Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • A61K38/1751—Bactericidal/permeability-increasing protein [BPI]
• • 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
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • 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
  • A61P31/12—Antivirals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates

Definitions

• This application relates to the prevention and treatment of adverse oral conditions. More particularly, this application relates to the prevention and treatment of adverse oral conditions such as peridontal disease, gingivitis, plaque, halitosis, and dental caries, by employing a biologically active peptide.
• a process for preventing and/or treating an adverse dental condition(s) comprising administering to a host at least one biologically active amphiphilic peptide or biologically active protein.
• the peptide or protein is an ion channel-forming peptide or protein.
• the composition is administered in an amount effective to prevent or treat adverse oral conditions in a host.
• adverse oral conditions as used herein is intended to mean any condition which adversely affects the oral cavity, including but not limited to teeth, gums, tongue, and the oral imicosa.
• adverse dental conditions include, but are not limited to, plaque; peridontitis, including chronic peridontitis, adult
• gingivitis including acute necrotizing ulcerative gingivitis, halitosis
• dental caries including root surface caries and superficial caries.
• An ion channel-forming peptide or protein or ionophore is a peptide or protein which increases the permeability for ions across a natural or synthetic lipid membrane.
• B. Christensen et al. PNAS Vol. 85 Pgs. 5072-76 (July, 1988) describes methodology which indicates whether or not a peptide or protein has ion channel-forming properties and is therefore an ionophore.
• an ion channel-forming peptide or ion channel forming protein is a peptide or protein which has ion channel-forming properties as determined by the method of Christensen et al.
• amphiphilic peptide is a peptide which includes both hydrophobic and hydrophilic peptide regions.
• the ion channel-forming peptid ⁇ s employed in the present invention are generally water soluble to a concentration of at least 20 mg/ml at neutral pH in water.
• the structure of such peptide provides for flexibility of the peptide molecule. Vfhen the peptide is placed in water, it does not assume an amphiphilic structure. When the peptide encounters an oily surface or membrane, the peptide chain folds upon itself into a rod-like structure.
• such peptides have at least 7 amino acids, and preferably at least 20 amino acids. In most cases, such peptides do not have in excess of 40 amino acids.
• the peptide or protein is generally employed in the form of an oral composition for oral hygiene.
• Such an oral composition may be incorporated into a wide variety of compositions and materials used for oral hygiene purposes, which include, but are not limited to, toothpastes, mouthwashes, tooth gels, and tooth powders.
• Such a compositon may thus be used to treat or prevent periodontal disease, to prevent or reduce plaque, and/or to prevent or treat or reduce dental caries.
• the peptide or protein thus may be used to inhibit, prevent, or destroy the growth of bacteria associated with plaque, periodontal disease, dental caries, and other dental or oral diseases or conditions.
• bacteria include, but are not limited to Streptococcus mutans. Actinobacillus ac inomvcetemcomitans.
• the peptides may also be employed in inhibiting, preventing, or destroying the growth of Enterobacter cloacae, which is associated with dental implant infections, such as periimplantitis.
• the peptides may also be used to inhibit, prevent, or destroy the growth of viruses which adversely affect the oral cavity, and of virally-infected cells found in the oral cavity.
• the peptides may also be employed to prevent, inhibit, or destroy the growth of fungi in the oral cavity.
• the peptides may be employed to prevent, inhibit, or destroy the growth of the fungus Candida albicans. which is associated with thrush.
• the peptides may also be employed in promoting or stimulating the healing of wounds in the oral cavity.
• wound healing includes various aspects of the wound healing process.
• These aspects include, but are not limited to, increased contraction of wounds in the oral cavity, increased deposition of connective tissue, as evidenced by, for example, increased deposition of collagen in the wound, and increased tensile strength of the wound; i.e., the peptides increase wound breaking strength.
• the peptides may also reverse the inhibition of wound healing caused by a depressed or compromised immune system.
• the peptide or protein is administered topically in an amount of from about 2 mg/ml to about 20 mg/ml.
• the peptide is a basic (positively charged) polypeptide having at least sixteen amino acids wherein the pol y peptide includes at least eight hydrophobic amino acids and at least eight hydrophilic amino acids.
• the hydrophobic amino acids are in groups of two adjacent amino acids, and each group of two hydrophobic amino acids is spaced from another group of two hydrophobic amino acids by at least one amino acid other than a hydrophobic amino acid (preferably at least two amino acids) and generally by no greater than four amino acids, and the amino acids between pairs of hydrophobic amino acids may or may not be hydrophilic.
• the hydrophilic amino acids are generally also in groups of two adjacent amino acids in which at least one of the two amino acids is a basic hydrophilic amino acid, with such groups of two hydrophilic amino acids being spaced from each other by at least one amino acid other than -a hydrophilic amino acid (preferably at least two amino aeida) and generally no greater than four amino acids, and the amino acids between pairs of hydrophilic amino acids may or may not be hydrophobic.
• the polypeptide comprises a chain of at least
• each group consisting of four amino acids.
• Two of the four amino acids in each group are hydrophobic amino acids, and two of the four amino acids in each group are hydrophilic, with at least one of the hydrophilic amino acids in each group being a basic hydrophilic amino acid and the other being a basic or neutral hydrophilic amino acid.
• the hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, lie, Leu, Met, Pro, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
• the neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gin, Ser, and Thr.
• the basic hydrophilic amino acids may be selected from the class consisting of Lys, Arg, His, Orn, hoaoarginine (Har), 2, 4-diaminobutyric acid (Dbu), and p-aminophenylalanine.
• Each of the groups of four amino acids may be of the sequence ABCD, BCDA, CDAB, or DABC, wherein A and B are each hydrophobic amino acids and may be the same or different, one of C or D is a basic hydrophilic amino acid, and the other of C or D is a basic or neutral hydrophilic amino acid and may be the same or different.
• the polypeptide chain may comprise 5 or 6 groups of this sequence. In each group, each of A, B, C and D may be the same in some or all of the groups or may be different in some or all of the groups.
• the polypeptide chain preferably has at least 20 amino acids, and no greater than 50 amino acids. It is to be understood, however, that the polypeptide does not have to consist entirely of the groups described above.
• the polypeptide may have amino acids extending from either or both ends of the noted groups forming the polypeptide chain and/or there may be amino acids between one or more of the at least four groups and still remain within the scope of the invention.
• the groups of amino acids may be repeating groups of amino acids, or the amino acids in the various groups may vary provided that in each group of the at least four groups of amino acids there are two hydrophobic and two hydrophilic amino acids as hereinabove noted.
• the biologically active polypeptide comprises a chain including at least four groups of amino acids, each containing four amino acids. Two of the four amino acids in each group are hydrophobic, at least one amino acid is basic hydrophilic, and the remaining one is basic or neutral hydrophilic, with the polypeptide chain preferably having at least 20 amino acids but no greater than 50 amino acids.
• each of the at least four groups of amino acids which are in the peptide chain is of the sequence A-B-C-D, B-C-D-A, C-D-A-B or D-A-B-C wherein A and B are hydrophobic amino acids, one of C or D is a
• the resulting polypeptide chain may have one of the following sequences:
• X 2 is A-, D-A- or C-D-A- £ is -B, -B-C or B-C-D 3 is B-, A-B-, D-A-B-
• Y 3 is -C, -C-D, -C-D-A
• X 4 is C-, B-C-, A-B-C-
• Y 4 is -D, -D-A, -D-A-B a is o or 1; b is o or 1 and n is at least 4.
• the peptide may have amino acids extending from either end of the chain.
• the chains may have a Ser-Lys sequence before the "Ala” end, and/or an Ala-Phe sequence after the "Lys" end.
• Other amino acid sequences may also be attached to the "Ala” and/or the "lys" end.
• the chain may have, for example, a C-D sequence
• amino acid sequences may be attached to the "A” and/or the "D" end of one of these polypeptide chains. Also there may be amino acids in the chain which space one or more groups of the hereinabove noted four amino acids from each other.
• the peptides may be produced by known techniques and obtained in substantially pure form.
• the peptides may be synthesized on an automatic synthesizer. Journal of American Chemical Society, Vol. 85 Pages 2149-54(1963). It is also possible to produce such peptides by genetic engineering techniques.
• the codons encoding the amino acids are known to those skilled in the art, and thus one may construct DNA encoding any of the peptides by accepted techniques, and clone such DNA into an expression vehicle such as, for example, a plasmid, and transfeet such an expression vehicle into a cell which will express the peptide.
• the peptide may be a againin peptide.
• a magainin peptide is either a magainin such as magainin I, II or III or an analogue or derivative thereof.
• the magainin peptides preferably include the following basic peptide structure X._
• R.. is a hydrophobic amino acid
• R is a basic hydrophilic amino acid
• R 13 is a hydrophobic, neutral hydrophilic, or basic hydrophilic amino acid
• R and R,- are hydrophobic or basic hydrophilic amino acids
• R.. is glutamic acid or aspartic acid, or a hydrophobic or a basic hydrophilic amino acid
• n is 0 or 1.
• R._ is a hydrophobic or neutral hydrophilic amino acid
• R.. is a hydrophobic amino acid
• R._ is glutamic acid or aspartic acid.
• a magainin peptide may include the following structure:
• X.. is as previously defined and Z._ is: (i) R. fi where R-, is a basic hydrophilic amino acid or asparagine or glutamine.
• R ⁇ is a neutral hydrophilic amino acid, a hydrophobic amino acid, or a basic hydrophilic amino acid.
• R._ is a neutral hydrophilic amino acid.
• a magainin peptide may also have the following structure:
• X.., Y._ and Z-_ are as previously defined and a is 0 or 1 and b is 0 or 1.
• the magainin peptides may also include the following basic peptide structure X._:
• R,14.a are amino acids as hereinabove described.
• the magainin peptide may also include the following structure X,_-Z,_; wherein X, is the hereinabove 13 13' 13 described basic peptide structure and 7... is
• CR ⁇ V (R llV (R llV (R 14aV (R 15V (R 14aV (R 14V- Wh ⁇ r ⁇ n R ll « R 14' R 14a' R 15' R 16' " R 17 are as hereinabove described, and n is 0 or 1, and each n may be the same or different.
• the magainin peptides generally include at least fourteen amino acids and may include up to forty amino acids.
• a magainin peptide preferably has 22 or 23 amino acids. Accordingly, the hereinabove described basic peptide structures of a magainin peptide may include
• magainin peptides having the following primary sequences as given in the accompanying sequence listing as well as appropriate analogues and derivatives thereof:
• Magainin peptides are described in Proc. Natl. Acad Sci. Vol. 84 pp. 5449-53 (Aug. 87).
• magainin peptides refers to the basic magainin structure as well as derivatives and analogs thereof, including but not limited to the representative derivatives or analogs.
• the peptide may be a PGLa peptide or an XPF peptide.
• a PGLa peptide is either PGLa or an analogue or derivative thereof.
• the PGLa peptides preferably include the following basic peptide structure X , :
• R. . , R. _ , R, » , and R_ 7 are as previously defined.
• the PGLa peptides generally include at least seventeen amino acids and may include as many as forty amino acids. Accordingly, the hereinabove described basic peptide structure for a PGLa peptide may include additional amino acids at the amino end or at the carboxyl end or at both the amino and carboxyl end.
• a PGLa peptide may have the following structure:
• a PGLa peptide may also have the following structure:
• a PGLa peptide may also have the following structure:
• a is 0 or 1 and b is 0 or 1.
• An XPF peptide is either XPF or an analogue or derivative thereof.
• the XPF peptides preferably include the following basic peptide structure X.,:
• R. j , R j _, R.,, R._ and R j _ are as previously defined and R, g is glutamine or asparagine or a basic hydrophilic, or hydrophobic amino acid and, n is 0 or 1.
• the XPF peptides generally include ⁇ t least nineteen amino acids and may include up to forty amino acids. Accordingly, the hereinabove described basic peptide structure of XPF may include additional amino acids at the amino end, or at the carboxyl end or at both the amino and carboxyl ends.
• an XPF peptide may include the following structure:
• An XPF peptide may include the following structure:
• X., Y., and Z. are as previously defined: a is 0 or 1 and b is 0 or 1.
• XPF or PGLa peptides which are characterized by the following primary amino acid sequences as given in the accompanying sequence listing:
• the peptide may be a CPF peptide or appropriate analogue or derviative thereof.
• CPF peptides as well as analogues and derivatives thereof are herein sometimes referred to collectively as CPF peptides.
• the CPF peptide may be one which includes the following basic peptide structure X..:
• R_. is a hydrophobic amino acid
• R_. is a hydrophobic amino acid or a basic hydrophilic amino acid
• R__ is a basic hydrophilic amino acid
• R_ is a hydrophobic or neutral hydrophilic amino acid
• R_ is a basic or neutral hydrophilic amino acid.
• hydrophobic amino acids are Ala, Cys, Phe, Gly, lie, Leu, Met, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cycloh ⁇ xylalanine (Cha).
• the neutral hydrophilic amino acids are Asn, Gin, Ser, and Thr.
• the basic hydrophilic amino acids are Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu), and p-amin ⁇ phenylalanine.
• the CPF peptide may include only the hereinabove noted amino acids or may include additional amino acids at the amino and/or carboxyl end or both the amino and carboxyl end. In general, the peptide does not include more than 40 amino acids.
• the CPF peptides including the above basic structure preferably have from 1 to 4 additional amino acids at the amino end.
• the carboxyl end of the basic peptide structure may also have additional amino acids which may range from 1 to 13 additional amino acids.
• the basic structure may have from 1 to 7 additional amino acids at the carboxyl end, which may be represented as follows:
• X 2 _, Y__ and Z 2Q are as previously defined and a is 0 or 1 and b is 0 or 1.
• CPF peptides which may be employed, some of which have been described in the literature, include the following sequences as given in the accompanying sequence listing: (SEQ ID NO:14) (SEQ ID NO:15) (SEQ ID NO:16) (SEQ ID NO:17) (SEQ ID NO:18) (SEQ ID NO:19)
• the peptide may include one of the following basic structures X 31 through X__ wherein:
• X 36 is ⁇ IR 32 -R 32 "R 3l 'R 32 "R 32 “R 33 “R 31 ,” n X 37 is "[R 32 "R 3l “R 32 “R 32 “R 33 “R 3l 'R 32 I” n wherein . ⁇ is a basic hydrophilic amino acid, R__ is a hydrophobic amino acid, R._ is a neutral hydrophilic, basic hydrophilic, or hydrophobic amino acid, and n is from 2 to 5.
• the basic hydrophilic amino acids may be selected from the class consisting of Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu), and p-aminophenylalanine.
• the hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, lie, Leu, Met, Pro, Val, Trp and Tyr,norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
• the neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gin, Ser and Thr.
• the peptide when the peptide includes the structure X,., the peptide may include the following structure:
• Y_.-X_. wherein X,- is as hereinabove described, and Y_ j is:
• the peptide when the peptide includes the structure X, j , the peptide may include the following structure:
• the peptide may include the following structure:
• the peptide may include the following structure:
• the peptide when the peptide includes the structure X,., the peptide may include the following structure:
• the peptide may include the following structure:
• Y 32 and z 3 are as previously defined, a is 0 or 1, and b is 0 or 1.
• the peptide when the peptide includes the structure X__, the peptide may include the following structure:
• the peptide when the peptide includes the structure X, the peptide may include the following structure:
• the peptide may include the following structure:
• the peptide when the peptide includes the structure X_, the peptide may include the following structure:
• the peptide when the peptide includes the structure X 34 , the peptide may include the following structure:
• the peptide may include the following structure:
• the peptide when the peptide includes the structure X__, the peptide may include the following structure:
• the peptide when the peptide includes the structure X,_, the peptide may include the following structure:
• the peptide may include the following structure:
• the peptide when the peptide includes the structure X, fi , the peptide may include the following structure:
• the peptide when the peptide includes the structure X, the peptide may include the following structure:
• the peptide may include the following structure:
• the peptide when the peptide includes the structure X,_, the peptide may includes the structure Y,_-X,_, wherein X,_ is as hereinabove described, and Y__ is:
• the peptide when the peptide includes the structure X 3 -, the peptide may include the following structure:
• the peptide may include the following structure:
• n 3
• peptide is of one of the following structures as given in the accompanying sequence listing:
• Xaa is p-aminoph ⁇ ylalanine.
• the biologically active amphiphilic peptide includes the following basic structure X,_:
• R 3 R 32 "R 32 'R 33 "R 34 "R 32 "R 32 'R 3 R 32 "R 32 "R 32 “R 34 "R 32 “R 32' wherein R_., R__, and R are as hereinabove described, and R_, is a basic hydrophilic or hydrophobic amino acid.
• the peptide may include the following structure:
• R 33 and R 34 are as hereinabove described.
• the peptide may include the following structure:
• n 0 is:
• R.,., R 32 , R,, and R_ are as hereinabove described.
• peptide has the following structural formula as given in the accompanying sequence listing:
• the peptide has the following structural formula as given in the accompanying sequence listing:
• the peptide has one of the one of the following structural formulae as given in the accompanying sequence listing:
• the peptide may include the following structural formula:
• n is from 2 to 5.
• n is 3, and the peptide has the following structural formula:
• the peptide may include the following structural formula:
• n is from 2 to 5.
• n 3
• the peptide has the following structural formula:
• the peptide may include the following structural formula:
• n is from 2 to 5.
• n is 3, and the peptide has the following structural formula:
• the peptide may be selected from the group consisting of the following structural formulae as given in the accompanying sequence listing:
• the peptide may be a cecropin or sarc ⁇ toxin.
• cecropins includes the basic structure as well as analogues and derivatives thpreof.
• the cecropins and analogues and derivatives thereof are described in Ann. Rev. Microbiol. 1987, Vol. 41, pages 103-26, in particular page 108, and in Christensen, et al., PNAS Vol. 85, pgs. 5072-76, which are hereby incorporated by reference.
• sarcotoxins includes the basic materials as well as analogues and derivatives thereof.
• the sarcotoxins and analogues and derivatives thereof are described in Molecular Entomology, pages 369-78, in particular page 375, Alan R. Liss, Inc. (1987), which is hereby incorporated by reference.
• amphiphilic peptide includes the following basic structure X ( - ⁇ '
• R,. is a hydrophobic amino acid, and R,. is a basic hydrophilic or neutral hydrophilic amino acid.
• the peptide includes the basic structure Y_ -X_. wherein X- 0 is as hereinabove described and Y_ 0 is:
• R,. is leitcine.
• R,_ is lysine.
• Representative examples of peptides in accordance with this aspect of the present invention include those having the following structures:
• amphiphilic peptide includes the following basic structure X__:
• R 42 R 4 R 2 'R 42 "R 4r R r R 2 "R 2 "R r R 2 ⁇ R 2' wherein R,. is a hydrophobic amino acid and R,_ is a basic hydrophilic or neutral hydrophilic amino acid.
• R 4 is leucine.
• R,Thr is lysine.
• the peptide includes the basic is as hereinabove
• the peptide may have the following structure; Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu Lys Lys Leu
• the peptide includes the basic structure X_. - Z_., wherein X_ is as hereinabove described, and Z_. is:
• the peptide may have the following structure:
• the peptide may include the structure:
• CT 52 J a " X 52 ⁇ (Z 52V whe " in X 52' Y 52 and Z 52 are as hereinabove described, and a is 0 or 1, and b is 0 or
• the peptide includes the following basic structure X_,:
• R 43' wherein R, , and R,note are as hereinabove described, and R.. 41 42 43 is a natural hydrophilic amino acid.
• the peptide may have the following structure:
• the peptide may have the following structure:
• the peptide includes the following basic structure X.,:
• R 4r R 42 "R 4 R 4l “R 42 “R 42 “R 4r R 4r R 42 “R 42 “R 44' wh ⁇ “i” R 41 and R,_ are as hereinabove described, and R, , is a neutral hydrophilic amino acid or proline.
• the peptide may include the following structure Y 56 " ⁇ e6» wherein X_, is the basic peptide structure hereinabove described, and Y_, is:
• the peptide may include the structure:
• the peptide may have one of the following structures: (SEQ ID NO:103); or (SEQ ID NO:104).
• the peptide may have the structure ⁇ g fl " X 56"f Z 5fi)b' wherein X 5fi» Y 56' and Z 5 are as hereinabove described, a is 0 or 1, and b is 0 or 1.
• the peptide includes the following basic structure X__:
• the peptide may include the structure ⁇ o ' ⁇ ⁇ g- wherein X, is as hereinabove described, and Y_. is:
• the peptide includes the structure X,_-Z__, wherein X, g is as hereinabove described, and Z_ is:
• the peptide has the following structure:
• the peptide may have the structure
• the peptide includes the following basic structure X,_;
• the peptide may have the following structure:
• the peptide may include the structure x gn" Z 60' wherein X.. is as hereinabove described, and Z,_ is:
• the peptide has the structure (a), and a representative example of such a structure is (SEQ ID NO:107), which is given in the accompanying sequence listing.
• the peptide has the structure (b), and a representative example of such a structure is (SEQ ID NO:108), which is given in the accompanying sequence listing.
• the peptide has the structure (c), and a representative example of such a structure is (SEQ ID NO:109) as given the accompanying sequence listing.
• the peptide has the structure (d), and a representative example of such a structure is (SEQ ID NO:110) as given in the accompanying sequence listing.
• the peptide has the structure (e), and representative examples of such a structure are (SEQ ID NO:111) and (SEQ ID NO:112) as given in the accompanying sequence listing.
• the peptide has the following structural formula:
• the peptide is melittin.
• -Melittin is an amphipathic peptide consisting of 26 amino acid residues, and is isolated from honeybee (Apis mellifera) venom.
• the peptide is known to be cytolytic. See Hab ⁇ rmann, et ⁇ l., Hoppe-Seyler's Zeitschrift Physiol. Chem.. Vol. 348, pgs. 37-50 (1987).
• the peptide purified in accordance with the present invention is an apidaecln.
• apidaecln as used herein includes the basic structure as well as analogues and derivatives thereof. Apidaecins are further described in European Patent Application No. 299,828.
• amphiphilic peptide or protein may be an ion channel-forming peptide or protein.
• Ion channel-forming proteins or peptides which may be employed include defensins, also known as human neutrophll antimicrobial peptides (HNP), major basic protein (MBP) of eosinophils, bactericidal permeability-increasing protein (BPI), and a pore-forming cytotoxin called variously perforin, cytolysin, or pore-forming protein.
• HNP human neutrophll antimicrobial peptides
• MBP major basic protein
• BPI bactericidal permeability-increasing protein
• a pore-forming cytotoxin called variously perforin, cytolysin, or pore-forming protein.
• Defensins are described in Selsted, et al. , J. Clin. Invest.. Vol. 76, pgs. 1436-1439 (1985).
• MBP proteins are described in Wasmoen,
• ion channel-forming proteins includes the basic structures of the ion channel-forming proteins as well as analogues and derivatives.
• each of the amino acid residues of the peptide or protein may be a D-amino acid residue or glycine.
• peptides or proteins may be administered in combination with one another.
• the peptides or proteins of the present invention may be employed in combination with an ion having pharmacological properties for the purposes hereinabove described.
• An ion having pharmacological properties is an ion which when introduced into a target cell inhibits and/or prevents and/or destroys the growth of a target cell.
• Such an ion having pharmacological properties is one which in the absence of an ion channel-forming peptide or protein is unable to cross a natural or synthetic lipid
• -43- membrane in particular a cell membrane, in sufficient amounts to affect a cell adversely.
• the peptide or protein and ion having pharmacological properties may be administered as a single composition or in separate compositions, and the single or separate compositions may include additional materials, actives and/or inactives, in addition to the peptide or protein and anti-target ion.
• ions having pharmacological properties there may be mentioned fluoride, peroxide, bicarbonate, silver, zinc, mercury, arsenic, copper, platinum, antimony, gold, thallium, nickel, selenium, bismuth, and cadmium ions.
• the ion having pharamacological properties is a fluoride ion.
• the peptide or protein and the ion having pharmacological properties are employed in amounts effective to inhibit and/or prevent and/or destroy the growth of oral bacteria, or of viruses or fungi which adversely affect the oral cavity.
• the ion potentiates the action of the peptide or protein, i.e., the amount of ion is effective to reduce the minimum effective concentration of the peptide or protein for inhibiting growth of oral bacteria, fungi, viruses, or
• the ion having pharmacological properties is generally employed in a concentration of from about 1 mg/ml to about 10 mg/ml.
• the peptide or protein could be administered in an amount of up to about 25 weight to weight and the ion delivered in an amount of about 0.1% to 1.0%.
• the peptide may be administered in combination with other oral hygiene agents such as, but not limited to, chlorhexidine (peridex), sanguinarine HCl, antiseptic mouthwashes, tricolssn, etc.
• oral hygiene agents such as, but not limited to, chlorhexidine (peridex), sanguinarine HCl, antiseptic mouthwashes, tricolssn, etc.
• the peptides or proteins of the present invention may be administered to a host in combination with an antibiotic selected from the class consisting of bacitracins, gramacidins, polymyxin, vancomycin, teichoplanin, amin ⁇ glycosides, hydrophobic antibiotics, penici... --onobactams, or derivatives or analogues thereof.
• an antibiotic selected from the class consisting of bacitracins, gramacidins, polymyxin, vancomycin, teichoplanin, amin ⁇ glycosides, hydrophobic antibiotics, penici... --onobactams, or derivatives or analogues thereof.
• the bacitracins, gramacidins, polymyxin, vancomycin, and teichoplanin, and derivatives - -liogues thereof, are a group of polypeptide antibiotics.
• a preferred bacitracin is bacitracin A.
• Amino lycoside antibiotics include tobramycin, kanamycin, amikacin, the ge ⁇ tamicins (e.g., gentamicin C., gentamicin C_, gentamicin C ), netilmicin, kanamycin, and derivatives and analogues thereof.
• the preferred aminoglycosides are tobramycin and the gentamicins.
• the aminoglycosides, and the bacitracins hereinabove described, tend to be hydrophilic and water-soluble.
• Penicillins which may be employed include, but are not limited to benzyl penicillin, amplcillin, methiclllln (dimethoxyphenyl penicillin), tic ⁇ ricillin, penicillin V (phenoxy ethyl penicillin), oxacillin, cloxacillin, diclox ⁇ cillln, flucloxacillin, amoxicillln, and amidinocillln.
• Preferred penicillins which may be employed are benzyl penicillin and amplcillin.
• a preferred monobacta which may be employed is aztreonam.
• hydrophobic antibiotics which may be used in the present invention, there may be mentioned macrolides such as erythromyein, roxythromycin, clarithromycin, etc. ; 9-N-alkyl derivatives of erythromyein; midecamycin acetate; azithromycin; flurithromycin; rif ⁇ butin; rokitamycin; a 6-0-ra ⁇ thyl erythromyein A known as TE-031 (Taisho); rifapentine; benzypiperazinyl rif ⁇ mycins such as CGP-7040, CGP-5909, CGP-279353 (Ciba-Geigy); an erythromyein A derivative with a cyclic carbamate fused to the C../C-. position of a macrolide ring known as
• rifamycin carbenicillin, and nafcillin may be employed as well.
• antibiotics which may be used are antibiotics which are 50-S rlbosome inhibitors such as lincomycin; cUndamycin; and chloramph ⁇ nicol; etc.; antibiotics which have a large lipid like 1actone ring, such as mystatin; pimaricin, etc.
• the peptide or protein and antibiotic may be adminstered orally In order to prevent, destroy or inhibit the growth of bacteria, such as but not limited to those hereinabove described, which may be associated with adverse oral conditions. Bacteria whose growth may be prevented, inhibited, or destroyed by the
• -47- administration of the peptides and antibiotic include Gram-positive and Gram-negative bacteria.
• antibiotic such as those hereinabove described, or derivatives or analogues thereof, is generally employed in a concetration of about 0.2% to about 2.0%.
• Peptide or protein dosages may be those as hereinabove described.
• a few colonies of each organism to be tested are picked up with a wire loop and introduced into a test tube containing broth.
• a broth containing 28g Brucella broth, 2g sodium formate, 3g sodium fumarate, 50 ml defibrinated sheep blood, 2 ml hemolyzed sheep blood cells, 10 ml herain-menadione solution, and 1 liter of distilled H.O was prepared for the following organisms:
• the hemin-menadione solution used in the above-described broth was prepared by dissolving 50 mg of hemin in 1 ml IN NaOH, and brought to 100 ml with distilled H 2 0 to form a hemin solution. 100 mg of menadione was added to 20 ml distilled H.O to form a menadione solution. 1 ml of the menadione solution is then added to 100 ml of the hemin solution to form the hemin-menadione solution 10 ml of which is added to the broth.
• a broth containing 22g trypticase soy broth, lg yeast extract, 100 ml horse serum, 5 ml of a bacitracin solution of 75 mg bacitracin in 5 ml distilled H.0, 5 ml of a vancomycin solution of 5 mg vancomycin in 5 ml distilled H 2 0, and 1 liter of distilled 1 0 is prepared for Actinobacillus actinomycetemcomitans.
• the tubes are incubated to produce a bacterial suspension of moderate cloudiness.
• the bacteria-containing broth is then diluted, If necessary, with water or saline solution to a density virtually equivalent to that of a 0.5 McFarland standard prepared by adding 0.5 ml' of 1% BaCl, to 99.5 ml of 1% H Constant,
• the bacterial broth suspension is streaked evenly in three planes onto the surface of the culture medium of 5 to 6 mm in depth, in a Petri dish, with a cotton swab.
• An agar medium of 3.0% tryptlease soy broth, 1.5% agarose, 2.5 mg/1 hemin, and 0.25 mg/1 menadione is used for the following organisms: Eikenella corrodens: Peptostreptococcus micros; Bacteroides gingivalis; Bacteroides fragilis; Bacteroides intermedius; and Actinomvces viscosus; and Actinobaeillus actinomycetemeomitans.
• disks saturated with peptide or peptide and NaF are placed on the agar with flamed forceps or a disk applicator.
• the disks have been saturated with 20 ⁇ l of a peptide solution to deliver a final dose of from 0.25 ⁇ g to 128 ⁇ g on the disk or with peptide in the heretofore mentioned dosages and 1,000 ppm NaF.
• the disks may be laid down in a circle to form an outer ring of about 9 disks, and the remaining disks may be placed in the center.
• zone diameters including the 6mm disk
• the zone diameters are measured on the undersurface of the Petri disk with a ruler or with calipers near the agar surface. A reading of 6mm indicates no inhibition. The end point is taken as the lowest dose causing inhibition of growth as determined by the naked eye.
• Peptides 1 through 4 were tested for the minimal inhibitory concentration (MIC) against A. actinomycetemcomitans strain Y4, Eikene11a corrodens. S. mutans strain 6714, S. san ius strain M-5, and A. viscosus strain T14V.
• Peptide 1 has the following structural formula:
• Peptide 2 has the following structural formula:
• Peptide 3 has the following structural formula: (SEQ ID NO: 116)
• Peptide 4 has the following structural formula: (SEQ ID NO:117)
• Example 2 The procedure of Example 1 was repated to determine the HIC of peptides 1 through 4 against A.viscosus strain T14, S.mutans. S.sangius. A.actinomycetemcomitans strain Y4, E.corrodens. Bacteroides gingivalis, Wolinella recta strain 371, and Baeterioides intermediu . The results are given below in Table II.
• Example 3 The MIC of Peptides 1 through 4 alone, and of Peptides 1 through 4 in combination with 1,000 ppm NaF was tested against A.viscosus. S.sangius strain M4, S.mutans strain 6715, E.corrodens strain 28371, A.actinomycetemcomitans strain Y4, B. ingivalis strain 33277, B.intermedius strain 25611, and W.recta strain 371.
• Table III gives the MIC of each peptide alone against each organism, while Table IV gives the MIC of each peptide in combination with 1,000 ppm NaF against each organism.
• peptides 1, 4, 5, 6, and 7 were tested for minimum inhibitory concentration (MIC) against Bacteroides fragilis.
• Peptide 5 has the following structural formula:
• Peptide 7 has the following structural formula:
• Table V gives the zones of inhibition at various concentrations of peptide per disc.
• Table V - Zones of Inhibition ⁇ g peptide/disc Peptide 0 I 1 4 ji JJ5 32 64 1 - 8mm 11mm 12mm
• the MIC of Peptide 1 is 16 ⁇ g/ml
• the MIC of Peptide 5 is B ⁇ g/ml
• the MIC of Peptide 6 is 64 ⁇ g/ml
• the MTC of Peptide 7 is 32 ⁇ g/ml.
• peptides 5, 8, 9, and 10 were tested for minimum inhibitory concentration (MIC) against the following organisms:
• Peptide 8 has the following structural formula:
• Peptide 9 has the following structural formula:
• Peptide 10 has the following structural formula:
• Example 6 -58- saliva, with its native bacterial flora intact, is isolated from a human volunteer, and is then passed continuously over germanium crystals for 20 minutes prior to treatment. Germanium crystals have surface properties similar to tooth enamel.
• water, or Peptide 11 also known as (SEQ ID NO:121)
• Peptide 12 also known as D-(SEQ ID NO:121) (In which each amino acid residue is a D-amino acid residue or glycine); chlorhexidine; or triclosan in a concentration of O.lmg/ml in water, were applied for a period of 30 seconds, two times per day for three days.
• the germanium crystals were analyzed for bacterial count, plaque area, plaque score, and extracellular carbohydrate (ex-CHO, which is indicative of bacterial concentration), by use of a Fourier transformed infrared speetrophotometer.
• the results are shown in Figure 1.
• the Peptide 11 and Peptide 12 are more effective against bacteria and plaque than chlorhexidine or triclosan.
• peptides of the present invention are effective against oral bacteria associated with plaque, gingivitis, halitosis, dental caries, periodontal disease, and other adverse oral conditions.
• ADDRESSEE Carella, Byrne, Bain, Gilfillan,
• NAME/KEY Magainin II peptide.
• NAME/KEY magainin peptide

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• 1992
  • 1992-07-09 EP EP92915677A patent/EP0671930A4/de not_active Withdrawn
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