EP0750632A1 - Peptides amphiphiliques formant des canaux ioniques et presentant des modifications n-terminales - Google Patents

Peptides amphiphiliques formant des canaux ioniques et presentant des modifications n-terminales

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Publication number
EP0750632A1
EP0750632A1 EP95909267A EP95909267A EP0750632A1 EP 0750632 A1 EP0750632 A1 EP 0750632A1 EP 95909267 A EP95909267 A EP 95909267A EP 95909267 A EP95909267 A EP 95909267A EP 0750632 A1 EP0750632 A1 EP 0750632A1
Authority
EP
European Patent Office
Prior art keywords
peptide
amino acid
lys
ala
basic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95909267A
Other languages
German (de)
English (en)
Inventor
U. Prasad Kari
Taffy J. Williams
Michael Mclane
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Magainin Pharmaceuticals Inc
Original Assignee
Magainin Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Magainin Pharmaceuticals Inc filed Critical Magainin Pharmaceuticals Inc
Publication of EP0750632A1 publication Critical patent/EP0750632A1/fr
Withdrawn legal-status Critical Current

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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
    • A61P31/04—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
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • 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
    • 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 . More particularly, this invention relates to biologically active peptides having N-terminal (or amino- terminal ) substitutions .
  • T is a biologically active peptide or protein.
  • the peptide or protein is preferably an ion channel-forming peptide or protein.
  • T is a lipophilic moiety, and W is T or hydrogen.
  • lipophilic means that the lipophilic moiety enhances the interaction of the peptide or protein with a lipid membrane, such as, for example, a cell membrane.
  • Lipophilic moieties which may be employed include, but are not limited to, any moiety which may be placed on the N-terminal of the peptide through a condensation reaction with nitrogen.
  • the lipophilic moiety T may be, for example, a carboxylic acid, a phosphoric acid, preferably an alkylphosphoric acid, a phosphonic acid, preferably an alkylphosphonic acid, a sulfonic acid, preferably an alkylsulfonic acid, or an alkyl group.
  • T is: O
  • R -C - wherein R is a hydrocarbon having at least two and no more than 16 carbon atoms.
  • R is an alkyl group.
  • the alkyl group may be a straight chain or branched chain alkyl group; or a
  • R may be CH 3 (CH_) -, wherein n is from 1 to 14.
  • n is from 3 to 12, more preferably from 4 to 11, still more preferably from 6 to 11, and most preferably n is 6, whereby T is an octanoyl group.
  • R is an aromatic (including phenyl and naphthyl) , or an alkyl aromatic group.
  • R may be 0-(CH_) -, wherein z is from 0 to 6.
  • R is
  • n is from 1 to 5.
  • n is 1, whereby R is-an ibuprofyl group.
  • T is: O
  • HOOC- (CH ) -C wherein x is from 1 to 14.
  • x is 2, and T is a succinyl group.
  • T is:
  • T is a sphingosine group.
  • T is:
  • x and y are hereinabove described.
  • x is 2, and y is 12.
  • W is hydrogen
  • the biologically active peptides or proteins of the present invention are preferably ion channel-forming peptides.
  • 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 . 5072-5076 (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 .
  • An amphophilic peptide or protein is a peptide or protein which includes both hydrophobic and hydrophilic peptide or protein 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 structure. When the peptide is placed in water, it does not assume an amphophilic structure. When the peptide encounters an oily surface or membrane, the peptide chain folds upon itself into a rodlike structure.
  • such peptides have at least 7 amino acids, and in many cases have at least 20 amino acids. In most cases, such peptides do not have in excess of 40 amino acids.
  • the peptides and/or analogues or derivatives thereof may be administered to a host; for example a human or non-human animal, in am amount effective to inhibit growth of a target cell, virus, or virally-infected cell.
  • a host for example a human or non-human animal
  • the peptides and/or analogues or derivatives thereof may be used as antimicrobial agents, anti-viral agents, anti ⁇ bacterial agents, anti-tumor agents, anti-parasitic agents, spermicides, as well as exhibiting other bioactive functions.
  • antimicrobial means that the polypeptides or proteins of the present invention inhibit, prevent, or destroy the growth or proliferation of microbes such as bacteria, fungi, viruses, or the like.
  • anti-bacterial means that the peptides or proteins employed in the present invention produce effects adverse to the normal biological functions of bacteria, including death or destruction and prevention of the growth or proliferation of the bacteria when contacted with the peptides or proteins.
  • antibiotic means that the peptides or proteins 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 or proteins.
  • spermicidal as used herein means that the peptides or proteins employed in the present invention, inhibit, prevent, or destroy the motility of sperm.
  • anti-fungal means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of fungi.
  • anti-viral means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of viruses, or of virally-infected cells.
  • anti-tumor means that the peptides or proteins inhibits the growth of or destroys tumors, including cancerous tumors.
  • anti-parasitic means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of parasites.
  • the peptides or proteins 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 or proteins of the present invention allow a method for treating or controlling microbial infection caused by organisms which are sensitive to the peptides or proteins. 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 or proteins.
  • antibiotics because of the antibiotic, antimicrobial, antiviral, and antibacterial properties of the peptides or proteins, they may also be used as preservatives or sterilants or disinfectants of materials susceptible to microbial or viral contamination.
  • the peptides or proteins and/or derivatives or analogues thereof may be administered in combination with a non-toxic pharmaceutical carrier or vehicle such as a filler, non- oxic buffer, or physiological saline solution.
  • a non-toxic pharmaceutical carrier or vehicle such as a filler, non- oxic 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.
  • the peptide or protein compositions 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, and the like, as well as by parasites.
  • the peptides or proteins of the present invention may be administered to a host; in particular a human or non-human animal, in an effective antibiotic and/or anti-tumor and/or anti-fungal and/or anti-viral and/or anti-microbial and/or antibacterial and/or anti-parasitic and/or spermicidal amount.
  • composition in accordance with the invention will contain an effective anti-microbial amount and/or an effective spermicidal amount and/or an effective anti-fungal amount and/or an effective anti-viral amount and/ or an effective anti-tumor amount and/or an effective anti-parasitic and/or an effective antibiotic amount of one or more of the peptides or proteins of the present invention which have such activity.
  • the peptides or proteins may be administered by direct application of the peptides or proteins to the target cell or virus or virally-infected cell, or indirectly applied through systemic administration.
  • the peptides or proteins 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 wound healing process.
  • peptides or proteins increase wound breaking strength*.
  • the peptides or proteins of the present invention may also be employed so as to reverse the inhibition of wound healing caused by conditions which depress or compromise the immune system.
  • the peptides or proteins 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 or proteins may be used to treat skin and burn infections caused by organisms such as, but not limited to, P. aeruqinosa and S. aureus.
  • SUBSTITUTE SHEET(RULE28) The peptides or proteins are also useful in the prevention or treatment of eye infections. Such infections may be caused by bacteria such as, but not limited to, P. aeru ⁇ inosa, S aureus, and N. onorrhoeae. by fungi such as but not limited to C. albicans and A fumiqatus. by parasites such as but not limited to A. castellani. or by viruses.
  • the peptides or proteins 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. fumiqatus, which forms spores as well.
  • the peptides or proteins may also be administered to plants in an effective antimicrobial or antiviral or antiparasitic amount to prevent or treat microbial or viral or parasitic contamination thereof.
  • the peptides or proteins may also be employed in treating septic shock in that such peptides neutralize bacterial endotoxins.
  • the peptides or proteins are positively charged, while in general, the bacterial endotoxins are negatively charged.
  • the peptides or proteins are particularly useful in that such compounds neutralize bacterial endotoxins without neutralizing essential proteins in plasma (such as heparin, for example) .
  • the peptides or proteins when used in topical compositions, are generally present in an amount of at least 0.1%, by weight. In most cases, it is not necessary to employ the peptide in an amount greater than 2.0%, by weight.
  • the active peptide or protein is present in an amount to achieve a serum level of the peptide of at least about 5 ug/ml .
  • the serum level of peptide or protein need not exceed 500 ug/ ml.
  • a preferred serum level is about 100 ug/ml.
  • Such serum levels may be achieved by incorporating the peptide or protein in a composition to be administered systemically at a dose of from 1 to about 10 mg/kg.
  • SUBSTITUTE SHEET(RULE28) or protein(s) need not be administered at a dose exceeding 100 mg/kg.
  • the peptides or proteins may be produced by known techniques and obtained in substantially pure form.
  • the peptides may be synthesized on an automatic peptide synthesizer. Journal of the American Chemical Society. Vol. 85, pgs . 2149-54 (1963) . It is also possible to produce such peptides or proteins by genetic engineering techniques.
  • the codons encoding specific amino acids are known to those skilled in the art, and therefore DNA encoding the peptides may be constructed by appropriate techniques, and one may clone such DNA into an appropriate expression vehicle (e.g., a plasmid) which is transfected into an appropriate organism for expression of the peptide or protein.
  • an appropriate expression vehicle e.g., a plasmid
  • the N-terminal (NH_ or amino terminal) of the peptide is reacted such that the lipophilic moiety is attached to the N-terminal of the peptide.
  • the reaction may be a condensation reaction with an amine.
  • T is O
  • R - C - the N-terminal is reacted with a carboxylic acid of the formula R-COOH, wherein R is a hydrocarbon having at least 2 carbon atoms.
  • the reaction may be carried out in the presence of a coupling agent, such as, for example, DCC, or DIC, and HOBT, or in the presence of an acid chloride.
  • a coupling agent such as, for example, DCC, or DIC, and HOBT
  • x is a peptide which is a basic (positively charged) polypeptide having at least sixteen 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, 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 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
  • SUBSTITUTE SHEET(RULE28) be the same or different.
  • the polypeptide chain may comprise 5 or 6 groups of this sequence.
  • 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 it 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 may comprise 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 amin ⁇ -aci.ds 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 therefore, 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 X 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 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 groups 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 "O" 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.
  • X is 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 peptides preferably include the following basic peptide structure X_. »
  • 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:
  • a magainin peptide may also have the following structure:
  • R _ is a basic hydrophilic amino acid or asparagine or glutamine.
  • R g -R.- 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: where X 12 . ⁇ 12 an ⁇ Z 12 are as P rev; *- ous ly 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 described basic peptide structure and Z _ is
  • R n' R i4 ' R l4a' R 15' R 16' and 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 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) .
  • the term "magainin peptides" as used herein refers to the basic magainin structure as well as derivatives and analogs thereof, including but not limited to the representative derivatives or analogs .
  • X 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_ . :
  • 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 :
  • Y 14 is ( i ) R l:L ;
  • a PGLa peptide may also have the following structure:
  • a PGL have the following structure: where X 14 ; Y ' 14 and Z . 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.lb- : --R 11 -R 17 -R 12 -R 11 -R 14 -R 18 -R 17 -
  • R ll ' R 12 ' R 14 ' R 15 and R 17 are as P rev:* - ous ly defined and R- o 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.
  • the hereinabove described basic peptide structure for 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:
  • R 11 -R 18 -P oline-R 12 An XPF peptide may also have the following structure:
  • X.lb ., Y.lb_ and Z.lb 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:
  • X is a CPF peptide or appropriate analogue or derivative thereof.
  • CPF peptides as well an 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 2 n : "R 2l “R 2l “R 22 “R 22 _R 2l “R 2l “R 23 “R 2l "
  • R_ is a hydrophobic acid
  • R 2 _ 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- j - 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 cyclohexylalanine (Cha) .
  • the neutral hydrophilic amino acids are Asn, Gin, Ser, Thr, and homoserine (Hse) .
  • 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.
  • X__ is the hereinabove described basic peptide structure and Y_ Q is ( i ) R 25-' or ( ii ) R 22 -R 2 5'- or
  • R 22- R 21- R 22 'R 25 Preferably Glycine - 21 -R 22 - 25 - wherein L ⁇ , R and R 2 _ are as previously defined.
  • 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 1 to 7 additional amino acids at the carboxyl end, which may be represented as follows:
  • X is the hereinabove defined basic peptide structure
  • Preferred peptides may be represented by the following structural formula
  • 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:
  • SUBSTITUTE SHEET (RULE 28) (SEQ " ID NO:20) (SEQ ID NO:21) (SEQ ID NO:22) (SEQ ID NO:23) (SEQ ID NO:24) (SEQ ID NO:25) (SEQ ID NO:26)
  • X is a peptide which includes one of the following basic structures X., through X_schreib wherein:
  • X 36 is " [R 32 "R 32 “R 3l”R 32 "R 32 “R 33 “R 31 and
  • X 3.7 . is -[ R 32 - R 3 1 - R 3 2 - R 3 2 - R 33- R 3 1 - R 32 wherein R _._. is a basic hydrophilic amino acid, R_ is a hydrophobic amino acid, R 3 -. is a neutral hydrophilic, basic hydrophilic, or hydrophobic amino acid, and n is from 1 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 Try, norleucine (Nle) , norvaline (Nva) , and cyclohexylalanine (Cha) .
  • the neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gin, Ser, Thr, and homoserine (Hse) .
  • 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 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 ⁇ -, the peptide may include the following structure:
  • R 33 -R 31 -R 32 -R 32 -R 31 -R 32 wherein R 31 , R 32 , and R 33 are as hereinabove described.
  • the peptide when the peptide includes the structure X-,-,. the peptide may include the following structure:
  • the peptide may include the following structure:
  • the peptides when the peptides includes the structure X 34 .
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X--, 4 .
  • the peptide when the peptide includes the structure X--, 4 .
  • the peptide may include the following structure :
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X, j ., the peptide may include the following structure:
  • Y 35 is:
  • R 32 "R 32- R 31- R 32- R 32- R 33' wher ein R 31 , R 32 and R 33 are as hereinabove described.
  • the peptide when the peptide includes the structure X 3 ⁇ » the peptide may include the following structure:
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure the peptide may include the following structure: is as hereinabove described, and
  • the peptide when the peptide includes the structure X 36.
  • the peptide may include the following structure : wherein X,., Jb, is as hereinabove described, and
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X-, 7 .
  • the peptide may includes the structure Y- j _-X 37 , wherein X_ 7 is as hereinabove described, and Y is:
  • the peptide when the peptide includes the structure 37 » 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:
  • He Ala Lys He Ala Gly Lys 3 (SEQ ID NO:54) .
  • X is a peptide which includes the following basic structure X 4r) :
  • R 32 » an d R 3 _ are as hereinabove described, and R 34 is a basic hydrophilic or hydrophobic amino acid.
  • the peptide may include the following structure:
  • X is a peptide which includes the following structure:
  • 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:
  • X is a peptide which includes one of the following structural formulae:
  • n is from 1 to 5.
  • n is 3
  • the peptide has one of the following structural formulae:
  • the X is a peptide which is selected from the group consisting of the following structural formulae as given in the accompanying sequence listing:
  • X is 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.
  • X is melittin or an analogue or derivative thereof.
  • Melittin is an amphipathic peptide consisting of 26 amino acid residues, and is isolated from honeybee (Apis mellifera) venom. Habermann, et al . , Hoppe-Seyler' s Zeitschrift Phvsiol. Chem.. Vol. 348, pgs 37-50 (1987) . Melittin has the following structural formula as represented by the three-letter amino acid code: Gly He Gly Ala Val Leu Lys Val. Leu
  • X is a amphiphilic peptide which includes the following basic structure X C -
  • R.. is a hydrophobic amino acid
  • R 42 is a basic hydrophilic or neutral hydrophilic amino acid.
  • the peptide includes the basic structure Y t - 0 -X c - n wherein X ⁇ . is as hereinabove described and
  • R is leucine.
  • R._ is lysine .
  • Representative examples of peptides in accordance with this aspect of the present invention include those having the following structures :
  • X is an amphiphilic peptide which includes the following basic structure X-._ :
  • R 41 is a hydrophobic amino acid and R 4 disturbance is a basic hydrophilic or neutral hydrophilic amino acid.
  • R.- is leucine. In another embodiment, R 4 _ is lysine.
  • the peptide includes the basic structure Y ⁇ 2 -X.- 2 , where X__ is as hereinabove described, and
  • 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_ 2 ⁇ Z 2 , where X ⁇ 2 is as hereinabove described, and
  • the peptide may have the following structure :
  • the peptide may include the structure:
  • X is a biologically active amphiphilic peptide which includes the following basic structure X-- 4 :
  • the peptide may have the following structure :
  • the peptide may have the following structure:
  • X is a biologically active amphiphilic peptide which includes the following basic structure X__ :
  • R4.2_ are as hereinabove described, and R4.4. is a neutral hydrophilic amino acid or proline.
  • the peptide may include the structure:
  • Z 56 is ( i ) - R 42 ; (ii) - R 42 "R 42 ; ( iii ) - 42 - R 42 - R 41
  • the peptide may have one of the following structures:
  • X is a biologically active amphiphilic peptide which includes the following basic structure X D_.O 0 :
  • R4 * ,1 . ' R4,2' and R4.3_, are as hereinabove described.
  • the peptide includes the structure
  • Xb c o o -Z 5 b c8 o o , l wwhheerreein Xb c o o is as hereinabove described, and Zb r o o is
  • the peptide has the following structure:
  • X is a biologically active amphiphilic peptide which includes the following basic structure X c bU n : R 4l "R 4l 'R 43 "R 42 “R 4l “R 4l “R 4l “R 4l “R 4l “R 4l “R 4l “R 42 “R 4l “R 42 “R 4l “R 42 “R 4l “R 42 “R 4l “ ' wherein R 41 ' R 42 ' and R 43 are as hereinabove described.
  • the peptide may have the following structure:
  • X is a peptide which includes the following basic structure X 62 "
  • R 4 -]_ and R4 2 are as hereinabove described.
  • the peptide includes the following structure Y g2 - g2 , where X g is as hereinabove described, and Y 62 is:
  • the peptide includes the structure X 62 ⁇ Z 62 ' wherein X fi _ is as hereinabove described, and Z fimony is (i) R 41
  • a representative example includes the following peptide having the structural formula given below and listed in the accompanying sequence listing: (SEQ ID NO: 112)
  • the peptide has the structure
  • X is a peptide having the following structural formula: (SEQ ID NO: 117)
  • X is a biologically active amphiphilic peptide including the following basic structure
  • the peptide may include the structure
  • Y6-4.-X6,4.- wherein X6 r 4. is as hereinabove described, and Y6_4. is: d ) - R 41 ; or ( ii ) R 42 -R 41 -
  • the peptide may include the structure wherein X6,4. is as hereinabove described,
  • the peptide has the structure:
  • X is a biologically active amphiphilic peptide including the following basic structure
  • X 66 R 41 "R 42- R 42- R 41- R 41- R 46- R 42- R 41- R 42- R 42- R 41' wherein R 41 and R are hereinabove described and R , is glutamic acid.
  • a representative example of such a peptide is the following:
  • X is a biologically active amphiphilic peptide including the following basic structure
  • the peptide includes the following basic structure Y gg -X gg , wherein X _ is as hereinabove described, and Y gg is:
  • X is a biologically active amphiphilic peptide including the following basic structure
  • X is a biologically active amphiphilic peptide including the following basic structure X 72 :
  • a representative example of such a peptide has the following structure:
  • X is a biologically active amphiphilic peptide having the following structure:
  • X is a biologically active amphiphilic peptide including the following structure X 7 4 :
  • X is a biologically active amphiphilic peptide including the following structure X / c b -.
  • the peptide includes the structure Y 7g -X 7g -, wherein X 7g is as hereinabove described, and Y g is:
  • the peptide includes the structure -X 7g -Z 7g , wherein X 7g is as hereinabove described, and Z 7g is:
  • R ft is a basic hydrophilic, neutral hydrophilic, or hydrophobic amino acid.
  • the peptide has the following structural formula:
  • X is a biologically active amphiphilic peptide including the following structural formula X 7g :
  • X has the following structure:
  • X is a biologically active amphiphilic peptide including the following structural formula X fl0 :
  • R4.-1, R4.2town, and R4.6_ are as hereinabove described.
  • a representative example of such a peptide has the following structure:
  • X is an ion channel-forming peptide or protein.
  • 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, cytolysin, or pore-forming protein.
  • HNP human neutrophil 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 asmoen, et al .
  • 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.
  • each of the amino acid residues of the peptides or proteins may be a D- amino acid or glycine.
  • the scope of this particular embodiment is not to be limited to any theoretical reasoning, it is believed that the above-mentioned peptides or proteins, when consisting entirely of D-amino acid or glycine residues, may have increased resistance to proteolytic enzymes while retaining their activity. Such peptides thus may be administered orally.
  • all of the amino acid residues may be D-amino acid or glycine residues, or L-amino acid or glycine residues.
  • peptides or proteins may be administered in combination with one another.
  • N-terminal substituted peptides or proteins of the present invention may be employed in combination with an ion having phamacological properties for the purposes hereinabove described.
  • An ion having pharmacological properties is one which when introduced into a target cell or virus or virally-infected cell inhibits and/or prevent 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 is unable to cross a natural or synthetic lipid membrane; in particular a cell or virus membrane, in sufficient amounts to affect a cell or virus 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, actives and/or inactives in addition to the peptide or protein and ion having pharmacological properties.
  • ions having pharmacological properties which may be employed, there may be mentioned fluoride, peroxide, bicarbonate, silver, zinc, mercury, arsenic, copper,
  • 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, i.e., the amount of ion is effective to reduce the maximum effective concentration of the peptide or protein for inhibiting growth of 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 having pharmacological properties may be delivered or administered in different forms; for example, the ion may be administered orally, while the peptide may be administered by IV or IP.
  • the peptide 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 or protein.
  • 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 subject.
  • SUBSTITUTE SHEET(RULE28) host in combination with an antibiotic selected from the class consisting of bacitracins, gramacidin, polymyxin, vancomycin, teichoplanin, aminoglycosides, hydrophobic antibiotics, penicillins, monobactams, or derivatives or analogues thereof.
  • an antibiotic selected from the class consisting of bacitracins, gramacidin, polymyxin, vancomycin, 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_ , gentamicin C. ) , netilmicin, 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) , oxacillin, 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; azithromycin; 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.
  • macrolides such as erythromycin, roxythromycin, clarithromycin, etc.
  • 9-N-alkyl derivatives of erythromycin midecamycin acetate
  • azithromycin flurithromycin
  • 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 administered by direct administration to a target cell or by systemic or tropical 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 concentration 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.
  • Table I which follows, indicates the Minimal Inhibitory Concentration (MIC) in ⁇ g/ml of various peptides, against S.aureus strain ATCC 25923 (S) , P. aeru ⁇ inosa strain ATCC 27853 (P) , and E. coli ATCC strain 25922(E) , and C.albicans (CA) .
  • a "D” indicates that each amino acid residue is a D-amino acid residue or a glycine residue.
  • SUBSTITUTESHEET(RULE28) unsubstituted at the N-terminal, substituted with an acetyl group at the N-terminal as indicated by Ac-; substituted with an octanoyl group at the N-terminal as indicated by Oct-, substituted with sphingosine as indicated by Sph- ; substituted with a succinyl group, as indicated by Sue-; substituted with a hexanoyl group, as indicated by Hex-; substituted with a heptanoyl group, as indicated by Hep-; substituted with a valeryl group, as indicated by Val-; substituted with a myristryl group, as indicated by Myr- ; or substituted with an ibuprofyl group, as indicated by Ibu- .
  • 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/ml of either S.aureus ATCC 25923, E. coli ATCC 25922, P. aeru ⁇ inosa ATCC 27853, or C.albicans, were added to the wells in full strength Mueller Hinton broth (BBL 11443) from a mid-log culture.
  • the inoculum is standardized spectrophotometrically at 600 nm and is verified by colony counts.
  • the plates are incubated for 16-20 hours at 37°C, and the minimal inhibitory concentration (MIC) for each peptide is determined.
  • Minimal inhibitory concentration is defined as the lowest concentration of peptide which produces a clear well in the microtiter plate.
  • the minimal inhibitory concentration of each of the peptides with and/or without the appropriate substitutions is given in Table I below.
  • SUBSTITUTE SHEET (RULE 28) Oct- (SEQ ID NO 120) - H 2 Oct- (SEQ ID NO 121) -NH Oct- (SEQ ID NO 122) -NH Oct- (SEQ ID NO 123) -NH Oct- (SEQ ID NO 124) -NH Oct- (SEQ ID NO 125) -NH Oct- (SEQ ID NO 126) -NH Oct- (SEQ ID NO 127) -NH Oct- (SEQ ID NO 128) -NH Oct- (SEQ ID NO 129) -NH Oct- (SEQ ID NO 130) -NH Oct- (SEQ ID NO 131) -NH 2 Oct- (SEQ ID NO 132) -NH Oct- (SEQ ID NO 133) -NH Oct- (SEQ ID NO 134) -NH Oct- (SEQ ID NO 135) -NH Oct- (SEQ ID NO 136) -NH Oct- (SEQ ID NO 137) -NH Oct- (SEQ ID NO 138) -NH Oct- (SEQ ID NO 139
  • CFUs colony-forming units
  • Antimicrobial susceptibility tests are performed according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) (Document M11-T2, 1989) .
  • Microtiter plates (Corning, Corning, NY) are filled aseptically with BHI broth (plus hemin plus vitamin K ) to a volume of 100 ⁇ l by the use of a Beckman Biomek 1000 robotic instrument (Beckman Instruments, Palo Alto, CA) .
  • Peptides are tested in duplicate lanes by adding manually 100 ⁇ l of a 1.024 mg/ml peptide solution in water (w/v) to the top wells of a microtiter plate lane.
  • the peptide is diluted serially 1:2 by mixing and transferring 100 ⁇ l from the top well down to the bottom well in the lane by use of the Beckman Biomek 1000 (Beckman Instruments, Palo Alto, CA) . The last 100 ⁇ l from the bottom well is discarded. One hundred microliters of the bacterial are added in BHI (plus hemin plus vitamin K ) to each test well to give final peptide dilutions from 0.25 ⁇ g/ml. The plates are incubated in the anaerobic
  • Oct.- (SEQ ID NO:143)-NH_ then was injected intravenously via the tail vein at 1 and 5 hours post-inoculation.
  • Control mice were inoculated and treated with 0.9% saline. Each different treatment group had 10 mice per group. All control mice died.
  • Treatment doses of Oct- (SEQ ID No:143)-NH 2 were 1, 5, 10, and 20 mg/kg in toto, and resulted in 20%, 40%, 90%, and 90% survival at six days post-inoculation, respectively.
  • Oct- (SEQ ID NO:143)-NH was injected intravenously into male C57BL/6J mice (average body weight, 20. Ig) approximately two minutes prior to intraperitoneal injection of a solution of lipopolysaccharide (either 0.1 ⁇ g or 0.5 ⁇ g mouse) from E.coli serotype 0111 :B4 and galactosamine (8 mg/mouse) .
  • Treatment doses of Oct.- (SEQ ID NO:143)-NH were 0, 5, 7.5, 10, 12.5, or 15 mg/kg (10 mice/group) , and when administered prior to 0.5 ⁇ g lipopolysaccharide/mouse resulted in 10%, 0%, 30%, 0%, 50%, and 60% survival at five days post-lipopolysaccharide administration, respectively.
  • the results were 40%, 90%, 100%, 100%, 100%, and 100% survival at five days post-lipopolysaccharide administration, respectively.
  • a stock solution (lOx) of 0.6 mM dye is prepared by adding 1.68 mg of (l-ethyl-2- (3- [1-ethylnapthol (1, 2-d) - thiazolin-2- ylidene] -2-methylpropenyl) naphtho- (1, 2-d) - thiazolium bromide (Signa E-7762) to 5 ml of 200 proof ethanol . 1 ml of this solution was added to 9 ml ethanol to give 0.06 mM of dye (60 ⁇ M dye) .
  • LPS lipopolysaccharide
  • Row 1 and rows 3 through 12 of a microtiter plate were filled with 100 ⁇ l of pyrogen free water or with 10 mg/ml of bovine serum albumin. 200 ⁇ l of peptide then is added to row 2 of the microtiter plate at a concentraiton of 1 ml/ml. 200 ⁇ l of pyrogen free water is added to each of the control wells in two lanes (having dye and LPS but no peptide or having dye and no LPS and no peptide) . 100 ⁇ l then is serially diluted from row 2 through row 12 of the microtiter plate. 50 ⁇ l of PBS (pH 7.4) and 50 ⁇ l of the LPS solution then are added to row 1 of the plate (blank wells) .
  • PBS pH 7.4
  • 50 ⁇ l of the LPS solution then are added to row 1 of the plate (blank wells) .
  • the dye-LPS-buffer mixture then is added to every well of the microtiter plate except to the blank wells and to the control lane that does not have LPS or peptide.
  • the plate is incubated for 10 minutes at room temperature in the dark and the absorbance at 460 nm and 510 nm is read. From these absorbances, the LPS50 value, which is the concentration in ⁇ g/ml of peptide necessary to inhibit the binding of 50% of the lipopolysaccharide to the dye, is calculated.
  • peptides or proteins of the present invention may be employed in a wide variety of agents such as ions having pharmacological properties, antibiotics, or other biologically active peptides or proteins as hereinabove described, may be employed in a wide variety of agents such as ions having pharmacological properties, antibiotics, or other biologically active peptides or proteins as hereinabove described, may be employed in a wide variety of agents such as ions having pharmacological properties, antibiotics, or other biologically active peptides or proteins as hereinabove described, may be employed in a wide variety of agents such as ions having pharmacological properties, antibiotics, or other biologically active peptides or proteins as hereinabove described, may be employed in a wide variety of agents such as ions having pharmacological properties, antibiotics, or other biologically active peptides or proteins as hereinabove described, may be employed in a wide variety of agents such as ions having pharmacological properties, antibiotics, or other biologically active peptides or
  • SUBSTITUTE SHEET (RULE 28) 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.
  • the peptides or proteins 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 peptides or proteins may be administered 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, or in an amount effective in treating septic shock in a host.
  • the peptides or proteins may be administered 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.
  • the peptide or protein is administered in combination with an agent as hereinabove described, it is possible to administer the peptide 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 administered 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.
  • a water-soluble vehicle which may be employed include, but are
  • SUBSTITUTESHEET(RULE28) not limited to, glycols, such as polyethylene glycol, hydroxycellulose, and KY Jelly.
  • the water-soluble vehicle is preferably free of an oil substance.
  • the peptide or protein may also be employed alone, or in combination with an ion having pharmacological properties, 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, gingivitis, and/or to prevent or treat or reduce dental caries.
  • the peptide and ion having pharmacological properties may be used to inhibit, prevent, or destroy the growth of Streptococcus mutans. which is associated with dental caries and periodontal disease.

Abstract

Peptide ou protéine à substitution N-terminale correspondant à la formule (1), dans laquelle X est un peptide ou protéine biologiquement actif formant un canal ionique amphiphilique, T est une fraction lipophile et, de préférence, représente (2) (où R est un hydrocarbure (alkyle ou aromatique ou alkylaromatique) comportant au moins 2 et au maximum 10 atomes de carbone. T est de préférence un groupe octanoyle. W est T ou hydrogène. Ces peptides et protéines à substitution N-terminale présentent une activité biologique améliorée contre des cellules cibles, des virus et des cellules infectées par un virus.
EP95909267A 1994-01-18 1995-01-18 Peptides amphiphiliques formant des canaux ioniques et presentant des modifications n-terminales Withdrawn EP0750632A1 (fr)

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EP1001800A2 (fr) * 1997-07-15 2000-05-24 Magainin Pharmaceuticals Inc. Peptides biologiquement actifs a toxicite reduite chez l'animal et procedes d'elaboration
KR100314721B1 (ko) * 1998-01-22 2001-11-23 김일웅 생물학적 활성이 있는 신규한 펩타이드
NL1008745C2 (nl) * 1998-03-30 1999-10-01 Stichting Tech Wetenschapp Therapeutisch werkzame verbinding voor behandeling van gist- en/of schimmelinfecties in de mondholte.
WO2001060162A2 (fr) * 2000-02-15 2001-08-23 Ohio University Peptides cationiques presentant une structure secondaire amphipatique de feuillet beta et leurs utilisations
WO2001098362A2 (fr) * 2000-06-16 2001-12-27 Hercules Incorporated Peptides modifies chimiquement, compositions et leur procede de production et d'utilisation
GB0024428D0 (en) * 2000-10-05 2000-11-22 King S College Absorption enhancers
CN101155825B (zh) * 2005-02-09 2014-11-26 赫里克斯生物医疗公司 抗微生物六肽

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JPH09507669A (ja) 1997-08-05
AU693518B2 (en) 1998-07-02
WO1995019370A1 (fr) 1995-07-20
AU1728895A (en) 1995-08-01
CA2180748A1 (fr) 1995-07-20

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