and include those described in Merrifield, in Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds. 1973); Merrifield, J- Am. Chem. Soc.. 85.2149 (1963); Davis et al., Biochem. Ihtl 10. 394-414 (1985); Stewart and Young, Solid Phase Peptide Synthesis (1969); U.S. Patents Nos. 3, 5 941,763 and 5,786,335; Finn et al., in The Proteins. 3rd ed., vol. 2, pp. 105-253 (1976); and Erickson et al. in The. Proteins. 3rd ed., vol. 2, pp. 257-527 (1976). See also, Polish Patent 315474(synthesisofHMS-contoiningpeptides)andShullenbergeretal., J.Am. Chem.Soc., 115, 1103811039 (1993) (synthesis of (8)-Orn-containmg peptides). Alternatively, the peptides may be synthesized by recombinant DNA techniques if they contain only L-amino 10 acids. Recombinant DNA methods and- suitable host cells, vectors and other reagents for use therein, are well known in the art. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1982), Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989). The invention further comprises derivatives of the peptide Pj - whether composed 15 of L~amino acids, D-amino acids, or a combination of L- and D-amino acids, which are more resistant to proteolytic enzymes, more lipid soluble (to allow the peptides to more readily penetrate cell membranes), or both. As illustrated in Figure 1 A, P, can be modified in the regions indicated by the arrows without altering the metal binding function of P,. In particular, Pt can be substituted at carbons 1 or 2 with R„ and the terminal -COOH of Pj can 20 be substituted with protecting group Rj (Figures 1B-D). P2 can be modified in ways similar to those described for P, to make P2 more resistant to proteolytic enzymes, more lipid soluble, or both. Ri can be a straight-chain or branched-chain alkyl containing from 1 to 16 carbon atoms, and the term "alkyl" includes the R and S isomers. R, can also be an aryl or heteroaryl 25 containing 1 or 2 rings. The term "aryl" means a compound containing at least one aromatic ring (e.g., phenyl, naphthyl, and diphenyl). The term "heteroaryl" means an aryl wherein at least one of the rings contains one or more atoms of S, N or O. These substitutions do not substantially decrease the ability of P, to bind metal ions. In particular, the ability of Pj to bind copper with high affinity is not decreased by these substitutions. For instance, some of 30 the substituents, such as a n-butyl attached to carbon 2 (see Figure 1C, Rt is n-butyl) should increase the affinity of the peptide for metal ions, such as copper, due to the inductive effect WO 03/043518 PCT/US02/37136 16 of the alkyl group. Substitution of carbon 2 (Figure 1C) with an aryl, heteroaryl, or a long chain alkyl (about 6-16 carbon atoms) should enhance transport of the peptide across lipid membranes. As noted above, methods of synthesizing peptides by solid phase synthesis are well 5 known. These methods can be modified to prepare the derivatives shown in Figures 1B-C. For example, the derivative of Pt illustrated in Figure 1C, wherein R, is octyl, can be prepared as illustrated in Figure 2A. In Figure 2A, the elliptical element represents the polymer resin and Rp is a standard carboxyl protecting group. As illustrated in Figure 2A, octanoic acid (freshly distilled) is treated with dry bromine followed by phosphorus 10 trichloride. He mixture is heated to about 100°C and kept at that temperature for 4 hours. a-Bromooctanoic acid is obtained as a colorless liquid upon distillation. Amination of the bromoacid is achieved by allowing the acid and an ammonia solution to stand at40-50° C for 30 hours. The octyl derivative of the amino acid is obtained by removing ammonium bromide with methanol washes. Classical resolution methods give the desired optically-pure 15 D-form. Other derivatives wherein R, is an alkyl, aryl or heteroaryl can be prepared in the maimer illustrated in Figure 2A In addition, the derivative of Pi illustrated in Figure IB, wherein Rj is phenyl, can be prepared as illustrated in Figure 2B. In Figure 2B, Polymer is the resin, t-Bu is t-butyl, and Bz is benzyl, Other derivatives wherein Rj is an alkyl, aryl or heteroaryl can be prepared in 20 the manner illustrated in Figure 2B. Rg can be -NH & -NHRl5 -NfR,^, -OR„ or R, (see Figure ID), wherein R, is defined above. These derivatives can be prepared as the last step of a solid-phase peptide synthesis before the peptide is removed from the resin by methods well known in the art. Substitutions with Rj do not substantially decrease the ability of P, to bind metal ions. 25 In addition, P, and P2 can be substituted with non-peptide functional groups that bind metal ions. These metal-binding functional groups can be attached to one or more pendent groups of the peptide, and the resulting peptide derivatives will possess one or more sites that are capable of binding metal ions, in addition to the binding site provided by P, and, optionally, the binding site provided by P2. As a consequence, the ability of such peptide 30 derivatives to bind metal ions is improved as compared to the corresponding unmodified peptide. For instance, the peptide derivative can bind two of the same type of metal ion WO 03/043518 PCT/US02/37136 17 instead of one (e.g., two Cu(n)), the peptide derivative can bind two different metal ions instead of one type of metal ion (e.g., one Cu(IT) and one Fe(ffl)), or the peptide derivative can bind one metal ion better (e.g., with greater affinity) than the corresponding unmodified peptide. S Metal-binding functional groups include polyamines (e.g., diamines, triamines, etc.). Suitable diamines include 1,2-alkyldiamines, preferably alkyl diamines wherein the alkyl contains 2-10 carbon atoms (e.g., H2N - (CHj),, - NH2s wherein n=2-10). Suitable diamines also include 1,2-aryldiamines, preferably benzene diamines (e.g., 1,2-diaminobenzene). Suitable diamines further include 1,2-cyclic alkane diamines. "Cyclic alkanes" are 10 compounds containing 1-3 rings, each containing 5-7 carbon atoms. Preferably the cyclic alkane diamine is 1,2-diaminocylcohexane (cyclohexane diamine). A particularly preferred diamine is 1,2-diaminocyclohexane (Figures 3A-B). Previous studies carried out by Rao & P. Williams (J. Chromatography A, 693,633 (1995)) have shown that a cyclohexane diamine derivative (Figure 3A, where PYR is pyridine) binds 15 to a variety of metal ions. The resulting metal chelator has been successfully used to resolve amino acids and peptides, showing that the molecule has a very high affinity for a-amino acids, forming a very stable coordination complex, which is unique in many respects. 1,2-Diaminocyclohexane possesses a reactive amino functional group to which a peptide of the invention can be attached. See Figure 3B, where M is a metal ion and at least one R4 is 20 -alkyl-CO-peptide, -aiyl-CO-peptide, -aryl-alkyl-CO-peptide, or-alkyl-aryl-CO-peptide (see also Figures 3C-D). The other R4 may be the same or may be -alkyl-COOH, -aryl-COOH, -aryl-alkyl-COOH, or alkyl-aryl-COOH. Derivatives of the type shown in Figure 3B will have several metal-binding sites and can, therefore, be expected to bind metal ions more readily than Hie unsubstituted peptide. Further, due to the presence of the cyclohexane 25 functionality, the compound will possess lipid-like characteristic which will aid its transport across lipid membranes. Cyclohexane diamine derivatives of the peptides of the invention can be prepared by two distinct routes. The first involves initial condensation with an aldehyde followed by reduction (see Figure 3C; in Figure 3C Bz is benzyl). A number of aldehydes (alkyl and aryl) 30 react readily with cyclohexane diamine at room temperature, forming an oxime. The oxime can be reduced with sodium borohydride under anaerobic conditions to give the diacid WO 03/043518 PCT/US02/37136 18 derivative. The caiboxyl moieties are then reacted with die free amino groups present in carboxy-piotectedP] to give the cyclohexane diamine derivative ofthepeptide. The second route is a direct alkylation process which is illustrated in Figure 3D. For example, cyclohexane diamine is treated with bromoacetic acid to give the diacetic acid derivative. 5 The carboxyl moieties are then reacted with the free amino groups present in carboxy-protected P, to give the derivative. In Figure 3D, R5 is H or another peptide. When RjisH, the derivative can be further reacted to produce typical caiboxylic acid derivatives, such as esters, by methods well known in the art. Metal binding experiments have indicated that the presence or absence of this group does not have a bearing on the metal binding capacity of 10 the whole molecule. However, these groups would either make the molecule hydrophobic or hydrophilic, depending upon the substituent, and this may, in turn, have an effect on delivery of the molecule across membranes. These two synthetic routes will work for the synthesis of diamine peptide derivatives using the other diamines described above. Additional suitable polyamines and polyamine derivatives and methods of attaching IS them to peptides are described in U.S. Patents Nos. 5,101,041 and 5,650,134, the complete disclosures of which are incorporated herein by reference. Other polyamine chelators suitable for attachment to peptides are known. See, e.g., U.S. Patents Nos. 5,422,096, 5,527,522, 5,628,982,5,874,573, and5,906,996 andPCT applications WO 97/44313, WO 97/49409, and WO 99/39706. 20 It is well known that vicinal diacids bind to metal ions, and the affinity for copper is particularly high. It is therefore envisaged that a peptide having a vicinal diacid functional group will be extremely effective in metal binding. Suitable vicinal diacids include any 1,2-alkyldiacid, such as diacetic acid (succinic acid), and any 1,2-aiyldiacid, The amino groups ofthe peptide can be reacted with diacetic acid to produce a diacid 25 derivative (see Figure 4). This can be conveniently accomplished by reacting the amino groups of the resin-bound peptide with a halogenated acetic acid (e.g., bromoacetic acid or chloroacetic acid) or a halogenated acetic acid derivative (e.gbenzyloxy ester). Solid phase synthetic procedures enable removal of unreacted materials by washing with solvent The final product is released from the resin by hydrolytic cleavage. Other diacid derivatives of 30 the peptides of the invention can be made in the same manner. WO 03/043518 PCT/US02/37136 19 Polyaminopolycarboxylic acids are known to bind metals, such as copper and iron. Suitable polyaminopolycarboxylic acids for making derivatives of the peptides of the invention and methods of attaching them to peptides are described in U.S. Patents Nos. 5,807,535 and. 5,650,134, and PCT application WO 93/23425, the complete disclosures of 5 which are incorporated herein by reference. See also, U.S. Patent No. 5,739,395. Vicinal polyhydroxyl derivatives are also included in the invention. Suitable vicinal polyhydroxyls include monosaccharides and polysaccharides disaccharide, trisaccharide, etc.)- Presentlypreferred are monosaccharides. See Figure 1. The monosaccharides fall into two major categories - furanoses and pyranoses. One of the prime examples of a fiiranose 10 ring system is glucose. The hydroxy! groups of ghicose can be protected as benzyl or labile t-bulyloxy functional groups, while leaving the aldehyde free to react with an amine group (e.g., that of lysine) of the tetrapeptide. Mild reduction/hydrolysis produces the monosaccharide peptide derivative. Other monosaccharide peptide derivatives can be prepared in this manner. 15 Bispyridylethylamine derivatives are known to form strong complexes with divalent metal ions. When attached to the peptide, this functional group would provide additional chelating sites for metal ions, including copper. The bispyridylethyl derivative of the tetrapeptide Asp Ala His Lys [SEQ ID NO: 1] is shown in Figure 5. It is anticipated that the metal-binding capacity of this tetrapeptide derivative will be increased by at least three-fold 20 as compared to the underivatized peptide. The preparation of this bispyridylethylamine derivative shares some similarities with the synthesis of diacid derivatives. The two amino groups of the tetrapeptide (one at Asp and the other at Lys) are reacted with 2-bromoethylpyridine to give the testa-substituted peptide derivative. The reaction is accomplished by reacting theresin-bound tetrapeptide with the bromoethylpyridine, followed 25 by cleavage of the product from the resin. Phenanthroline is another heterocyclic compound capable of binding divalent metal ions. Phenanthroline derivatives of the peptides can be synthesized in the same manner as for the bispyridylethylamine derivatives. Porphyrins are a group of compounds found in all living matter and contain a 30 tetrapyrrolic macrocycle capable of binding to metals. Heme, chlorophyll and corrins are prime examples of this class of compounds containing iron, magnesium and cobalt, WO 03/043518 PCT/US02/37136 20 respectively. Mesoporphyrin IX (Figure 6A-B, where M is a metal ion) is derived from heme and has been observed to possess specific affinity for copper. Addition of this structure to a peptide of the invention would produce a porphyrin-peptide derivative possessing several sites for binding of copper (see Figure 6C). In addition to their roles in metal binding, the 5 imidazole residues at positions 3 and 3' of the tetrapeptide shown in Figure 6C may provide a binding site for metals other than copper, thereby stabilizing the porphyrin-metal complex. In particular, cyanocobalamine (vitamin B-12) contains cobalt as the metal in the porphyrin nucleus, and the contplex is stabilized by the imidazole groups. On the basis of this analogy it is anticipated that the porphyrin-tetrapeptide derivative would bind cobalt (or other metals) 10 at normal physiological conditions in the prophyrin nucleus and that the complex would be stabilized by the His imidazole groups. To prepare the porphyrin-peptide derivative shown in Figure 6C, the carboxyl groups of mesoporphyrin IX can be activated and coupled with the amino groups of the peptide employing standard solid-phase peptide synthesis. Typically, the free amino group of the 15 lysine residue of the resin-bound peptide can be coupled with carboxy activated porphyrin nucleus. The condensation product can be cleaved off the resin using standard methods. This method can be used to synthesize other porphyrin derivatives of peptides of the invention. Other suitable porphyrins and macro cyclic chelators and methods of attaching them 20 to peptides are described in U.S. Patents Nos. 5,994,339 and 5,087,696, the complete disclosures ofwhich are incorporated herein by reference. Other porphyrins ahdmacrocyclic chelators that could be attached to peptides are known. See, e.g., U. S. Patents Nos. 5,422,096,5,527,522, 5,628,982, 5,637,311, 5,874,573, and 6,004,953, PCT applications WO 97/44313 and WO 99/39706. 25 A variety of additional metal chelators and methods of attaching them to proteins are described in U.S. Patent No. 5,683,907, the complete disclosure of which is incorporated herein by reference. Dithiocarbamates are known to bind metals, including iron. Suitable dithiocarbamates formaking derivatives ofthepeptides ofthe invention are described in U.S. 30 Patents Nos. 5,380,747 and 5,922,761, the complete disclosures of which are incorporated herein by reference. WO 03/043518 PCT/US02/37136 21 Hydroxypyridones are also known to be iron chelators. Suitable hydroxypyridones for making derivatives of the peptides of the invention are described in U.S. Patents Nos. 4,912,118 and 5,104,865 and PCT application WO 98/54138, the complete disclosures of which are incorporated herein by reference. 5 Additional non-peptide metal chelators are known in the art or will be developed. Methods of attaching chemical compounds to proteins and peptides are well known in the art, and attaching non-peptide metal chelators to the peptides of the invention is within the skill in the art. See, e.g., those patents cited above describing such attachment methods. As can be appreciated, the non-peptide, metal-binding functional groups could be 10 attached to another metal-binding peptide (MBP) in the same manner as they are to peptide Pj - P2. The resulting peptide derivatives would contain one or more metal-binding functional groups in addition to the metal-binding site of MBP. Preferably, MBP contains from 2-10, more preferably 3-5, amino acids. Preferably MBP contains one or more D-amino acids; most preferably all ofthe amino acids of MBP are D-amino acids. As described above, the 15 sequences of many metal-binding peptides are known. These peptides and peptides comprising the metal-binding sites of these peptides can be prepared in the same ways as described above for peptide Pj-Pj. Derivatives of these peptides having one or more metal-binding functional group attached to the peptide can be prepared in the same ways as described above for derivatives of pqptide Pt - P2. 20 The invention also provides metal-binding peptide dimers of the formula; Pj - L - P3. P3 is any peptide capable of binding a metal ion, and each P3 may be the same or different Each P3 preferably contains 2-10, more preferably 3-5, amino acids. As described above, metal-binding peptides are known, and each P3 may comprise the sequence of one or 25 more of the metal-binding sites of these peptides. Although each P3 may be substituted as described above for P, andPj, including with a non-peptide, metal-bindingfenctional group, both P3 peptides are preferably unsubstituted. P3 may also comprise any amino acid sequence substituted with a non-peptide, metal-binding functional group as described above to provide the metal-binding capability of P3. Preferably, each P3 is an unsubstituted metal-binding 30 peptide (i.e., an unsubstituted peptide comprising a peptide sequence which binds metal WO 03/043518 PCT/US02/37136 22 ions). Most preferably, one or both of the P3 groups is P, (i.e., the dimers have the sequence P3 - L - Pj, Pj - L - P3 or, most preferably, Pj - L - Pj). Pj is defined above. L is a linker which is attached to the C-terminal amino acid of each P3. L may be any physiologically-acceptable chemical group which tan connect the two P3 peptides through 5 their C-terminal amino acids. By "physiologically-acceptable" is meant that a peptide dimer containing the linker L is not toxic to an animal (including a human) or an organ to which the peptide dimer is administered as a result of the inclusion of the linker L in the peptide dimer. Preferably, L links the two P3 groups so that they can cooperatively bind metal ions (similar to a 2:1 peptide:metal complex; see Example 8). L is also preferably neutral. Most 10 preferably, L is a straight-chain or branched-chain alkane or alkene residue containing from 1-18, preferably from 2-8, carbon atoms (e.g., -CH2-, -CH2CH2- -CH2CH2CH2-, -CH2CH2(CH3)CH2-, -CHCH-, etc.) or a cyclic alkane or alkene residue containing from 3-8, preferably from 5-6, carbon atoms (see Figure 12A, compound D,)s preferably attached to a P3 by means of an amide linkage. Such linkers are particularly preferred because they impart 15 hydrophobicity to the peptide dimers. In another preferred embodiment, L is a nitrogen-containing heterocyclic alkane residue (see Figure 12A, compounds D2, D3 and D4); preferably a piperazide (see Figure 12A, compound Dj). hi another preferred embodiment L is a glyceryl ester (see Figure 12A, compound D5; in formula DSs R is an alkyl or aryl containing, preferably containing 1-6 carbon atoms). Finally, L could be a metal-binding porphyrin (see 20 Figure 6C). These preferred linkers L will allow the two peptides P3 to bind metal ions cooperatively and are biocompatible, and the peptide dimers containing these preferred linkers can be made easily and in large quantities. By "biocompatible" is meant that a peptide dimer containing the linker L does not produce any undesirable side-effects due to the linker L in an animal (including a human) to which the peptide dimer is administered. 25 Methods of synthesizing the peptide dimers are illustrated in Figures 12B-D. In general, the C-terminal amino acids (protected by methods and protecting groups well known in Hie art) of the two P3 groups are attached to L, and the resulting amino acid dimers used in standard peptide synthetic methods to make the peptide dimers. For instance, a peptide dimer, where each peptide has the sequence Asp Ala His Lys, 30 [SEQ ID NO: 1] can be synthesized by coupling protected lysines to a free diamine functional group, either as an acid chloride or by using standard coupling agents used in peptide synthesis WO 03/043518 PCT/US02/37136 23 (see Figures 12B-C). Many suitable diamines are available commercially or suitable diamines can be readily synthesized by methods known in the art For instance, the lysine dimer 2 (Figure 12B) can be prepared as follows. To a stirred solution of9-fluorenylmethyloxycarbonyl (Fmoc)- and t-benzyloxycarbonyl(Boc)- protected 5 D-Lys (Fmoc-D-Lys(Boc)-OH) (20 mmole) in dry dimethylfonnamide (DMF; 100 mL; dry argon flushed) are added butane-1,4-diamine 1 and 2-(lH-benzotriazole-l-yl> 1,2,3,3-tetraxnethyluronmmtetrafluoroborate (TBTU; 0.5 mmole). The solution is stirred for 36 hours at room temperature. The bis-protected lysine 2 is isolated by flash chromatography over silica and elution with mixtures of ethyl acetate/methanol. The peptide dimer 3 is then 10 prepared from the protected lysine dimer 2 employing classical peptide synthesis methodology (see Figure 12B). Another peptide dimer, where each peptide has the sequence Asp Ala His Lys [SEQ ID NO:l], can be synthesized as follows. First, a different protected lysine dimer 4 is synthesized by acylating the two amino centers of a piperazine 5 (see Figure 12C; see also 15 Chambrier et al., Proc. Natl. Acad. Set, 96,10824-10829 (1999)). Then, the remainder ofthe amino acid residues are added employing standard peptide synthesis methodology to give the peptide dimer 6 (see Figure 12C). Peptide dimers, where eachpeptide has the sequence Asp Ala His Lys [SEQ ID NO: 1] and where L is a glyceryl ester, can be synthesized as follows. The 3-substituted propane-1,2-20 diols of formula 7 in Figure 12D, wherein R is an alkyl or aryl, ebb commercially available. A lysine diester 8, wherein R is methyl, can be prepared as follows (see Figure 12D). To a stirred solution of Fmoc-D-Lys(Boc)-OH (20 mmole) in dry toluene (100 mL; dry argon flushed) is added 3-methoxypropane-l ,2-diol (200 mmole) and imidazole (15 mmole). The solution is stirred for 36 hours at room temperature. The solvent is removed in vacuo, and the 25 residue is dissolved in ethyl acetate. This solution is washed with citric acid solution (2%), water, 0.5 N NaHC03 solution, and again with water; then the organic layer is dried over magnesium sulphate (removal of the solvent gives a pale yellow residue). The bis-protected lysine 8 is isolated by flash chromatography ova: silica and elution with mixtures of ethyl acetate/methanol. The peptide dimer 9 is then prepared from the protected lysine dimer 8 30 employing classical peptide synthesis methodology (see Figure 12D). WO 03/043518 PCT/US02/37136 24 The physiologically-acceptable salts ofthe metal-binding compounds are also included in the invention. Physiologically-acceptable salts include conventional non-toxic salts, such as salts derived from inorganic acids (such as hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, and the like), organic acids (such as acetic, propionic, succinic, glycolic, stearic, lactic, 5 malic, tartaric, citric, glutamic, benzoic, salicylic, and the like) or bases (such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation). The salts are prepared in a conventional manner, e.g., by neutralizing the free base form of the compound with an acid. The invention also provides oral care products comprising a metal-binding compound 10 or compounds of the invention. Oral care products include oral care compositions and oral care devices. Oral care compositions of the invention include washes, rinses, gargles, solutions, drops, emulsions, suspensions, liquids, pastes, gels, ointments, creams, sprays, powders, tablets, gums, lozenges, mints, films, patches, and tooth whitening compositions. Oral care 15 compositions of the invention include compositions intended for use by consumers and patients and compositions intended for use by dental professionals (e.g.r dental hygienists, dentists and oral surgeons). The oral care compositions of the invention will comprise a metal-binding compound or compounds of the invention as active ingredient(s) in admixture with one or more 20 pharmaceutically-acceptable carriers. Oral care compositions of the invention will generally comprise from about 0.001% to about 20% by weight of a metal-binding compound or a combination of metal-binding compounds ofthe invention. The oral care compositions ofthe invention may also comprise one or more other acceptable ingredients, including other active compounds and/or other ingredients conventionally used in oral care compositions. Each 25 carrier and ingredient must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the animal. Suitable ingredients, including pharmaceutically-acceptable earners, for use in oral care compositions, and methods of making and using oral care compositions, are well known in the art See, e.g„ U.S. PatentsNos. 4,847,283,5,032,384,5,043,183,5,180,578,5,198,220, 30 5,242,910, 5,286,479, 5,298,237, 5,.328,682, 5,407,664, 5,466,437, 5,707,610, 5,709,873, 5,738,840, 5,817,295, 5,858,408, 5,876,701, 5,906,811, 5,932,193, 5,932,191, 5,951,966, WO 03/043518 PCT/US02/37136 25 5,976,507,6,045,780,6,197,331,6,228,347,6,251,372, and6,350,438,PCT applications WO 95/32707, WO 96/08232 and WO 02/13775, and EP applications 471,396, the complete disclosure of all of which are incoiporatedhereia by reference. Conventional ingredients used in oral care compositions include water, alcohols, humectants, surfactants, thickening agents, 5 abrasives, flavoring agents, sweetening agents, antimicrobial agents, anti-caries agents, anti-plaque agents, anti-calculus agents, pH-adjusting agents, and many others. The water used in oral care compositions should preferably be of low ion content It should also be free of organic impurities. The alcohol must be nontoxic. Preferably the alcohol is ethanol. Ethanol is a solvent 10 and also acts as an antibacterial agent and as an astringent Humectants suitable for use in oral care compositions include edible polyhydric alcohols such as glycerol, sorbitol, xylitol, butylene glycol, polyethylene glycol, propylene glycol, mannitol, raid lactitol. Humectants help keep oral care compositions, such as pastes, from hardening upon exposure to air, give oral care compositions a moist feel to the mouth, 15 and may impart desirable sweetness. Surfactants include anionic, nonionic, amphoteric, zwitterionic and cationic synthetic detergents. Anionic surfactants include the water-soluble salts of alkyl sulfates having 8-20 carbon atoms in the alkyl radical (such as sodium alkyl sulfate), the water-soluble salts of sulfonated monoglycerides of fatty acids having from 8-20 carbon atoms (such as sodium 20 lauiyl sulfate and sodium coconut monoglyceride sulfonates), sarcosinates (such as sodium and potassium salts of lauroyl sarcosinate, myristoyl sarcosinate, palmitoyl sarcosinate, stearoyl sarcosinate andoleoyl sarcosinate), taurates, higher alkyl sulfoacettes (such as sodium lauryl sulfoacetate), isethionates (such as sodium lauroyl isethionate), sodium laureth carboxyiate, sodium dodecyl benezesulfonate, and mixtures of the foregoing. Preferred are 25 the sarcosinates since they inhibit acid formation in the mouth due to carbohydrate breakdown. Nonionic surfactants include poloxamers (sold under the tradename Plutonic), polyoxyethylene sorbitan esters (sold under the tradename Tween), fatty alcohol ethoxylates, polyethylene oxide condensates of alkyl phenols, products derived from the condensation of ethylene oxide with fatty acids, fatty alcohols, fatty amides, polyhydric alcohols, and 30 polypropyleneoxide, ethylene oxide condensates of aliphatic alcohols, long-chain tertiary amine oxides, long-chain tertiaryphospine oxides, long-chain dialkyl sulfoxides, and mixtures WO 03/043518 PCT/US02/37136 26 of such materials. Amphoteric surfactants include betaines (such as cocamidopropylbetaine), derivatives of aliphatic secondary and tertiary amines in which the aliphatic radical can be a straight or branched chain and wherein one of the aliphatic substituents contains about 8-18 carbon atoms and one contains an anionic water-solubilizing group (such as carboxylate, 5 . sulfonate, sulfate, phosphate or phosphonate), and mixtures of such materials. Zwitterionic surfactants include derivatives of aliphatic quaternary ammonium; phosphonium and sulfonium compounds in which the aliphatic radical can be a straight or branched chain and wherein one of the aliphatic substituents contains about 8-18 carbon atoms and one contains an anionic water-solubilizing group (such as carboxy, sulfonate, sulfate, phosphate or 10 phosphonate). Cationic surfactants include aliphatic quaternary ammonium compounds having one long alkyl chain containing about 8-18 carbon atoms (such as lauiyl trimethylammonium chloride, cetylpyridinium chloride, cetyltrimethylammonium bromide, diisobuytylphenoxyethyldimethylbenzylammoniiun chloride, coconut alkyltrimetylammonium nitrite, cetylpyridinium fluoride). Certain cationic surfactants can also act as antimicrobials. 15 Thickening agents include carboxyvinyl polymers, polyvinylpyrrolidone, polyacrylates, carrageenan, cellulose derivatives (e.g.,. hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, and hydroxyethyl cellulose), laponite, water-soluble salts of cellulose ethers (such as sodium carboxymethylcellulose and sodium carboxymethyl hydroxyethyl cellulose), natural gums (such as gum karaya, xanthan gum, gum arabic and gum 20 tragacanth), polymeric polyether compounds (such as polyethylene oxide and polypropylene oxide), homopolymers of acrylic acid crosslinked with an alkyl ether of pentaerythritol, alkyl ether of sucrose, carbomers (sold under the tradename Carbopol®), starch, copolymers of lactide and glycolide monomers (the copolymer having an average molecular weight of about 1,000-120,000), colloidal magnesium aluminum silicate and finely divided silica. Thickening 25 agents will be added in amounts sufficient to give a desired consistency to an oral care composition. Abrasives include silicas (including gels and precipitates), aluminas, calcium carbonates, calcium phosphates, dicalcium phosphates, tricalcium phosphates, hydroxyapatites, calcium pyrophosphates, trimetaphosphates, insoluble polymetaphopsphates 30 (such as insoluble sodium polymetaphosphate and calcium polymetaphosphate), magnesium carbonates, magnesium oxides, resinous abrasive materials (such as particulate condensation WO 03/043518 PCTYU S02/37136 27 products of urea and formaldehyde), particulate thermosetting polymerized resins (suitable resins include melamines, phenolics, ureas, melamine-ureas, melamine-formaldehydes, urea-formaldehydes, melamine-urea-formaldehydes, cross-linked epoxides and cross-linked polyesters), and combinations of the foregoing. Silica abrasives are preferred because they 5 provide excellent dental cleaning and polishing performance without unduly abrading tooth enamel or dentine. Flavoring agents include peppermint, oil, spearmint oil, wintergreen oil, clove, menthol, dihydroanethole, estragole, methyl salicylate, eucalyptol, cassia, 1-menthyl acetate, sage, eugenol, parsley oil, menthone, oxanone, alpha-irisone, alpha-ionone, anise, maqoram, 10 lemon, orange, propenyl guaethol, cinnamon, vanillin, ethyl vanillin, thymol, linalool, limonene, isoamylacetate, benzaldehyde, ethylbutyrate, phenyl ethyl alcohol, sweet birch, cinnamic aldehyde, cinnamaldehyde glycerol acetal (known as CGA), and mixtures of the foregoing. Sweetening agents include sucrose, glucose, saccharin, dextrose, levulose, lactose, 15 mannitol, sorbitol, fructose, maltose, xylitol, saccharin salts, thaumatin, aspartame, D-tryptophan, dihydrochalcones, acesulfame, cyclamate salts, and mixtures of the foregoing, In addition to the flavoring and sweetening agents, the oral care compositions may include coolants, salivating agents, warming agents and numbing agents as optional ingredients. Coolants include carboxamides, menthol, paramenthan carboxamides, 20 isopropylbutanamide, ketals, diols, 3-1-menthoxypropane-1,2-diol, menthone glycerol acetal, menthyl lactate, and mixtures thereof. Salivating agents include Jambu® (manufactured by Takasago). Warming agents include capsicum and nicotinate esters (such as benzyl nicotinate). Numbing agents include benzocaine, lidocaine, clove bud oil and ethanol. Antibacterial and anti-plaque agents include triclosan, sanguinarine and sanguinaria, 25 quaternary ammonium compounds, cetylpyridinium chloride, tetradecylpyridinium chloride and N-tetradecyl-4-ethylpyridinium chloride, benzalkonium chloride, bisquanides, chlorhexidine, chlorhexidine digluconate, hexetidine, octenidine, alexidine, halogenated bisphenolic compounds, 2,2- methylenebis-(4-chloro-6-bromophenol). 5-chloro-2-(2,4-dichlorqphenoxy)-phenol, salicylanilide, domiphen bromide, delmopinol, octapinol, other 30 piperadino derivatives, nicin, zinc stannous ion agents, antibiotics (such as augimentin, WO 03/043518 PCT/US02/37136 28 amoxicillin, tetracycline, doxydcycline, minocycline, and metronidazole), analogs and salts of the foregoing, and mixtures of the foregoing. Anti-caries agents include sodium fluoride, stannous fluoride, potassium fluoride, amine fluorides, indium fluoride, sodium monofluorophosphate, calcium lactate, calcium 5 glycerophosphates, strontium salts, and strontium polyacrylates. Anti-calculus agents include pyrophosphate salts such as dialkali metal pyrophosphate sails and tetraalkali metal pyrophosphate salts (e.g., disodium dihydrogen pyrophosphate, -tetrasodium pyrophosphate and tetrapotassium pyrophosphate, in their hydrated and unhydrated forms). Other anti-calcuhis agents which can be used instead of, or in addition to, 10 the pyrophosphate salts include synthetic anionic polymers (such as polyacrylates and copolymers of maleic anhydride or acid and methyl vinyl ether), polyaminopropane sulfonic acid, zinc citrate trihydrate, polyphosphates (such as tripolyphosphate and hexametaphosphate), polyphosphonates (such as disodium ethane-l-hydroxy-1,1-diphosphonate (EHDP), methanedisphosphonic acid, and 2-phosphonobutane-1 ,2,4-15 tricarboxylic acid), and polypeptides (such as polyaspartic acid and polyghitamic acid). The pH of the oral compositions of the invention should not be acidic, since acidic conditions will lessen the effectiveness of the metal-binding compounds of the invention. Thus, the pH of the oral care compositions of the invention should be greater than about 6.5, preferably from about 7.0 to about 8.5, more preferably from about 7.2 to about 7.6. Thus, 20 a pH-adjusting agent and/or a buffering agent or agents may need to be included in the oral care compositions. The pH-adjusting agent may be any compound or mixture of compounds that will achieve the desired pH. Suitable pH-adjusting agents include organic and inorganic acids andbases, such as benzoic acid, citric acid, potassium hydroxide, and sodium hydroxide. Buffering agents include acetate salts, borate salts, carbonate salts, bicarbonate salts (e.g., an 25 alkali metal bicarbonate, such as sodium bicarbonate (also known as baking soda)), gluconates, tartrates, sulfates, citrates (such as sodium citrate), benzoate salts, nitrate salts (such as sodium and potassium nitrate), phosphate salts (such as potassium and sodium phosphate), and combinations of the foregoing as needed to achieve and maintain the desired pH. 30 The oral care compositions of the invention may further include one or more antioxidants, anti-inflammatory compounds, and/or metal-binding compounds in addition to WO 03/043518 PCT/DS02/37136 29 the metal-binding compounds of the invention (which, as noted above, are anti-inflammatory and reduce the damage done by ROS, in addition to binding metal ions). Suitable anti-inflammatory agents include ibuprofen, flurbiprofen, ketoprofen, aspirin, kertorolac, naproxen, indomethacin, piroxicam, meclofenamic acid, steroids, and mixtures of 5 (he foregoing. Suitable antioxidants include superoxide dismutase, catalase, ghrtathioneperoxidase, ebselen, glutathione, cysteine, N-acetyl cysteine, penicillamine, allopurinol, oxypurinol, ascorbic acid, a-tocopherol, Trolox (water-soluble a-tocopherol), vitamin A, {J-carotene, fatty-acid binding protein, fenozan, probucol, cyanidanol-3, dimercaptopropanol, indapamide, 10 emoxipine, dimethyl sulfoxide, and others. See, e.g., Das et al., Methods Enzymol, 233,601-610 (1994); Stohs, J. Basic Clin. Physiol Pharmacol, 6,205-228 (1995). Suitable metal-binding compounds include metal-binding peptide and/ornon-peptide chelators. Metal-binding peptides and non-peptide chelators are described above, and others are known in the art. For instance, a peptide P, (i.e., peptide P, - P2 wherein n = 0 in the 15 formula of P2), which binds Cu(D) tightly, could be given in combination with a separate peptide suitable for binding CuQ) (suitable Cu(T)-binding peptides are described above). As another example, a peptide P, could be given in combination with a separate peptide or non-peptide chelator capable of binding iron. Suitable iron-binding peptides and non-peptide chelators are described above and others are known in the art (e.g., deferoxamine mesylate). 20 The oral care compositions of the invention may advantageously contain a protease inhibitor to prevent degradation of the metal-binding compounds of the invention and/or for an additional therapeutic effect (certain proteases are involved in inflammatory processes and others have been implicated in tissue breakdown in the mouth). Suitable protease inhibitors include metalloproteinase and serine protease inhibitors, such as those described in U.S. 25 Patents Nos. 6,403,633, 6,350,438, 6066673, 5,622,984, and 4,454,338, the complete disclosures of which are incorporated herein by reference. Many other ingredients are known that may be incorporated into oral care compositions. These include suspending agents (such as a polysaccharide - see U.S. Patent No. 5,466,437), polymeric compounds which can enhance the delivery of active ingredients 30 (such as copolymers of polyvinylmethylethsr with maleic anhydride and those delivery enhancing polymers described in DE 942,643 andU.S. PatentNo. 5,466,437), materials which WO 03/043518 PCT/US02/37136 30 allow for a strong and continuing adherence of the oral care composition to the tissues of the mouth, thereby providing for a protracted topical therapeutic effect (such as natural gums, plant extracts, animal extracts (e.g., gelatin), natural and synthetic polymers, and starch derivatives; see, e.g., U.S. Patents Nos. 5,032,384, 5,298,237, and 5,466,437), oils, waxes, 5 silicones, coloring agents (such as FD&C dyes), color change systems, preservatives (such as methylparaben, propylparaben, and sodium benzoate), opacifying agents (such as titanium dioxide), plant extracts, solubilizang agents (such as propylene glycol; see, e.g., U.S. Patent No. 5,466,437), enzymes (such as dextranase and/or mutanase, amyloglucosidase, glucose oxidase with lactoperoxidase, and neuraminidases), synthetic or natural polymers, tooth 10 whitening agents (such from about 0.1 % to about 10%by weight of a peroxygen compound; see additional discussion of tooth whitening compositions below), an alkali metal bicarbonate (such as sodium bicarbonate (also known as baking soda), generally present at from about 0.01% to about 30% by weight), desensitizers (such as potassium salts (e.g., potassium nitrate, potassium citrate, potassium chloride, potassium tartrate, potassium bicarbonate, and 15 potassium oxalate), and strontium salts), analgesics (such as lidocaine or benzocaine), antifungal agents, antiviral agents, etc. The presence of a significant amount of copper and iron salts is preferably avoided. The presence of significant amounts of copper and iron ions in the oral care compositions could reduce the ability of the metal-binding compounds of the invention to bind copper and 20 iron ions found in the mouth. It will be appreciated that a wide variety of different oral care compositions can be prepared utilizing the above described ingredients and other ingredients known in the art or which will be developed. It is within the skill in the art to chose appropriate ingredients and combinations of ingredients and to determine an effective amount of the metal-binding 25 compound(s) of the invention to include in a particular oral care composition, given the knowledge in the art and the guidance provided herein. What follows are a few examples of oral care compositions into which a metal-binding compound or a combination of metal-binding compounds of the invention could be incorporated. It will be understoodby those skilled in the art that additional types of oral care 30 compositions and additional oral care compositions having different ingredients and/or WO 03/043518 PCT/US02/37136 31 different amounts of ingredients can be prepared utilizing the knowledge and skill in the art and the guidance provided herein. Dentrifices include toothpastes, tooth gels, tooth powders and liquid dentrifices. Toothpastes and tooth gels generally include a dental abrasive, a surfactant, a thickening 5 agent, a humectant, a flavoring agent, a sweetening agent, a coloring agent and. water. Toothpastes and tooth gels may also include opacifying agents, anti-caries agents, anti-calculus agents, tooth whitening agents, and other optional ingredients. Typically, a toothpaste or tooth gel will contain from about 5% to about 70%, preferably from about 10% to about 50%, of an abrasive, from about 0.5% to about 10% of a surfactant, from about 0.1% 10 to about 10% of a thickening agent, from about 10% to about 80% of a humectant, from about 0.04% to about 2% of a flavoring agent, from about 0.1% to about 3% of a sweetening agent, from about 0.01% to about 0.5% of a coloring agent, from about 0.05% to about 0.3% of an anti-caries agent, from about 0.1% to about 13% of an anti-calculus agent, and from about 2% to about 45% water. Tooth powders of course contain substantially all non-liquid components 15 and typically contain from about 70% to about 99% abrasive. Liquid dentrifices may comprise water, ethanol, a humectant, a surfactant, a thickening agent, an abrasive (if an abrasive is included, a suspending agent (e.g., a high molecular weight polysaccharide) must be included; see U.S. Patent No. 5,466,437), an antibacterial agent, an anti-caries agent, a flavoring agent and a sweetening agent. A typical liquid dentrifice will comprise from about 20 50% to about 85% water, from about 0.5% to about 20% ethanol, from about 10% to about 40% of a humectant, from about 0.5% to about 5% of a surfactant, from about 0.1% to about 10% of a thickening agent, and may contain from about 10% to about 20% of an abrasive, from about 0.3% to about "2% of a suspending agent, from about 0.05% to about 4% of an antibacterial agent, from about 0.0005% to about 3% of an anti-caries agent, from about 0.1% 25 to about 5% of a flavoring agent, and from about 0.1% to about 5% of a sweetening agent Gels include dentrifice gels (see description above), non-abrasive gels and subgingival gels. Non-abrasive gels and subgingival gels generally include a thickening agent, a humectant, a flavoring agent, a sweetening agent, a coloring agent, and water. Such gels may also include one or more anti-caries agents and/or anti-calculus agents. Typically, such a gel 30 will contain from about 0.1 % to about 20% of a thickening agent, from about 10% to about 55% of a humectant, from about 0.04% to about 2% of a flavoring agent, from about 0.1% to WO 03/043518 PCT/USO 2/37136 32 about 3% of a sweetening agent, from about 0.01 % to about 0.5% of a coloring agent, and the balance water. Such gels may also contain from about 0.05% to about 0.3% of an anti-caries agent and from about 0.1% to about 13% of an anti-calculus agent Creams generally include a thickening agent, a humectant and a surfactant, and may 5 include a flavoring agent, a sweetening agent, a coloring agent Typically, a cream will contain from about 0.1% to about 30% of a thickening agent, from about 0% to about 80% of a humectant, from about 0.1% to about 5% of a surfactant, from about 0.04% to about 2% of a flavoring agent, from about 0.1% to about 3% of a sweetening agent, from about 0.01% to about 0.5% of a coloring agent, and from about 2% to about 45% of water. 10 Ointments suitable for oral use are described in, e.g., U.S. Patents Nos. 4,847,283, 5,855,872 and 5,858,408, the complete disclosures of which are incorporated herein by reference. Ointments generally include one or more of the following: fats, oils, waxes, parafins, silicones, plastibase, alcohols, water, humectants, surfactants, thickening agents, talc, bentonites, zinc oxide, aluminum compounds, preservatives, antiviral compounds, and 15 other ingredients. For instance, the ointment may comprise from about 80% to about 90% petrolatum and from about 10% to about 20% ethanol or propylene glycol. As another example, the ointment may comprise about 10 % petrolatum, about 9% lanolin, about 8% talc, about 32% cod liver oil, and about 40% zinc oxide. As a third example, the ointment may comprise from about 30% to about 45% water, from about 10% to about 30% oil 20 (e.g.,petrolatum or mineral oil), from about 0.1% to about 10% emulsifier (e.g., wax NF), from about 2% to about 20% humectant (eg., propylene glycol), from about 0.05% to about 2% preservatives (e.g., methyl paraben and propyl paraben), and from about 10% to about 40% sterol alcohol. Mouthwashes, rinses, gargles and sprays generally include water, ethanol, and/or a 25 humectant, and preferably also include a surfactant, a flavoring agent, a sweetening agent, and a coloring agent, and may include a thickening agent and one or more anti-caries agents and/or anti-calculus agents. A typical composition contains from about 0% to about 80% of a humectant, from about 0.01% to about 7% of a surfactant, from about 0.03% to about 2% of a flavoring agent, from about 0.005% to about 3% of a sweetening agent, from about 0.001% 30 to about 0.5% of a coloring agent, with the balance being water. Another typical composition contains from about 5% to about 60%, preferably from about 5% to about 20%, ethanol, from WO 03/043518 PCT/US02/37136 33 about 0% to about 30%, preferably from about 5% to about 20%, of a humectant, from about 0% to about 2% emulsifying agents, from about 0% to about 0.5% of a sweetening agent, from about 0% to about 0.3% of a flavoring agent, and the balance water. A further typical composition contains from about 45% to about 95% water, from about 0% to about 25%, 5 ethanol, from about 0% to about 50% of a humectant, from about 0.1 % to about 7% of a surfactant, from about 0.1% to about 3% of a sweetening agent, from about 0.4% to about 2% of a flavoring agent, and from about 0.001% to. about 0.5% of a coloring agent. These compositions may also comprise from about 0.05% to about 0.3% of an anti-caries agent, and from about 0.1% to about 3% of an anti-calculus agent 10 Solutions generally include water, a preservative, a flavoring agent, and a sweetening agent, and may include a thickening agent and/or a surfactant. Typically, solutions contain from about 85% to about 99% water, from about 0.01% to about 0.5% of a preservative, from about 0% to about 5% of a thickening agent, from about 0.04% to about 2% of a flavoring agent, from about 0.1% to about 3% of a sweetening agent, and from about 0% to about 5% 15 of a surfactant. Another simple solution that can be used is a saline solution, a buffer solution, or buffered saline, optionally containing a preservative, a thickening agent and/or a surfactant. Lozenges and mints generally include a base, a flavoring agent and a sweetening agent. The base may be a candy base (hard sugar candy), glycerinated gelatin or a combination of sugar with sufficient mucilage to give it form. See U.S. Patent No. 6,350,438 and Remington, 20 The Science And Practice Ofpharmacy, 19th edition (1995). Lozenge compositions also typically include one or more fillers (e.g., a compressible sugar) and lubricants. Chewing gums, chewable tablets and chewable lozenges are described in U.S. Patents Nos. 6,471,991, 6,296,868, 6,146,661, 6,060,078, 5,869,095, 5,709,873, 5,476,647, and 5,312,626, PCT applications WO 84/04453 and WO 99/02137, and Lieberman et al., 25 Pharmaceutical Dosage Forms, 2n<l ed. (1990), the complete disclosures of which are incorporated here in by reference. As one example, a compressed chewable tablet comprises a water-disintegratable, compressible carbohydrate (such as mannitol, sorbitol, maltitol, dextrose, sucrose, xylitol, lactose and mixtures thereof), a binder (such as cellulose, cellulosic derivatives, polyvinyl 30 pyrrolidone, starch, modified starch and mixtures thereof), and, optionally, a lubricant (such as magnesium stearate, stearic acid, talc, and waxes), sweetening, coloring and flavoring WO 03/043518 PCT/US02/37136 34 agents, a surfactant, a preservative, and other ingredients. All ofthe ingredients, including the metal binding compound(s) of the invention, are dry blended and compressed into a tablet As another example, a chewable tablet may comprise a core surrounded by an outer layer wrapping the core. The core may comprise a metal binding compound or compounds 5 of the invention and, optionally, other active ingredients in a jelly base or a chewable base. The outer layer may be a chewable base. The jelly base may comprise pectin, sorbitol, maltitol, isomalt, liquid ghicose, sugar, citric acid and/or a flavoring agent The chewable base of the core or outer layer may be a gum, soft candy, nougat, caramel or hard candy. The tablels are formed by extrusion of the core and outer layer to form a rope, fallowed by cutting 10 the rope into tablets. Chewing gam compositions generally include a gum base, a flavoring agent and a sweetening agent Suitable gum bases include jelutong, rubber, latex, chicle, and vinylite resins, desirably with conventional plasticizers or softeners. Plasticizers include triacetin, acetyl tributyl citrate, diethyl sebacetate, triethyl citrate, dibutyl sebacetate, dibutyl succinate, 15 diethyl phthalate and acetylated monoglycerides. Typically, chewing gum compositions contain from about 50% to about 99% gum base, from about 0.4% to about 2% of a flavoring agent and from about 0.01% to about 20% of a sweetening agent. The metal-binding compound(s) of the invention and other active ingredients may be incorporated into a gum base by, e.g., stirring than into a warm gum base or coating them onto the outer surface of the 20 gum base. Films and sheets, and gels which form solids in the mouth, made of lactide/glycolide copolymers are described in U.S. Patents Nos. 5,198,220,5,242,910 and 6.350,438. Another polymer film suitable for use in the mouth is described in PCT application WO 95/32707. Patches that adhere to hard dental surfaces, such as teeth and dentures, and which degrade in 25 the mouth, are described in U.S. Patent No. 6,197,331. All of these materials slowly release active agents contained in them into the mouth. Other compositions (including pastes, gels, ointments, liquids and films) providing for slow release of active agents are also known. See, e.g., U.S. Patents Nos. 5,032,384,5,298,237,5,466,437,5,709,873, and 6,270,781. Tooth whitening compositions will comprise a tooth whitening agent Tooth 30 whitening agents include peroxides, percarbonates and perborates of the alkali and alkaline earth metals or complex compounds containing hydrogen peroxide. Tooth whitening agents WO 03/043518 PCT/US02/37136 35 also include peroxide salts ofthe alkali or alkaline earth metals. The most commonly used tooth whitening agent is carbamide peroxide. Other commonly used tooth whitening agents arehydrogenperoxide, peroxyacetic acid and sodium perborate. These tooth whitening agents liberate active oxygen and hydrogen peroxide. Tooth whitening agents can be present in tooth 5 whitening compositions at a concentration of from about 0.1% to about 90%; typically, the concentration of carbamide peroxide in tooth whitening compositions is from about 10% to about 25%. Many tooth whitening compositions are known in the art, including aqueous solutions, gels, pastes, liquids, films, strips, one-part systems, two-part systems, compositions that 10 require activation of the tooth whitening agent (e.g., by inclusion of a radiant-energy or heat-energy absorbing substance, such as substantially conjugated hydrocarbons, which activates the bleaching agent when irradiated), etc. See, e.g.} U.S. Patents Nos. 5,302,375,5,785,887, 5,858,332,5,891,453,5,922,307,6,322,773,6,419,906, andPCT applications WO 99/37236, WO 01/89463 and WO 02/07695, the complete disclosures of which are incorporated herein 15 by reference. Also, many other oral care compositions (e.g., toothpastes) and devices (e.g., dental flosses) comprise a tooth whitening agent Theuse of tooth whitening compositions, or of one of the many oral care compositions and devices which comprise a tooth whitening agent, results in the production of ROS and can cause inflammation of the tissues of the mouth. Incorporation of a metal-binding compound 20 or compounds of the invention in tooth whitening compositions or other oral care compositions and devices comprising a tooth whitening agent will reduce or prevent the inflammation and/or the production of ROS. The inclusion of a metal-binding compound or compounds ofthe invention in such compositions may also result in more effective whitening, since hydrogen peroxide, which is responsible for the whitening of teeth by the hydrogen 25 peroxide-type whitening agents, will not be converted into hydroxyl radicals (see Examples 8 and 9) and will, therefore, remain active longer. Alternatively, an oral care composition or device comprising a metal-binding compound or compounds of the invention can be used before or after the tooth whitening composition or oral care composition or device comprising a tooth whitening agent to reduce or prevent the inflammation and/or the production of ROS. 30 WO 03/043518 PCT/US02/37136 36 For instance, teeth are commonly whitenedby applying a tooth whitening composition to the teeth by means of a dental tray or trough. A metal-binding compound or compounds ofthe invention couldbe incorporated into the tooth whitening composition that is used in the tray or trough. Alternatively, a separate composition comprising a metal binding compound 5 or compounds of the invention could be applied to the teeth in a cleaned or different tray or trough after the application of the tooth whitening composition is completed. In a further alternative, a wash or rinse comprising a metal binding compound or compounds of the invention could be used to rinse the mouth before and/or after the application of the tooth whitening composition. 10 A recently developed product for applying a tooth whitening composition to the teeth is a flexible strip. See, e.g., U.S. Patents Nos.5,891,453 and 6,419,906. A metal-binding compound or compounds of the invention could be incorporated into such strips. For instance, the metal-binding compound(s) could be incorporated into the tooth whitening composition, which is then applied to the strips, or a solution, gel or other composition 15 comprising the metal-binding compound(s) could be separately applied to the strips, either during their manufacture or just prior to use by the patient la yet another alternative, strips comprising a tooth whitening composition and strips comprising the metal binding compound(s) could both be supplied to file patient and would be used sequentially. The oral care compositions ofthe invention may comprise a single phase or a plurality 20 of phases. A plurality of phases will be used, e.g., where some of the ingredients are incompatible, some ofthe ingredients are unstable, or the ingredients are best combined at the time of use. Thus, one of the phases will include some ofthe ingredients, and the remainder of the ingredients will be contained in one or more additional phases. The plurality of phases may be a plurality of separate compositions, in which case the plurality of phases will be 25 provided in a plurality of separate containers or in a plurality of compartments in a single container, and the plurality of phases will be combined at the time of use. As an alternative the plurality of phases maybe formed by encapsulating some ofthe ingredients, in which case the plurality of phases may all be contained in a single container. Multi-phase oral care compositions are described in, e.g., U.S. Patents Nos. 5,302,.375, 5,906,811, 5,976,507, 30 6,228,347 and 6,350,438 and PCT application number WO 99/37236. WO 03/043518 PCT/US02/37136 37 The invention also provides oral care devices comprising a metal-binding compound or compound(s). Oral care devices of the invention include devices intended for use by consumers and patients and devices intended for use by dental professionals (e.g., dental hygienists, dentists and oral surgeons). 5 The oral care devices of the invention include surgical materials (such as sutures and sponges), flosses, tapes, chips, strips, fibers, a toothpick or rubber tip, dental implants and dental appliances (such as trays and troughs that fit over and cover the teeth and, optionally, the periodontal tissue) having a metal-binding compound or compounds of the invention adhered to, absorbed into, bound to, attached to, entrapped in, coated onto, or otherwise 10 incorporated into, them. See, e.g., U.S. Patents Nos. 5,709,873, 5,863,202, 5,891,453, 5,967,155,5,972,366,5,980,249,6,026,829,6,080,481,6,102,050,6,350,438,6,419,906, PCT application WO 02/13775, andEP application 752833, which describe such oral care devices and methods of incorporating compounds into them (the complete disclosures of all of these patents and applications are incoiporated herein by reference). For instance, a metal-binding 15 compound or compounds of the invention can be incoiporated into a binder (e.g., a wax or polymer) and coated onto dental floss, dental floss can be soaked in a bath of a liquid containing a metal-binding compound or compounds of the invention to impregnate or coat the floss with the compound(s), a metal-binding compound or compounds ofthe invention in solid (e.g., freeze-dried) form can be incorporated into a polymer film suitable for application 20 to the teeth, a metal-binding compound or compounds ofthe invention in a solution or gel can be applied to a flexible strip suitable for application to teeth, or a suture or other surgical material can be soaked in a solution containing a metal-binding compound or compounds of the invention followed by removal of the solvent so that the compound(s) become associated with (bound to, entrapped in, coated onto, etc.) the suture or surgical material. See,e.g.,U.S. 25 Patents Nos. 5,891,453, 5,967,155, 5,972,366, 6,026,829, 6,080,481, 6,102,050, and 6,419,906. Also included within the scope of the invention ore oral care products for animals, such as foods, chews, and toys. Suitable products are described in U.S. PatentNo. 6,350,438. A metal-binding compound or compounds ofthe invention can be used to treat a tissue 30 of an animal's mouth. ,eMouth" is used herein to mean the cavity bounded externally by the lips and internally by the pharynx that encloses the tongue, gums and teeth. Thus, the tissues WO 03/043518 PCT/US02/37136 38 of the mouth include the lips, tongue, gums, buccal tissue, palate and teeth. A single tissue, a plurality of tissues, a portion of one or more tissues, all or substantially all of the tissues of the mouth, or combinations of the foregoing, may be treated according to the invention. 'Treat" and variations thereof are used herein to mean to cure, ameliorate, alleviate, inhibit, 5 prevent, reduce the likelihood of, or reduce the severity ofj a disease or condition, or of at least some of the symptoms or effects thereof. To treat a tissue of the mouth, the tissue is contacted with a metal-binding compound or compounds of the invention. For instance, the tissue may be contacted with an oral care composition comprising the metal-binding compound^). Methods of contacting tissues of 10 the mouth with oral care compositions are well known in the art. Suitable methods include rinsing the tissue with a solution (e.g., a mouthwash, rinse, spray, liquid dentrifice, or other solution), brushing the teeth with a dentrifice (e.g., a toothpaste, tooth gel, or powder), applying a non-abrasive solution, gel, paste, cream or ointment directly to the tissue (with or without the me of an applicator), chewing gum, chewing or sucking a lozenge, mint or tablet, 15 and many other means of topical application. Suitable applicators for applying oral care compositions, such as solutions, gels, pastes, creams and ointments, to a tissue include a swab, a stick, a plastic paddle, a dropper, a syringe, a strip (such as those described in U.S. Patents Nos.5,891,453 and 6,419,906), a finger, or a dental tray or appliance (such as those shown in U.S. Patents Nos. 5,863,202 and 5,980,249 and BP application 752833) which allows for 20 immersion of the teeth and, optionally, the periodontal tissue in, e.g., a gel or solution. In addition, to treat a tissue of the mouth, the tissue may be contacted with an oral care device comprising the metal-binding compound(s). Methods of contacting tissues of the mouth with oral care devices are well known in the art For instance, sutures can be used to close a surgical wound or a wound resulting from a tooth extraction, dental floss can be used to floss 25 the teeth, etc. The treatment ofthe tissue can be prophylactic treatment For instance, the tissue may be treated as part of a prophylactic oral care regimen. The metal-binding compound(s) of the invention can be incorporated into an oral care composition or device, such as a toothpaste, a tooth gel, a mouthwash or rinse, or a dental floss, that is employed in such a regimen and 30 will be used preferably at least once per day, more preferably two or three times per day. In another alternative, the metal-binding compound(s) of the invention may be contained in a WO 03/043518 PCT/US02/37136 39 separate oral care composition or device which will be used separately from other compositions and devices employed in the prophylactic oral care regimen. For instance, the metal-binding compound(s) of the invention can be incoiporated into a mouthwash or rinse, a gum, a lozenge or a chewable tablet, which would preferably be used at least once per day, 5 more preferably at least two or three times per day. Itmaybeparticularlybeneficialforthose patients who utilize tobacco products to use the metal-binding compound(s) ofthe invention as part of a prophylactic oral care regimen to attempt to ameliorate the damage done to tissues of the mouth by such products. It is known to include metal salts, particularly copper salts, in toothpastes and other 10 oral care compositions, generally as antibacterial, anti-plaque, anti-caries, and anti-gingivitis agents. See, e.g„ U.S. Patents Nos. 5,286,479, 5,298,237, and 6,355,706, EP application 658,565, PCT application WO 92/08441, Japanese application 4159211, Waerhaug et al., J, Clin. Periodontal., 11:176-180 (1984). The use of oral care compositions containing copper salts could be harmful to the tissues of the mouth, since free copper ions catalyze the 15 formation of ROS. Thus, the use of an oral care composition of the present invention at an appropriate time after the use of the copper-containing compositions (i.e„ allowing sufficient time for the copper salts to exert their activity) could be very beneficial in reducing the damage done by ROS generated by copper ions present in the mouth as a result of use of these products. For instance, the metal-binding compound(s) could conveniently be supplied in a 20 gum, lozenge or chewable table which would be chewed or sucked after use of the copper-containing compositions. Tissues may also be treated prophylactically in connection with a variety of dental procedures, including surgeries and tooth extractions. For instance, the tissue(s) on which surgery is being performed, those tissues near the area where the surgery is being performed 25 or, for ease of treatment, all or substantially of the tissues of the mouth, can be treated prior to surgery, during surgery, after the surgery, or combinations thereof. Similarly for a tooth extraction, the tissues) surrounding the tooth which is to be extracted, adjacent tissues or, for ease of treatment, all or substantially of the tissues of the mouth, can be treated prior to tooth extraction, during the tooth extraction, after the tooth extraction, or combinations thereof. For 30 instance, the mouth could be rinsed prior to surgery or tooth extraction with a solution comprising the metal-binding compound^), die wound(s) caused by the surgery or tooth WO 03/043518 PCT/DS02/37136 40 extraction could be closed with sutures haying the metal-binding compound(s) incoiporated into them, and/or the mouth could be rinsed immediately after the surgery or tooth extraction, and/or at intervals thereafter, with a solution comprising (he metal-binding compound(s). Tissues can also be treated prophylactically in connection with radiation, such as 5 dental x-rays. Finally, as described above, tissues may be treated prophylactically in connection with the whitening of the teeth of an animal. A metal-binding compound or compounds of the invention can be used to treat a disease or condition of a tissue of an animal's mouth. Diseases and conditions treatable according to the invention include infections (bacterial infections, viral infections and fungal 10 infections), inflammation due to any cause, and any disease or condition involving or caused by metal ions and/or ROS. Specific diseases and conditions treatable according to the invention include diseases of the periodontal tissue, such as gingivitis and periodontitis, ulcers, cold sores, canker sores, other viral infections, bacterial infections and yeast and fungal infections. 15 It is understood by those skilled in the art that the dosage amount of the metal-binding compound(s) of the invention needed to treat a tissue of an animal's mouth will vary with the particular metal-binding compound employed, whether the treatment is prophylactic or for the treatment of a disease or condition, the identity of the disease or condition to be treated, the severity ofthe disease or condition, the type of oral care composition used, the duration of the 20 treatment, the identify of any other drugs being administered to the animal, the age, size and species of the animal, and like factors known in the medical and veterinary arts. In general, a suitable daily dose of a compound of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect As noted above, it is expected that usage of oral care compositions comprising from about 0.001% to about 25 20% of a metal binding compound or compounds of the invention one or more times per day will provide effective daily dosages. However, the actual daily dosage to be employed, the number of treatments per day, and the length of treatment will be determined by an attending physician or veterinarian within the scope of sound medical judgment The invention also provides a kit comprising an oral care product according to the 30 invention. In the case where the oral care product is an oral care composition, the kit may also include an applicator for applying the oral care composition to a tissue of an animal's mouth, WO 03/043518 PCT/US02/37136 41 such as a swab, a stick, a plastic paddle, a dropper, a syringe, a strip (such as that described in U.S. Patents Nos.5,891,453 and 6,419,906) or a dental toy or appliance (such as those shown in U.S. Patents Nos. 5,863,202 and 5,980,249 and EP application 752833) which allows for immersion of the teeth and, optionally, the periodontal tissue in, e.g., a gel or 5 solution. The kit could also include a cup, vial or other device for dispensing and/or measuring the amount of the oral care composition ofthe invention needed for the intended use. Of course, the kits could include both an oral care composition and an oral care device according to the invention, k addition to an oral care composition and/or device of the invention, the kits could also comprise another type of oral care composition or device, such 10 as a tooth whitening composition, strips comprising a tooth whitening agent, applicators for applying oral care compositions, etc. Kits according to the invention will also include instructions for using the kit aiid/or the oral care product of the invention and may include any other desired items. • It is to be noted that "a" or "an" entity refers to one or more of that entity. For 15 example, "a cell" refers to one or more cells. This application incorporates by reference U.S. application Serial No. 10/186,168, filed June 27,2002; U.S. application 10/076,071, filed February 13,2002; U.S. Provisional Application 60/268,558, filed February 13, 2001; U.S. Provisional Application (formerly 09/816,679), filed March22,2001; U.S. Provisional Application 60/281,648, filed 20 April 4, 2001; U.S. Provisional Application 60/283,507, filed April 11, 2001; U.S. application Serial No. 09/678,202, filed September 29,2000; U.S. Provisional Application 60/157,404, filed October 1,1999; U.S. Provisional Application 60/211,078, filed June 13, 2000; U.S. Provisional Application 60/331,665, filed November 19, 2001; and U.S. Provisional Application 60/360,736, filed February 27,2002. 25 WO 03/043518 PCT/US02/37136 42 EXAMPLES EXAMPLE 1: Synthesis of Tetrapeptide Asp Ala His Lvs [SEP ID NO: 11 This example describes the synthesis of the tetrapeptide Asp Ala His Lys [SEQ ID 5 NO: 1] composed of all L-amino acids using standard solid-phase synthesis techniques. First, 9-fluorenylmethyloxycarbonyl (Fmoc)-protected Asp (v COO- ester, Tolsulfonyl) on Wang resin (0.6 mmole; Nova Biochem) was suspended in a solution of piperidine/dimethylformamide (DMF) (40% v/v; 3 ml) for 30 min with occasional agitation. At the end of this period, the solvent was drained, and the resin was washed sequentially with 10 DMF and dichloromethane (DCM; 5x3 ml). A ninhydrin test was used to monitor the reaction. Ihe resin was swollen with DMF (~1 ml). The C-protected t-benzyloxycarbonyl (Boc) ester of alanine in DMF was added, followed by a mixture of diisopropylamine (8 equivalent) and 2-(lH-benzotriazole-l-yl)-ls2,3,3-tetramethyluroniumtetra£luoroborate (TBTU-) (4 equivalents). The resin was shaken for about 24 hours, and the reaction was 15 monitored by the ninhydrin test At the end of this period, DMF was drained, and the resin was washed with DMF and DCM. The solution was drained, and the beads were washed with DCM (3x2 ml). The protecting group of the dipeptide-resin was removed, and the beads were suspended in DMF. Amino protected (benzyloxy) derivative of histidine (4 mmole) was added, followed by mixture of diisopropylamine (8 equivalent) and TBTU- (4 equivalent). 20 The resin was shaken for about 24 hours, and the reaction monitored by ninhydrin test. At the raid of this period, DMF was drained, and the resin was washed with DMF and DCM. The tripeptide-resin was briefly dried in a gentle stream of nitrogen and suspended in nitrogen-saturated DMF. Protected lysine was added, followed by a mixture of diisopropylamine (8 equivalent) and TBTU- (4 equivalent). The resin was shaken for about 24 hours, and the 25 reaction monitored by the ninhydrin test. At the end of this period, DMF was drained and the resin was washed with DMF and DCM. The Boc protecting group was carefully removed to give the tetrapeptide bound to the resin, with a typical loading of 5 mmole/g. The resin bound tetrapeptide (0.25 gm; 5 xnmolar) was treated with trifluoroacetic acid (TFA) and was shaken for 24 hours. At the end of this period, the ninhydrin test gave a blue color, indicating the 30 release of the tetrapeptide from the resin. Ia some circumstances, addition of 5%(V/V) of DMF to TFA accelerated the rate of release of the peptide from the resin. Removal of TFA WO 03/043518 PCT/US02/37136 43 at reduced pressure gave the tetrapeptide (all D) as TFA salt and was dried under vacuum at 5°C for 24 hours. The residue was a white powder and was characterized by spectrometry methods. A number of enantiomers of the tetrapeptide can be prepared in this manner. For example, use of D-amino acids in the peptide synthesis forms the tetrapeptide containing all D-amino acids. Also, combinations of L-amino acids and D-amino acids can be used. EXAMPLE 2: Preparation of Cyclohexanediamine Derivative of Asp Ala His Lvs ["SEP ID NO: 1]. Trans-diaminocyclohexane was prepared by resolving cis/trans 1,2-diaminocyclohexane (Aldrich-Sigma) as the tartaric acid salt. The R-trans isomer melts at 75°C and the S-trans isomermelts between43-45°C (PLD. Thesis, P.D. Newman, University College, Cardiff U.K., 1994). The trans-diaminocyclohexane (10 gm) was then suspended in anhydrous toluene (30mL) and cooled to 5°C in an ice bath, and bromoacetic acid (8 gm) in toluene (25 mL) was added dropwise. At the end of the addition, the reaction temperature was raised to 30°C andkept at that temperature for a further 5 hours. Toluene was evaporated, and theR-trans 1,2-diaminocyclohexane diacetic acid was crystallized fromhexane/tolueneto give a white solid (yield 70%). The product was characterized by spectroscopic methods. The resin-bound tetrapeptide prepared in Example 1 (20mg) was suspended in DMF (5 mL) and was treated with the R-trans 1,2-diaminocycIohexanediacettc acid (20 mg) followed by addition of a mixture of diisopropylamine (8 equivalent) and TBTU-(4 equivalent). Theresinwasshakenforabout24honaroller. Then, the resin was washed with DMF followed by DCM (5x3mL) and partially dried Hydrolysis of the resin linkage was effected by treating the resin-bound reaction product with TFA (5mL; 5 hr). The resin was separated and washed with DCM. The washings were combined with TFA and concentrated under vacuum. The residue (cyclohexanediamine tetrapeptide; formula given in Figure 3D whore Rj is H) was characterized by spectrometry analysis. EXAMPLE 3: Preparation of Tetrapeptide Tetracetic Acid The resin-bound tetrapeptide prepared in Example 1 (20 mg) was suspended in DMF (5 mL) and treated with excess (10-fold) chloroacetic acid. The resin was shaken at room temperature for 48 hours, followed by heating to 60°C for a further hour. DMF was removed WO 03/043518 PCT/US02/37136 44 byfiltration, and the resin was washed with DMF followed by DCM (5x3mL). Partially dried resin was used without further treatment in the next stage. Hydrolysis of the resin linkage was effected by treating the resin-bound reaction product with TFA (5mL; 5 hr). The resin was separated and washed with DCM. The washings were combined with TFA and concentrated 5 under vacuum (yield 30%). The product (formula given in Figure 4) was characterized by spectrometric methods. EXAMPLE 4: Preparation of Mesoporphyrin IX Tetrapeptide The resin-bound tetrapeptide prepared in Example 1 (20 mg) was suspended in DMF 10 (5 mL) and treated with mesoporphyrin IX dicarboxylic acid (10 (imole; formula given in Figure 6A), followed by addition of a mixture of diisopropylamine (8 equivalent) andTBTU-(4 equivalent). The resin was shaken for about 24 hours on a roller kept in a dark chamber. The resin was washed with DMF followed by DCM (5x3mL) and partially dried Hydrolysis ofthe resin linkage was effected by treating the resin-bound reaction product with TFA (5mL; 15 5hr). The resin was separated and washed with DCM/TFA mixture (1:1.5mL). The washings were combined and concentrated under vacuum. The porphyrin tetrapeptide (formula given in Figure 6C) was purified by semi-preparative HPLC (yield 60%). The structure was confirmed by spectrometric methods. This procedure can be used to synthesize other porphyrin-peptides, such as 20 mesoporphyrin I and related molecules. EXAMPLE 5: Preparation of Tetrabispiridvlethvl Tetrapeptide The resin-bound tetrapeptide prepared in Example 1 (20 mg) was suspended in DMF (5 mL) and treated with bromoethylpyridine (20 /imole). This was followed by the addition 25 of pyridine (0.5 mL). The resin was shaken for about 48 hours on a roller. The resin was washed with DMF, followed by DCM (5x3 mL) to remove all ofthe unreacted monomers, and then dried under vacuum for 30 minutes. Hydrolysis of the resin linkage was effected by treating the resin-bound reaction product with TFA (5mL; 5 hr). The resin was separated and washed with DCM/TFA mixture (1:1.5mL). The washings were combined and concentrated 30 under vacuum. The pyridylethyl tetrapeptide derivative (formula given in Figure 5) was WO 03/043518 PCT/US02/37136 45 purified by semi-preparative HPLC (yield 50 %). The structure was confirmed by spectrometric methods. This procedure can be applied to other heterocycles, such as phenanthroline and related molecules. 5 EXAMPLE 6: Preparation of Arvl Derivative of Asp Ala His Lvs [SEP ID NO:l] A derivative having the formula shown in Figure IB, wherein Ri is phenyl, was prepared. Diethylacetamidomalonate (10 gm) in anhydrous ethanol (100 mL) was added to a slurry of sodium ethoxide in ethanol (5 gm; 50 mL) and heated to reflux for 30 min. The 10 product was cooled (10°C)and reacted with ethyl a-bromophenyl acetate (5 gm). The reaction was allowed to proceed to completion (24 h), and excess sodium ethoxide was neutralized with dilute acid The triester was extracted into ethylacetate and, upon removal of solvent, gave a viscous liquid The crude product was hydrolyzed with hydrochloric acid (100 mL) and decarboxylated to give phenyl substituted aspartic acid (10 gm). The N-benzoyloxy t-butyl 15 derivative was prepared using a standard reaction sapience. To the resin-bound tripeptide (Lys His Ala) prepared as described in Example 1 (20 mg) in DMF was added the N-benzoyloxy-t-butyl aspartic acid derivative, followed by a mixture of diisopropylamine (8 equivalent) and TBTU- (4 equivalent). The resin was shaken for about 24 h, and the reaction monitored by the ninhydrin test At the end of this period, DMF was drained, and the resin 20 was washed with DMF and DCM. The solution was drained, and the beads washed with DCM (3x2 ml). The tetrapeptide derivative was isolated by careful hydrolysis. Stereoisomers of the tetrapeptide were separated by preparative-scale HPLC. EXAMPLE 7: Inhibition Of The Generation Of ROS 25 Bv The Tetrapeptide Asp Ala His Lvs TSEO ID NO: 11 A tetrapeptide having the sequence L-Asp L-Ala L-His L-Lys [SEQ ID NO: 1] (the L-tetrapeptide) was obtained from one or more companies that provide custom synthesis of peptides, including Ansynth Services, QCB, Genosys and Bowman Research. The peptide was prepared by standard solid phase synthesis methods (see also Example 1). 30 The ability of the L-tetrapeptide to inhibit the generation of ROS was tested as described in Gutteridge and Wilkins, Biochim. Biophys. Acta, 759, 38-41 (1983) and Cheeseman et al., Biochem. J., 252,649-653 (1988). Briefly, Cu(H) and H202 were mixed WO 03/043518 PCT/US02/37136 46 causing the generation of hydroxy radicals in a Fenton-type reaction. The hydroxyl radicals attack the sugar 2-deoxy-D-ribose (the sugar residue of DNA) to produce fragments. Heating the fragments at low pH produces malonaldehyde that, upon the addition of 2-thiobarbituiic acid, yields a pink chromogen which can be measured spectrophotometrically at 532 nm. Thus, the absorbance at 532 nm is a measure of the damage to 2-deoxy-D-ribose. The assay was performed with and without the L-tetrapeptide. The results are summarized in Table 1. As can be seen from Table 1, when the L-telrapeptide was present at Cu(D):tetrapeptide ratios of 1:1.2 and 1:2, the degradation of 2-deoxy-D-ribose was inhibitedby 38% and 73%, respectively. Clearly, the L-tetrapeptide inhibited the degradation of 2-deoxy-D-ribose by hydroxyl radicals. TABLE 1 CuCla (mM) H202 (mM) Tetra-peptltie (mM) OD at 532 nm Percent Inhibition Control 0.1 2.0 0.0 0.124 Tetrapeptide 0.1 2.0 0.12 0.077 38 Control 0.1 2.0 0.0 0.175 Tetrapeptide 0.1 2.0 0.2 0.048 73 A similar assay was also performed using a tetrapeptide having the sequence Asp Ala His Lys composed of all D-amino acids (D-tetrapeptide). The D-tetrapeptide was obtained from one or more companies that provide custom synthesis of peptides, including Ansynth Services and QCB. The peptide was prepared by standard solid phase synthesis methods (see Example 1) The ability of the D-tetrapeptide to inhibit the generation of ROS was tested as described by Zhao and Jung, Free Radic Res, 23(3), 229-43 (1995). Briefly, Cu(H) and ascorbic acid were mixed causing the generation of hydroxyl radicals in a Fenton-type reaction. The advantage of using ascorbic acid instead ofhydrogen peroxide is that ascorbic acid does not interfere with other assays (e.g., LDH assay) which is not the casewith peroxide. The hydroxyl radicals attack the sugar 2-deoxy-D-ribose to produce fragments. Heating the fragments at low pH produces malonaldehyde that, upon the addition of 2-thiobarbituric acid, WO 03/043518 PCT/US02/37136 47 yields a pink chromogen which can be measured spectrophotometrically at 532 nm. Thus, the absorbance at 532 nm is a measure of the damage to 2-deoxy-D-ribose. Establishing optimal Cu(H) and ascorbic acid concentrations was the first step in developing this protocol. First, a constant Cu(IT) concentration of 1 OjuM was used based on 5 this level being the physiological concentration found in the body (bound and unbound Cu(H)). The ascorbic acid concentrations were varied in order to establish a linear range. The ascorbic acid concentration chosen was 500[xM since it gave the most absorbance at 532 nm and still Ml in the linear range. Interestingly, at ascorbic acid concentrations greater than SOOjiM, there was a steady decrease in hydroxyl radicals presumably due to ascorbic acid's 10 dual effect as a hydroxyl radical generator at low concentrations and an antioxidant at high concentrations. Using the aforementioned concentrations forCu(II) and ascorbic acid, a titration curve was established for the D-tetrapeptide. Briefly, the D-tetrapeptide was pre-incubated with .Cu(II) for 15 minutes at room temperature prior to adding ascorbic acid. This was done to 15 permit the D-tetrapeptide to bind with the Cu(H) and therefore inhibit ROS generation. As can be seen from the table, when the Cu(E):D-tetrapeptide ratio was between 4:1 to 4:7, there was little to no inhibition of hydroxyl radical generation. When the ratio was 1:2 or higher, there was total inhibition of hydroxyl radical production. 20 TABLE 2 25 30 Cu(ll):D- cu(iq Ascorbic D-Tetrapeptide Tetra peptide (MM) Acid (uM) (gM) A532 % Inhibition 1:0 10 500 0 0.767 4:1 10 500 2.5 0.751 2:1 10 500 5 0.743 1:1 10 500 10 0.751 4:5 10 500 12.5 0.789 2:3 10 500 15 0.774 4:7 10 500 17.5 0.737 1:2 10 500 20 0.029 96.2 1:4 10 500 40 0.016 97.9 WO 03/043518 PCT/US02/37136 48 EXAMPLE 8: Inhibition Of The Generation Of ROS The ability of the tetrapeptide L-Asp L-Ala L-His L-Lys [SEQ ID NO: 1] and other peptides and compounds to inhibit the production of ROS was tested. The other peptides tested were: L-Asp L-Ala L-His L-Lys L-Ser L-Glu L-Val L-Ala L-His L-Arg L-Phe L-Lys [SEQ ID NO:3]; L-Ala L-His L-Lys L-Ser L-Glu L-Val L-Ala L-ffis L-Arg L-Phe L-Lys [SEQ ID NO:4]; L-His L-Lys L-Ser L-Glu L-Val L-Ala L-His L-Arg L-Phe L-Lys [SEQ ID NO:5]; and Acetylated-L-Asp L-Ala L-His L-Lys L-Ser L-Glu L-Val L-Ala L-His L-Arg L-Phe L-Lys [SEQ ID NO:6]. Hie peptides were obtained from one or more companies that provide custom synthesis of peptides, including Ansynth Services, QCB, Genosys and Bowman Research. The other compounds tested were histidine (Sigma Chemical Co.), catalase (Sigma Chemical Co.), and superoxide dismutase (Sigma Chemical Co.). 1. Tnhfhitirm OfHvdroxvl Radical Production The hydroxyl radical is probably the most reactive oxygen-derived species. The hydroxyl free radical is very energetic, short-lived and toxic. Some researchers suggest that the toxicity of hydrogen peroxide and superoxide radical may be due to their conversion to the hydroxyl free radical. The superoxide radical can be directly converted to the hydroxyl radical via the Haber-Weiss reaction. Alternatively, it can be converted to hydrogen peroxide which, in turn, is converted into the hydroxyl radical via the Fenton reaction. Both pathways require a transition metal, such as copper (Acworth and Bailey, The Handbook Of Oxidative Metabolism (ESA, Inc. 1997)). It is also known that copper, in the presence of ascorbate, produces hydroxyl radicals. The following reaction scheme has been suggested: Ascorbate + 2Cu2+ -+ 2Cu+ + dehydroascorbate + 2H* (Eq. 1) Biaglow et al., Free Radio. Biol. Med., 22(7): 1129-1138 (1997). The ability ofthe compounds listed above to inhibit the generation ofhydroxyl radicals was tested as described in Gutteridge and Wilkins, Biochim. Biophys. Acta, 759:38-41 (1983). Briefly, Cu(B) and ascorbic acid were mixed causing the generation ofhydroxyl radicals. Cu+ + 02-02- + Cu2* Cu+ + 02" + 2H+ -» Cu2+ + H202 Cu+ + HA - 0H'+ Off + Cu2+ (Bq.2) (Eq.3) (Eq.4) WO 03/0435X8 PCT/US02/37136 49 Then, deoxyribose was added, and the hydroxyl radicals, if present, attacked the deoxyribose to produce fragments. Heating the fragments at low pH produced malonaldeliyde that, upon the addition of 2-thiobarbituric acid (TBA), yielded a pink chromogen which was measured spectrophotometrically at 532 nm. Thus, absorbance at 532 nm is a measure ofthe damage 5 to deoxyribose and, therefore, ofhydroxyl radical formation. To perform the assay, CuCl2 in buffer (20 mM KH2P04 buffer, pH 7.4) and either one of the test compounds in buffer or buffer alone were added to test tubes (final concentration of CuCl2 was 10|iM). The test tubes were incubated for 15 minutes at room temperature. Then, 0.5 mM ascorbic acid in buffer and 1.9 mM 2-deoxy-D-ribose in buffer were added to 10 each test tube, and the test tubes were incubated for 1 hour at 37°C. Finally, 1 ml of 1% (w/v) TBA in 50 mM NaOH and 1 ml of concentrated acetic acid were added to each test tube, and the test tubes were incubated in boiling water for 15 minutes. After the test tubes bad cooled for 15 minutes, the absorbance at 532 nm was read. It was found that the tetrapeptide L-Asp L-Ala L-His L-Lys [SEQ ID NO:l] caused 15 complete inhibition of the formation ofhydroxyl radicals in this assay at tetrapeptide/copper ratios of 2:1 or higher. Tetrapeptide/copper ratios less than 2:1 were ineffective. The results of a time course are presented in Figure 8 A As can be seen in Figure 8 A, copper and ascorbate (no added peptide) produced TBA-reactive substances quickly and reached a maximum in 30 minutes. The tetrapeptide at a tetrapeptide/copper ratio of 2:1 20 prevented all formation of TBA-reactive substances. Interestingly, the tetrapeptide at a tetrapeptide/copper ratio of 1:1 slowed the production of TBA-reactive substances. These data suggest that the tetrapeptide at a 1:1 tetrapeptide/copper ratio is able to offer some protection from hydroxyl radicals by binding copper which results in site-directed hydroxyl attack on (he tetrapeptide. Once enough of the tetrapeptide is destroyed, then copper is 25 released, which allows it to produce hydroxyl radicals that attack the dexoyribose. When the tetrapeptide at a tetrapeptide/copper ratio of 2:1 was incubated for longer periods of time, its ability to prevent the formation ofTBA-reactive substances slowly eroded. See Figure 8B. As can be seen from Figure 8B, the production of TBA-reactive substances was inhibited by 95% during the first 4 hours of incubation. By 24 hours, the level of 30 inhibition had dropped to 50% and, by 48 hours, the level of inhibition had dropped to 20%. These data suggest that TBA-reactive substances are still being produced even in the presence WO 03/043518 PCT/US02/37136 50 ofthe tetrapeptide. They also suggest that the tetrapeptide is being degraded during the time course of the experiment This degradation is more than likely due to the formation of free radicals in close proximity to the tetrapeptide/copper complex which attack and degrade the tetrapeptide, with release of the copper. Since free radicals, such as the hydroxyl radical, are 5 very reactive, they will attack the first electron rich molecule they come into contact with, which would be the tetrapeptide in this case. The effect of pH on the inhibition of hydroxyl radical formation by the tetrapeptide was tested at a tetrapeptide/copper ratio of 2:1. At this ratio, the tetrapeptide gave >95% inhibition of the formation of TBA-reactive species at pH 7.0-8.5. These are physiological 10 pH levels andpH levels that would be expected during ischemia (acidosis occurs in ischemic tissues). At pH 6.0, the tetrapeptide was ineffective at preventing the formation of TBA-reactive species, possibly due to the reduced ability of the histidine to hind copper. The nitrogen atom on the imidazole ring of histidine participates in binding copper with a pKa of 6.0. Therefore, at a pH of 6.0, histidine is only able to bind 50% of the copper. The other 15 50% of fee copper would be unbound or loosely bound to the tetrapeptide by the other amino acids and would, therefore, be able to participate in the production of TBA-reactive species. Histidine and several peptides with histidine in different positions were tested at 1:1 and 2:1 peptide:copper ratios for their ability to inhibit the production ofhydroxyl radicals. Also, a peptide having an acetylated aspartic acid (Ac-Asp) as the N-terminal amino acid was 20 also tested. The results are presented in Table 3. In Table 3, the % inhibition is the percent decrease in absorbance compared to buffer alone divided by the absorbance of the buffer alone. As can be seen from the results in Table 3, the peptides with histidine in the second and third positions gave >95% inhibition at a 2:1 peptide:copper ratio, while these peptides 25 at a 1:1 peptidercopper ratio were ineffective. Interestingly, at a 2:1 peptidexopper ratio, the peptide with histidine in the first position and the peptide with acetylated aspartic acid as the N-terminal amino acid provided some protection (about 47% and about 28% inhibition, respectively), although this protection might be attributable to the histidine in the seventh and ninth p ositions, respectively, of these peptides. Histidine alone at a 2:1 histidine: copper ratio 30 provided some protection (about 20% inhibition). Catalase has been shown to prevent hydroxyl radical formation. Gutteridge and WilkinSj Biochim. Biophys. Acta, 759:38-41 (1983); Facchinetti et "al., Cell. Molec. WO 03/043518 51 PCT/US02/37136 Newobiol., 18(6):667-682 (1998); Samuni et al., Eur. J. Biochem., 137:119-124 (1983). Catalase (0-80 nM) was, therefore, tested in this assay, and it was found to prevent the formation of the pink chromogen (data not shown). This finding suggests that hydrogen peroxide is formed in this assay, since catalase breaks down hydrogen peroxide to water and 5 agrees with Equations 3 and 4 above. Catalase also prevents the formation of the pink chiomogen when the L-Asp L-Ala L-His L-Lys [SEQ ID NO:l] tetrapeptide at a tetrapeptide/copper ratio of 1:1 is present (data not shown). As shown above; at this ratio, the copper is still able to participate in the redox, reactions to produce hydroxyl radicals. These experiments show that hydrogen peroxide is an important precursor to the formation of the 10 hydroxyl radical. TABLE 3 15 20 25 30 Compound (Ratio)8 Absorbance at 532 nm Absorbance at 532 nm % Inhibition Copper only (buffer control) 0.767* 0.954 0 Hlstindine/copper (2:1) 0.760 20.3 His Lys Ser Glu Val Ala His Arg Phe Lysb/copper (1:1) 0.716 24.9 His Lys Ser Glu Val Ala His Arg Phe LysVcopper (2:1) 0.509 46.6 Ala His Lys Ser Glu Val Ala His Arg Phe Lys7copper(1:1) 0.843 11.6 Ala His Lys Ser Glu Val Ala His Arg Phe Lys7copper (2:1) 0.047 95.1 Asp Ala His Lys Ser Glu Val Ala His Arg PheLysd/copper(1:1) 0.645 13.2 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lysd/copper (2:1) 0.040 95.6 Ac-Asp Ala His Lys Ser Glu Val Ala His Arg Phe LysVcopper (1:1) 0.633 16.9 Ac-Asp Ala His Lys Ser Glu Val Ala His Arg Phe LysVcopper (2:1) 0.692 27.5 Asp Ala His LysVcopper (1:1) 0.751* 1.3 Asp Ala His Lys'/copper (2:1) 0.029* 96.2 35 a All amino acids are L-amino acids. b SEQ ID NO:5 0 SEQ ID NO:4 d SEQ ID NO:3 8 SEQ ID NO:6 40 fSEQ ID NO:1 * Data taken from Table 2 in Example 7. WO 03/043518 PCT/US02/37136 52 B. Assay For Superoxide Dismutase (SOD) Activity The enzyme superoxide dismutase (SOD) is a naturally-occurring enzyme which, is responsible for the breakdown in the body of superoxide to hydrogen peroxide (similar to Equation 3). Hydrogen peroxide can then be detoxified by catalase. SOD was assayed for activity in the assay described in the previous section and was found to have none (data not shown). This result is not surprising since SOD actually converts superoxide radical into hydrogen peroxide. Hydrogen peroxide can then be converted into the hydroxyl radical by reduced copper. There axe reports in the literature that copper complexes have SOD activity. Athar et al., Biochem. Mol. Biol Int., 39(4):813-821 (1996); Ciuffi et al., Pharmacol Res., 38(4):279-287 (1998); Pogni et al., J. Inorg. Biochem., 73:157-165 (1999); Willingham and Sorenson, Biochem. Biophys. Res. Commun., 150(l):252-258 (1988); Konstantinova et al., Free Rod. Res. Comms., 12-13:215-220 (1991); Goldstein et al., J. Am. Chem. Soc., 112:6489-6492 (1990). This finding is not surprising since SOD itself has copper in its active site. The SOD activity of copper complexes of the tetrapeptide L-Asp L-Ala L-His IrLys [SEQ ID NO: 1] was assayed. Superoxide radicals were produced using the xanthine oxidase assay ofBeauchamp and Fridovich, Anal. Biochem., 44:276-287 (1971). Xanthine oxidase converts xanthine into uric acid, with oxygen acting as an electron acceptor. This causes superoxide radical to be produced. Superoxide radical is able to reduce nitro blue tetrazolium (NBT). Reduced NBT has a knax of 560 nm. It is known that copper inhibits xanthine oxidase activity (Konstantinova et al., Free Rad. Res. Comms., 12-13:215-220 (1991)), so all experiments containing copper also contained ethylenediaminetetracetic acid (EDTA), a known copper chelator. The EDTA-copper complex was tested for SOD activity and was shown to have no SOD activity (data not shown). To perform the assay for SOD activity, 0.1 mM xanthine (Sigma Chemical Co.), 25 jiM NBT (Sigma Chemical Co.), 50 mM sodium carbonate, and 1,2 jiM EDTA (Sigma Chemical Co.), were mixed in a cuvette (all final concentrations, final pH 10.2). The reaction was started by the addition of various amounts of a tetrapeptide-copper complex (tetrapqrtide/copper ratios of 1:1 and 2:1) and 20 nMxanthine oxidase (Sigma Chemical Co.). The tetrapeptide-copper complex was prepared by mixing the tetrapeptide and copper (as CuClj) and allowing the mixture to incubate for 15 minutes at room temperature immediately WO 03/043518 PCT/USII2/37136 53 before addition to the cuvette. The samples were read at time 0 and every 60 seconds for five minutes at 560 nm. The complex of the tetrapeptide with copper at a ratio of 1:1 was shown to have SOD activity, as evidenced by inhibition of NBT reduction (see Figure 9). However, the complex 5 was about 500 times less effective than SOD itself, based on IC50 values (amount that gives 50% inhibition) in this assay. The complex of the tetrapeptide with copper at a ratio of 2:1 was found to have no SOD activity (data not shown). To verify that the 1:1 tetrapeptide-copper complex did not interfere with xanthine oxidase activily, uric acid production was measured at 295 nm. Athar et al., Biochem. Mol. 10 Biol Int., 39(4):813-821 (1996); Ciuffi et al., Pharmacol Res., 38(4):279-287 (1998). This assay is similar to the SOD assay, except that NBT is not present Instead, uric acid is assayed at 295 nm every 60 seconds for 5 minutes. It was found that the 1:1 tetrapeptide-copper complex only inhibited uric acid production by 11% at a concentration of600 nM (data not shown). Therefore, the 1:1 tetrapeptide-copper complex has true SOD activity. Since 15 superoxide is converted to hydrogen peroxide by the complex, this could help to explain why it is not effective at preventing hydroxyl radical production. Superoxide radical production was measured in solutions containing the 1:1 or 2:1 tetrapeptide-copper complexes. The assay combined techniques from the TBA assay and the xanthine oxidase assay. NBT was added to all test tubes in order to quantitate its reduction 20 by superoxide radical. The samples also contained ascorbate and copper and were incubated at 37°C. At 5,15,30 and 60 minutes, the samples were removed from the incubator and read at560nm. The results are shown in Figure 10. In the sample containing the 2:1 tetrapeptide-copper complex, NBT reduction increased over time and reached a maximum at 30 minutes. The sample containing the 1:1 tetrapeptide-copper complex also showed an increase in NBT 25 reduction, with a decreased maximum reached at 60 minutes. These data suggest that superoxide accumulates in the sample containing the 2:1 tetrapeptide-copper contplex, while the 1:1 tetrapeptide-copper complex mimics superoxide dismutase. The likely sequence of events that occurs in the production ofhydroxyl radicals is as follows: 30 02 - 02" - H202 - Off (Eq. 5). WO 03/043518 PCT/US02/37136 54 It has already been shown that the 1:1 tetrapeptide-copper complex can convert superoxide radical (02*~) into hydrogen peroxide (HjOj). This is the SOD activity of the complex. The 2:1 tetrapeptide-copper complex cannot facilitate this conversion since the two molecules of the tetrapeptide fill all six coordination bonds of copper. This explains why the 2:1 5 tetrapeptide-copper complex is so effective because it inhibits the formation of hydrogen peroxide, which could in turn react with reduced copper to produce hydroxyl radicals via the Fenton reaction. The 1:1 tetrapeptide-copper complex also provides a valuable service by eliminating the superoxide radical. Even though it produces hydrogen peroxide, most compartments of the human body have sufficient quantities of the enzyme catalase that can 10 eliminate hydrogen peroxide. In the brain, however, catalase activity is reported to be minimal. Halliwell et al., Methods in Enzymol, 186:1-85 (1990). Therefore, the brain is a particularly vulnerable organ during periods of ischemia, since copper is released due to the acidosis that accompanies ischemia. 15 C. Protection of DNA DNA strandbreaks were measured according to the methodof Asaumi et al., Biochem. Mol. Biol. Int., 39(l):77-86 (1996). Briefly, 17 (Xg/ml of plasmid pBR322DNA was allowed to pre-incubate for 15 minutes at room temperature with 50 fiM CuCl2 and concentrations of the tetrapeptide of 0-200 (iM. Then, 2.5 mM ascorbate was added to each reaction, and the 20 mixture was incubated for 1 hour at 37°C. The total volume of the mixture was 16jiL. Next, 3 jjJL of loading buffer containing 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanole FF, and 40% (w/v) sucrose in water was added. The samples were separated by electrophoresis in a 0.8% agarose gel for 90 minutes at 70 Volts. The gel was stained in IX TBE (Tris-Borate-EDTA buffer) containing 2 (ig/ml ethidium bromide for 30 minutes. The 25 gel was then destained in IX TBE for 5 minutes prior to photographing the gel. The results showed that the tetrapeptide was very effective at preventing the formation of DNA strand breaks. See Figure 11. Optimal protective tetrapeptide:copper ratios were 2:1 and greater, since superhelical circular DNA was still visible on the gel at these ratios. At a tetrapeptide: copper ratios of 1:1 or less, nicked circular DNA, linear DNA and more damaged 30 DNA (smears) were visible. WO 03/043518 PCT/US02/37136 55 EXAMPLE 9: Reduction nf the Damage Done to DNA bv ROS ROS damages DNA by causing strand breaks, base modifications, point mutations, altered methylation patterns, and DNA-protein cross linking (Marnett, Carcinogenesis 21:361-370 (2000); Cerda et al., Mutat. Res. 386:141-152 (1997)). Copper, iron, and other transition 5 metals, in the presence of reducing agents, catalyze the production of ROS such as superoxide (02*), hydrogen peroxide (HjOj) and the hydroxyl radical (OH) through both the Haber-Weiss and Fenton reactions (Stoewe et al., Free Radic. Biol. Med. 3:97-105 (1987)). OH« is considered the most reactive and damaging ROS and is capable of producing all the above DNA lesions (Marnett, Carcinogenesis 21:361-370 (2000)). Previous investigations have 10 reported that OH» induced, single- and double-strand DNA breaks occur during site-specific copper ion reactions in vitro and during excessive copper exposure in vivo (Chiu et al., Biochemistry 34:2653-2661 (1995); Kim et al., Free Radic. Res. 33:81-89 (2000); Hayashi et al., Biochem. Biophys. Res. Comm. 276:174-178, doi: 10.1006/bbrc.2000.3454 (2000)). Telomeres, which are repeats of the hexanucleotide TTAGGG, exist at the ends of 15 DNA to form a "protective cap" against degradation, chromosomal rearrangement, and allow the replication of DNA without the loss of genetic information (Reddel, Carcinogenesis 21:477-484 (2000)). The classical theory of cellular aging, or senescence, involves the telomere aid replication problem (Olovnikov, J. Theor. Biol. 41:181-190 (1973)). DNA polymerase is unable to replicate the terminal end of the lagging strand during DNA 20 replication resulting in the loss of 30-500 base pairs (Harley et al., Nature 345:458-460 (1990); vonZglinicki etal.,£^. Cell Res. 220:186-193, doi:10.1006/excr.l995.1305 (1995)). Somatic cells are unable to replace these lost telomeric repeats, leading to progressive telomere shortening during a cell's replicative life. Senescence is manifested when telomere length reaches a critical threshold (Reddel, Carcinogenesis 21:477-484 (2000)). Premature 25 senescence has been documented in human fibroblasts exposed to oxidative stress (Chen et al., Proc. Nail. Acad. Sci. USA 91:4130-4134 (1994)). Examination of telomere length in fibroblasts after several population doublings under conditions of higher oxidative stress reveals shortened telomere lengths similar to senescence under normal conditions (von Zglimckietal.,JEg>. CellRes. 220:186-193, doi:10.1006/excr.l995.1305 (1995)). Thesedata 30 suggest that ROS-induced DNA damage in the telomere sequence may play an important role in telomere shortening. WO 03/043518 PCT/US02/37136 56 In this study, the ability of Asp Ala His Lys [SEQ ID NQ:1] to protect DNA and telomeres from ROS damage induced by copper coupled with ascorbic acid was examined. A Materials and Methods 5 Reagents: The synthetic D-analog of Asp Ala His Lys (D-Asp Ala His Lys) was obtained from Bowman Research Ltd (Newport, Wales, UK). TeloTAGG Telomere Length Assay and X-ray film were purchased from Roche Molecular Biochemicals (Mannheim, Germany). DNeasy genomic isolation kits were purchased from Qiagen (Valencia, CA). Hybond-N+ nylon membrane was ordered from Amersham Pharmacia Biotech (Piscataway, 10 NJ). All other chemicals were obtained from Sigma (St Louis, MO). DNA treatments: DNA strand breaks were measured using a modified method of Asaumi (Asaumi et al., Biochem, Mol. Biol. Int. 39:77-86 (1996)). Raji cells, a Burkitt lymphoma derived cell line (obtained from American Type Culture Collection (ATCC), Rockville, MD, ascension number CCL-86), were grown in Iscove's modified Dulbecco's 15 medium (1MDM) with 10% fetal calf serum (PCS) at 10% C02 and 37 °C. Genomic DNA was isolated using DNeasy spin columns (Qiagen) following the manufacturer's protocol. Then, 1 jig genomic DNA was incubated per reaction with CuCl^ ascorbic acid, and/or the tetrapeptide in 10 mM sodium phosphate buffer, pH 7.4. Final concentrations were as follows: CuCI2 = 10 fiM, 25 fiM, and 50 fiM; ascorbic acid = 25 (iM, 50 JiM, and 100 fiM; 20 D-Asp Ala His Lys = 50 fiM, 100 jiM, and 200 fiM. Total reaction volumes of 20 p.1 in 0.2 ml PCR tubes were incubated at 37°C for 2 hours. Following the incubation, strand breaks were visualized by immediately adding 5 jxl of loading dye [0.25% (w/v) bromophenol blue and 40% (w/v) sucrose] and loading on a 0.5% tris acetic acidEDTA (TAB) agarose gel. Gels were then run at 70Y for 90 min and stained using 2|ig/ml ethidium bromide for 30 minutes. 25 Prior to photographing, gels were rinsed in TAE for 10 minutes. Cell treatments: Raji cells were washed with PB S (10 mM phosphate buffered saline; 138 mM NaCl; 2.7 mM KC1 pH 7.4). Then, 1.5 x 10s cells were put into 5 ml PBS containing CuCl^ ascorbic acid, and/or D-Asp Ala His Lys. Final concentrations were as follows: CuCl2 = 10 fiM, 25 fiM, and 50 fiM; ascorbic acid =100 jiM, 250 fiM, and 500 fiM; 30 D-Asp Ala His Lys = 50 fiM, 100 fiM, and 200 fiM. The cells were then incubated at 37 °C WO 03/043518 PCT/US02/37136 57 for 2 hours. Following the incubation, genomic DNA was isolated using DNeasy columns. DNA damage was visualized by 0.5% TAE agarose gel electrophoresis. Telomere Length Assay: To examine telomere damage, the TeloTAGG Telomere Length Assay (Roche) was used according to manufacturer's recommendations: digesting Ifig 5 of genomic DNA per reaction using Hinfl and RBAI. Samples were then run on a 0.8% TAE agarose gel at 70V for 2 hours. Southern blots were performed and probed using a digoxigenin (DIG) labeled telomere specific oligonucleotide. For cell treated samples, genomic DNA was used as described above. For DNA treated samples, reactions were setup as above, brought to 200 ja.1 with PBS, and isolated using DNeasy columns prior to restriction 10 digestions. B. Results and Discussion Copper ions, an essential part of chromatin (Dijkwel et al., J. Cell Set 84:53-67 (1986)), are present within DNA (Wacker et al., J. Biol. Chan. 234:3257-3262 (1959)) and may participate in oxidative DNA damage (Chiu et al., Biochemistry 34:2653-2661 (1995); 15 Hayashi et al., Biochem, Biophys. Res. Comm. 276:174-178, doi:10.1006/bbrc.2000.3454 (2000); Kagawa et aL, J. Biol. Chem. 266:20175-20184 (1991)). In the presence of ascorbate or other reducing agents, copper can lead to the production of ROS by catalyzing the following reactions (Biaglow et al., Free Radic. Biol. Med. 22:1129-1138 (1997)): 1) 2 Cu/* + ascorbate - 2 Cu++dehydroascorbate + 2H1" 20 2) Cu+ + 02 -»02*" + Cu2* 3) Cu+ + 02»" + 2H1" -♦ Cu2+ + H202 4) Cu+ + H202 - OH" + OH* + Cu2+ While iron is found at higher concentrations physiologically, oxidation by copper and H202 is 50 times faster than iron (Stoewe et al., Free Radic. Biol Med. 3:97-105 (1987); Halliwell 25 J. Neurochem. 59:1609-1623 (1992)). Due to the negative charge of the sugar phosphate backbone, cations can loosely bind DNA Site-specific binding of copper ions within base pains may be important to the regulation of DNA biosynthesis (Minchenkova et al., Biopolymers 5:615-625 (1967)). Unlike iron-catalyzed reactions, OH* scavengers do not prevent copper-mediated oxidative damage suggesting that oxidative DNA damage occurs in 30 close proximity to the copper ions (Oikawa et al., Biochim. Biophys. Acta 1399:19-30(1998)). The reactivity of OH* is so great that, presumably, OH* interactions only occur at or near the WO 03/043518 PCT/US02/37136 58 site of OH* production (Marnett, Carcinogenesis, 21, 361-370 (2000)). Oikawa, et al., (Oikawa et al., Biochim. Biophys. Acta 1399:19-30 (1998)) have shown that the following copper-mediated ROS reaction also occurs, and that the resulting DNA-copper-peroxide complex may be even more damaging to DNA than OH*: 5 Cu+ + H202 - Cu+OOH + IT As expected, the results of the above-described experiments showed that copper and ascorbic acid alone were unable to cause strand breaks. When CuCl2 and ascorbic acid were combined, a dose dependent accumulation of Iowa* molecular weight DNA fragments was seen, the result of double strandbreaks. These double strand breaks wore attenuated by D-Asp 10 Ala His Lys in a dose dependent manner (Figure 13). At molar ratios of 1:1(50 pM copper to 50 pM D-Asp Ala His Lys) and 1:2S some strand breaks were apparent. By elevating the ratio to 1:4, no strand breaks were detected. Similar results were observed in Raji cells treated with copper and ascorbic acid (Figure 14). A lower ratio of 1:2 (copper to D-Asp Ala His Lys) provided complete protection to DNA in cell samples. It is reasonable to expect that 15 DNA samples would require higher D-Asp Ala His Lys levels due to competition for copper with DNA and proximal OH* attack. The separation of DNA and copper would be critical in these samples necessitating the need for elevated D-Asp Ala His Lys. In cell samples, damage would be attributable to H202. H202 is freely diffusible, can penetrate to the nucleus, and has been shown to damage DNA in fibroblasts (Chen et al., Proc. Natl, Acad. Sci. USA 20 91:4130-4134 (1994); vonZglinicki et&l,FreeRadic. Biol Med. 28:64-74 (2000)). Entrance ofH202into the cell may lead either to the formation of DNA peroxide complexes with native metals or to the release of sequestered metal stores that, combined with endogenous reducing agents (reduced glutathione (GSH), reduced nicotinamide dinucleotide (NADH), and ascorbic acid), would drive the production of OH*. One possible mechanism of D-Asp Ala His Lys 25 protection, would be the chelation of copper ions, thereby preventing production of OH* and H202. Another mode of protection may be the formation of D-Asp Ala His Lys-copper-peroxide complexes which would absorb the OH* damage rather than DNA, "mop-up" peroxides, and perhaps, in cell samples, keep H202 outside the cell. Prior reports suggest that oxidative DNA damage may be directed at G-C rich areas, 30 including telomeres. Rodriguez, et. al., reported that copper induced ROS damage primarily targeted DNA guanine (Rodriguez et al., Cancer Res. 57:2394-2403 (1997)). Strong, WO 03/043518 PCT/U502/37136 59 preferential binding of Cu (II) to the G-C pair has been reported at the N-7 and 0-6 of the guanine bases plus the N-3 of cytosine (Kagawa et al., J Biol. Chem. 266:20175-20184 (1991)). DNA peroxides complexes formed at these positions are believed to direct OH« attack to adjacent bases (Oikawa et al., Biochim. Biophys. Acta 1399:19-30 (1998)). In 5 addition, CjCjCj in tolorncnc 15NA. has been shown to be sensitive to copper mediated ROS damage (Oikawa et al., FEBSLett 453:365-368 (1999)). Examination of the telomere in the genomic DNA samples in the present study showed double strand breaks in response to oxidative stress. DNA samples examined by Southern blot showed severely depleted and shortened telomere sequences (Figure 15). Cell treatments 10 showed damage to the telomere with some conservation of the sequence, even at the highest levels of copper and ascorbic acid used (Figure 16), which may be attributed to ROS production outside the cells with the DNA sheltered inside the nucleus. D-Asp Ala His Lys protected the telomere from copper-mediated damage in these samples. In addition to the double strand breaks detected in the experiments, other DNA lesions 15 may be involved in ROS disease processes. Some cations, including copper, bound loosely to the phosphate backbone have been implicated in strand breaks while those coordinated in the helix cause base modifications (Marnett, Carcinogenesis 21:361-370 (2000); Rodriguez et al., Cancer Res. 57:2394-2403 (1997)). Episodes of increased copper and oxidative stress may direct DNA damage to G-C rich areas. In addition to telomeres, G-C rich areas exist at 20 the 5' end of many genes (Bird, Afeifire 321:209-213 (1986)) hinting toward a site of oxidative damage involved in gene regulation. 8-Oxo-deoxyguanosine (8-oxo-dG) is a common DNA adduct produced by ROS, which can result in G -»• T point mutations widely seen in mutated oncogenes (Marnett, Carcinogenesis 21:361-370 (2000)). Conditions such as acidosis occurring during myocardial ischemia or alterations of ceruloplasmin have been shown to 25 mobilize free copper to catalyze local oxidative tissue and DNA damage (Kim et al., Free Radic. Res, 33:81-89 (2000); Chevion et al., Proc. Natl. Acad. Sci. XJSA 90:1102-1106 (1993)). Levels of 8-oxo-dG are reported to be three to four times higher in the DNA of ischemic rat hearts than in controls (You et al., J. Mol. Cell Cardiol. 32:1053-1059, doi: 10.1006/jmcc.2000.1142 (2000)). In addition, chronic inflammation can produce areas 30 of localized oxidative damage. Inflammatory cells, such as macrophages and neutrophils, release ROS that have been shown to damage the DNA of nearby cells (Shacter et al., WO 03/043518 PCT/US02/37136 60 Carcinogenesis 9:2297-2304 (1988)). Nitric oxide and superoxide released from activated leukocytes can lead to the production of peroxynitrite, which is more reactive with 8-oxo-dG than unmodified bases andpossibly exacerbates the damage (Marnett, Carcinogenesis 21:361-370(2000)). 5 C. Summary Both DNA and the telomeric sequence are susceptible to copper-mediated ROS damage, particularly damage attributed to hydroxyl radicals. In (his study, ROS-induced DNA double strand breaks and telomere shortening were produced by exposure to copper and ascorbic acid. D-Asp-Ala-His-Lys, a copper chelating tetrapeptide D-analog of the N~ 10 terminus ofhuman albumin, attenuated DNA strand breaks in a dose dependent manner. The D-tetrapeptide, at a ratio of 4:1 (peptide:Cu), provided complete protection of isolated DNA and, at a ratio of 2:1 (peptide: Cu.), completely protected Raji Burkitt cells' DNA exposed to copper/ascorbate. Southern blots of DNA treated with copper/ascorbate showed severe depletion mid shortening of telomeres with some conservation of telomere sequences. The 15 D-tetrapeptide provided complete telomere length protection at a ratio of2:l (peptide:Cu). While the exact mechanisms for ROS DNA damage have yet to be fully elucidated, D-Asp Ala His Lys inhibited copper-induced DNA double-strand breaks by ROS in both genomic DNA and in the telomere sequence. 20 EXAMPLE 10: Inhibition Of IL-8 Release Interleukin 8 (IL-8) is a pro-inflammatory cytokine and a potent chemoattractant and activator of neutrophils. It has also been reported to be a chemoattractant and activator of T-lymphocytes and eosinophils. IL-8 is produced by immune cells (including lymphocytes, neutrophils, monocytes and macrophages), fibroblaste and epithelial cells. Reports indicate 25 an important role for IL-8 in the pathogenesis of respiratory viral infections, asthma, bronchitis, emphysema, cystic fibrosis, acute respiratory distress syndrome, sepsis, multiple organ dysfunction syndrome, and other inflammatory disorders. The IL-8 release by Jurkat cells (American Type Culture Collection (ATCC), Rockville, MD) exposed to copper and ascorbic acid (to produce ROS - see Examples 7, 8 30 and 9) was investigated. To do so, 1 x 106 Jurkat cells were incubated at 37 °C and 5% C02 in 0.5 mllMDM medium (ATCC) (serum-free) with insulin transferin selenite solution (ITSS; Sigma) for 24 hours with the following additives. WO 03/043518 PCT/US02/37136 61 Experiment 1: a. None (control); b. Asp Ala His Lys [SEQ ID NO: 1] C'DAHK") - 200 jiM and ascorbic acid - 500 |iM; c. CuCl2- 10 fiM and ascorbic acid - 500 fiM; d. CuCl2- 25 fiM and ascorbic acid - 500 (iM; e. CuClj - 50 jiM and ascorbic acid - 500 fiM; £ CuClj-100 fiM and ascorbic acid - 500 fiM; g. CuCl2 - 50 fiM and DAHK - 50 fiM and ascorbic acid - 500 fiM; L CuCl2 - 50 fiM and DAHK -100 fiM and ascorbic acid - 500 fiM; and i. CuCl2 - 50 fiM and DAHK - 200 fiM and ascorbic acid - 500 fiM. Experiment 2: a. None (control); b. CuCl2-100 fiM; c. DAHK - 200 jiM and ascorbic acid - 500 fiM; d. CuClj - 25 fiM and ascorbic acid - 500 fiM; e. CuCl2- 50 jiM and ascorbic acid - 500 (iM; f. CuCl2- 100 fiM and ascorbic acid - 500 fiM; g. CuCl2 - 50 (iM and DAHK - 50 fiM and ascorbic acid - 500 fiM; h. CuClj - 50 fiM and DAHK -100 fiM and ascorbic acid - 500 fiM; and i. CuCl2-50 fiM and DAHK-200 (iM and ascorbic acid-500 fiM. Experiments: a. None (control); b. CuCVlOOfiM; c. DAHK - 400 fiM and ascorbic acid - 250 fiM; d. CuCl2 - 25 fiM and ascorbic acid - 250 fiM; e. CuCl2- 50 (iM and ascorbic acid - 250 fiM; f. CuCl2- 100 fiM and ascorbic acid - 250 fiM; h. CuCl2 -100 jiM and DAHK - 200 fiM and ascorbic acid - 250 fiM; and i. CuCl2 - 100 fiM and DAHK - 400 fiM and ascorbic acid - 250 fiM. WO 03/043518 PCT/US02/37136 62 After the 24-hour incubation, supematants were collected and the concentration oflL-8 in each supernatant was determined by an BLXSA using human IL-8 matched pair antibodies (Endogen, Cambridge, MA). The ELISA was performed using an ELISA kit from Endogen, Cambridge, MA according to the manufacturer's instructions with the following exceptions: 5 (1) coating antibody at 1 (xg/ml; (2) detecting antibody 30 ng/ml; StrepAvidin HRP diluted 1:32,000. The results are presented in Figure 17A (Experiment 1), Figure 17B (Experiment 2), and Figure 17C (Experiment 3). As can be seen, copper and ascorbic acid caused the release of IL-8 from the cells in a dose-dependent manner. As can also be seen, DAHK inhibited the 10 release of IL-8, with the best results being obtained with an 8:1 DAHK:Cu ratio. EXAMPLE 11: Inhibition Of Oxidation Of CoA Coenzyme A (CoA) is essential for acetylation reactions in the body and, as a consequence, plays a critical role in the metabolism of carbohydrates and fatty acids. CoA can IS be oxidized to a disulfide which cannot participate in acetylation reactions. As a result, metabolism and energy utilization are inhibited. hi this example, it was investigated whether Cu(II) could oxidize CoA and, if so, whether the tetrapeptide Asp Ala His Lys [SEQ ID NO: 1] (Bowman Research, Inc., United Kingdom) could protect CoA (Sigma) from oxidation by Cu(II). The experimental setup and 20 results are presented in Table 4 below. All ofthe ingredients were added simultaneously and, after a 15-minute incubation, absorbance at 412 nm (A412) was measured. Free thiol groups were measured using DTNB. DTNB is dithionitrobenzoic acid (Sigma). As can be seen from Table 4, Cu(U) oxidized CoA. As can also be seen, fee tetrapeptide at a 1:1 tetrapeptide:Cu(H) ratio provided some protection of CoA, and the 25 tetrapeptide at a 2:1 tetrapeptide:Cu(H) ratio provided 100% protection. WO 03/043518 PCT/US02/37136 63 Table 4 1 2 3 4 5 6 7 Asp Ala 50 jil 50 jxl 50 jil 50 fil 50 fil His Lys (190 (190 (190 (190 (190 (2mM) (iM) |iM) fiM) fiM) jiM) CoA (2 50 ill 50 fil 50 jxl 50 nl 50 fil mM) (190 (190 (190 (190 (190 fiM) fiM) jiM) jiM) jiM) CuC^l 100jil 100 fil 100 jil 50 fil mM) (190 fiM) (190 MM) (190 fiM) (85 fiM) Tris 200 jil 300 jil 250 fil 350 jil 350 fil 250 jil 250 jil buffer, 50 mM, pH 8.0 DTNB 125 (il 125 |il 125 Jul 125 jil 125 fil 125 jil 125 jil (3 mM) A412 0.279 1.119 0.127 0.888 0.142 0.113 1.111 EXAMPLE 12: Inhibition Of IL-8 Secretion Bv d-DAHK Systemic inflaxnmatoryresponse syndrome (SIRS) can occur following severe trauma, sepsis, or major surgery and frequently progresses to multiple organ failure, the most common cause of death in surgical intensive care units. Vascular endothelial cells lining Mood vessels have been shown to adversely contribute to early SIRS by secreting excessive amounts of interleukin-8 (IL-8), a potent pro-inflammatory cytokine associated with an increased risk of multiple organ failure and death after severe trauma. McGill et al., World J. Surg. 22,171 (1998); Patrick et al., Am. J. Surg., 172, 425 (1996). Interestingly, endogenous copper is reported to play a central role in post-ischemic reperfixsion injury (Powell et al., Am. J. Physiol 277(3 Pt 2), H956 (1999)), which is also associated with increased IL-8 levels and endothelial dysfunction. However, the role of copper in activating IL-8 secretion from human WO 03/043518 PCT/US02/37136 64 endothelial cells has not previously been identified and maybe important in the pathogenesis of SIRS and multiple organ failure. This example presents data showing for the first time that endothelial cells secrete markedly elevated levels of IL-8 after exposure to a physiologically relevant concentration of 5 copper. Further, addition of a high-affinity Cu(H)-bmding peptide significantly inhibits copper-induced IL-8 secretion from endothelial cells. Copper is an essential human trace element that is closely regulated by plasma proteins, such as ceruloplasmin and albumin, during homeostatic conditions and normal pH. Major trauma or sepsis can lower the pH and produce microvascular and tissue acidosis due 10 to increased tissue oxygen requirements, impaired oxygen extraction, maldistributed blood flow, and diminished energy stores. Mizock et al., Crit. Care Med. 20,80 (1992). The acidic environment subsequently allows Cu(H) ions to be released from carrier proteins (Lamb etal., FEBS Lett. 338,122 (1994)) and to be free to participate in various biochemical pathways such as oxidative stress, inactivation of activated protein C, and inhibition of endothelial 15 nitric-oxide synthase. Bar-Or, et al., Biochem. Biophys. Res. Commun. 290, 1388 (2002); Biancbini et al., J. Biol. Chem. 274,20265 (1999). Human umbilical vein endothelial cells (HUVEC) (5.0 x 104 cells) were incubated in serum-free and ascorbate-free endothelial cell basal medium-2 (EGMJ medium (BioWhittaker) with ITS supplement with (i) copper, (ii) a tetrapeptide analogue of the high-20 affinity, N-terminal Cu(H) binding site ofhuman albumin (D-Asp D-Ala D-His D-Lys or d-DAHK), or (iii) both of them (n=3, in duplicate). IL-8 was determined by ELISA (see Example 10). HUVEC incubated for 24 hours with 25 |iM CuCl2 showed a > 5,5-fold higher IL-8 secretion as compared to controls incubated with water (P < 0.007, t test) (Figure 25). Hie tetrapeptide d-DAHK at 1:1 and 2:1 molar ratios (d-DAHK:copper) inhibited IL-8 25 secretion by 86.1% (P = 0.007) and 102.4% (P = 0.002), respectively, after 24 hours of incubation (Figure 18). Decreased IL-8 secretion by 100 jiM d-DAHK alone after 24 hours of incubation compared to controls was not significant (P = 0.16) (Figure 18). Upon visual examination, all cells appeared to be viable at 24 hours. Preliminary experiments with both human lung microvascular and human iliac artery endothelial cells demonstrated similar 30 results after exposure to copper and d-DAHK Additional HUVEC data showed no copper-induced secretion of tumor necrosis factor-a (TNF-a), prostaglandin Ej or prostacyclin and WO 03/043518 PCT/US02/37136 65 no increase in IL-8 levels after three hours exposure to copper (data not shown). Hie latter result indicates that the copper-induced increase in IL-8 results from synthesis of the IL-8, rather than release of IL-8 from pre-existing storage sites. Analysis of the culture medium after exposure to copper and d-DAHK, alone and 5 together, but without any cells, detected primarily Cu(II) and < 7% Ca(T). This was determined as follows. Cupric(E!) chloride (10-50hM) and d-DAHK (6.25-IOOjj.M) were incubated alone and together in EGM2 medium for 24 hours with 5% C02, then filtered and combined with 400p.M bicinchoninic acid for 1 hour (all at 37°C). Cuprous© chloride standards (0.5-50jiM) were made in water from ImM CuCl stock containing 20mM ascorbate 10 to insure predominance of Cu(I). CuQ was read at 562nm in duplicate (Shimadzu spectrophotometer, Model UV160U). Cu(I) ions catalyze the generation of reactive oxygen species resulting in IL-8 secretion from other cell types. However, the results presented here provide evidence that Cu(D) ions stimulate IL-8 secretion from human endothelial cells independent of oxidative sire®. In 15 addition, a high-affinity Cu(II)-binding compound significantly inhibited copper-induced endothelial cell IL-8 secretion. These data suggest that sequestration of unbound Cu(H) ions could have human therapeutic potential. A possible mechanism for the Cu(II)-induced endothelial IL-8 secretion may be activation of serine-threonine kinase Akt (protein kinase B), which has been reported in 20 human fibroblasts. Ostrakhovitch et al., Arch. Biochem. Biophys. 397, 232 (2002). If a similar pathway is stimulated in human endothelium in vivo, copper could be a major contributor in the development of systemic inflammation by activating nuclear factor-kappaB (NF-kappaB). NF-kappaB is an inflammation transcription factor well known to stimulate high levels of cytokines that significantly augment vascular and cellular inflammatory 25 responses. Additionally, it is possible that sustained or recurring post-ischemic reperfusion injmy and acidosis in the hours after the initial injury could result in persistent Cu(H)-induced IL-8 secretion. It should also be noted that there is evidence that Asp Ala His Lys [SEQ ID NO:l]is degraded in vivo to produce Asp Ala diketopiperazine (DA-DKP) (datanot shown). DA-DKP 30 has been shown to be anti-inflammatory (see U.S. patent application number 09/922,234, filed August 2,2001, and PCT application WO 02/11676, the complete disclosures of which are WO 03/043518 PCT/US02/37136 66 incoiporated herein by reference.), and Asp Ala His Lys [SBQ ID NO: 1 ] may cause additional anti-inflammatory effects as a result of being degraded to produce DA-DKP. EXAMPLE 13: T-nhihitinn Of IL-8 Bv Asp Ala His Lvs in Crevicular Gingival Fluid 5 Cervicular gingival fluid (CGF) was obtained from normal controls (11 individuals) and patients with gingivitis (7 individuals) and periodontitis (9 individuals). Crest Whitestrips™ were applied to the teeth of 5 of the normal controls according to the package instructions. CGF samples were obtained at various times after the Crest Whitestrips™ were applied to the teeth, as indicated below. 10 In a separate experiment, a 4 mM solution of the tetrapeptide L-Asp L-Ala L-His L- Lys [SEQ ID NO: 1] (referred to herein as DAHK-1199) was prepared in phosphate buffered saline (0.1 M sodium phosphate and 0.15 M sodium chloride, pH 7.2), and the pH was adjusted to 7.4. Then 0.1 ml of this solution of DAHK was applied evenly over the surface of each Crest Whitestrip™. The Crest Whitestrips™ were then applied to the teeth of 3 ofthe 15 normal controls according to the package instructions. CGF samples were obtained before the Crest Whitestrips™ with the DAHK-1199 were applied to the teeth and at various times thereafter as indicated below. CGF was collected using dental wicks (Sigma, St Louis, MO). The dental wicks containing the CGF were placed in 150 (i.1 storage buffer (phosphate buffered saline (0.1 M 20 sodium phosphate and 0.15 M sodium chloride, pH 7.2) containing4% bovine serum albumin (Sigma, St Louis, MO), 0.1 mg/ml phenylmethylsulfonyl fluoride (PMSF; Sigma, St Louis, MO), and 0.1 mg/ml aprotinin (Sigma, St Louis, MO) and frozen at -20°C until used. The frozen CGF samples ware thawed on ice and kept at 4°C. Then ELISA assays were performed to determine the amounts of interleukin 8 (IL-8), tumor necrosis factor-a 25 (TNFa), and soluble tumor necrosis factor-a receptor (sTNFR75). Not enough CGF was obtained to perform all of the ELISA assays on all of the samples; the number of samples assayed will be indicated below. The IL-8 ELISA was performed as follows. Anti-human IL-8 antibody (Pierce Endogen, Rockford, IL; catalogue number M801-E, lot number CK41959) was diluted to 1 30 ) ig/ml in phosphate buffered saline, pH 7.2-7.4, and 100 jil ofthe diluted antibody was added to each well of Nunc Maxisorb ELISA strip plates. The plates were incubated overnight at 15 20 25 WO 03/043518 PCT/USG2/37136 67 room temperature. The liquid was aspirated from the wells, and the plates ware blotted on a paper towel. Then, 200 jil of assay buffer (phosphate buffered saline, pH 7.2-7.4, containing 4% bovine serum albumin (Sigma, St Louis, MO; ELIS grade=low fatty acid and IgG)) were added to each well, and the plates were incubated for 1 hour at room temperature. The liquid was aspirated from the wells, and the wells were washed 3 times with wash buffer (50 mM Tris, 0.2% Tween-20, pH 7.9-8.1) and were then blotted on a paper towel. Standards and CGF samples (50 fil/well; standards were diluted in storage buffer) were added to the wells, and the plates were incubated for 1 hour at room temperature with gentle shaking. The liquid was aspirated, the wells were washed 3 times with wash buffer, and the plates were then blotted on a paper towel. Then, 100 jil of biotm-labeled anti-human IL-8 (Pierce Endogen, Rockford, EL; catalogue number M802-E, lot number CE49513), diluted to 60 ng/ml in assay buffer, were added to each well. The plates were incubated for 1 hour at room temperature, the liquid was aspirated, the wells were washed 3 times with wash buffer, and the plates were blotted on a paper towel. Thai, 100 jil of HRP-conjugated streptavidin (Pierce Endogen, Rockford, IL; catalogue number N100) in assay buffer, were added to each well. The plates were incubated for 30 minutes at room temperature, the liquid was aspirated, the wells were washed 3 times with wash buffer, and the plates were blotted on a paper towel. Finally, 100 fil ofTMB substrate solution (Pierce Endogen, Rockford, IL; catalogue number N301) were added to each well. The plates were incubated for 30 minutes at room temperature. The reaction was stopped by adding 100 fil/well of 0.18 M H2S04. The optical densities at 450 nm and 530 nm were read on an ELISA plate reader and the difference (OD 450 - OD 530) calculated. The TNFa ELISA was performed as follows. Anti-human TNFa antibody (Pierce Endogen, Rockford, IL; catalogue number M303-E, lot number 018334) was diluted to 2 ftg/inl in phosphate buffered saline, pH 12-1 A, and 100 jal of the diluted antibody was added to each well of Nunc Maxisorb ELISA strip plates. The plates were incubated overnight at room temperature. The liquid was aspirated from the wells, and the plates were blotted on a paper towel. Then, 200 fil of assay buffer were added to each well, and the plates were incubated for 1 hour at room temperature. The liquid was aspirated from the wells, and the wells were washed 3 times with wash buffer, and the plates were then blotted on a paper towel. Standards and CGF samples (50 fil/well; standards were diluted in storage buffer) were WO 03/043518 PCT/US02/37136 68 added to the wells, and the plates were incubated for 1 hour at room temperature with gentle shaking. The liquid was aspirated, the wells were washed 3 times with, wash buffer, and the plates were then blotted on a paper towel. Then, 100 jul ofbiotin-labeled anti-human TNFa (Pierce Endogen, Rockford, IL; catalogue number M302-B, lot number 017005), diluted to 5 250 ng/ml in assay buffer, were added to each well. The plates were incubated for 1 hour at room temperature, the liquid was aspirated, the wells were washed 3 times with wash buffer, and the plates were blotted on a paper towel. Then, 100 p.1 of HRP-conjugated streptavidin in assay buffer, were added to each well. The plates were incubated for 30 minutes at room temperature, the liquid was aspirated, the wells were washed 3 times with wash buffer, and 10 the plates were blotted on a paper towel. Finally, 100 p.1 of TMB substrate solution were added to each well. The plates were incubated for 30 minutes at room temperature. The reaction was stopped by adding 100 (il/well of 0.18 M H2S04. The optical densities at 450 nm and 530 nm were read on an ELBA plate reader and the difference (OD 450 - OD 530) calculated 15 The STNFR75 ELISA assay was performed using the Quantikine sTNFR75 ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Briefly, 50 pi of each standard and of each CGF sample were added to the wells of plates coated with anti-sTNFR75 antibody, and the plates were incubated for 2 hours atroom temperature. Then, the liquid was aspirated from each well, and the wells were washed three times with 400 20 (U/well of supplied wash buffer. Next, 200 (al sTNFR75 conjugate solution were added to each well, and the plates were incubated at room temperature for 1 hour. Again, the liquid was aspirated from each well and the wells were washed three times with 400 (il/well of supplied wash buffer. After washing was completed, 200 fil of the supplied substrate solution were added to each well, and the plates were incubated for 20 minutes at room temperature. 25 Finally, 50 (i.1 of stop solution were added to each well, and the optical density at 450 nm minus the optical density at 530 nm was determined. With respect to the CGF samples taken without the application of Crest Whitestrips, the IL-8 ELISA was performed on all of the samples (samples from 11 normal controls, 9 periodontitis patients and 7 gingivitis patients), the TNFa EIISA was performed on CGF 30 samples from 2 of the normal controls, 1 of the periodontitis patients and 3 of the gingivitis patients, and the sTNFR75 ELISA was performed on CGF samples from 1 normal control, WO 03/043518 PCT/US02/37136 69 5 periodontitis patients, and 3 gingivitis patients. The IL-8 ELISA was performed on all of the CGF samples taken from the 5 normal controls who had the Crest Whitestrips™ applied to their teeth and on all ofthe CGF samples taken from the 3 normal controls to who had the Crest Whitestrips™ with DAHK 1199 applied to their teeth. AH samples were assayed in duplicate. The results are presented in Figures 19A-B. As can be seen in Figures 19A-C, IL-8 and sTNFR.75 were elevated, and TNFa was decreased in patients with gingivitis and periodontitis, as compared to normal controls. Treatment with Crest Whitestrips™ increased the amount of IL-8 in the CGF of the normal controls by six hours after treatment (see Figure 19D). Treatment with Crest Whitestrips™ with DAHK-1199 reduced the amount of IL-8 in the CGF ofthe normal controls at six hours compared to Crest Whitestrips™ withoutDAHK-1199 (compare Figure 19E with Figure 19D). </div>

Claims

<div class="application article clearfix printTableText" id="claims"> Received @ IPONZ 17 June 2009 70 WE CLAIM:

1. An oral care product comprising one or more dosages of a peptide present in an amount effective for oral care, wherein a single dosage is less than lOOmg of the peptide, and wherein the peptide has the formula: P1-P2, wherein: Pi is: Xaai Xaa2His: or Xaai XaajHis Xaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, [3-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof.

2. An oral care product, wherein the product is a composition comprising a pharmaceutically-acceptable canier and one or more dosages of a peptide present in an amount effective for oral care, wherein a single dosage is less than lOOmg of the peptide, and wherein the peptide has the formula: Pi - P2, wherein: Pi is: Xaai Xaa2His: or Received @ IPONZ 17 June 2009 71 Xaai Xaa2His Xaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof.

3. An oral care product, wherein the product is a composition comprising a pharmaceutically-acceptable carrier and one or more dosages of a peptide present in an amount effective for oral care, wherein a single dosage is less than lOOmg of the peptide, and wherein the peptide has the formula: P1-P2, wherein: Pi is: Xaai Xaa2 His: or Xaai Xaa2His Xaaa; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, Received @ IPONZ 17 June 2009 72 arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof; and the composition is a spray, a solution or an emulsion.

4. An oral care product, wherein the product is a composition comprising a pharmaceutically-acceptable carrier and one or more dosages of a peptide present in an amount effective for oral care, wherein a single dosage is less than lOOmg of the peptide, and wherein the peptide has the formula: Pi -P2, wherein: Pi is: Xaai Xaa2His: or Xaaj Xaa2 His Xaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof; and Received @ IPONZ 17 June 2009 73 the composition is a gel, a paste, an ointment or a cream.

5. An oral care product, wherein the product is a composition comprising a pharmaceutically-acceptable carrier and one or more dosages of a peptide present in an amount effective for oral care, wherein asingle dosage is less than 1 OOmg of the peptide, and wherein the peptide has the formula: P1-P2, wherein: Pi is: Xaai Xaa2His; or Xaai Xaa2His Xaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof; and the composition is a mouth wash, a mouth rinse or a gargle.

6. An oral care product, wherein the product is a composition comprising a pharmaceutically-acceptable carrier and one or more dosages of a peptide present in an amount effective for oral care, wherein a single dosage is less than lOOmg ofthe peptide, and wherein the peptide has the formula: Pi-P2s wherein: Received @ IPONZ 17 June 2009 74 Pi is: Xaai Xaa2His: or Xaai Xaa2His Xaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof; and the composition is a gum, a lozenge or a mint.

7. An oral care product, wherein the product is a composition comprising a pharmaceutically-acceptable carrier and one or more dosages of a peptide present in an amount effective for oral care, wherein a single dosage is less than lOOmg of the peptide, and wherein the peptide has the formula: P1-P2, wherein: Pi is: Xaai Xaa2His: or Xaai Xaa2His Xaa?; P2 is (Xaa4)„; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Received @ IPONZ 17 June 2009 75 Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof; and the composition is a film, a sheet, a patch or a gel that forms a solid in the mouth.

8. An oral care product, wherein the product is a composition comprising a pharmaceutically-acceptable carrier and one or more dosages of a peptide present in an amount effective for oral care, wherein a single dosage is less than lOOmg of the peptide, and wherein the peptide has the formula: Pi - P2, wherein: Pi is: Xaai Xaa2His: or Xaai Xaa2 His Xaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, p-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; Received @ IPONZ 17 June 2009 76 or a physiologically-acceptable salt thereof; and the composition is a dentrifice.

9. An oral care product, wherein the product is a composition comprising: (a) a pharmaceutically-acceptable carrier; (b) a whitening agent; and (c) one or more dosages of a peptide present in an amount effective for oral care, wherein a single dosage is less than lOOmg of the peptide, and wherein the peptide has the formula: Pi-Pa, wherein: Pi is: Xaai XaaiHis; or Xaai Xaa2His Xaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, p-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof.

10. An oral care product, wherein the product is a composition comprising: (a) a pharmaceutically-acceptable carrier; (b) an anti-plaque agent, an anti-calculus agent, an anti-caries agent or an antibacterial agent, or a combination of one or more of the foregoing agents; and Received @ IPONZ 17 June 2009 77 (c) one or more dosages of a peptide present in an amount effective for oral care, wherein a single dosage is less than lOOmg of the peptide, and wherein the peptide has the formula: Pi - P2, wherein: Pi is: Xaai Xaa2His: or Xaai Xaa2His Xaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaai is glycine, alanine, p-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof.

11. An oral care product, wherein the product is a composition comprising a pharmaceutically-acceptable carrier and one or more dosages of a peptide present in an amount effective for oral care, wherein a single dosage is less than lOOmg ofthe peptide, and wherein the peptide has the formula: Pi-Pi, wherein: Pi is: Xaai Xaa2His: or Xaai Xaa2His Xaa3; P2 is (Xaa4)n; Received @ IPONZ 17 June 2009 78 Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, p-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof; and wherein the composition is formulated to provide for slow release of the peptide in the mouth.

12. An oral care product, wherein the product is a device comprising one or more dosages of a peptide present in an amount effective for oral care, wherein a single dosage is less than lOOmg of the peptide, and wherein the peptide has the formula: P1-P2, wherein: Pi is: Xaai Xaa2His: or Xa^ Xaa2His Xaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Received @ IPONZ 17 June 2009 79 Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof.

13. The product of Claim 12 wherein the device is a surgical material.

14. The product of Claim 13 wherein the surgical material is a suture or a sponge,

15. The product of Claim 12 wherein the device is a strip.

16. The product of Claim 15 wherein the strip further comprises a tooth whitening agent.

17. The product of Claim 12 wherein the device is a dental implant.

18. The product of Claim 12 wherein the device is a dental appliance.

19. The product of Claim 18 wherein the device is a dental tray or trough.

20. The product of Claim 12 wherein the device is a floss.

21. The product of any one of Claims 1-20 wherein Xaai is aspartic acid, glutamic acid, arginine, threonine, or a-hydroxymethylserine.

22. The product of any one of Claims 1-20 wherein Xaa2 is glycine, alanine, valine, leucine, isoleucine, threonine, serine, asparagine, methionine, histidine or a-hydroxymethylserine.

23. The product of any one of Claims 1-20 wherein Xaa3 is lysine.

24. The product of any one of Claims 1-20 wherein Xaai is aspartic acid, glutamic acid, arginine, threonine, or a-hydroxymethylserine, Xaa2 is glycine, alanine, valine, leucine, isoleucine, threonine, serine, asparagine, methionine, histidine or a-hydroxymethylserine, and Xaa3 is lysine.

25. The product of Claim 24 wherein Xaai is aspartic acid or glutamic acid and Xaa2 is alanine, glycine, valine, threonine, serine, leucine, or a-hydroxymethylserine.

26. The product of Claim 25 wherein Xaa2 is alanine, threonine, leucine, or a-hydroxymethylserine.

27. The product of Claim 26 wherein Xaai is aspartic acid and Xaa2 is alanine.

28. The product of an one of Claims 1-27 wherein n is 0-5.

29. The product of Claim 28 wherein n is 0. Received @ IPONZ 17 June 2009 80

30. The product of any one of Claims 1-27 wherein P2 comprises a metal-binding sequence.

31. The product of Claim 30 wherein P2 comprises one of the following sequences: (Xaa4)m Xaas Xaa2 His Xaa3; or (Xaa4)m Xaas Xaa2 His; wherein: m is 0-5; and Xaas is an amino acid having a free side-chain -NH2, and (Xaa4)m , if present, or Pi is attached to Xaas by means of the side-chain amino group.

32. The product of Claim 31 wherein Xaas is Orn or Lys.

33. The product of Claim 30 wherein P2 comprises a sequence which binds Cu(I),

34. The product of Claim 33 wherein P2 comprises one of the following sequences: Met Xaa4Met, Met Xaa4Xaa4Met, Cys Cys, Cys Xaa4 Cys, Cys Xaa4 Xaa4 Cys, Met Xaa4 Cys Xaa4 Xaa4 Cys, Gly Met Xaa4 Cys Xaa4 Xaa4 Cys [SEQ ID NO:7], Gly Met Thr Cys Xaa4 Xaa4 Cys [SEQ ID NO:8], Gly Met Thr Cys Ala Asn Cys [SEQ ID NO:9], or y-Glu Cys Gly.

35. The product of Claim 34 wherein P2 is Gly Met Thr Cys Ala Asn Cys [SEQ IDNO;9].

36. The product of any one of Claims 1-27 wherein P2 comprises a sequence which enhances the ability ofthe peptide to penetrate cell membranes.

37. The product of Claim 36 wherein P2 is hydrophobic or an arginine oligomer.

38. The product of any one of Claims 1-37 wherein at least one of the amino acids of Pi other than p-alanine, when present, is a D-amino acid. Received @ IPONZ 17 June 2009 81

39. The product of any one of Claims 1-28 or 30-37 wherein at least one of the amino acids of P2 other than p-alanine, when present, is a D-amino acid.

40. The product of any one of Claims 1-27 wherein at least one amino acid of Pi, at least one amino acid of P^, or at least one amino acid of Pi and at least one amino acid of P2 is substituted with (a) a substituent that increases the lipophilicity of the peptide without altering the ability of Pi to bind metal ions, (b) a substituent that protects the peptide from proteolytic enzymes without altering the ability of Pi to bind metal ions, or (c) a substituent which is a non-peptide, metal-binding functional group that increases the ability of the peptide to bind metal ions.

41. The product of any one of Claims 1-27 wherein n is 0 and Pi has one of the following formulas: ch2c02h chc02h co I nh I co nh r1—ch I co h cooh h cooh Received @ IPONZ 17 June 2009 82 ch2co2h h2n-ch I co I nh I H3C—CH I co nh I H2c—CH cor2 ch2co2h (r3)2n-ch co I nh I h3c—ch co I nh I h2c—ch I cooh h2n-ch co I nh chc02h hac- h2c~ -ch I co nh -ch I co I nh I ch~(ch2)4nh2 co2h ch2co2h h2n-ch co I nh I ri—ch I co I nh I H2c—ch co I nh ch-(ch2)4nh2 co2h Received @ IPONZ 17 June 2009 83 ch2c02h ch2co2h (r3)2n-ch co I nh I co I nh I H3c—CH I co Oh N nh h2c—ch h nh h2c—ch co h nh I ch-(ch2)4n(r3)2 co2h nh ch-(ch2)4nh2 cor2 wherein: Ri is an alkyl, aryl, or heteroaryl; R2 is -NH2, -NHRj, N(Ri)2, -ORi, or Ri; and R3 is H, a non-peptide, metal-binding functional group or the two R3 groups together form a non-peptide, metal-binding functional group.

42. The product of any one of claims 1 to 41, wherein the oral care product is adapted to provide single dosage of less than 30mg.

43. A kit comprising a product according to any one of Claims 1-42 or a combination of such products.

44. A use of a peptide in the manufacture of an oral care product for treating an inflammatory disease or condition of an animal's mouth, the peptide being present in the product in an amount effective for oral care, the peptide having the formula: Pi - P2, wherein: Pi is: Xaai Xaa2His: or Xaai Xaa2 His Xaa3; P2 is (Xaa4)„; Received @ IPONZ 17 June 2009 84 Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof,

45. The use of Claim 44 wherein the disease or condition is a disease or condition of the periodontal tissue.

46. The use of Claim 45 wherein the disease or condition is gingivitis or periodontitis.

47. The use of Claim 44 wherein the disease or condition is an infection.

48. A use of a peptide in the manufacture of an oral care product for treating inflammation of a tissue of an animal's mouth, the peptide being present in the oral care product in an amount effective for oral care, the peptide having the formula: Pi-P2, wherein: Pi is: Xaai Xaa2His: or Xaai Xaa2His Xaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Received at IPONZ on 533595 85 Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof.

49. The use of Claim 48 wherein the inflammation is inflammation of the periodontal tissue.

50. The use of any one of Claims 48 or 49 wherein the oral care product is to be used prophylactically.

51. The use of Claim 50 wherein the oral care product is to be used as part of a prophylactic oral care regimen.

52. The use of Claim 48 wherein the oral care product is to be used prior to surgery, during surgery, after surgery, or combinations thereof.

53. The use of Claim 48 wherein the oral care product is to be used prior to a tooth extraction, during a tooth extraction, after a tooth extraction, or combinations thereof.

54. The use of Claim 48 wherein the tissue is all or substantially all of the tissue of the mouth.

55. A use of a peptide in the manufacture of an oral care product for reducing the damage done by reactive oxygen species to a tissue of an animal's mouth, the peptide being present in the oral care product in an amount effective for oral care, the peptide having the formula: P1-P2, wherein: Pi is: Xaai Xaa2His: or Xaai Xaa2His Xaaa; P2 is (Xaa4)n; Received @ IPONZ 17 June 2009 86 Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof.

56. A use of a peptide in the manufacture of an oral care product for reducing the concentration of a metal in or on a tissue of an animal's mouth, the peptide being present in the oral care product in an amount effective for oral care, the peptide having the formula; P1-P2, wherein; Pt is: Xaai Xaa2His: or Xaai Xaa2His Xaas; P2 is (Xaa4)„; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Received @ IPONZ 17 June 2009 87 Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and nis 0-10; or a physiologically-acceptable salt thereof.

57. A use of a peptide in the manufacture of an oral care product to be employed in connection with the whitening of one or more teeth of an animal, the peptide being present in the oral care product in an amount effective for oral care, the peptide having the formula: P1-P2, wherein: Pi is: Xaai Xaa2His: 01* Xaai XaaaHisXaaj; P2 is (Xaa4)„; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof.

58. The use of Claim 57 wherein the product is contacted with a tissue of the animal's mouth prior to whitening of the teeth, during whitening of the teeth, after whitening of the teeth, or combinations thereof. Received @ IPONZ 17 June 2009 88

59. The use of Claim 57 wherein the tissue is all or substantially all of the tissue of the mouth.

60. The use of any one of Claims 57-59 wherein the product also comprises a tooth whitening agent.

61. The use of any one of Claims 43-60 wherein Xaai is aspartic acid, glutamic acid, arginine, threonine, or a-hydroxymethylserine.

62. The use of any one of Claims 43-60 wherein Xaa2 is glycine, alanine, valine, leucine, isoleucine, threonine, serine, asparagine, methionine, histidine or a-hydroxymethylserine,

63. The use of any one of Claims 43-60 wherein Xaa3 is lysine.

64. The use of any one of Claims 43-60 wherein Xaai is aspartic acid, glutamic acid, arginine, threonine, or a-hydroxymethylserine, Xaa2 is glycine, alanine, valine, leucine, isoleucine, threonine, serine, asparagine, methionine, histidine or a-hydroxymethylserine, and Xaa3 is lysine,

65. The use of Claim 64 wherein Xaai is aspartic acid or glutamic acid and Xaa2 is alanine, glycine, valine, threonine, serine, leucine, or a-hydroxymethylserine.

66. The use of Claim 65 wherein Xaa2 is alanine, threonine, leucine, or a-hydroxymethylserine.

67. The use of Claim 66 wherein Xaai is aspartic acid and Xaa2 is alanine.

68. The use of any one of Claims 43-67 wherein n is 0-5.

69. The use of Claim 68 wherein n is 0.

70. The use of any one of Claims 43-67 wherein P2 comprises a metal-binding sequence,

71. The use of Claim 70 wherein P2 comprises one of the following sequences: (Xaa4)m Xaa5 Xaa2 His Xaa3; or (Xaa4)m Xaas Xaa2 His; wherein: m is 0-5; and Xaas is an amino acid having a free side-chain -NH2, and (Xaa4)m , if present, or Pi is attached to Xaas is by means of the side-chain amino group.

72. The use of Claim 71 wherein Xaa5 is Orn or Lys.

73. The use of Claim 70 wherein P2 comprises a sequence which binds Cu(I). Received @ IPONZ 17 June 2009 89

74. The use of Claim 73 wherein P2 comprises one of the following sequences: Met Xaa4Met, Met Xaa4Xaa4Met, Cys Cys, Cys Xaa4 Cys, Cys Xaa4 Xaa4 Cys, Met Xaa4 Cys Xaa4 Xaa4 Cys, Gly Met Xaa4 Cys Xaa4 Xaa4 Cys [SEQ ID NO:7], Gly Met Thr Cys Xaa4 Xaa4 Cys [SEQ ID NO:8], Gly Met Thr Cys Ala Asn Cys [SEQ ID NO:9], or y-Glu Cys Gly.

75. The use of Claim 74 wherein P2 is Gly Met Thr Cys Ala Asn Cys [SEQ ID NO:9].

76. The use of any one of Claims 43-67 wherein P2 comprises a sequence which enhances the ability ofthe peptide to penetrate cell membranes.

77. The use of any one of Claims 76 wherein P2 is hydrophobic or an arginine oligomer.

78. The use of any one of Claims 43-77 wherein at least one of the amino acids of Pi other than p-alanine, when present, is a D-amino acid.

79. The use of any one of Claims 43-68 or 70-78 wherein at least one of the amino acids of P2 other than P-alanine, when present, is a D-amino acid.

80. The use of any one of Claims 43-67 wherein at least one amino acid of Pi, at least one amino acid of P2, or at least one amino acid of Pi and at least one amino acid of P2 is substituted with (a) a substituent that increases the lipophilicity of the peptide without altering the ability of Pi to bind metal ions, (b) a substituent that protects the peptide from proteolytic enzymes without altering the ability of Pi to bind metal ions, or (c) a substituent which is a non-peptide, metal-binding functional group that increases the ability of the peptide to bind metal ions.

81. The use of any one of Claims 43-67 wherein n is 0 and Pi has one of the following formulas: Received @ IPONZ 17 June 2009 90 chco2h h2n-ch co nh h3c—ch co I NH I H2c—CH cooh ch2c02h h2n-ch I co I nh I r-,—ch co I nh I h2c—ch cooh ch2c02h h2n-ch co I nh ! h3c—ch I co n—a nh < )-h2c-ch h cor2 ch2co2h (r3)2n-ch co I nh I h3c—ch 3 I co nh I h2c—ch cooh Received @ IPONZ 17 June 2009 91 chco2h h2n-ch I co I nh hac- -ch co nh I H2c—ch I co nh I ch-(ch2)4nh2 co2h N h ch2co2h h2n-ch co nh I r-i—ch I co I nh I -H2c—ch I co I nh I ch-(ch2)4nh2 co2h ch2co2h h2n-ch co nh I H3c—ch co n—nh 7-H2C-CH XNX h co I nh I ch-(ch2)4nh2 cor2 ch2c02h I (r3)2n-ch co I nh I h3c—ch ^ ^h2C-i co nh I ch I co I nh I ch-(ch2)4n(r3)2 co2h wherein: Ri is an alkyl, aryl, or heteroaryl; R2 is -NH2, -NHRi, N(Ri)2, -ORi, or Ri; and R3 is H, a non-peptide, metal-binding functional group or the two R3 groups together form a non-peptide, metal-binding functional group.

82. The use of any one of Claims 43-81 wherein the product is an oral care device. Received @ IPONZ 17 June 2009 92

83. The use of any one of Claims 43-81 wherein the product is an oral care composition.

84. The use of Claim 83 wherein the composition is a gel, a paste, an ointment, a cream, a powder, a dentrifice, a wash, a rinse, a gargle, a spray, a solution, a tablet, a gum, a lozenge, a mint, a film, or a patch.

85. A method of treating a tissue of a nonhuman animal's mouth comprising contacting the tissue with an oral care product comprising a peptide, the peptide being present in the oral care product in an amount effective for oral care, the peptide having the formula: P1-P2, wherein: Pi is: Xaai Xaa2His: or Xaai Xaa2 His Xaas; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof.

86. The method of Claim 85 wherein the tissue is treated prophylactically.

87. The method of Claim 86 wherein the tissue is treated as part of a prophylactic oral regimen. Received @ IPONZ 17 June 2009 93

88. The method of Claim 85 wherein the tissue is treated prior to surgery, during surgery, after surgery, or combinations thereof.

89. The method of Claim 85 wherein the tissue is treated prior to a tooth extraction, during a tooth extraction, after a tooth extraction, or combinations thereof.

90. The method of Claim 85 wherein the tissue is all or substantially all of the tissue of the mouth.

91. A method of treating a disease or condition of a tissue of a nonhuman animal's mouth comprising contacting the tissue with an oral care product comprising a peptide, the peptide being present in the oral care product in an amount effective for oral care, the peptide having the formula: P1-P2, wherein: Pi is: Xaai Xaa2His: or Xaai Xaa2 His Xaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, p-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof.

92. The method of Claim 91 wherein the disease or condition is a disease or condition of the periodontal tissue. Received @ IPONZ 17 June 2009 94

93. The method of Claim 92 wherein the disease or condition is gingivitis or periodontitis.

94. The method of Claim 91 wherein the disease or condition is an infection.

95. A method of treating inflammation of a tissue of a nonhuman animal's mouth comprising contacting the tissue with an oral care product comprising a peptide, the peptide being present in the oral care product in an amount effective for oral care, the peptide having the formula: Pi - P2, wherein: Pi is: Xaai Xaa2His: or Xaai Xaa2 His Xaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, p-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof.

96. The method of Claim 95 wherein the inflammation is inflammation of the periodontal tissue,

97. A method of whitening one or more teeth of a nonhuman animal comprising contacting a tissue of the animal's mouth with an oral care product comprising a peptide, the peptide being present in the oral care product in an amount effective for oral care, the peptide having the formula: Received @ IPONZ 17 June 2009 95 Pi " P2, wherein: Pi is: Xaai Xaa2His: or Xaai Xaa^ His Xaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof.

98. The method of Claim 97 wherein the tissue is all or substantially all of the tissue of the mouth.

99. The method of Claim 97 or 98 wherein the tissue is contacted with the product prior to whitening of the teeth, during whitening of the teeth, after whitening of the teeth, or combinations thereof.

100. The method of any one of Claims 97-99 wherein the product also comprises a tooth whitening agent.

101. A method of reducing the damage done by reactive oxygen species to a tissue of a nonhuman animal's mouth comprising contacting the tissue with an oral care product comprising a peptide, the peptide being present in the oral care product in an amount effective for oral care, the peptide having the formula: P1-P2, wherein: Received @ IPONZ 17 June 2009 96 Pi is: Xaai Xaa2His: or Xaai Xaa2His Xaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof.

102. A method of reducing the concentration of a metal in or on a tissue of a nonhuman animal's mouth comprising contacting the tissue with an oral care product comprising a peptide, the peptide being present in the oral care product in an amount effective for oral care, the peptide having the formula: P1-P2, wherein: Pi is: Xaai Xaa2 His: or Xaai Xaa2His Xaas; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Received @ IPONZ 17 June 2009 97 Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; and n is 0-10; or a physiologically-acceptable salt thereof.

103. The method of any one of Claims 85-102 wherein Xaai is aspartic acid, glutamic acid, arginine, threonine, or a-hydroxymethylserine.

104. The method of any one of Claims 85-102 wherein Xaa2 is glycine, alanine, valine, leucine, isoleucine, threonine, serine, asparagine, methionine, histidine or a-hydroxymethylserine,

105. The method of any one of Claims 85-102 wherein Xaa3 is lysine.

106. The method of any one of Claims 85-102 wherein Xaai is aspartic acid, glutamic acid, arginine, threonine, or a-hydroxymethylserine, Xaa2 is glycine, alanine, valine, leucine, isoleucine, threonine, serine, asparagine, methionine, histidine or a-hydroxymethylserine, and Xaa3 is lysine.

107. The method of Claim 106 wherein Xaai is aspartic acid or glutamic acid and Xaa2 is alanine, glycine, valine, threonine, serine, leucine, or a-hydroxymethylserine.

108. The method of Claim 107 wherein Xaa2 is alanine, threonine, leucine, or a-hydroxymethylserine.

109. The method of Claim 108 wherein Xaai is aspartic acid and Xaa2 is alanine.

110. The method of any one of Claims 85-109 wherein n is 0-5.

111. The method of Claim 110 wherein n is 0.

112. The method of any one of Claims 85-109 wherein P2 comprises a metal-binding sequence.

113. The method of Claim 112 wherein P2 comprises one of the following sequences; (Xaa4)m Xaa5 Xaa2 His Xaa3; or (Xaa4)m Xaas Xaa2 His; Received @ IPONZ 17 June 2009 98 wherein: m is 0-5; and Xaas is an amino acid having a free side-chain -NH2, and (Xaa4)m , if present, or Pi is attached to Xaas by means ofthe side-chain amino group.

114. The method of Claim 113 wherein Xaas is Orn or Lys.

115. The method of Claim 112 wherein P2 comprises a sequence which binds Cu(I).

116. The method of Claim 115 wherein P2 comprises one of the following sequences: Met Xaa4Met, Met Xaa4Xaa4Met, Cys Cys, Cys Xaa4 Cys, Cys Xaa4 Xaa4 Cys, Met Xaa4 Cys Xaa4 Xaa4 Cys, Gly Met Xaa4 Cys Xaa4 Xaa4 Cys [SEQ ID NO:7], Gly Met Thr Cys Xaa4 Xaa4 Cys [SEQ ID NO:8], Gly Met Thr Cys Ala Asn Cys [SEQ ID NO:9], or y-Glu Cys Gly.

117. The method of Claim 116 wherein P2 is Gly Met Thr Cys Ala Asn Cys [SEQ ID NO:9].

118. The method of any one of Claims 85-109 wherein P2 comprises a sequence which enhances the ability of the peptide to penetrate cell membranes.

119. The method of any one of Claims 85-109 wherein P2 is hydrophobic or an arginine oligomer.

120. The method of any one of Claims 85-119 wherein at least one of the amino acids of Pt other than P-alanine, when present, is a D-amino acid.

121. The method of any one of Claims 85-110 or 112-120 wherein at least one of the amino acids of P2 is a D-amino acid.

122. The method of any one of Claims 85-109 wherein at least one amino acid of Pi, at least one amino acid of P2, or at least one amino acid of Pi and at least one amino acid of P2 is substituted with (a) a substituent that increases the lipophilicity of the Received @ IPONZ 17 June 2009 99 peptide without altering the ability of Pi to bind metal ions, (b) a substituent that protects the peptide from proteolytic enzymes without altering the ability of Pi to bind metal ions, or (c) a substituent which is a non-peptide, metal-binding functional group that increases the ability of the peptide to bind metal ions.

123. The method of any one of Claims 85-109 wherein n is 0 and Pi has one of the following formulas: ch2c02h chco2h co I nh I nh r.)—ch I co HoC—CH I CO h cooh h cooh ch2c02h ch2co2h (r3)2n-ch CO I nh I co I nh I H cor2 Received @ IPONZ 17 June 2009 100 \ chco2h h2n-ch co I nh 1 h3c—ch I co nh I h2c—ch co I nh ch-(ch2)4nh2 co2h ch2co2h h2n-ch I co I nh I r-t—ch I co I nh I h2c—ch co I nh I ch-(ch2)4nh2 co2h ch2co2h h2n-ch co I nh I h3c—ch I co I nh I h2c—ch co I nh I ch-(ch2)4nh2 cor2 ch2co2h (r3)2n-ch co I nh I H3c—ch co nh I h2c—ch I co I nh I ch-(ch2)4n(r3)2 co2h wherein: Ri is ail alkyl, aryl, or heteroaryl; R2 is -NH2, -NHR1; N(Ri)2> -ORi, or Ri; and R3 is H. a non-peptide, metal-binding functional group or the two R3 groups together form a non-peptide, metal-binding functional group.

124. The method of any one of Claims 85-123 wherein the product is an oral care device. Received @ IPONZ 17 June 2009 101

125. The method of any one of Claims 85-123 wherein the product is an oral care composition.

126. The method of Claim 125 wherein the composition is a gel, a paste, an ointment, a cream, a powder, a wash, a rinse, a gargle, a spray, a solution, a tablet, a gum, a lozenge, a mint, a film, or a patch.

127. A peptide having the formula: P1-P2, wherein: Pi is: Xaai Xaa2His: or Xaai Xaa2HisXaa3; P2 is (Xaa4)n; Xaai is glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, isoaspartic acid, asparagine, glutamic acid, isoglutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa2 is glycine, alanine, P-alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, or a-hydroxymethylserine; Xaa3 is glycine, alanine, valine, lysine, arginine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine or tryptophan; Xaa4 is any amino acid; n is 0-10; and P2 comprises a sequence that binds Cu(I).

128. The peptide of Claim 127 wherein Xaai is aspartic acid, glutamic acid, arginine, threonine, or a-hydroxymethylserine.

129. The peptide of Claim 127 wherein Xaa2 is glycine, alanine, valine, leucine, isoleucine, threonine, serine, asparagine, methionine, histidine or a-hydroxymethylserine.

130. The peptide of Claim 127 wherein Xaa3 is lysine. Received @ IPONZ 17 June 2009 102

131. The peptide of Claim 127 wherein Xaai is aspartic acid, glutamic acid, arginine, threonine, or a-hydroxymethylserine, Xaa2 is glycine, alanine, valine, leucine, isoleucine, threonine, serine, asparagine, methionine, histidine or a-hydroxymethylserine, and Xaa3 is lysine.

132. The peptide of Claim 131 wherein Xaai is aspartic acid or glutamic acid and Xaa2 is alanine, glycine, valine, threonine, serine, leucine, or a-hydroxymethylserine.

133. The peptide of Claim 132 wherein Xaa2 is alanine, threonine, leucine, or a-hydroxymethylserine.

134. The peptide of Claim 133 wherein Xaai is aspartic acid and Xaa2 is alanine.

135. The peptide of any one of Claims 127-134 wherein P2 comprises one ofthe following sequences: Met Xaa4Met, Met Xaa4 Xaa4 Met, Cys Cys, Cys Xaa4 Cys, Cys Xaa4 Xaa4 Cys, Met Xaa4 Cys Xaa4 Xaa4 Cys, Gly Met Xaa4 Cys Xaa4 Xaa4 Cys [SEQ ID NO:7], Gly Met Thr Cys Xaa4 Xaa4 Cys [SEQ ID NO:8], Gly Met Thr Cys Ala Asn Cys [SEQ ID NO:9], or y-Glu Cys Gly.

136. The peptide of Claim 135 wherein P2 is Gly Met Thr Cys Ala Asn Cys [SEQ IDNO:9].

137. The peptide of any one of Claims 127-136 wherein at least one of the amino acids of Pi other than p-alanine, when present, is a D-amino acid.

138. The peptide of any one of Claims 127-137 wherein at least one of the amino acids of P2 is a D-amino acid.

139. An oral care product comprising a peptide according to any one of Claims 127-138.

140. The oral care product of Claim 139 wherein the product is an oral care device. Received @ IPONZ 17 June 2009 103

141. The oral care product of Claim 139 wherein the product is an oral care composition.

142. An oral care product as claimed in claim 1, substantially as herein described with reference to the accompanying Examples.

143. A use of a peptide in the manufacture of an oral care product for treating a disease or condition of a tissue of an animal's mouth as claimed in claim 44 or 48, substantially as herein defined with reference to the accompanying Examples.

144. A use of a peptide in the manufacture of an oral care product for: treating inflammation of a tissue damage done by reactive oxygen species; reducing the concentration of a metal or whitening teetch in or on the tissue of an animals's mouth as claimed in claims 55, 56, 57 or 53 respectively, substantially as herein defined with reference to the accompanying Examples.

145. A method of treating a tissue of a non-human animals's mouth as claimed in claim 85, substantially as herein defined with reference to the accompanying Examples.

146. A method of whitening one or more teeth in a non-human's mouth as claimed in claim 97, substantially as herein defined with reference to the accompanying Examples.

147. A method of reducing damage done by reactive oxygen species to a tissue of a non-human animal's mouth or reducing the concentration of a metal in or on a tissue of a non-human animal's mouth as claimed in claims 101 or 102 respectively substantially as herein defined with reference to the accompanying Examples.

148. A peptide according to claim 127 and substantially as herein defined with reference to the accompanying Examples. </div>