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
  • 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
• • 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
  • C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to biologically active peptides and proteins , and more particularly to compositions rind uses involving biological ly active peptides having C- terminal substitutions .
• composition comprising a C-termin ⁇ ] substituted peptide or protein of the formula : O
• X is a biologically active amphiphiiic peptide or protein.
• the peptide or protein is an ion channel-forming peptide or protein.
• T is selected from the group consisting of:
• R is a substituted or unsubstituted aliphatic, aromatic, or aralkyl group having from 1 to 10 carbon atoms.
• R is an alkyl group.
• the resulting C-terminal substituted peptide is sometimes hereinafter referred to as a C-terminal ester.
• R' and R" are hydrogen or selected from the class consisting of group (i) and .group (ii), wherein; group (i) is a hydroxy-substituted aliphatic, aromatic, or aralkyl group having no more than 10 carbon atoms.
• group (i) is a hydroxy-substituted alkyl group.
• group (i) is CH-CH-OH.
• Group (ii) is an amino-substituted aliphatic or aromatic, aralkyl, or alkylaromatic group.
• the amino-substituted group has no more than 10 carbon atoms.
• group (ii) is an amino-substituted alkyl group.
• group (ii) is CH-CH-NH,.
• the peptides terminated with group (d) are sometimes hereinafter referred to as substituted C-terminal amides.
• the peptide or protein is an ion channel-forming peptide or protein.
• 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 protei is a peptide or protein which has ion channel-forming properties as determined by the method of Christensen et al.
• An a phiphilic peptide is a peptide which includes both hydrophobic and hydrophilic peptide regions.
• the ion channel-forming peptides 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.
• Such peptides are capable of forming an alpha-helical s 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 administered topically at a concentration of from .05% to 10%.
• the peptide or protein is employed to provide peptide or protein dosages of from l g to 500mg per kilogram of host weight.
• compositions may be administered to a host; for example a human or non-human animal, in an amount effective to inhibit growth of a target cell.
• a host for example a human or non-human animal
• the compositions may be used as antimicrobial agents, anti-viral agents, antibiotics, anti-tumor agents, antiparasitic agents, antifungal agents, spermicides, as well as exhibiting other bioactive functions.
• antimicrobial means that the peptides of the present invention inhibit, prevent, or destroy the growth or proliferation of microbes such as bacteria, fungi, or the like.
• antibiotic means that the peptides employed in the present invention produce effects adverse to the normal biological functions of the non-host cell, tissue, or organism including death or destruction and prevention of the growth or proliferation of the non-host cell, tissue, or organism when contacted with the peptides.
• spermicidal as used herein means that the peptides employed in the present invention, inhibit, prevent, or destroy the motility of sperm.
• antiviral means that the peptides employed in the present invention inhibit, prevent, or destroy the growth or proliferation of viruses, or virally-infected cells.
• anti-tumor means that the peptide inhibits the growth of or destroys tumors.
• antifungal means that the peptide of the present invention may be used to inhibit the growth of or destroy fungi.
• antiparasitic means that the peptides of the present invention may be used to inhibit the growth of or destroy parasites.
• the peptides of the present invention have a broad range of potent antibiotic activity against a plurality of microorganisms including Gram-positive and Gram-negative bacteria, fungi, protozoa, and the like, as well as parasites.
• the peptides of the present invention allow a method for treating or controlling microbial infection caused by organisms which are sensitive to the peptides. Such treatment may comprise administering to a host organism or tissue susceptible to or affiliated with a microbial infection an antimicrobial amount of at least one of the peptides.
• antibiotics because of the antibiotic, antimicrobial, and antiviral properties of the peptides, they may also be used as preservatives or sterilants of materials susceptible to microbial or viral contamination.
• peptide and/or derivatives or analogues thereof may be administered in combination with a non-toxic pharmaceutical carrier or vehicle such as a filler, non-toxic buffer, or physiological saline solution.
• a non-toxic pharmaceutical carrier or vehicle such as a filler, non-toxic buffer, or physiological saline solution.
• Such pharmaceutical compositions may be used topically or systemic lly and may be in any suitable
• SUBSTITUTESHEET form such as a liquid, solid, semi-solid, injectable solution, tablet, ointment, lotion, paste, capsule, or the like.
• the peptide compositions may also be used in combination with adjuvants, protein inhibitors, or compatible drugs where such a combination is seen to be desirable or advantageous in controlling infection caused by harmful microorganisms including protozoa and the like, as well as by parasites and viruses.
• the peptide(s) of the present invention may be administered to a host; in particular an animal, in an effective antibiotic and/or anti-tumor and/or anti-viral and/or anti-microbial and/or anti-parasitic and/or an antispermicidal amount.
• composition in accordance with the invention will contain an effective anti-microbial amount and/or an effective antispermicidal amount and/or an effective anti-viral amount and/or an effective anti-tumor amount and/or an effective antibiotic amount and/or anti-parasitic amount of one or more of the hereinabove described peptides which have such activity.
• the peptide of the present invention may also be employed in promoting or stimulating healing of a wound in a host.
• wound healing includes various aspects of the would healing process.
• peptides increase wound breaking strength.
• the peptides of the present invention may also be employed so as to reverse the inhibition of wound healing caused by a depressed or compromised immune system.
• compositions of the present invention may also be used in the treatment of external burns and to treat and/or prevent skin and burn infections.
• the compositions may be any suitable compositions of the present invention.
• SUBSTITUTE SHEET used to treat skin and burn infections caused by organisms such as, but not limited to, P. eruginosa and S.aureus.
• the peptides of the present invention may be used in the treatment of external burns and to treat and/or prevent skin and burn infections.
• the peptides may be used to treat skin and burn infections cause by organisms such as, but not limited to, P. aeruginosa and S. aureus.
• the peptides are also useful in the prevention or treatment of eye infections.
• infections may be caused by bacteria such as, but not limited to, P. aeruginosa, S. aureus, and N. gonorrhoeae, by fungi such as but not limited to C. albicans and A. fumigatus, by parasites such as but not limited to A. castellani, or by viruses.
• the peptides may also be effective in killing cysts, spores, or trophozoites of infection - causing organisms.
• Such organisms include, but are not limited to Acanthamoeba which forms trophozoites or cysts, C. albicans, which forms spores, and A. fumigatus, which forms spores as well.
• the peptides may also be administered to plants in order to prevent, inhibit, or destroy the growth of microbes, bacteria, fungi, viruses, cysts, spores, or parasites in plants.
• each of the amino acid residues of the peptide or protein is a D-amino acid residue or glycine.
• the scope of this particular embodiment is not to be limited to any theoretical reasoning, it is believed that the peptide or protein, when consisting entirely of D-amino acid or glycine residues, may have increased resistance to proteolytic enzymes while retaining their biological activity. Such peptides or proteins thus may be administered orally.
• all of the amino acid residues are D-amino acid or glycine residues, or -amino acid or glycine residues.
• the peptide is a basic (positively charged) polypeptide having at least sixteen
• SUBSTITUTESHEET amino acids wherein the polypeptide includes at least eight hydrophobic amino acids and at least eight hydrophilic amino acids. Still more particularly, 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 acids) 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 four groups of amino acids, with 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 and homoserine (Hse).
• 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.
• 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 o C or D is a basic hydrophilic amino acid, and the other of C or 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, eac 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 ⁇ rroups 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 provide 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 havin 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 basic hydrophilic amino acid, and the other of C or D is basic or neutral hydrophilic amino acid.
• the resulting polypeptide chain may have one of the following sequences:
• X- is A-, D-A- or C-D-A-
• Y- is -B, -B-C or B-C-D
• Y- 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 0 or 1; b is 0 or 1 and n is at least 4.
• the peptide chain may include amino acids between the hereinabove noted groups of four amino acids provided that the spacing between such groups and the charge on the amino acids does not change the characteristics of the peptide chain which provide amphiphilicity and a positive charge and do not adversely affect the folding characteristics of the chain to that which is significantly different from one in which the hereinabove noted group of four amino acids are not spaced from each other.
• 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 before the first A-B-C-D group.
• other amino acid sequences may be attached to the "A" and/or the "D" end of one of these polypeptide chains.
• amino acids in the chain which space one or more groups of the hereinabove noted four amino acids from each other.
• the peptides with the substituted C-terminals may be synthesized by either solid phase peptide synthesis or solution methods of peptide synthesis.
• solid phase peptide synthesis the peptide, with appropriate side chain protection, is synthesized by established methods on a suitable resin.
• the protected peptide resin is suspended in methanol (25 ml/g of resin) and stirred with a large excess of an appropriate amino component for several days in order to produce a C-terminal substituted hydrazide, hydroxylamine, or amide having one of the structures hereinabove described.
• a diamino compound is used, one of the amino groups is suitably protected.
• the peptide plus resin is filtered, washed with methanol, and dried.
• the resin and peptide mixture is subjected to HE treatment and the deprotected peptide amide, hydroxylamine, or hydrazide is extracted with dilute acetic acid and lyophilized.
• the C-terminal esters can be obtained by transesterification of the peptide with an appropriate alcohol as solvent in the presence of a tertiary base such as triethylamine.
• a tertiary base such as triethylamine.
• the work up and the isolation of the peptide ester is similar to the procedure for isolating the other C-terminal substituted peptides.
• the C-terminal substituted ester peptide may be made by beginning the solution synthesis with an appropriate amino acid ester. If other modified C-terminal • peptides are desired, the protected peptide C-terminal ester may be reacted with hydroxylamine or hydrazine to produce an appropriate peptide C-terminal hydroxylamine or peptide C-terminal hydrazide, respectively.
• the C-terminal ester is hydrolyzed by standard techniques, and the protected peptide acid is reacted with the desired amino component (suitably protected) by standard coupling procedures. The protected peptide derivative is then deprotected and purified.
• the peptides in unsubstituted form by genetic engineering techniques.
• the codons encoding the amino acids of the peptides 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 transfect such an expression vehicle into a cell which will express the peptides.
• an expression vehicle such as, for example, a plasmid
• the peptide may be substituted at the C-terminal in accordance with the present invention by techniques such as those hereinabove described.
• the peptide may be a magainin peptide.
• a magainin peptide is either a magainin such as magainin I, II or III or an'analogue or derivative thereof.
• the magainin is either a magainin such as magainin I, II or III or an'analogue or derivative thereof.
• SUBSTITUTE SHEET peptides preferably include the following basic peptide structure
• R-- is a hydrophobic amino acid
• R_- is a basic hydrophilic amino acid
• R,- 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:
• R ⁇ / R i2' R i4 ancl R l4a are as P reviousi y defined.
• a magainin peptide may also have the following structure:
• R. fi where R, g is a basic hydrophilic amino acid or asparagine or glutamine.
• R 16 -R 17 where 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:
• the magainin peptides may also include the following basic peptide structure X--:
• the magainin peptide may also include the following structure X._-2- 3 ; wherein X__ is the hereinabove described basic peptide structure and Z.- is
• R ll n- ⁇ R ll>n- ⁇ R ll)n ⁇ R 14a)n- ⁇ R 15)n- ⁇ R 14a)n ⁇ R 14 ) n- ⁇ R 16)n- ⁇ R 17)n
• R ⁇ , R n ⁇ R i a ' R 15* R 16' and R 17 are as nereinaDove 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 additional amino acids at the amino end or at the carboxyl end, or at both ends.
• 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 14 *
• 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:
• PGLa peptide may also have the following structure:
• a PGLa peptide may also have the following structure:
• X 14 ; Y 14 and ⁇ 14 are as previously defined, 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 lg *
• R 11 "R 14 'R 12 "R 11 "R 11 “R 12 “ R ⁇ _R ⁇ r wherein R ll' R 12' R 14' R 15 and R 17 are as P reviousl y defined and R ⁇ a is glutamine or asparagine or a basic hydrophilic, or hydrophobic amino acid and, n is 0 or 1.
• the XPF peptides generally include at 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:
• An XPF peptide may also have the following structure: • ⁇ i6>a "Xl6 ⁇ 2l6) b where lg / Y 16 and Z lg 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 2 : "*R 2l "R 21 ⁇ R 22 ⁇ R 22 "R 2l “R 2l “R 23 "R 2l "
• R 77 is a hydrophobic amino acid or a basic hydrophilic amino acid
• R, 3 is a basic hydrophilic amino acid
• R_. is a hydrophobic or neutral hydrophilic amino acid
• R ?t - is a basic or neutral hydrophilic amino acid.
• hydrophobic amino acids are Ala, Cys, Phe, Gly, lie, Leu, Met, Val, Trp, Tyr, norleucine (Nle), norvaiine (Nval), and cyclohexylalanine (Cha).
• SUBSTITUTESHEET 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-aminophenylalanine.
• 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 is the hereinabove defined basic peptide structure and Z 20 is
• Preferred peptides may be represented by the following structural formula Y 20 a " X 20 " (Z 20 ) b
• 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) (SEQ ID NO:20) (SEQ ID N0:21) (SEQ ID NO:22) (SEQ ID N0:23) (SEQ ID NO:24) (SEQ ID NO:25) (SEQ ID NO:26)
• the peptide may include one of the following basic structures X 3 - through X 3 _ wherein X 31 is -[R 31 -R 32 -R 32 -R 33 -R 31 -R 32 -R 32 n 32 is " - R 32 "R 32 ⁇ R 33 "R 3l “R 32 “R 32 “R 31 “ n 33 is " - R 32 "R 33 -R 3l “R 32 “'R 32 “R 3l “R 32 " n
• X 34 is ⁇ - R 33 ⁇ R 31 ⁇ R 32 ⁇ R 32 ⁇ R 31 ⁇ R 32 ⁇ R 32 " n
• : 35 is -[ 3 1 - R 32 - 3 2 - R 3 1 - R 3 2 - R 3 2 - 33 " n
• X 36 is ⁇ [R 32 ⁇ R 32 ⁇ R 3l “R 32 ⁇ R 32 ⁇ R 33 ⁇ R 31 " n and X '3.7 is "[R 32 "R 3l “R 32 “R 32 “R 33 “R 3l “R 32 “ n wherein R 31 is a basic hydrophilic amino acid, R,_, IS a hydrophobic amino acid, R 33 is a neutral 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, Val, Trp and Tyr,norleucine (Nle), norvaiine (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 31 the peptide may include the following structure:
• the peptide when the peptide includes the structure 31 / the peptide may include the following structure:
• the peptide may include the following structure:
• X 32 / tne peptide may include the following structure:
• the peptide when the peptide includes the structure X 32r the peptide may include the following structure:
• the peptide may include the following structure: t - Y 32' ) a " X 32 " Z 32* , b' wherein Y 32 and Z 32 re as reviously defined, a is 0 or 1, and b is 0 or 1.
• the peptide when the peptide includes the structure 33 .
• the peptide may include the following structure:
• the peptide when the peptide includes the structure X33 / the peptide may include the following structure: 33 ⁇ Z 33 wherein X 33 is as hereinabove described, and Z 33
• 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 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 X35.
• the peptide may include the following structure:
• the peptide when the peptide includes the structure X 35 .
• the peptide may include the following structure:
• the peptide may include the following structure:
• the peptide when the peptide includes the structure X 3g .
• the peptide may include the following structure:
• the peptide when the peptide includes the structure X 3g / the peptide may include the following structure:
• the peptide may include the following structure: Y 36>a" X 36 ⁇ 36 ⁇ ' wherein Y 36 and Z 36 are aS Previously defined, a is 0 or 1, and b is 0 or 1.
• the peptide when the peptide includes the structure X 37 .
• the peptide may includes the structure
• Y 37 -X 37 wherein X 37 is as hereinabove described, and Y 37 is:
• the peptide when the peptide includes the structure X 37 .
• the peptide may include the following structure:
• the peptide may include the following structure:
• n 3
• peptide has one of the following structures:
• Xaa is p-aminophenylalanine.
• the biologically active amphiphilic peptide which includes the following basic structure X4. rt 0:
• R 34 is a basic hydrophilic or hydrophobic amino acid.
• the peptide may include the following structure:
• the peptide may include the following structure:
• X 4 ⁇ " Z 40' w erein X 4Q is as hereinabove described and Z 40 is: (i) R 31 ; (ii) R 31 - R 32 ; (iii) R 31 - 32 - 32 ;
• 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 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 is selected from the group consisting of the following structural formulae as given in the accompanying sequence listing:
• the peptide includes the following basic structure ⁇ o : R 4l "R 42 “R 42 “R 4l “R 42 “R 42 “R 4l “ 4l “R 42 “R 4l “ 4l “R 42 “R 4l “R 41 ]
• R 4 - is a hydrophobic amino acid, and R 4 , is a basic hydrophilic or neutral hydrophilic amino acid.
• the peptide includes the basic structure
• R 4.1. is leucine. In another embodiment, R is lysine.
• Representative examples of peptides in accordance with this aspect of the present invention include those having the following structures:
• R 41 is leucine. In another embodiment,
• R 42 is lysine
• the peptide includes the basic structure Y__ - c2 ' wherein X-.-, is as hereinabove described, and Y g2 is: (i) R 42 ; ( ⁇ ) R 41 - 42 ;
• the peptide may have the following structure
• the peptide includes the basic structure X 52 -Z 52 , wherein X-- is as hereinabove described, and
• the peptide may have the following structure:
• the peptide may include the structure:
• X 52' Y 52' and Z 52 are S hereinabove described, and a is 0 or 1, and b is 0 or 1.
• the peptide includes the following basic structure ⁇ 4 :
• the peptide may have the following structure:
• the peptide may have the following structure:
• the peptide includes the following basic structure X 5g :
• the peptide may include the following structure Y- g -X-. ⁇ , wherein X-. g is the basic peptide structure hereinabove described, and Y-. g is:
• the peptide may have one of the following structures:
• the peptide may have the structure (Y- g ) -X 5 g-(Z-. g ) b , wherein ⁇ g / ⁇ ⁇ g- and z -. g are s hereinabove described, a is 0 or 1, and b is 0 or 1.
• the peptide includes the following basic structure X ⁇ a : R 41 ⁇ R 41 ⁇ R 42 ⁇ R 42 ⁇ R 41 ⁇ R 42 ⁇ R 42 ⁇ R 41 ⁇ R 41 ⁇ R 42 ⁇ R 42 ⁇ R 41 ⁇ R 43' wherein 41 / R 42 and R.- are as hereinabove described.
• the peptide may include the structure Yc g -Xc g ; wherein X_. ⁇ is as hereinabove described, and Y-. g is:
• the peptide includes the structure X- g -Z 58 , wherein X-. tenu is as hereinabove described, and Z 58 is:
• the peptide has the following structure:
• the peptide may have the structure (Y 58 ) a -X 5Q -(Z 58 ) b , wherein X 5 g. Y 5 g, and Z-.g are as hereinabove described, a is 0 or 1, and b is 0 or 1.
• the peptide includes the following basic structure X g0 ;
• the peptide may have the following structure:
• the peptide may include the structure X g0 -Zg 0 wherein X g0 is as hereinabove described, and Z g0 is:
• the peptide has a structure selected from the group consisting of:
• 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: (SEQ ID NO:113).
• the peptide may be a cecropin or sarcotoxin.
• cecropins includes the basic structure as well as analogues and derivatives thereof.
• 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.
• Ion channel-forming proteins or peptides which may be employed include defensins, also known as human neutrophil antimicrobial peptides (HNP), major basic protein (MBP) of eosinophils, bactericidal permeability-increasing protein (BPI), and a pore-forming cytotoxin called variously perforin,
• defensins also known as human neutrophil antimicrobial peptides (HNP), major basic protein (MBP) of eosinophils, bactericidal permeability-increasing protein (BPI), and a pore-forming cytotoxin called variously perforin
• HNP human neutrophil antimicrobial peptides
• MBP major basic protein
• BPI bactericidal permeability-increasing protein
• variously perforin a pore-forming cytotoxin
• SUBSTITUTESHEET cytolysin, or pore-forming protein 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, et al., J. Biol. Chem. , Vol. 263, pgs 12559-12563. (1988).
• BPI proteins are described in Ooi, et al, J. Biol. Chem. , Vol. 262, pgs. 14891-14894 (1987).
• Perforin is described in Henkart, et al., J. Exp. Med. , 160: 75 (1984), and in Podack, et al., J. Exp. Med. , 160:695 (1984). The above articles are hereby incorporated by reference.
• ion channel-forming proteins includes the basic structures of the ion channel-forming proteins as well as analogues and derivatives.
• 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 one which when introduced into a target cell, virus, or virally-infected cell, inhibits and/or prevents and/or destroys the growth of the target cell, virus, or virally-infected 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 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 ion having pharmacological properties.
• additional materials such as sodium, sodium, sodium, sodium, sodium, sodium, sodium, sodium, sodium, sodium, sodium, sodium, sodium, sodium, sodium, sodium, sodium, sodium sulfate, sodium bicarbonate, sodium ions.
• 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 the target cell, virus, or virally-infected cell.
• 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 or a target cell, virus, or virally-infected cell.
• the ion having pharmacological properties when used topically, is generally employed in a concentration of from 0.05% to 2.0%. When used systemically, the ion is generally employed in an amount of from 1 to 10 mg. per kg. of host weight. Peptide or protein dosages may be within the ranges hereinabove described.
• the peptide or protein and ion may be delivered or administered in different forms; for example, the ion may be administered orally, while the peptide or protein may be administered by IV or IP.
• the peptide or protein could be administered in an amount of up to about 1% weight to weight and the ion delivered in an amount of about 50mM (about 0.1%).
• the ion, in the form of a salt such as sodium fluoride could be administered orally in conjunction with systemic administration of the peptide.
• the peptide or protein may be administered IV or IP to achieve a serum dose of 100 micrograms per milliliter (10 milligrams per kilogram) in conjunction with an oral dose of ion, in particular, sodium fluoride, of 10 meq per kilogram.
• 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, gramacidin, polymyxin, vancomycin,
• SUBSTITUTESHEET teichoplanin aminoglycosides, hydrophobic antibiotics, penicillins, monobactams, or derivatives or analogues thereof.
• the bacitracins are a group of polypeptide antibiotics.
• a preferred bacitracin is bacitracin A.
• Aminoglycoside antibiotics include tobramycin, kanamycin, amikacin, the gentamicins (e.g., gentamicin C., gentamicin C 7 , gentamicin C J.lEL), 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, ampicillin, methicillin (dimethoxyphenyl penicillin), ticaricillin, penicillin V (phenoxymethyl penicillin), ⁇ xacillin, cloxacillin, dicloxacillin, flucloxacillin, amoxicillin, and amidinocillin.
• Preferred penicillins which may be employed are benzyl penicillin and ampicillin.
• a preferred monobactam which may be employed is aztreonam.
• hydrophobic antibiotics which may be used in the present invention, there may be mentioned macrolides such as erythromycin, roxythromycin, clarithromycin, etc.; 9-N-alkyl derivatives of erythromycin; midecamycin acetate; azithro ycin; flurithromycin; rifabutin; rokitamycin; a 6-0-methyl erythromycin A known as.TE-031 (Taisho); rifapentine; benzypiperazinyl rifamycins such as CGP-7040, CGP-5909, CGP-279353 (Ciba-Geigy); an erythromycin A derivative with a cyclic carbamate fused to the c ⁇ / c ⁇ 2 Position of a macrolide ring known as A-62514 (Abbott); AC-7230 (Toyo Jozo); bbYenzoxazinorifamycin; difficidin; dirithromycin
• SUBSTITUTESHEET (Abbott); 3-formylrifamycin SV-hydrazones with diazabicycloalkyl side chains such as CGP-27557 and CGP-2986 (Ciba-Geigy); and 16-membered macrolides having a 3-0-alpha-L-cladinosyl moiety, such as 3-0-alpha-L-cladinosyldeepoxy rosaramicin; tylosins and acyl demycinosyl tylosins.
• rifamycin carbenicillin, and nafcillin may be employed as well.
• antibiotics which may be used are antibiotics which are 50-S ribosome inhibitors such as lincomycin; clindamycin; and chloramphenicol; etc.; antibiotics which have a large lipid like lactone ring, such as mystatin; pimaricin, etc.
• the peptide or protein and antibiotic may be adminstered by direct administration to a target cell or by systemic or topical administration to a host which includes the target cell, in order to prevent, destroy or inhibit the growth of a target cell.
• Target cells whose growth may be prevented, inhibited, or destroyed by the administration of the peptides and antibiotic include Gram-positive and Gram-negative bacteria as well as fungal cells.
• the antibiotic such as those hereinabove described, or derivatives or analogues thereof, when used topically, is generally employed in a concetration of about 0.1% to about 10%.
• the antibiotic or derivative or analogue thereof when used systemically, is generally employed in an amount of from 1.25 mg. to about 45 mg. per kg. of host weight per day.
• Peptide or protein dosages may be those as hereinabove described.
• the peptide or protein could be administered in an amount of from about 0.1% to about 10% weight to weight, and the antibiotic is delivered in an amount of from about 0.1% to about 10% weight to weight.
• the peptides or proteins of the present invention may be administered in combination with an antiparasitic agent or an antifungal agent.
• Antiparasitic agents which may be employed include, but are not limited to, anti-protozoan agents.
• Examples of specific anti-parasitic agents which may be employed include, but are not limited to, pentamidine isethionate, and propamidine isethionate (Brolene) .
• Anti-fungal agents which may be employed include, but are not limited to, ketoconazole. It is also to be understood that certain anti-parasitic agents, may also have anti-fungal activity, and that certain anti-fungal agents may have anti-parasitic activity.
• the peptides or proteins of the present invention may be administered in combination with an antibiotic which inhibits DNA gyrase, which is an enzyme involved in the formation of bonds between individual coiling strands of replicating bacterial DNA.
• DNA gyrase is necessary for the normal replication of bacterial DNA, and, therefore, antibiotics which inhibit DNA gyrase inhibit the normal replication of bacterial DNA.
• antibiotics which inhibit DNA gyrase include nalidixic acid, oxolinic acid, cinoxacin, and quinolone antibiotics which include ciprofloxacin, norfloxacin, ofloxacin, enoxacin, pefloxacin, lomefloxacin, fleroxacin, tosulfloxacin, temafloxacin, and rufloxacin.
• the peptides or proteins of the present invention may be administered for the purposes hereinabove described in combination with one another.
• Peptide 1 has the following structural .formula:
• Peptide 2 has the following structural formula:
• Peptide 3 has the following structural formula:
• the stock peptide solution is diluted in serial dilutions (1:2) down the wells of a microtiter plate so that the final concentrations of peptides in the wells are 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, 128, and 256 ⁇ g/ml.
• 1-5 X 10 5 CFUs/ l of either S. aureus ATCC 25923, E. coli ATCC 25922, or P. aeruginosa ATCC 27853 were added to the wells in full strength Mueller Hinton broth (BBL 11443) from a mid-log culture.
• the inoculum is standarized spectrophotometrically at 600nm and is verified by colony counts.
• MIC minimal inhibitory concentration
• S is the MIC of the peptide against S.aureus
• P is the MIC of the peptide against P. eruginosa
• E is the MIC of the peptide against E.coli.
• peptides or proteins of the present invention may be employed in a wide variety of pharmaceutical compositions in combination with a non-toxic pharmaceutical carrier or vehicle such as a filler,- non-toxic buffer, or physiological saline solution.
• a non-toxic pharmaceutical carrier or vehicle such as a filler,- non-toxic buffer, or physiological saline solution.
• Such pharmaceutical compositions may be used topically or systemically and may be in any suitable form such as a liquid, solid, semi-solid, injectable solution, tablet, ointment, lotion, paste, capsule or the like.
• peptide or protein and/or agent as hereinabove described may also be used in combination with adjuvants, protease inhibitors, or compatible drugs where such a combination is seen to be desirable or advantageous in controlling infection caused by harmful microorganisms including protozoa, viruses, parasites, fungi, and the like.
• the peptide or protein may be administerd to a host in particular an animal, in an effective antibiotic and/or anti-tumor and/or antiviral and/or antimicrobial and/or antispermicidal and/or antifungal and/or antiparasitic amount, or in an amount effective to stimulate wound healing in a host.
• the peptides or proteins may be administerd either alone or in combination with an ion having pharmacological properties, antibiotic, or ion channel forming peptide or protein as hereinabove described. When the peptide or protein is administered in combination with an ion having pharmacological properties, the activity of the peptide or protein is potentiated.
• SUBSTITUTE SHEET administer the peptide or protein and agent in separate forms.
• the agent may be administered systemically and the peptide or protein may be administered topically.
• the peptide or protein When the peptide or protein is adminitered topically, it may be administered in combination with a water-soluble vehicle, said water-soluble vehicle being in the form of an ointment, cream, lotion, paste or the like.
• water-soluble vehicles which may be employed include, but are not limited to, glycols, such as polyethylene glycol, hydroxycellulose, and KY Jelly.
• the water-soluble vehicle is preferably free of an oily substance.
• the peptide or protein may also be employed in combination with a toxic ion as hereinabove described in the form of an oral composition for oral hygiene.
• a 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 composition 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 and toxic ion may be used to inhibit, prevent, or destroy the growth of Streptococcus mutans, which is associated with dental caries and periodontal disease.
• ADDRESSEE Carella, Byrne, Bain, Gilfillan,
• NAME/KEY Magainin II peptide.
• NAME/KEY magainin peptide
• NAME/KEY magainin peptide
• NAME/KEY magainin peptide

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• 1992
  • 1992-06-01 EP EP92913961A patent/EP0590044A4/en not_active Withdrawn
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