Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
  • A61P39/04—Chelating agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
  • A61P39/06—Free radical scavengers or antioxidants

Definitions

• the invention relates to methods, oral care products and kits for treating mouth tissues.
• 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.
• ROS reactive oxygen species
• ROS Reactive oxygen species
• free radicals e.g., superoxide anion and hydroxyl, peroxyl, and alkoxyl radicals
• 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 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).
• Metal ions can cause the production and accumulation of ROS.
• copper and iron ions released from storage sites are one of the main causes of the production of ROS following injury, including ischemia/reperfusion injury and injury due to heat, cold, trauma, excess exercise, toxins, radiation, and infection.
• Copper and iron ions, as well as other transition metal ions have been reported to catalyze the production of ROS. See, e.g., Stohs, J. Basic Clin. Physiol.
• 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.
• ROS may also be present in the mouth as a result of 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.
• 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. Pharmacol, 6, 205-228 (1995); Dunphy et al, Am. J. Physiol, 276, H1591-H1598 (1999)).
• albumin The antioxidant character of albumin has 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 steroids, to bind and transport bilirubin, to scavenge HOC1, and others. See, e.g., Halliwell and Gutteridge, Arch. Biochem. Biophys., 280, 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.
• Albumin contains several metal binding sites, including one at the N-terminus.
• albumin is, and is not, neuroprotective in animal models of cerebral ischemia. Compare Huh et al., Brain Res., 804, 105-113 (1998) and Remmers et al., Brain Res. , 827, 237-242 (1999), with Little et al., Neurosurgery, 9, 552- 558 (1981) and Beaulieu et al., J. Cereb. Blood Flow. Metab., 18, 1022-1031 (1998).
• albumin has been characterized as an antioxidant, it has also been reported to enhance superoxide anion production by microglia (Si et al., GLIA, 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/reperfusion and some neurodegenerative diseases.
• the N-terminal metal-binding sites of several albumins exhibit high- affinity for Cu(H) and NiQI). These sites have been studied extensively, and a general amino terminal Cu(II)- and Ni( ⁇ )-binding (ATCUN) motif has been identified. See, e.g., Harford and Sarkar, Ace. Chem. Res., 30, 123-130 (1997).
• the ATCUN motif can be defined as being present in a protein or peptide which has a free -NH 2 at the N-terminus, a histidine residue in the third position, and two intervening peptide nitrogens. See, e.g., Harford and Sarkar, Ace. Chem. Res., 30, 123-130 (1997).
• 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, Ace. Chem. Res., 30, 123-130 (1997).
• the Cu(H) and Ni(II) are bound by four nitrogens provided by the three amino acids of the ATCUN motif (the nitrogen of the free -NH 2 , the two peptide nitrogens, and an imidazole nitrogen of histidine) in a slightly distorted square planar configuration. See, e.g., Harford and Sarkar, Ace. Chem. Res., 30, 123-130 (1997).
• the sequence ofthe N-terminal metal-binding site of human serum albumin is Asp Ala His Lys [SEQ ID NO:l], and the free side-chain carboxyl ofthe N-terminal Asp and the Lys residue have been reported to be involved in the binding of Cu( ⁇ ) and Ni(H), in addition to the four nitrogens provided by Asp Ala His. See Harford and Sarkar, Ace. Chem. Res., 30, 123-130 (1997); Laussac etal.,5/oc/zem., 23,2832- 2838 (1984); and Sadler et al., Eur. J. Biochem., 21SS, 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, Ace. Chem. Res., 30, 123-130 (1997); Bal etal., Chem. Res. ToxicoL, 10, 906-914 (1997); M ⁇ ynarz, eta ⁇ ., Speciation 98: Abstracts, http://www.jate.u-szeged.hu/ ⁇ spec98/abstr/mlynar.html.
• 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 Pj - P 2 or a physiologically-acceptable salt thereof.
• the invention also provides an oral care product comprising the peptide P, - P 2 , 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 j - P 2 :
• P is Xaa, Xaa 2 His or Xaaj Xaa ⁇ is Xaa 3 ; and P 2 is (Xaa 4 ) n .
• Xaaj 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 Xaa 2 through its ⁇ -carboxyl, hereinafter “isoAsp”), asparagine (Asn), glutamic acid (Glu), isoglutamic acid (i.e., Glu attached to Xas ⁇ through its ⁇ -carboxyl, hereinafter “isoGlu”), glutamine (Gin), lysine (Lys), hydroxylysine (Hylys), histidine (His), arginine (Arg), omithine (Orn), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), cysteine (Cys), methionine
• Xaa can be an amino acid which comprises a ⁇ -amino group (e.g., Orn, Lys) having another amino acid or a peptide attached to it (e.g., Gly ( ⁇ )-Orn).
• Xaa is preferably Asp, Glu, Arg, Thr, or HMS. More preferably, Xaa ! is Asp or Glu. Most preferably Xaa j is Asp.
• Xaa 2 is Gly, Ala, ⁇ -Ala, Val, Leu, He, Ser, Thr, Asp, Asn, Glu, Gin, Lys, Hylys, His, Arg, Orn, Phe, Tyr, Trp, Cys, Met or HMS.
• Xaa 2 is preferably Gly, Ala, Val, Leu, He, Thr, Ser, Asn, Met, His or HMS. More preferably Xaa 2 is Ala, Val, Thr, Ser, Leu, or HMS. Even more preferably Xaa 2 is Ala, Thr, Leu, or HMS. Most preferably Xaa 2 is Ala.
• Xaa 3 is Gly, Ala, Val, Lys, Arg, Orn, Asp, Glu, Asn, Gin, or Trp, preferably Lys.
• Xaa 4 is any amino acid.
• n is 0-100, preferably 0-10, more preferably 0-5, and most preferably 0.
• One or more of the amino acids of P, and/or P 2 can be substituted with (a) a substituent that increases the lipophilicity ofthe peptide without altering the ability of P ! to bind metal ions, (b) a substituent that protects the peptide from proteolytic enzymes without altering the ability of P j to bind metal ions, or (c) a substituent which is a non-peptide, metal- binding functional group that improves the ability ofthe 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 (MBP) having attached thereto a non-peptide, metal-binding functional group.
• MBP metal-binding peptide
• the metal- binding peptide MBP maybe any metal-binding peptide, not just P, - P 2 .
• 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.
• 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 ofthe formula P 3 - L - P 3 , wherein each P 3 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 P 3 peptides through their C-terminal amino acids.
• a metal-binding peptide dimer ofthe formula P 3 - L - P 3 wherein each P 3 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 P 3 peptides through their C-terminal amino acids.
• one or both ofthe two P 3 peptides is P,.
• the invention also provides an oral care product comprising the metal-binding peptide dimer ofthe formula P 3 - L - P 3 and a kit comprising the oral care product.
• Figures 2 A-B Schematic diagrams ofthe synthesis of derivatives ofthe tetrapeptide Asp Ala His Lys [SEQ ID NO: 1] coming within the formula of Figure 1C ( Figure 2 A) and Figure IB ( Figure 2B).
• Figure 3 A-B Formulas of cyclohexane diamine derivatives.
• Figures 3C-D Schematic diagrams of syntheses of cyclohexane diamine derivatives ofthe tetrapeptide Asp Ala His Lys [SEQ ID NO:l].
• Figure 4 Formula of a tetraacetic acid derivative ofthe tetrapeptide Asp Ala His Lys [SEQ ID NO:l].
• Figure 5 Formula of a bispyridylethylamine derivative ofthe tetrapeptide Asp Ala
• Figures 6 A-B Formulas of mesoporphyrin LX with ( Figure 6B) and without ( Figure 6A) a bound metal ion M.
• Figure 6C Formula of mesopo hyrin LX derivative of the tetrapeptide Asp Ala His Lys [SEQ ID NO: 1].
• Figure 7 Formulas of monosaccharides.
• Figure 8 A-B Graphs of absorbance at 532 nm (A532) versus incubation time in an assay for the production of hydroxyl radicals.
• ⁇ ascorbate only
• ⁇ copper and ascorbate
• ⁇ 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).
• Figure 9 Graph of % inhibition versus concentration tetrapeptide (L-Asp L-Ala L- His L-Lys [SEQ ID NO:l])-copper complex 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.
• Figure 11 Gel after electrophoresis of DNA treated in various ways.
• Lane 1 - 17 ⁇ g/ml plasmid DNA (untreated control); Lane 2 - 17 ⁇ g/ml plasmid DNA and 50 ⁇ M CuCl 2 ; Lane 3 - 17 ⁇ g/ml plasmid DNA and 2.5 mM ascorbate; Lane 4 - 17 ⁇ g/ml plasmid DNA, 2.5 mM ascorbate, 50 ⁇ M CuCl 2 , and 200 ⁇ M tetrapeptide (L-Asp L-Ala L-His L-Lys [SEQ ID NO:l]) (4:1 ratio tetrapeptide/copper); Lane 5 - 17 ⁇ g/ml plasmid DNA, 2.5 mM ascorbate, 50 ⁇ M CuCl 2 , and 100 ⁇ M tetrapeptide (2:1 ratio tetrapeptide/copper); Lane 6 -17 ⁇ g/ml plasmid DNA, 2.5 mM ascorbate, 50 ⁇ M Cu
• Figures 12B-C Diagrams illustrating the synthesis of peptide dimers according to the invention.
• FIG. 13 TAE (tris acetic acidEDTA (ethylenediamine tetracetic acid)) agarose gel visualized with ethidium bromide showing attenuation of ROS-induced DNA double strand breaks in genomic DNA by D-Asp Ala His Lys.
• TAE tris acetic acidEDTA (ethylenediamine tetracetic acid)
• Figure 14 TAE agarose gel 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 2 , 50 ⁇ M; Lane 3 - ascorbic acid, 500 ⁇ M; Lane 4 - D-Asp Ala His Lys, 200 ⁇ M; Lane 5 - CuCl 2 , 10 ⁇ M + ascorbic acid, 500 ⁇ M; Lane 6 - CuCl 2 , 25 ⁇ M + ascorbic acid, 500 ⁇ M; Lane 7 - CuCl 2 , 50 ⁇ M + ascorbic acid, 500 ⁇ M; Lane 8 - CuCl 2 , 50 ⁇ M + ascorbic acid, 100 ⁇ M; Lane 9 - CuCl 2 , 50 ⁇ M + ascorbic acid, 250 ⁇ M; Lane 10 - CuCl 2 , 50 ⁇ M + ascorbic acid, 500 ⁇ M +
• 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 - CuCl 2 , 50 ⁇ M; Lane 3 - ascorbic acid, 100 ⁇ M, Lane 4 - D-Asp Ala His Lys, 200 ⁇ M; Lane 5 - CuCl 2 ,
• 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 - CuCl 2 , 50 ⁇ M; Lane 3 - ascorbic acid, 500 ⁇ M; Lane 4 - D-Asp Ala His Lys, 200 ⁇ M; Lane 5 - CuCl 2 ,
• FIGs 17A-C Graphs of interleukin-8 (IL-8) concentration versus various 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).
• IL-8 interleukin-8
• 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 error.
• Figure 19 A Bar graph showing IL-8 concentrations for normal controls and gingivitis and peridontitis patients.
• Figure 19B Bar graph showing tumor necrosis factor- ⁇ (TNF ⁇ ) concentrations for normal controls and gingivitis and peridontitis patients.
• Figure 19C Bar graph showing soluble tumor necrosis factor- ⁇ 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 WhitestripsTM to the teeth of five normal 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 WhitestripsTM to which the tetrapeptide L-Asp L-
• P is Xaa, Xaa ⁇ is or is Xaa, Xaa j His Xaa 3 , wherein Xaa,
• Xaa j , and Xaa 3 are defined above.
• P is a metal-binding peptide sequence that binds transition metal ions of Groups ⁇ b-7b or 8 ofthe Periodic Table of elements (including V,
• P ! (including As, Sb and Pb).
• the binding of metal ions by P ! inhibits (i. e. , reduces or prevents) 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.
• the damage that can be caused by ROS in the absence ofthe binding of the metal ions to P is reduced.
• P ! binds Cu(LI), Ni(H), Co(H), and Mn(H) with high affinity.
• Xaa ! is most preferably Asp
• Xaa 2 is most preferably Ala
• Xaa 3 is most preferably Lys (see above).
• the preferred sequences of P j are Asp Ala His and Asp Ala His Lys [SEQ ID NO: 1].
• Most preferably the sequence of P, is Asp Ala His Lys [SEQ ID NO : 1 ] .
• Asp Ala His is the minimum sequence ofthe N-terminal metal-binding site of human serum albumin necessary for the high-affinity binding of Cu(H) and Ni(H), and Lys has been reported to contribute to the binding of these metal ions to this site.
• Asp Ala His Lys [SEQ ID NO:l] has been found by mass spectometry to bind Fe( ⁇ ).
• Other preferred sequences for P j include Thr Leu His (the N-terminal sequence of human ⁇ -fetoprotein), Arg Thr His (the N-terminal sequence of human sperm protamin HP2) and HMS HMS His (a synthetic peptide reported to form extremely stable complexes with copper; see Mlynarz et al., Speciation 98: Abstracts, http://www.jate.u-szeged.hu/spec98/abstr/mlynar.html 4/21/98).
• P 2 is (Xaa 4 ) n , wherein Xaa 4 is any amino acid and n is 0-100.
• 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 less subject to proteolysis. Therefore, in P 2 , preferably n is 0-10, more preferably n is 0-5, and most preferably n is 0.
• P 2 may have any sequence
• P 2 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.
• P 2 may comprise the sequence of one or more ofthe metal-binding sites of these peptides.
• P 2 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 2 , so that the metal-binding sites of P j and P 2 may potentially cooperatively bind metal ions (similar to a 2:1 peptide:metal complex; see Example 8).
• the spacer sequence is composed of 1-5, preferably 1-3, neutral amino acids.
• the spacer sequence maybe Gly, Gly Gly, Gly Ala
• P 2 when P 2 comprises a metal-binding site, it preferably comprises one of the following sequences: (Xaa 4 ) m Xaa 5 Xaa 2 His Xaa 3 or (Xaa 4 ) m Xaa 5 Xaa 2 His. Xaaj, Xaa 3 and Xaa 4 are defined above, and m is 0-5, preferably 1-3.
• Xaa 5 is an amino acid which comprises a ⁇ -amino group (preferably Orn or Lys, more preferably Orn) having the Xaa 4 amino acid(s), if present, or Pj attached to it by means ofthe ⁇ -amino group.
• a ⁇ -amino group preferably Orn or Lys, more preferably Orn
• the ⁇ -amino group of Xaa 5 can still participate in binding metals by means ofthe ATCUN motif).
• P, - P 2 could be Asp Ala His Gly Gly ( ⁇ )-Orn Ala His [SEQ ID NO:2].
• P 2 may comprise one ofthe following sequences : [(Xaa 4 ) m Xaa 5 Xaa j His
• P 2 comprises a peptide sequence that can bind
• Cu(I) As discussed in more detail below, Cu(H) is converted to Cu(I) 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). P, can bind Cu(H) 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 P 2 which could bind Cu(I).
• 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 Xaa 4 Met, Met Xaa 4 Xaa 4 Met, Cys Cys,
• Glutathione ⁇ -Glu Cys Gly
• the Cu(i)-binding peptide comprises the sequence Cys Xaa 4 Xaa 4 Cys (e.g., Gly Met Xaa 4 Cys Xaa 4 Xaa 4 Cys [SEQ ID NO:7], more preferably Gly Met Thr Cys Xaa 4 Xaa 4 Cys [SEQ ID NO:8], most preferably Gly Met Thr Cys Ala Asn Cys [SEQ ID NO:9]).
• Cys Xaa 4 Xaa 4 Cys e.g., Gly Met Xaa 4 Cys Xaa 4 Xaa 4 Cys [SEQ ID NO:7], more preferably Gly Met Thr Cys Xaa 4 Xaa 4 Cys [SEQ ID NO:8], most preferably Gly Met Thr Cys Ala Asn Cys [SEQ ID NO:9].
• P 2 is preferably hydrophobic or an arginine oligomer (see Rouhi, Chem. & Eng. News, 49-50 (January 15, 2001)).
• P 2 is hydrophobic, it preferably contains 1-3 hydrophobic amino 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 P 2 which is an arginine oligomer may be particularly desirable when P, - P 2 is to be administered topically.
• the amino acids of the peptide may be L-amino acids, D-amino acids, or a combination thereof.
• at least one of the amino acids of P is a D-amino acid (preferably Xaa, and/or His), except for ⁇ -Ala, when present.
• all of the amino acids of P l5 other than ⁇ -Ala, when present, are D-amino acids.
• preferably about 50% ofthe amino acids of P 2 are D-amino acids, and most preferably all ofthe amino acids of P 2 are D-amino acids.
• D-amino acids are preferred because peptides containing D-amino 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 ofthe peptide to bind metal ions, including the ability ofthe peptide to bind copper with high affinity.
• the peptides ofthe invention may be made by methods well known in the art.
• 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 methods. Suitable techniques are well known in the art, and include those described in Merrifield, in Chem. Polvpeptides. pp. 335-61 (Katsoyannis and Panayotis eds. 1973); Merrifield, J. Am. Chem. Soc. 85, 2149 (1963); Davis et al., Biochem. Intl.
• the peptides may be synthesized by recombinant DNA techniques if they contain only L-amino 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 ofthe peptide P, - P 2 , whether composed 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.
• P can be modified in the regions indicated by the arrows without altering the metal binding function of P,.
• P can be substituted at carbons 1 or 2 with R rent and the terminal -COOH of P, can be substituted with protecting group R 2 ( Figures IB-D).
• P 2 can be modified in ways similar to those described for P, to make P 2 more resistant to proteolytic enzymes, more lipid soluble, or both.
• R 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 containing 1 or 2 rings.
• aryl means a compound containing at least one aromatic ring (e.g., phenyl, naphthyl, and diphenyl).
• heteroaryl means an aryl wherein at least one of the rings contains one or more atoms of S, N or O.
• substituents such as a n-butyl attached to carbon 2 (see Figure IC, R f is n-butyl) should increase the affinity ofthe peptide for metal ions, such as copper, due to the inductive effect ofthe alkyl group.
• substituents such as a n-butyl attached to carbon 2 (see Figure IC, R f is n-butyl) should increase the affinity ofthe peptide for metal ions, such as copper, due to the inductive effect ofthe alkyl group.
• Substitution of carbon 2 ( Figure IC) with an aryl, heteroaryl, or a long chain alkyl (about 6-16 carbon atoms) should enhance transport ofthe peptide across lipid membranes.
• the derivative of P illustrated in Figure IC, wherein R, is octyl
• Figure 2A the elliptical element represents the polymer resin and R p is a standard carboxyl protecting group.
• octanoic acid freshly distilled
• the mixture is heated to about 100°C and kept at that temperature for 4 hours.
• ⁇ -Bromooctanoic acid is obtained as a colorless liquid upon distillation.
• Amination ofthe bromoacid is achieved by allowing the acid and an ammonia solution to stand at 40-50° 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 D-form.
• Other derivatives wherein R, is an alkyl, aryl or heteroaryl can be prepared in the manner illustrated in Figure 2A.
• the derivative of P, illustrated in Figure IB, wherein R, is phenyl can be prepared as illustrated in Figure 2B.
• Polymer is the resin
• t-Bu is t-butyl
• Bz is benzyl.
• Other derivatives wherein R, is an alkyl, aryl or heteroaryl can be prepared in the manner illustrated in Figure 2B.
• R 2 can be -NH 2 , -NHR l5 -N(R,) 2 , -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 R 2 do not substantially decrease the ability of P, to bind metal ions. In addition, P , and P 2 can be substituted with non-peptide functional groups that bind metal ions.
• metal-binding functional groups can be attached to one or more pendent groups ofthe 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 P 2 .
• the ability of such peptide derivatives to bind metal ions is improved as compared to the corresponding unmodified peptide.
• the peptide derivative can bind two of the same type of metal ion instead of one (e.g., two Cu(lT)), the peptide derivative can bind two different metal ions instead of one type of metal ion (e.g., one Cu(IJ) and one Fe(lTJ)), or the peptide derivative can bind one metal ion better (e.g. , with greater affinity) than the corresponding unmodified peptide.
• Metal-binding functional groups include polyamines (e.g. , diamines, triamines, etc.).
• a particularly preferred diamine is 1,2-diaminocyclohexane ( Figures 3 A-B).
• Previous studies carried out by Rao & P. Williams have shown that a cyclohexane diamine derivative ( Figure 3 A, where PYR is pyridine) binds 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 ⁇ -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 ofthe invention can be attached.
• R 4 is -alkyl-CO-peptide, -aryl-CO-peptide, -aryl-alkyl-CO-peptide, or -alkyl-aryl-CO-peptide (see also Figures 3C-D).
• the other R 4 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 the unsubstituted peptide. Further, due to the presence of the cyclohexane functionality, the compound will possess lipid-like characteristic which will aid its transport across lipid membranes.
• Cyclohexane diamine derivatives ofthe peptides ofthe 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) 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 derivative. The carboxyl moieties are then reacted with the free amino groups present in carboxy-protected P, to give the cyclohexane diamine derivative ofthe peptide.
• the second route is a direct alkylation process which is illustrated in Figure 3D.
• cyclohexane diamine is treated with bromoacetic acid to give the diacetic acid derivative.
• the carboxyl moieties are then reacted with the free amino groups present in carboxy- protected P, to give the derivative, hi Figure 3D, R 5 is H or another peptide.
• R 5 is H
• the derivative can be further reacted to produce typical carboxylic 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 the whole molecule.
• 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-aryldiacid.
• the amino groups ofthe peptide can be reacted with diacetic acid to produce a diacid derivative (see Figure 4).
• This can be conveniently accomplished by reacting the amino groups ofthe resin-bound peptide with a halogenated acetic acid (e.g., bromoacetic acid or chloroacetic acid) or a halogenated acetic acid derivative (e.g., benzyloxy ester).
• a halogenated acetic acid e.g., bromoacetic acid or chloroacetic acid
• a halogenated acetic acid derivative e.g., benzyloxy 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 the peptides of the invention can be made in the same manner.
• Polyaminopolycarboxylic acids are known to bind metals, such as copper and iron.
• Vicinal polyhydroxyl derivatives are also included in the invention. Suitable vicinal polyhydroxyls include monosaccharides andpolysaccharides (i.e., disaccharide, trisaccharide, etc.). Presently preferred are monosaccharides. See Figure 7. The monosaccharides fall into two major categories - furanoses and pyranoses. One of the prime examples of a furanose ring system is glucose. The hydroxyl groups of glucose can be protected as benzyl or labile t-butyloxy functional groups, while leaving the aldehyde free to react with an amine group (e.g., that of lysine) of the tetrapeptide.
• an amine group e.g., that of lysine
• 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 tetra-substituted peptide derivative.
• the reaction is accomplished by reacting the resin-bound tetrapeptide with the bromoethylpyridine, followed by cleavage of the product from the resin.
• Phenanthroline is another heterocyclic compound capable of binding divalent metal ions.
• Phenanthroline derivatives ofthe 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 tetrapyrrolic macrocycle capable of binding to metals. Heme, chlorophyll and corrins are prime examples of this class of compounds containing iron, magnesium and cobalt, respectively.
• Mesopo ⁇ hyrin LX ( 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 ofthe invention would produce a po ⁇ hyrin-peptide derivative possessing several sites for binding of copper (see Figure 6C).
• the imidazole residues at positions 3 and 3' ofthe tetrapeptide shown in Figure 6C may provide a binding site for metals other than copper, thereby stabilizing the porphyrin-metal complex.
• cyanocobalamine (vitamin B-12) contains cobalt as the metal in the po ⁇ hyrin nucleus, and the complex is stabilized by the imidazole groups.
• the po ⁇ hyrin-tetrapeptide derivative would bind cobalt (or other metals) at normal physiological conditions in the prophyrin nucleus and that the complex would be stabilized by the His imidazole groups.
• the carboxyl groups of mesopo ⁇ hyrin LX can be activated and coupled with the amino groups of the peptide employing standard solid-phase peptide synthesis.
• the free amino group of the lysine residue of the resin-bound peptide can be coupled with carboxy activated po ⁇ hyrin nucleus.
• the condensation product can be cleaved off the resin using standard methods.
• This method can be used to synthesize other po ⁇ hyrin derivatives of peptides of the invention.
• Dithiocarbamates are known to bind metals, including iron. Suitable dithiocarbamates for making derivatives ofthe peptides ofthe invention are described in U. S . Patents Nos. 5,380,747 and 5,922,761, the complete disclosures of which are inco ⁇ orated herein by reference. 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 inco ⁇ orated herein by reference. Additional non-peptide metal chelators are known in the art or will be developed.
• MBP metal-binding peptide
• MBP metal-binding peptide
• the resulting peptide derivatives would contain one or more metal-binding functional groups in addition to the metal-binding site of MBP.
• MBP contains from 2-10, more preferably 3-5, amino acids.
• MBP contains one or more D-amino acids; most preferably all ofthe amino acids of MBP are D-amino acids.
• 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 P, - P 2 . 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 peptide P, - P 2 .
• the invention also provides metal-binding peptide dimers ofthe formula:
• P 3 is any peptide capable of binding a metal ion, and each P 3 may be the same or different. Each P 3 preferably contains 2-10, more preferably 3-5, amino acids. As described above, metal-binding peptides are known, and each P 3 may comprise the sequence of one or more ofthe metal-binding sites of these peptides. Although each P 3 may be substituted as described above for P, and P 2 , including with a non-peptide, metal-binding functional group, both P 3 peptides are preferably unsubstituted. P 3 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 P 3 .
• each P 3 is an unsubstituted metal-binding peptide (i.e., an unsubstituted peptide comprising a peptide sequence which binds metal ions).
• P is an unsubstituted metal-binding peptide
• the dimers have the sequence P 3 - L - Pj, P, - L - P 3 or, most preferably, P, - L - P,).
• Pj is defined above.
• L is a linker which is attached to the C-terminal amino acid of each P 3 .
• L may be any physiologically-acceptable chemical group which can connect the two P 3 peptides through their C-terminal amino acids.
• 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 ofthe inclusion ofthe linker L in the peptide dimer.
• L links the two P 3 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.
• L is a straight-chain or branched-chain alkane or alkene residue containing from 1-18, preferably from 2-8, carbon atoms (e.g., -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 (CH 3 )CH 2 -, -CHCH-, etc.) or a cyclic alkane or alkene residue containing from 3-8, preferably from 5-6, carbon atoms (see Figure 12A, compound D j ), preferably attached to a P 3 by means of an amide linkage.
• Such linkers are particularly preferred because they impart hydrophobicity to the peptide dimers.
• L is a nitrogen- containing heterocyclic alkane residue (see Figure 12 A, compounds D 2 , D 3 and D 4 ), preferably a piperazide (see Figure 12A, compound D 2 ).
• L is a glyceryl ester (see Figure 12 A, compound D 5 ; in formula D 5 , R is an alkyl or aryl containing, preferably containing 1-6 carbon atoms).
• L could be a metal-binding po ⁇ hyrin (see Figure 6C).
• 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.
• Methods of synthesizing the peptide dimers are illustrated in Figures 12B-D. hi general, the C-terminal amino acids (protected by methods and protecting groups well known in the art) ofthe two P 3 groups are attached to L, and the resulting amino acid dimers used in standard peptide synthetic methods to make the peptide dimers.
• a peptide dimer where each peptide has the sequence Asp Ala His Lys, [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 (see Figures 12B-C).
• Many suitable diamines are available commercially or suitable diamines can be readily synthesized by methods known in the art.
• the lysine dimer 2 ( Figure 12B) can be prepared as follows. To a stirred solution of 9-fluorenylmethyloxycarbonyl (Fmoc)- and t-benzyloxycarbonyl(Boc)- protected D-Lys (Fmoc-D-Lys(Boc)-OH) (20 mmole) in dry dimethylformamide (DMF; 100 mL; dry argon flushed) are added butane- 1,4-diamine 1 and 2-(lH-benzotriazole-l-yl)-l,2,3,3- tetramethyluroniumtetrafluoroborate (TBTU; 0.5 mmole).
• DMF dry dimethylformamide
• TBTU 2-(lH-benzotriazole-l-yl)-l,2,3,3- tetramethyluroniumtetrafluoroborate
• 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 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 C mb ⁇ QX Qtal.,Proc. Natl.Acad. Sci., 96, 10824-10829 (1999)). Then, the remainder of the amino acid residues are added employing standard peptide synthesis methodology to give the peptide dimer 6 (see Figure 12C).
• Peptide dimers where each peptide has the sequence Asp Ala His Lys [SEQ ED NO : 1 ] and where L is a glyceryl ester, can be synthesized as follows.
• a lysine diester 8, wherein R is methyl, can be prepared as follows (see Figure 12D).
• the bis-protected lysine 8 is isolated by flash chromatography over silica and elution with mixtures of ethyl acetate/methanol.
• the peptide dimer 9 is then prepared from the protected lysine dimer 8 employing classical peptide synthesis methodology (see Figure 12D).
• 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, malic, tartaric, citric, glutamic, benzoic, salicylic, and the like) or bases (such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation).
• inorganic acids such as hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, and the like
• organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, glutamic, benzoic, salicylic, and the like
• bases such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation
• the invention also provides oral care products comprising a metal-binding compound or compounds ofthe 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 compositions of the invention include compositions intended for use by consumers and patients and compositions intended for use by dental professionals (e.g., dental hygienists, dentists and oral surgeons).
• the oral care compositions ofthe invention will comprise a metal-binding compound or compounds of the invention as active ingredient(s) in admixture with one or more pharmaceutically-acceptable carriers.
• Oral care compositions ofthe 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 carrier and ingredient must be "acceptable” in the sense of being compatible with the other ingredients ofthe formulation and not injurious to the animal.
• Suitable ingredients including pharmaceutically-acceptable carriers, 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. Patents Nos.4,847,283, 5,032,384, 5,043,183, 5,180,578, 5,198,220, 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, 5,976,507, 6,045,780, 6,197,331, 6,228,347, 6,251,372, and 6,350,438, 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 inco ⁇ orated herein by reference
• ingredients used in oral care compositions include water, alcohols, humectants, surfactants, thickening agents, 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.
• the alcohol is ethanol.
• Ethanol is a solvent 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, and 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, and may impart desirable sweetness.
• edible polyhydric alcohols such as glycerol, sorbitol, xylitol, butylene glycol, polyethylene glycol, propylene glycol, mannitol, and 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, 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 lauryl sulfate and sodium coconut monoglyceride sulfonates), sarcosinates (such as sodium and potassium salts of lauroyl sarcosinate, myristoyl sarcosinate, palmitoyl sarcosinate, stearoyl sarcosinate and oleoyl sarcosinate), taurates, higher alkyl sulfoacettes (such as sodium lauryl sulfoacetate), isethionates (such as sodium lau
• Nonionic surfactants include poloxamers (sold under the tradename Pluronic), 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 polypropyleneoxide, ethylene oxide condensates of aliphatic alcohols, long-chain tertiary amine oxides, long-chain tertiary phospine oxides, long-chain dialkyl sulfoxides, and mixtures 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 ofthe aliphatic substituents contains about 8-18 carbon atoms and one contains an anionic water-solubilizing group (such as carboxylate, sulfonate, sulfate, phosphate or phosphonate), and mixtures of such materials.
• betaines such as cocamidopropylbetaine
• 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 ofthe aliphatic substituents contains about 8-18 carbon atoms and one contains an anionic water-solubilizing group (such as carboxy, sulfonate, sulfate, phosphate or phosphonate).
• Cationic surfactants include aliphatic quaternary ammonium compounds having one long alkyl chain containing about 8-18 carbon atoms (such as lauryl trimethylammonium chloride, cetylpyridinium chloride, cetyltrimethylammonium bromide, diisobuytylphenoxyethyldimethylbenzylammonium chloride, coconut alkyltrimetylammonium nitrite, cetylpyridinium fluoride). Certain cationic surfactants can also act as antimicrobials.
• 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 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
• Abrasives include silicas (including gels and precipitates), aluminas, calcium carbonates, calcium phosphates, dicalcium phosphates, tricalcium phosphates, hydroxyapatites, calcium pyrophosphates, trimetaphosphates, insoluble polymetaphopsphates (such as insoluble sodium polymetaphosphate and calcium polymetaphosphate), magnesium carbonates, magnesium oxides, resinous abrasive materials (such as particulate condensation 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 ofthe foregoing. Silica abrasives are preferred because they provide excellent dental cleaning and polishing performance without unduly ab
• 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, marjoram, 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.
• CGA cinnamic alde
• Sweetening agents include sucrose, glucose, saccharin, dextrose, levulose, lactose, mannitol, sorbitol, fructose, maltose, xylitol, saccharin salts, thaumatin, aspartame, D- tryptophan, dihydrochalcones, acesulfame, cyclamate salts, and mixtures ofthe foregoing.
• the oral care compositions may include coolants, salivating agents, warming agents and numbing agents as optional ingredients.
• Coolants include carboxamides, menthol, paramenthan carboxamides, 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, 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- dichlorophenoxy)-phenol, salicylanilide, domiphen bromide, delmopinol, octapinol, other piperadino derivatives, irrigationn, zinc stannous ion agents, antibiotics (such as augimentin, amoxicillin, tetracycline, doxydcycline, minocycline, and
• Anti-caries agents include sodium fluoride, stannous fluoride, potassium fluoride, amine fluorides, indium fluoride, sodium monofluorophosphate, calcium lactate, calcium glycerophosphates, strontium salts, and strontium polyacrylates.
• Anti-calculus agents include pyrophosphate salts such as dialkali metal pyrophosphate salts and tetraalkali metal pyrophosphate salts (e.g., disodium dihydrogen pyrophosphate, tetrasodium pyrophosphate and tetrapotassium pyrophosphate, in their hydrated and unhydrated forms).
• pyrophosphate salts such as dialkali metal pyrophosphate salts and tetraalkali metal pyrophosphate salts (e.g., disodium dihydrogen pyrophosphate, tetrasodium pyrophosphate and tetrapotassium pyrophosphate, in their hydrated and unhydrated forms).
• anti-calculus agents which can be used instead of, or in addition to, 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- 1-hydroxy- 1,1- diphosphonate (EHDP), methanedisphosphonic acid, and 2-phosphonobutane- 1,2,4- tricarboxylic acid), and polypeptides (such as polyaspartic acid and polyglutamic acid).
• 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
• 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.
• the pH ofthe oral care compositions ofthe 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.
• 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 and bases, 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 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 ofthe foregoing as needed to achieve and maintain the desired pH.
• bicarbonate salts e.g., an alkali metal bicarbonate, such as sodium bicarbonate (also known as baking soda)
• gluconates such as sodium bicarbonate (also known as baking soda)
• tartrates such as sodium bicarbonate (also known as baking soda)
• sulfates such as sodium bicarbonate (also known as baking soda)
• citrates such as sodium citrate
• benzoate salts such as sodium citrate
• 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 the metal-binding compounds ofthe 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 the foregoing.
• Suitable antioxidants include superoxide dismutase, catalase, glutathioneperoxidase, ebselen, glutathione, cysteine, N-acetyl cysteine, penicillamine, allopurinol, oxypurinol, ascorbic acid, ⁇ -tocopherol, Trolox (water-soluble ⁇ -tocopherol), vitamin A, ⁇ -carotene, fatty-acid binding protein, fenozan, probucol, cyanidanol-3, dimercaptopropanol, indapamide, 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/or non-peptide chelators.
• Metal-binding peptides and non-peptide chelators are described above, and others are known in the art.
• 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).
• the oral care compositions ofthe invention may advantageously contain a protease inhibitor to prevent degradation ofthe metal-binding compounds ofthe 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. Patents Nos. 6,403,633, 6,350,438, 6066673, 5,622,984, and 4,454,338, the complete disclosures of which are inco ⁇ orated herein by reference.
• suspending agents such as a polysaccharide - see U.S. Patent No. 5,466,437), polymeric compounds which can enhance the delivery of active ingredients (such as copolymers of polyvinylmethylether with maleic anhydride and those delivery enhancing polymers described inDE 942,643 and U.S. PatentNo.5,466,437), materials which allow for a strong and continuing adherence ofthe oral care composition to the tissues ofthe 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.
• suspending agents such as a polysaccharide - see U.S. Patent No. 5,466,437
• polymeric compounds which can enhance the delivery of active ingredients such as copolymers of polyvinylmethylether with maleic anhydride and those delivery enhancing polymers described inDE 942,643 and U.S. PatentNo.
• Patents Nos. 5,032,384, 5,298,237, and 5,466,437) oils, waxes, 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, solubilizing agents (such as propylene glycol; see, e.g., U.S. Patent No.
• enzymes such as dextranase and/or mutanase, amyloglucosidase, glucose oxidase with lactoperoxidase, and neuraminidases
• synthetic or natural polymers such as tooth whitening agents (such from about 0.1 % to about 10% by weight of a peroxygen compound; see additional discussion of tooth whitening compositions below)
• tooth 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 potassium oxalate), and strontium salts
• analgesics such as lidocaine or benzocaine
• anti- fungal agents such as
• 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 ofthe metal-binding compounds ofthe invention to bind copper and iron ions found in the mouth.
• Dentrifices include toothpastes, tooth gels, tooth powders and liquid dentrifices.
• Toothpastes and tooth gels generally include a dental abrasive, a surfactant, a thickening 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.
• 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% to about 10% of athickening agent, from about 10% to about 80% of ahumectant, 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 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 suspending agent e.g., a high molecular weight polysaccharide
• a typical liquid dentrifice will comprise from about 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% 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.
• such a gel 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 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 include a flavoring agent, a sweetening agent, a coloring agent.
• 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.
• Ointments suitable for oral use are described in, e.g., U.S. Patents Nos. 4,847,283,
• 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 other ingredients.
• the ointment may comprise from about 80% to about 90% petrolatum and from about 10% to about 20% ethanol or propylene glycol.
• the ointment may comprise about 10 % petrolatum, about 9% lanolin, about 8% talc, about 32% cod liver oil, and about 40% zinc oxide.
• the ointment may comprise from about 30% to about 45% water, from about 10% to about 30% oil (e.g.jpetrolatum or mineral oil), from about 0.1% to about 10% emulsifier (e.g., wax NF), from about 2% to about 20% humectant (e.g., 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.
• oil e.g.jpetrolatum or mineral oil
• emulsifier e.g., wax NF
• humectant e.g., propylene glycol
• preservatives e.g., methyl paraben and propyl paraben
• Mouthwashes, rinses, gargles and sprays generally include water, ethanol, and/or a 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% 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 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%, 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 Solutions generally include water, a preservative, a flavoring agent, and a sweetening agent, and may include a thickening agent and/or a surfactant.
• 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% of a surfactant.
• a saline solution is 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, The Science And Practice Of pharmacy, 19 th 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., Pharmaceutical Dosage Forms, 2 nd ed. (1990), the complete disclosures of which are inco ⁇ orated here in by reference.
• 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 pyrrolidone, starch, modified starch and mixtures thereof), and, optionally, a lubricant (such as magnesium stearate, stearic acid, talc, and waxes), sweetening, coloring and flavoring agents, a surfactant, a preservative, and other ingredients.
• 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 pyrrol
• 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 ofthe 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 glucose, sugar, citric acid and/or a flavoring agent.
• the chewable base ofthe core or outer layer may be a gum, soft candy, nougat, caramel or hard candy.
• the tablets are formed by extrusion ofthe core and outer layer to form a rope, followed by cutting the rope into tablets.
• Chewing gum 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, diethyl phthalate and acetylated monoglycerides.
• 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 inco ⁇ orated into a gum base by, e.g., stirring them into a warm gum base or coating them onto the outer surface ofthe gum base.
• Tooth whitening compositions will comprise a tooth whitening agent. Tooth whitening agents include peroxides, percarbonates and perborates ofthe alkali and alkaline earth metals or complex compounds containing hydrogen peroxide. Tooth whitening agents 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 are hydrogen peroxide, peroxyacetic acid and sodium perborate. These tooth whitening agents liberate active oxygen and hydrogen peroxide. Tooth whitening agents can be present in tooth 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%.
• Tooth whitening compositions are known in the art, including aqueous solutions, gels, pastes, liquids, films, strips, one-part systems, two-part systems, compositions that require activation ofthe 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.
• tooth whitening compositions or of one ofthe many oral care compositions and devices which comprise a tooth whitening agent, results in the production of ROS and can cause inflammation ofthe tissues ofthe mouth.
• Inco ⁇ oration of a metal-binding compound 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 peroxide-type whitening agents, will not be converted into hydroxyl radicals (see Examples 8 and 9) and will, therefore, remain active longer.
• 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.
• teeth are commonly whitened by applying a tooth whitening composition to the teeth by means of a dental tray or trough.
• a metal-binding compound or compounds ofthe invention could be inco ⁇ orated into the tooth whitening composition that is used in the tray or trough.
• a separate composition comprising a metal binding compound 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.
• 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.
• 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 inco ⁇ orated into such strips.
• the metal-binding compound(s) could be inco ⁇ orated into the tooth whitening composition, which is then applied to the strips, or a solution, gel or other composition 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.
• strips comprising a tooth whitening composition and strips comprising the metal binding compound(s) could both be supplied to the patient and would be used sequentially.
• the oral care compositions ofthe invention may comprise a single phase or a plurality 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.
• one ofthe phases will include some ofthe ingredients, and the remainder ofthe 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 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.
• the plurality of phases may be 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, 6,228,347 and 6,350,438 and PCT application number WO 99/37236.
• 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).
• the oral care devices ofthe 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 inco ⁇ orated into, them. See, e.g., U.S. Patents Nos.
• a metal-binding compound or compounds of the invention can be inco ⁇ orated 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 ofthe 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 inco ⁇ orated into a polymer film suitable for application 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 ofthe solvent so that the compound(s) become associated with (bound to, entrapped in, coated onto, etc.) the suture or surgical material.
• a binder e.g., a wax or polymer
• oral care products for animals such as foods, chews, and toys. Suitable products are described in U.S. Patent No.6,350,438.
• a metal-binding compound or compounds ofthe invention can be used to treat a tissue of an animal's mouth.
• “Mouth” is used herein to mean the cavity bounded externally by the lips and internally by the pharynx that encloses the tongue, gums and teeth.
• the tissues ofthe 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 ofthe 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, prevent, reduce the likelihood of, or reduce the severity of, a disease or condition, or of at least some ofthe symptoms or effects thereof.
• the tissue is contacted with a metal-binding compound or compounds ofthe invention.
• the tissue may be contacted with an oral care composition comprising the metal-binding compound(s).
• Methods of contacting tissues of 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 use of an applicator), chewing gum, chewing or sucking a lozenge, mint or tablet, and many other means of topical application.
• a solution e.g., a mouthwash, rinse, spray, liquid dentrifice, or other solution
• a dentrifice e.g., a toothpaste, tooth gel, or powder
• a non-abrasive solution e.g., gel, paste, cream or ointment directly to the tissue (with or without the use of an applicator)
• chewing gum chewing or sucking a lozenge, mint or tablet, and many other means of
• Suitable applicators for applying oral care compositions such as solutions, gels, pastes, creams and ointments, to a tissue
• 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 EP application 752833) which allows for immersion of the teeth and, optionally, the periodontal tissue in, e.g., a gel or solution.
• the tissue may be contacted with an oral care device comprising the metal-binding compound(s).
• an oral care device comprising the metal-binding compound(s).
• Methods of contacting tissues ofthe 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 the teeth, etc.
• the treatment ofthe tissue can be prophylactic treatment.
• the tissue may be treated as part of a prophylactic oral care regimen.
• the metal-binding compound(s) ofthe invention can be inco ⁇ orated 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 will be used preferably at least once per day, more preferably two or three times per day.
• the metal-binding compound(s) of the invention may be contained in a separate oral care composition or device which will be used separately from other compositions and devices employed in the prophylactic oral care regimen.
• the metal-binding compound(s) ofthe invention can be inco ⁇ orated into a mouthwash or rinse, a gum, a lozenge or a chewable tablet, which would preferably be used at least once per day, more preferably at least two or three times per day. It may be particularly beneficial for those 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 ofthe mouth by such products.
• metal salts particularly copper salts
• toothpastes and other oral care compositions generally as antibacterial, anti-plaque, anti-caries, and anti-gingivitis agents.
• metal salts particularly copper salts
• the use of oral care compositions containing copper salts could be harmful to the tissues of the mouth, since free copper ions catalyze the formation of ROS.
• an oral care composition ofthe present invention at an appropriate time after the use ofthe 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.
• the metal-binding compound(s) could conveniently be supplied in a 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.
• the tissue(s) on which surgery is being performed those tissues near the area where the surgery is being performed or, for ease of treatment, all or substantially ofthe tissues ofthe mouth, can be treated prior to surgery, during surgery, after the surgery, or combinations thereof.
• the tissue(s) surrounding the tooth which is to be extracted adjacent tissues or, for ease of treatment, all or substantially ofthe tissues ofthe mouth, can be treated prior to tooth extraction, during the tooth extraction, after the tooth extraction, or combinations thereof.
• the mouth could be rinsed prior to surgery or tooth extraction with a solution comprising the metal-binding compound(s), the wound(s) caused by the surgery or tooth extraction could be closed with sutures having the metal-binding compound(s) inco ⁇ orated 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 the metal-binding compound(s).
• Tissues can also be treated prophylactically in connection with radiation, such as dental x-rays.
• tissues may be treated prophylactically in connection with the whitening ofthe 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 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.
• the dosage amount ofthe metal-binding compound(s) ofthe 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 ofthe disease or condition to be treated, the severity ofthe disease or condition, the type of oral care composition used, the duration ofthe treatment, the identify of any other drugs being administered to the animal, the age, size and species ofthe animal, and like factors known in the medical and veterinary arts.
• 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.
• oral care compositions comprising from about 0.001% to about 20% of a metal binding compound or compounds ofthe invention one or more times per day will provide effective daily dosages.
• 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 invention.
• the kit may also include an applicator for applying the oral care composition to a tissue of an animal's mouth, 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 tray 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 solution.
• an applicator for applying the oral care composition to a tissue of an animal's mouth 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,
• kits could also include a cup, vial or other device for dispensing and/or measuring the amount ofthe oral care composition ofthe invention needed for the intended use.
• the kits could include both an oral care composition and an oral care device according to the invention, h 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 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 and/or the oral care product ofthe invention and may include any other desired items.
• a or “an” entity refers to one or more of that entity.
• a cell refers to one or more cells.
• This example describes the synthesis of the tetrapeptide Asp Ala His Lys [SEQ ID NO: 1] composed of all L-amino acids using standard solid-phase synthesis techniques.
• Fmoc 9-fluorenylmethyloxycarbonyl
• DMF dimethylformamide
• a ninhydrin test was used to monitor the reaction.
• the 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- 1 -yl)- 1 ,2,3,3-tetramethyluroniumtetrafluoroborate (TBTU-) (4 equivalents).
• the resin was shaken for about 24 hours, and the reaction was 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 (3 x 2 ml).
• the protecting group ofthe 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).
• the resin was shaken for about 24 hours, and the reaction monitored by ninhydrin test. At the end 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.
• the ninhydrin test gave a blue color, indicating the release of the tetrapeptide from the resin, i some circumstances, addition of 5%(V/V) of DMF to TFA accelerated the rate of release ofthe peptide from the resin.
• Removal of TFA 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 spectrometric methods.
• a number of enantiomers of the tetrapeptide can be prepared in this manner.
• use of D-amino acids in the peptide synthesis forms the tetrapeptide containing all D-amino acids.
• combinations of L-amino acids and D-amino acids can be used.
• 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 isomer melts between 43-45°C (Ph.D. Thesis, P.D. Newman, University College, Edinburgh, 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 ofthe addition, the reaction temperature was raised to 30°C and kept at that temperature for a further 5 hours. Toluene was evaporated, and the R-trans 1 ,2-diaminocyclohexane diacetic acid was crystallized from hexane/toluene to 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-diaminocyclohexanediacetic acid (20 mg) followed by addition of a mixture of diisopropylamine (8 equivalent) and TBTU-(4 equivalent). The resin was shaken for about 24 h on a roller. 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 where R 5 is H) was characterized by spectrometric analysis.
• the resin-bound tetrapeptide prepared in Example 1 (20 mg) was suspended in DMF (5 mL) and treated with mesopo ⁇ hyrin LX dicarboxyhc acid (10 ⁇ mole; formula given in Figure 6A), followed by addition of a mixture of diisopropylamine (8 equivalent) and TBTU- (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 (5x3 mL) and partially dried. Hydrolysis ofthe resin linkage was effected by treating the resin-bound reaction product with TFA (5mL; 5 hr). Theresinwas separatedandwashedwithDCM/TFAmixture(l:1.5mL). Thewashings were combined and concentrated under vacuum. The po ⁇ hyrin 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 po ⁇ hyrin-peptides, such as mesopo ⁇ hyrin I and related molecules.
• the resin-bound tetrapeptide prepared in Example 1 (20 mg) was suspended in DMF (5 mL) and treated with bromoethylpyridine (20 ⁇ mole) . This was followed by the addition 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 under vacuum. The pyridylethyl tetrapeptide derivative (formula given in Figure 5) was 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.
• a derivative having the formula shown in Figure IB, wherein R ] 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 product was cooled ( 10 ° C) and reacted with ethyl ⁇ -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 derivative was prepared using a standard reaction sequence.
• the resin was shaken for about 24 h, and the reaction monitored by the ninhydrin test.
• a tetrapeptide having the sequence L-Asp L-Ala L-His L-Lys [SEQ ID NO: 1] 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). 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
• the assay was performed with and without the L-tetrapeptide.
• the results are summarized in Table 1.
• Table 1 As can be seen from Table 1, when the L-tetrapeptide was present at Cu( ⁇ ):tetrapeptide ratios of 1:1.2 and 1:2, the degradation of 2-deoxy-D-ribose was inhibited by 38% and 73%, respectively.
• the L-tetrapeptide inhibited the degradation of 2-deoxy-D-ribose by hydroxyl radicals.
• D-tetrapeptide having the sequence Asp Ala His Lys composed of all D-amino acids.
• 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 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.).
• the hydroxyl radical is probably the most reactive oxygen-derived species.
• the hydroxyl free radical is very energetic, short-lived and toxic.
• 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)).
• 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 ofthe formation of TBA-reactive species at pH 7.0-8.5. These are physiological pH levels and pH 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 bind copper. The nitrogen atom on the imidazole ring of histidine participates in binding copper with a pKa of 6.0.
• histidine is only able to bind 50% of the copper.
• the other 50% ofthe 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 of hydroxyl radicals.
• a peptide having an acetylated aspartic acid (Ac- Asp) as the N-terminal amino acid was also tested.
• the results are presented in Table 3.
• the % inhibition is the percent decrease in absorbance compared to buffer alone divided by the absorbance of the buffer alone.
• the peptides with histidine in the second and third positions gave >95% inhibition at a 2: 1 peptide:copper ratio, while these peptides at a 1 : 1 peptide:copper ratio were ineffective.
• 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 positions, respectively, of these peptides.
• Histidine alone at a 2: 1 histidine: copper ratio provided some protection (about 20% inhibition).
• Catalase has been shown to prevent hydroxyl radical formation. Gutteridge and Wilkins, Biochim. Biophys. Acta, 759:38-41 (1983); Facchinetti et al., Cell. Molec. Neurobiol., 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 agrees with Equations 3 and 4 above.
• Catalase also prevents the formation of the pink chromogen when the L-Asp L-Ala L-His L-Lys [SEQ ED 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 ofthe hydroxyl radical.
• SOD superoxide dismutase
• 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 su ⁇ rising since SOD actually converts superoxide radical into hydrogen peroxide. Hydrogen peroxide can then be converted into the hydroxyl radical by reduced copper.
• the reaction was started by the addition of various amounts of a tetrapeptide-copper complex (tetrapeptide/copper ratios of 1:1 and2:l) and 20 nM xanthine oxidase (Sigma Chemical Co.).
• the tetrapeptide-copper complex was prepared by mixing the tetrapeptide and copper (as CuCl 2 ) and allowing the mixture to incubate for 15 minutes at room temperature immediately before addition to the cuvette. The samples were read at time 0 and every 60 seconds for five minutes at 560 nm.
• the complex ofthe 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 was about 500 times less effective than SOD itself, based on IC 50 values (amount that gives
• uric acid production was measured at 295 nm. Athar et al., Biochem. MoL 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 of 600 nM (data not shown). Therefore, the 1:1 tetrapeptide-copper complex has true SOD activity. Since superoxide is converted to hydrogen peroxide by the complex, this could help to explain why it is not effective at preventing hydroxyl radical production.
• the sample containing the 1 : 1 tetrapeptide-copper complex also showed an increase in NBT reduction, with a decreased maximum reached at 60 minutes. These data suggest that superoxide accumulates in the sample containing the 2: 1 tetrapeptide-copper complex, while the 1 :1 tetrapeptide-copper complex mimics superoxide dismutase.
• the 1 : 1 tetrapeptide-copper complex also provides a valuable service by eliminating the superoxide radical. Even though it produces hydrogen peroxide, most compartments ofthe human body have sufficient quantities ofthe enzyme catalase that can eliminate hydrogen peroxide, hi 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.
• DNA strand breaks were measured according to the method of Asaumi et al., Biochem. MoL Biol. Int., 39(l):77-86 (1996). Briefly, 17 ⁇ g/ml of plasmid pBR322 DNA was allowed to pre-incubate for 15 minutes at room temperature with 50 ⁇ M CuCl 2 and concentrations of the tetrapeptide of 0-200 ⁇ M. Then, 2.5 mM ascorbate was added to each reaction, and the mixture was incubated for 1 hour at 37°C. The total volume ofthe mixture was 16 ⁇ L.
• 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)).
• 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 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.,
• Telomeres which are repeats of the hexanucleotide TTAGGG, exist at the ends of 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)).
• DNA polymerase is unable to replicate the terminal end of the lagging strand during DNA replication resulting in the loss of 30-500 base pairs (Harley et al., Nature 345:458-460 (1990); vonZglinicki etal.,Exp. C ⁇ //i?e5. 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)).
• D-Asp Ala His Lys 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, 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 medium (EMDM) with 10% fetal calf serum (FCS) at 10% CO 2 and 37°C.
• EMDM Iscove's modified Dulbecco's medium
• FCS fetal calf serum
• strand breaks were visualized by immediately adding 5 ⁇ l of loading dye [0.25% (w/v) bromophenol blue and 40% (w/v) sucrose] and loading on a 0.5% tris acetic acid EDTA (TAE) agarose gel. Gels were then run at 70V for 90 min and stained using 2 ⁇ g/ml ethidium bromide for 30 minutes. Prior to photographing, gels were rinsed in TAE for 10 minutes.
• loading dye 0.25% (w/v) bromophenol blue and 40% (w/v) sucrose
• TAE tris acetic acid EDTA
• Telomere Length Assay To examine telomere damage, the TeloTAGG Telomere Length Assay (Roche) was used according to manufacturer's recommendations: digesting 1 ⁇ g of genomic DNA per reaction using Hinfl and RSA I. 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 ⁇ l with PBS, and isolated using DNeasy columns prior to restriction digestions.
• DIG digoxigenin
• Copper ions an essential part of chromatin (Dijkwel et al, J. Cell Sci. 84:53-67 (1986)), are present within DNA (Wacker et al., J. Biol. Chem. 234:3257-3262 (1959)) and may participate in oxidative DNA damage (Chiu et al., Biochemisti ⁇ 34:2653-2661 (1995); Hayashi et al., Biochem. Biophys. Res. Comm. 276:174-178, doi:10.1006/bbrc.2000.3454 (2000); Kagawa etal.,J. 5zo/. Chem. 266:20175-20184 (1991)).
• copper can lead to the production of ROS by catalyzing the following reactions (Biaglow et al., Free Radic. Biol. Med. 22:1129-1138 (1997)):
• OH « scavengers do not prevent copper-mediated oxidative damage suggesting that oxidative DNA damage occurs in 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 site of OH* production (Marnett, Carcinogenesis, 21, 361-370 (2000)).
• Oikawa, et. al. (Oikawa et al., Biochim. Biophys.
• 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 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 H 2 O 2 . H 2 O 2 is freely diffusible, can penetrate to the nucleus, and has been shown to damage DNA in fibroblasts (Chen et al., Proc Natl. Acad. Sci.
• H 2 O 2 Entrance of H 2 O 2 into 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».
• endogenous reducing agents reduced glutathione (GSH), reduced nicotinamide dinucleotide (NADH), and ascorbic acid
• GSH reduced glutathione
• NADH reduced nicotinamide dinucleotide
• Ascorbic acid One possible mechanism of D-Asp Ala His Lys protection would be the chelation of copper ions, thereby preventing production of OH» and H 2 O 2 .
• 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 H 2 O 2 outside the cell.
• telomere Examination ofthe 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 showed damage to the telomere with some conservation ofthe 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.
• 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 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. USA 90:1102-1106 (1993)).
• 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 and possibly exacerbates the damage (Marnett, Carcinogenesis 21:361- 370 (2000)).
• Both DNA and the telomeric sequence are susceptible to copper-mediated ROS damage, particularly damage attributed to hydroxyl radicals.
• 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- terminus of human 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.
• Lnterleukin 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. EL-8 is produced by immune cells (including lymphocytes, neutrophils, monocytes and macrophages), fibroblasts and epithelial cells. Reports indicate an important role for EL-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 EL-8 release by Jurkat cells (American Type Culture Collection (ATCC),
• Experiment 1 a. None (control); b. Asp Ala His Lys [SEQ ED NO: 1] ("DAHK”) - 200 ⁇ M and ascorbic acid ⁇ 500 ⁇ M; c. CuCl 2 - 10 ⁇ M and ascorbic acid - 500 ⁇ M; d. CuCl 2 - 25 ⁇ M and ascorbic acid - 500 ⁇ M; e. CuCl 2 - 50 ⁇ M and ascorbic acid - 500 ⁇ M; f. CuCl 2 - 100 ⁇ M and ascorbic acid - 500 ⁇ M; g.
• Experiment 3 a. None (control); b. CuCl 2 - 100 ⁇ M; c.
• DAHK - 400 ⁇ M and ascorbic acid - 250 ⁇ M d. CuCl 2 - 25 ⁇ M and ascorbic acid - 250 ⁇ M; e. CuCl 2 - 50 ⁇ M and ascorbic acid - 250 ⁇ M; f. CuCl 2 - 100 ⁇ M and ascorbic acid - 250 ⁇ M; h. CuCl 2 - 100 ⁇ M and DAHK - 200 ⁇ M and ascorbic acid - 250 ⁇ M; and i. CuCl 2 - 100 ⁇ M and DAHK - 400 ⁇ M and ascorbic acid - 250 ⁇ M.
• Coenzyme A 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 be oxidized to a disulfide which camiot participate in acetylation reactions. As a result, metabolism and energy utilization are inhibited.
• Cu(EI) could oxidize CoA and, if so, whether the tetrapeptide Asp Ala His Lys [SEQ ED NO: 1] (Bowman Research, Inc., United Kingdom) could protect CoA (Sigma) from oxidation by Cu(ET).
• Table 4 The experimental setup and 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).
• EXAMPLE 12 Inhibition Of EL-8 Secretion By d-DAHK Systemic inflammatory response 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 blood vessels have been shown to adversely contribute to early SERS by secreting excessive amounts of interleukin-8 (EL-8), a potent pro-inflammatory cytokine associated with an increased risk of multiple organ failure and death after severe trauma.
• EL-8 interleukin-8
• This example presents data showing for the first time that endothelial cells secrete markedly elevated levels of EL-8 after exposure to a physiologically relevant concentration of copper. Further, addition of a high-affinity Cu(EE)-binding peptide significantly inhibits copper-induced EL-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 to increased tissue oxygen requirements, impaired oxygen extraction, maldistributed blood flow, and diminished energy stores. Mizocket al., Crit. CareMed. 20, 80 (1992). The acidic environment subsequently allows Cu(EI) ions to be released from carrier proteins (Lamb et al., FEBSLett. 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 nitric-oxide synthase. Bar-Or, et al., Biochem. Biophys. Res. Commun. 290, 1388 (2002); Bianchini et al, J. Biol. Chem. 274, 20265 (1999).
• carrier proteins Lib
• EL-8 was determined by ELISA (see Example 10).
• Cu(I) ions catalyze the generation of reactive oxygen species resulting in EL-8 secretion from other cell types.
• the results presented here provide evidence that Cu(H) ions stimulate EL-8 secretion from human endothelial cells independent of oxidative stress.
• a high-affinity Cu(H)-binding compound significantly inhibited copper-induced endothelial cell IL-8 secretion.
• Akt protein kinase B
• NF-kappaB nuclear factor-kappaB
• DA-DKP Asp Ala His Lys [SEQ JD NO: l]is degraded in vivo to produce Asp Ala diketopiperazine (DA-DKP) (data not shown).
• DA-DKP 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 inco ⁇ orated herein by reference.), and Asp Ala His Lys [SEQ ED NO : 1 ] may cause additional anti-inflammatory effects as a result of being degraded to produce DA-DKP.
• EXAMPLE 13 Inhibition Of EL-8 By Asp Ala His Lvs in Crevicular Gingival Fluid Cervicular gingival fluid (CGF) was obtained from normal controls (11 individuals) and patients with gingivitis (7 individuals) and periodontitis (9 individuals).
• Crest WhitestripsTM were applied to the teeth of 5 ofthe normal controls according to the package instructions. CGF samples were obtained at various times after the Crest
• Lys [SEQ JD 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 WhitestripTM. The Crest WhitestripsTM were then applied to the teeth of 3 ofthe normal controls according to the package instructions. CGF samples were obtained before the Crest WhitestripsTM 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 ⁇ l storage buffer (phosphate buffered saline (0.1 M sodium phosphate and 0.15 M sodium chloride, pH 7.2) containing 4% bovine serum albumin
• EL-8 interleukin 8
• TNF ⁇ tumor necrosis factor- ⁇
• sTNFR75 soluble tumor necrosis factor- ⁇ receptor
• the EL-8 ELISA was performed as follows. Anti-human EL-8 antibody (Pierce
• TMB substrate solution (Pierce Endogen, Rockford, EL; 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 ⁇ l/well of 0.18 M H 2 SO 4 . 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 TNF ⁇ ELISA was performed as follows. Anti-human TNF ⁇ antibody (Pierce Endogen, Rockford, EL; catalogue number M303-E, lot number 018334) was diluted to 2 ⁇ g/ml in phosphate buffered saline, pH 7.2-7.4, and 100 ⁇ l ofthe 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 ⁇ l 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 ⁇ l/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.
• 100 ⁇ l of biotin-labeled anti-human TNF ⁇ (Pierce Endogen, Rockford, EL; catalogue number M302-B, lot number 017005), diluted to 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 ⁇ l 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 the plates were blotted on a paper towel. Finally, 100 ⁇ l 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 ⁇ l/well of 0.18 M H 2 SO 4 .
• 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 sTNFR75 ELISA assay was performed using the Quantikine sTNFR75 ELISA kit
• the EL-8 ELISA was performed on all ofthe samples (samples from 11 normal controls, 9 periodontitis patients and 7 gingivitis patients), the TNF ⁇ ELISA was performed on CGF samples from 2 ofthe normal controls, 1 ofthe periodontitis patients and 3 ofthe gingivitis patients, and the sTNFR75 ELISA was performed on CGF samples from 1 normal control, 5 periodontitis patients, and 3 gingivitis patients.
• the EL-8 ELISA was performed on all of the CGF samples taken from the 5 normal controls who had the Crest WhitestripsTM applied to their teeth and on all ofthe CGF samples taken from the 3 normal controls to who had the Crest WhitestripsTM with DAHK 1199 applied to their teeth. All samples were assayed in duplicate.
• FIGS 19A-E The results are presented in Figures 19A-E.
• EL-8 and sTNFR75 were elevated, and TNF ⁇ was decreased in patients with gingivitis and periodontitis, as compared to normal controls.
• Treatment with Crest WhitestripsTM increased the amount of EL-8 in the CGF ofthe normal controls by six hours after treatment (see Figure 19D).
• Treatment with Crest WhitestripsTM with DAHK- 1199 reduced the amount of EL-8 in the CGF ofthe normal controls at six hours compared to Crest WhitestripsTM without D AHK- 1199 (compare Figure 19E with Figure 19D).

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